Potassium channel activator drugs: mechanism of action, pharmacological properties, and therapeutic potential.

Potassium Channel Activator Drugs: Mechanism of Action, Pharmacological Properties, and Therapeutic Potential Susan D. Longman and Thomas C. Hamilton SmithKline Beecham Pharmaceuticals, Medicinal Research Centre, The Pinnacles, Coldharbour Road, Harlow, Essex, United Kingdom

I.

D. Molecular Biology of K + Channels 11. Cardiovascular

n Vitro Studies . . . C. Cardiac.. . . . . . . . . . . . . . . . . . 111.

IV . V. Gastrointestinal Smo

........... ................................ ................................

VI . Uterine Smooth Mus VII. VIII. Central Neurones.. .. IX. Skeletal Muscle ................................ X. Other Effects of PCAs ................................................ ...................... XI. Concluding - Remarks . . . . . . . . . . . . . . References. ......................................................................

122 123

134 136

I. INTRODUCTION This review is primarily concerned with the properties of a new class of smooth muscle relaxant drug, namely, the potassium (K ) channel activators or openers (PCAs). These drugs appear to possess a novel mechanism of action, since they have been found to open membrane K + channels, causing an increase in outward K+ conductance. We have reviewed evidence for the influence of PCAs upon smooth muscle tone and their ability to alter K + efflux and hence membrane potential. The therapeutic potential of these smooth muscle relaxants for indications such as hypertension, asthma, and incontinence has been examined in animal models. Where available, relevant clinical data have also been reviewed. Additionally, the wider effects of PCAs in nonsmooth muscle tissues, such as skeletal muscle, neurones, pancreas, and the heart, have been examined. The rapid, recent expansion of interest in the role of K + channels in the +

Medicinal Research Reviews, Vol. 12, No. 2, 73-148 (1992) CCC 0198-6325/92/020073-76$04.00 0 1992 John Wiley & Sons, Inc.

74

LONGMAN AND HAMILTON

functioning of smooth muscle in health and disease has largely stemmed from the discovery that the benzopyran cromakalim (BRL 34915) relaxed vascular smooth muscle by a mechanism involving enhancement of membrane K + permeability.lf2 Subsequently, this property was shown to be stereospecific, with biological activity of cromakalim residing predominantly in the (-)enantiomer lemakalim (BRL 38227) rather than the (+)-enantiomer (BRL 38226)3 (Fig. 1). An early study with the nicotinamide derivative nicorandi14 demonstrated an ability to open K + channels in arteries. Since the description of a probable causal association between K+ channel opening and the relaxant activity of cromakalim and nicorandil, a number of other drugs-pinacidil, diazoxide, RP 49356, and minoxidil sulphate-have been found to relax smooth muscle by a mechanism attributed to the opening of K + channel(s). A similar mechanism has been proposed for other novel benzopyran analogs, including Ro 31-6930, EMD 52692 ( = SR 44866), EMD 56431, WAY 120491, SDZ PCO 400, S 0121, NIP 121, and HOE 234. Figure 1 shows the chemical structures of these drugs and emphasizes the heterogeneity of structural type between the family of cromakalimllemakalim congeners and other structures, including pyridylcyanoguanidines and tetrahydrothiopyrans. In addition, several published patents have disclosed other benzopyran structures (see Refs. 5 and 6). The evaluation of the mechanism of action of the PCAs has been aided by the availability of a variety of K +-channel-blockingdrugs (including venom toxins) and by advancement in electrophysiological techniques that has enabled the characterization of single K + channels. It is not the purpose of this article to review the wide distribution and complexity of K + channel types in all tissue-these have recently been comprehensively reviewed by others. A synopsis of some properties of K + channels in smooth muscle and known K + channel blockers is given below (see Ref. 7 and references therein). A. Voltage-Gated K + Channels Voltage-gated K + channels have been widely studied and consist of three subtypes of varying conductance-firstly, the delayed rectifier K + current ( IK; conductance 10-50 pS), which may be involved in repolarization of slow wave contraction. This channel is blocked by tetraethylammonium (TEA) and phencyclidine and is found in jejunum, portal vein, and bladder. Secondly, the inward rectifier K+ current ( I K I , 5-30 pS) is found in arterioles, intestine, and Tom C. Hamilton received his Ph.D degree in Pharmacology from Strathclyde University (Glasgow) in 1972, after graduating B.Sc in Pharmacy from the University of Glasgow. After appointments in the Pharmacology Department, Roche Products, Ltd., in Welwyn Garden City, he joined Beecham Research Laboratories in 1976 and became Manager of the Anti-Hypertension Research Project in 1981. He is currently Head of the Cardiovascular Product Support Group at SmithKline Beecham Pharmaceuticals. His research interests include the pharmacological actions of Kf channel activator drugs with particular emphasis on their effects in smooth muscle. Susan D. Longman received her Ph.D degree in Pharmacology from Bradford University in 1987, after gaining a B.Sc degree in Physiologyfrom the University of ShefF’eld. She joined Beecham Research Laboratories in 1978 and pursued research interests in adrenoceptors and angiotensin converting enzyme. Currently assigned to the Cardiovascular Support Group at SmithKline Beecham Pharmaceuticals, her research focuses on the influence of K + channel activator drugs at a cellular level.

POTASSIUM CHANNEL ACTIVATOR DRUGS

75

CROMAKALM

BRL 38226

LEMAKALM

NC

NC

Me Me Ro 3 1-6930

EMD 52692 (SR 44866)

Me EMD 56431

Me

W A Y 120491

do

0

Me

SDZ PCO 400

s 0121

N

Me NIP 121

Figure 1. Chemical structures of K+ channel activators (PCAs).

HOE 234


76

aNyM

c1

'

,NH

o/Ao

DIAZOXIDE

PINACIDIL

oso; CSNHMe

0

0-

RP 49356 RP 52891 [(-)-enantiorner]

MINOXIDIL SULPHATE

ONO,

0

QNoz

NICORANDIL

H

NCN KFW 2391

Figure 1. Continued

NIGULDIPINE


77

cerebral artery. This channel, which is blocked by TEA, is inactivated by depolarization and activated by hyperpolarization, thus serving to modulate resting K + flux. Thirdly, the transient outward K + current ( I A , 20 pS) is activated by depolarization, sensitive to 4-aminopyridine (4-AP)and has been described in pulmonary artery and is commonly found in neurones.

B. Calcium-Activated K Channels +

Calcium (Ca2+)-activatedK + channels appear to exist as three subtypes dependent upon their ability to conduct K + , that is, the large- (100-300 pS), intermediate- (15-30 pS), and small- (10-14 pS) conductance K + channels. The channel with high conductance is found in a number of smooth muscles (arteries, airways, intestine, and portal vein) and functions to repolarize the action potential. A rise in the intracellular concentration of Ca2+ (i[Ca’+]) opens this channel, and the same effect is produced by depolarization at any maintained i[Ca’+]. TEA and charybdotoxin are blockers of this channel. The K + channel with low conductance is voltage insensitive, although both this and the K + channel with intermediate conductance are activated by increases in i[ Ca2+ 1. Charybdotoxin and quinine block the intermediate-conductance K + channel and apamin (also quinine and methylene blue) blocks the lowconductance K + channel. Both channels are found in portal veid. The lowconductance K+ channel, which is also found in taenia caeci, may modulate hyperpolarization and is activated by noradrenaline (NA) and extracellular ATP. Hence, this K+ current modulates the relaxation of taenia caeci produced by these agents.

C. Metabolically Gated K + Channels Metabolically gated K + channels in smooth muscle have also been described. In vascular smooth muscle, K+ channels regulated by intracellular concentrations of ATP (i[ATP])have been described. This channel is blocked by drugs such as the sulphonylureas and TEA and may function in hypoxia to reduce muscle tone and energy requirements. Recent evidence suggests that PCA drugs may open this K + channel in a number of tissues (See Table IV). Finally, in stomach smooth muscle, a K + channel (23 pS) activated by arachidonic acid, and some other fatty acids, has been described; the presence of Ca2+ and nucleotides is not required for activation of this K + channel, which modulates stomach relaxation. Although many of the K + channels described above have been categorized as distinct groups, some characteristics of a defined K+ channel may be common to other K+ channels. Some K + channels may be voltage sensitive in addition to those already defined as voltage-dependent K + channels. In addition, it is worth noting that many K+ channel blockers [e.g., TEA, barium (Ba”)] are not totally selective for a single class of channel, but may show differential selectivity between K + channels. The exception to this is apamin, which is highly specific for the low-conductance Ca2+ -activated K + channel. Sulphonylurea drugs are generally considered to be specific blockers of ATPsensitive K+ channels, although glibenclamide has also been reported to block an ATP-insensitive K ~ h a n n e land ~ ~ to , ~possess ~ thromboxane-A2-blocking activity at high concentrations.26 +

78


D. Molecular Biology of K + Channels Further understanding of the structure and function of K + channels should be aided by advances in molecular biology. To date, the cloning of K + channels has been restricted mainly to those resembling the transient outward K+ channel and delayed rectifier K+ channel in neuronal tissue and largely stems from studies using the Shaker mutant of Drosophilu fruit fly.27

E. Principle of K + Channel Opening and Inhibitory Responses Whilst the emergence of PCAs has occurred comparatively recently, the physiological role of K+ channel opening by endogenous substances (neurotransmitters and hormones) is a recognized inhibitory mechanism. For example, relaxation of intestinal smooth muscle by noradrenaline (NA) (via aadrenoceptor stimulation) is due to K + channel opening." In the heart, the depressant effects of acetylcholine (ACh), mediated by muscarinic receptor stimulation, are due to the opening of K + channel^.'^ In excitable cells the resting membrane potential is determined by the permeability of the plasma membrane to various ions (K+, Caz+,Na+)including the transmembrane movement of ions determined by the activity of the Na+/K+ ATPase pump (for Na+ extrusion and K + influx). This pump helps to maintain intracellular K + (i[K+])at 150 mM while extracellular K + is approximately 25 times lower at 6 mM. However, if K + channels are opened, for instance by neurotransmitters, hormones, or synthetic drugs, then K effluxes from the cell and the membrane potential is raised (i.e., hyperpolarized) towards the K + equilibrium potential (Ek)(approximately -90 mV). Once this value is reached no further net loss of K + occurs. Addition of K + to a physiological buffer would alter the cell membrane potential to a new Ekr resulting in contraction only if the new Ekis less negative than the potential at which the voltage-dependent Ca2+ channel opens (approximately - 45 mV).30As a consequence of this hyperpolarization, the probability of other ion channels achieving the threshold voltage for allowing Ca2+ and Na+ to enter cells is greatly diminished. Thus PCA drugs should be able to relax contractions to 20 mM KC1 (Ek approximately -50 mV), but not contractions produced by 80 mM KCl (Ekapproximately -20 mV).l It has also been suggested that PCA drugs may also modulate intracellular Ca2+ release (see Secs. 1I.A and 111). The probable sequence of events from K+ channel opening to smooth muscle relaxation is depicted in Fig. 2 and demonstrates a link between K + channel opening, hyperpolarization, and reduction in both Ca2 influx and in the release of Ca2+from intracellular stores. +

+

11. CARDIOVASCULAR

A. Vascular Smooth Muscle-In Vift.0 Studies In a variety of vascular tissues, including rat portal vein,'^^^"^ rat and rabbit and rabbit mesenteric a r t e ~ y , ~PCA , ~ ' drugs display the ability to relax smooth muscle by inhibiting both spontaneous tone and/or spasmogeninduced contraction. Although these drugs are capable of influencing other


I

PCAdrugs

79

1

I I

K+ channel opening

Enhanced K+ efflux

Hyperpolarisation

I

Reduced Ca2+entry/release

I

Smooth muscle relaxation

I

Figure 2. Flow diagram to represent sequence of events from K + channel opening to smooth muscle relaxation.

tissues, such as cardiac muscle, they display selectivity for vascular smooth muscle (see Table I). PCA drugs have been shown to inhibit contractions to a wide range of agonists, including NA, angiotensin I1 (AII), 5-hydroxytryptamine (5-HT), and h i ~ t a m i n e . These ~ , ~ ~agonists , ~ ~ ~ vary ~ in the degree to which they utilize the influx of extracellular Ca2+ through voltage-sensitive ion channels. Thus studies investigating the ability of PCAs to relax agonist-induced contractions have been useful for distinguishing the characteristics of these drugs from other smooth muscle relaxants such as dihydropyridine (DHP) Ca2+ antagonists. In rabbit aorta, despite some similarity between the action of cromakalim and the Ca2+antagonist isradipine (the lack of influence upon contractions elicited by low concentrations of NA), they displayed distinct profiles in terms of antivasoconstrictor activity. For example, cromakalim (0.1-100

CRK 0.5-50 pM2 PIN 10 pM43 NIC 5-500 uM2

CRK 10 pM41 PIN 10 pM43

Hyperpolarization

CRK 0.410 fiMM

CRK 0.5-100 pM" PIN 30 pMM NIC 10-500 pM4'

CRK ICx, 0.14 pMW PIN ICm 0.064 pMM NIC ICm 7 wMM

Rabbit mesenteric artery

CRK ICx, 0.002 pM39 PIN ICm 0.046 pM39 NIC ICm 1.3 pM39

Rabbit portal vein

Rabbit papillary muscle

=

pinacidil, NIC =

CRK ICx, 44 pM39 PIN ICm 79 pM39 NIC -22% at 300 pM39

"Values are expressed as ICm or the concentration range over which such effects were observed. CRK = cromakalim, PIN nicorandil. Superscripts are reference numbers, not exponents.

CRK 0.5-50 pM2 PIN 3 3 0 pM43 NIC5-500 pM2

CRK 0.5-5 pM2 PIN ICx, 0.63 pM3' NIC 5-500 wM2

CRK 0.610 pM2r41 PIN 3-30 pM43 NIC slight effect 32 pM2

CRK 0.2-0.8 pM2 NIC 8-32 pM2

CRK 0.1-50 pMz PIN 0.3-30 p M 1 NIC 1-500 wMZ

Rat portal vein

Increase in =Rb+ efflux

Negative inotropic activity

Inhibition of contractions to KCL (5-30 mM)

Inhibition of spontaneous contraction

Rat aorta

Table I Effects of Cromakalim, Pinacidil, and Nicorandil upon Vascular Smooth Muscle and Cardiac Tissue"

8z

P

5z

00 0


81

pM) produced greater depression of A11 and 5-HT-induced contractions than isradipine (1 nM-10 F M ) . ~In~addition, cromakalim inhibited tonic contractions to NA, whereas isradipine was without effect.47Another contrast between the depressant activity of cromakalim (0.1-10 pM) and the Ca2+ antagonist nimodipine (1 nM-1 pM) upon 5-HT-induced contractions has been described in rabbit mesenteric and basilar arteries.49In rabbit mesenteric artery, 5-HT concentration-response curves were depressed by cromakalim but only slightly affected by nimodipine. However, in basilar artery, only the first component of the 5-HT concentration-response curve was cromakalim sensitive, unlike nimodipine, which depressed both components of the curve. Numerous studies conducted in rat aorta or portal vein have demonstrated that unlike Ca2+ antagonists, PCAs, including cromakalim,2RP 49356,% Ro 31-6930,32dia~oxide,~' WAY 120491,33and NIP 121%are capable of reducing contractions evoked by low (G30mM) but not high (40-80 mM) concentrations of KCl. Although the differential ability of these drugs to inhibit contractions to KC1 has been used to distinguish between the mode of action of PCAs and Ca2+ antagonists, the picture becomes complicated by virtue of the fact that other drugs such as nitrovasodilators also display differential inhibition of KC1-induced contra~tions.~~ Interestingly, although KRN 2391 and FA080 evoked preferential inhibition of contractions to 20 mM KCl, responses to higher concentrations (50-80 mM KC1) were also ir~hibited.~',~~ These drugs appear to possess mixed characteristics of a PCA and Ca2+ antagonist, a property discussed in greater detail at a later stage. Another example of a drug with mixed properties is niguldipine, which inhibits dihydropyridine (DHP)-sensitive Ca2+ channels and also opens a Ca2+-activatedK + PCA drugs such as pinacidil or cromakalim are effective in relaxing agonistinduced (NA) or K +-induced contractions in rat isolated mesenteric resistance vessels56or perfused mesenteric bed.57This is of interest because these vessels exhibit a major influence upon total peripheral resistance (TPR)and may differ from larger vessels in terms of agonist-induced depolarization and Ca2+handling.58,59 Despite distinct structural dissimilarity, a stereospecific mechanism appears to underlie the nature of vasorelaxation induced by PCA drugs, with biological activity residing predominantly in the (-)-enantiomers of cromakalim (namely le~nakalim),~ pinacidil," RP 49356 (i.e., RP 52891) (Cavero, personal communication, 1988) and SDZ PCO 400.35 Early electrophysiological studies in porcine and guinea pig coronary arteries demonstrated an increase in K + ion conductance and membrane hyperpolarization following administration of nicorandil(2-5 P M ) . Further ~ studies with nicorandil demonstrated a decrease in membrane resistance and abolition of spontaneous electrical discharge in a variety of tissues, including porcine and guinea pig mesenteric arteries and guinea pig and rat portal veins.2*60,61 Since nicorandil is a nitrate-containing nicotinic acid derivative, comparisons have been made with nitrate containing vasodilators such as glyceryl trinitrate and sodium nitroprusside. The hyperpolarizing effects of nicorandil (largely seen at concentrations >10 pM) appeared to di€fer from the effects of nitrovasodilators, which has little influence upon membrane potential or resistance.41*62,63 Nevertheless, a proportion of the vasorelaxation observed with

82


Tension

h

0.5g[

h n , l

t 0 . 1 pM Membrane Potential mV

Tension

O[

-90 0.5g[

I

n Min

)I__

, (b)

1 t5PM

1

Min

,

Figure 3. The effect of exposure to cromakalim [(a) 0.1 pM, (b) 5 pM] on membrane potential (upper traces) and tension (lower traces) in a single experiment in rat portal vein. The traces are parts of a continuous impalement of a single cell with washout of cromakalim and recovery between (a) and (b) (reproduced from Ref. 1 with permission).

nicorandil lies in its nitro-moiety and an ability to stimulate guanylate cyclase. Cromakalim (0.1-10 p M ) possesses hyperpolarizing activity in a wide range of vascular tissue, including rat portal vein and rabbit mesenteric artery, rat and rabbit aorta, and rabbit pulmonary a ~ - t e r y ' , ~(see ' , ~ Fig 3). Similar observations have also been made in smooth muscle cells isolated from rabbit portal vein and bovine and rabbit a ~ r t a . ~ ~ , ~ ~ P i n a c ipM), d i l ( llike O cromakalim, produced a marked hyperpolarization in rat portal vein.42 l'inacidil has also been shown to hyperpolarize rat caudal artery and mesenteric resistance vessel^^^,^' and rat azygous vein cultured smooth muscle cells.71Other drugs, by virtue of their ability to hyperpolarize vascular tissue, have been recognized as PCA drugs, including EMD 52692, minoxidil sulphate, diazoxide, E4080, and HOE 234.37,46,53,72,73 Studies conducted in rat portal vein have demonstrated that low concentrations of nicorandil (1-5 pM), cromakalim (0.1-0.5 p M ) and pinacidil (0.33 pM) abolished the characteristic spontaneous multispike complexes and inhibited (decreased frequency and amplitude) mechanical activity of this vessel. 1,2,31,41 These effects were generally not accompanied by a detectable hyperpolarization, which was noted at higher concentrations of the respective drugs (nicorandil50 pM, cromakalim 5 pM, and pinacidil 10 pM). Microelectrode studies, such as those conducted in rat portal vein,' have assisted in understanding the basis of the hyperpolarization evoked by cromakalim and other PCAs, in terms of increased K + ion conductance. In the above study, cromakalim (5 pM) changed the cell membrane potential to a

83


value (-88 mV) close to the calculated K + equilibrium potential (EJ. This suggested that the drug was able to open K+ channels that are normally closed at the resting membrane potential. The ability of drugs such as cromakalim to inhibit responses only to low concentrations of added KCl (as previously described) is entirely consistent with their ability to open K + channels. One notable discrepancy is the ability of nicorandil to inhibit contractions to high concentrations of KCl in rat portal vein,’ supporting other evidence that the drug has additional properties. Since cromakalim-evoked hyperpolarization in rat portal vein was observed only at concentrations higher than required to inhibit spontaneous electrical discharges (see above), this may indicate an interaction at two distinct K + channel populations. The inhibitory effects seen at low concentrations may represent an action upon K + channels of pacemaker cells (involved in spike repolarization) within the portal vein. 74 It is possible that hyperpolarization effects observed at higher concentrations may involve an interaction with additional K + channels. The electrophysiology studies outlined above indicate that drugs such as nicorandil and cromakalim influence K + ion movement. Thus, attempts have been made to monitor the effects of these drugs upon K + efflux in a variety of tissues, mostly by use of 86Rb+as a marker for K + . The validity for using 86Rb+has been described in canine trachea” and also in rabbit and guinea pig arterial tissue,76where no qualitative difference was noted between agonist (NA) and K + evoked 86Rb+and 42K+ efflux. In rat portal vein, but not thoracic aorta, nicorandil (5-500 pM) stimulated =Rb+ efflux.* Subsequent work with cromakalim, lemakalim, pinacidil, minoxidil sulphate, diazoxide, Ro 31-6930, WAY 120491, and SDZ PCO 400 has demonstrated increases in 86Rb+efflux in either rat portal vein or a wide variety of rat and rabbit arterial tissues1-3,3133,35,42,43,51,77 (see Fig 4). Studies employing 42K+ have also demonstrated increases in K e f f l u ~ , ~ ’ , ~ ~ substantiating results obtained using =Rb+. The ability of a PCA to display differential vasorelaxant potency in canine, rabbit, or rat arterial tissue,7941 coupled with a wide variation in the maximum increase in 86Rb+efflux evoked by NA or ~ r o m a k a l i m , ’ ~ ,has ~ ~ ,suggested ~~ that K + channels of differing vascular origin may exhibit considerable heterogeneity. This is supported by evidence that minoxidil sulphate may open a K + channel (evoking 4’Kf efflux) in rabbit mesenteric arteryn but not (using =Rb+ as a marker) in rat aorta.83 Similar results with %Rb+ in rat aorta have been reported for nicorandil.2 However, studies demonstrating minimal variation in the stimulation of %Rb efflux produced by cromakalim (1-10 pM) in different rabbit arterial preparationsQ tend to confound this proposal and indicate that a degree of homogeneity may exist between the K + channels opened by such drugs. Interestingly, the concentration of a PCA drug required to produce increases in K + ion efflux exceeds those concentrations necessary to induce vasorelaxation (see Table I). It was originally suggested that the hyperpolarization evoked by PCAs prevented the opening of voltage-dependent Ca’+ channels.’ This has been supported by evidence that the vasorelaxation produced by pinacidil and nifedipine may be a consequence of reduced i[Ca’+] (measured by fura-2 methodM). However, data have already been described above that allude to distinct differences between PCAs and Ca2 antagonists. Furthermore, evi+

+

+


d 5

2 X 3 -

-

' ' '

I ' , 0 ,

I

I

a,

Cromakalim Control

+

o

15

30

45

60 min

ii)

120 100

80 60 40 20

0

pM Nicorandil

pM Cromakalim

(c) Comparison of the effect of cromakalim on '%b+ efflux rate in different arterial preparations of the rabbit

Tissue Pulmonary artery Ear artery Brachial artery Abdominal aorta

Basal rate ("A) per 3 min) 1 92

* 0 23

211*019

*

1 52 0 10 1 78 t 0 18

Max stimulation ("10)

concentration

(Lw

(V

106 t 18

10

7

50 16 93 k 27 96 t 16

1

6 6 5

*

Cromakalim

10 3

Values are mean f SEM N = number of preparations

Figure 4. (a) A typical mean %-rubidium (%Rb') efflux curve from rabbit isolated mesenteric artery and the effect of cromakalim upon such a curve. Cromakalim (5 pM), when present, is indicated by the horizontal bar. Efflux points are the mean with standard error of 6 experiments. (b) Stimulation of ffiRb+efflux from rabbit isolated mesenteric artery by (i) cromakalim and (ii) nicorandil. Stimulation of efflux rate, at each drug concentration, is the maximum efflux rate observed during exposure to the drug divided by the basal efflux rate and is presented as mean with standard error bar from number of experiments shown in parenthesis. (c) Comparison of the effect of cromakalim upon &Rb+ efflux rate in different arterial preparations of the rabbit. (Reproduced from M.C. Coldwell and D.R. Howlett, Biochem. Pharmacol, 36, 3663 (1987), with permission. 0 1987 Pergamon Press PIC.)


85

dence that cromakalim-evoked increase in =Rb efflux or hyperpolarization of vascular tissue was not influenced by lanthanum (La3+),or the Ca2+ antagonist nifedipine, is indicative that the action of drugs such as cromakalim is not dependent upon the influx of external Ca2+.43*44,81,85 The relationship between PCAs and voltage-operated Ca2+channels may differ between vascular preparations, as judged by the ability of cromakalim to hyperpolarize rat aorta only after the tissue was depolarized.a The ability of these compounds to block dihydropyridine-insensitive influx of Ca2+ (as evidenced by inhibition of the sustained phase of an NA contraction) indicates that PCAs may prevent Ca2+ entry through receptor-operated Ca2 channel^.^^,^ Furthermore, preliminary studies have shown that PCA drugs may display a continuous inhibitory influence upon phasic responses to NA in rabbit aorta (followingre-exposure to Ca2+)after a prolonged Ca2+free period and also upon caffeine-induced contractions in rabbit renal artery. These observations have inferred that PCA drugs may possibly inhibit the refilling of intracellular Ca2+stores and also the release of Ca2+from such stores.46,87,88 In addition, cromakalim has been shown to inhibit the contraction and 45Ca2+entry produced by NA in rabbit aorta.89Inhibition of NAevoked 45Ca2+uptake was also observed with cromakalim and lemakalim in rat aortic smooth muscle,g0although cromakalim was without influence upon basal or NA-evoked 45Caz+release in rabbit aorta.91 In contrast, nicorandil has been shown to decrease i[Ca2+]and Ca2+ transients (quin-2 method) evoked by NAg2and also to inhibit NA-induced 45Ca2+effluxg1,although this latter effect may be due to an influence upon guanylate cyclase (see below). Pinacidil and nicorandil have been reported to inhibit basal levels or agonistinduced increases in i[Ca2+]in guinea pig femoral artery, rat aorta, or rat vascular smooth muscle cells.84,93,94 Few additional studies have been concerned with examining the effects of PCAs upon levels of i[Ca2+].However, a redistribution of Ca2+(measured using a fura-2 technique) has been reported within the sarcoplasmic reticulum (SR) of rat venous smooth muscle cells following treatment with p i n a ~ i d i l . ~ ~ The consensus of data indicates that PCA drugs share a common mechanism of action, although there appears to be some disparity between the concentrations of drug necessary to open K + channels and those at which vasorelaxation occurs (see Table I). In addition, drugs such as cromakalim have been reported to evoke antispasmogenic activity (in rat aorta) without causing hyperpolarization.2Such evidence highlights the deficit in knowledge regarding the mechanism underlying hyperpolarization-associatedvasorelaxation. Discrepancies such as the ability of high concentrations of pinacidil(l0-500 FM) to inhibit contractions to high concentrations (80-145 mM) of KC1,96,97 or to retain the ability to relax NA-induced contractions in rabbit aorta depolarized by 35 mM KCltg6has suggested that actions additional to K + channel opening may contribute to the vasorelaxant properties of pinacidil. Such additional properties of pinacidil are not considered to include stimulation of a Na+/K+pump, Na+/Ca2+exchange, or SR Ca2+uptake.97The ability of high concentrations of pinacidil to inhibit Ca2+-induced contractions in K+ depolarized strips of canine mesenteric artery has suggested that pinacidil may inhibit Ca2 influx.81Evidence that pinacidil produced a greater stimulation +

+

+

86


of =Rb+ efflux in the presence of nifedipine has inferred an inhibitory action upon Ca2+-activatedK + channels," although others have suggested an additional inhibitory influence upon receptor-mediated GTP-binding proteincoupled phosphatidyl inositol (PI) turnover.94 Evidence in guinea pig mesenteric artery and rat aorta that nicorandil induced mechanical inhibition2,60without change in membrane potential or in =Rb+ efflux, and also inhibited contractions evoked by high concentrations of KCl,2,38,98 have provided substantial information to infer that nicorandil possesses an additional property. This is believed to be stimulation of guanylate cyclase resulting in increases in c G M P , ~a~property ,~ common to other vasodilators such as sodium nitroprusside (SNP).41In contrast, other PCA drugs such as cromakalim and pinacidil do not change levels of cGMP and CAMPor stimulate guanylate cyclase or adenylate c y c l a ~ eHowever, . ~ ~ ~ ~ in ~ ~ ~ rabbit mesenteric artery, cromakalim-evoked increase in =Rb + efflux is inhibited by methylene blue (inhibits soluble guanylate cyclase)99and forskolin (activates adenylate cyclase) (Howlett, unpublished). In addition, methylene blue has been shown to inhibit cromakalim-induced relaxation of contractions to 5-HT in rat basilar artery,50suggesting that it may be a putative K + channel blocker. Forskolin may also act as a K + channel blocker in this instance, since it has previously been shown to block K + channels in pancreatic p-cells.'oo However, in contrast, forskolin has been reported to possess "PCA activity in a variety of vascular tissues (Howlett, unpublished; Ref. 101). Another additional action of nicorandil is the ability to cause extraneuronal accumulation of 3H-isoprenalinein segments of rabbit main pulmonary artery."' The consequence of this property in vivo is unknown, although it does suggest that resting membrane potential may modulate Uptake'. Although nicorandil and nitrovasodilators share some properties, PCAs in general exhibit dissimilarity in profile from such drugs. PCA drugs such as cromakalim do not require an intact endothelium to produce relaxation of vascular tissue.41Much interest has recently centered upon the contribution made by endothelium-derived relaxing and contracting factors to the maintenance and control of vascular tone. Current theory suggests that the vasorelaxant effect of some substances (e.g., Ach) is a consequence of the release of endothelium-derived relaxing factor (EDRF) (which may itself be nitric oxide) and subsequent stimulation of guanylate cyclase. Elevation in cGMP may activate cGMP-dependent protein kinase leading to dephosphorylation of myosin light chains and ultimately re1a~ation.l'~This vasorelaxation is a consequence of a lowered cytosolic concentration of Ca'+, which is thought to result predominantly from cGMP and G-kinase-enhanced plasmalemmal Ca'+-extrusion" and also by the sequestration of Ca'+ into an intracellular store, such as SR.lo5 Substances such as Ach and Substance P have been shown to evoke endothelium-dependent relaxation and also hyperpolarization. However, although nitrovasodilators may produce relaxation and activation of guanylate cyclase, in general they fail to hyperpolarize vascular smooth muscle. Discrepancies such as this have led to the suggestion that EDRF may consist of at least two distinct factors, one of which activates guanylate cyclase and the other which induces hyperpolarization (EDHF) (for a review, see Ref. 106). Although the vasorelaxant effects of PCAs are not endothelium dependent,


a7

recent studies in rat a ~ r t a ~ ~ have , ' ~ suggested ~,'~ that cromakalim (3-10 pM), lemakalim (0.3-3 pM), and HOE 234 (0.1 pM) inhibit contractions evoked by exogenous endothelin, a 21 amino acid peptide released by vascular endothelial cells.'09 Upon endothelial removal, the relaxant property of HOE 234 was significantly reduced. 73 The mechanisms involved in endothelin contraction are not fully understood. It appears to elicit contractions via the entry of extracellular Ca2+but not through direct interaction with a DHP-sensitive Ca2+ channel.'" The nature of any interaction between PCA drugs and endothelin requires further investigation and highlights a deficit in knowledge regarding the complex association between the release of substances from the endothelium and their subsequent influence upon vascular smooth muscle tone. Attempts have been made to investigate the possible influence of PCAs upon other second messenger and receptor mechanisms. Unfortunately, limited data are available for studies conducted in vascular tissue. However, in rabbit mesenteric artery cultured smooth muscle cells, cromakalim (30 p M ) had little effect in studies using 3H-nimodipine, thereby appearing to lack affinity for a DHP binding site (Longman, unpublished). Despite the lack of evidence for any direct interaction between PCAs and DHP binding sites, an initial report (in strips of rat portal vein) has indicated that the hyperpolarization produced by cromakalim may produce a change in the conformational state of voltage-dependent Ca2 channels and thereby inhibit 3H-desmethoxyverapamil binding. ll1 The ability of depolarization to produce similar effects is indicative that the binding of this ligand is highly dependent upon resting membrane potential. In rat cerebral membranes, cromakalim displayed negligible affinity of a100 pM) at a variety of other receptor binding sites, including 5-HT1, 5-HT2, P-adrenoceptors, dopamine receptors, and a'and az-adrenoceptors.43Thus the ability of phentolamine to modify the vasorelaxant effects and increase in =Rb+ efflux evoked by cromakalim (Table 11)has suggested that its activity may be unrelated to a1-adrenoceptorblockade but rather due to its effects as a putative K + channel blocker.'" Interestingly, the ability of diazoxide to open pancreatic K+ channels (leading to inhibition of insulin secretion) is inhibited by efaroxan (an az-adrenoceptor antagonist).l" Since guanine nucleotide-binding proteins (G-proteins) are known to provide a link between membrane receptors and intracellular events, the possibility exists that G-proteins may modulate the activity of a K + channel sensitive to PCA drugs. However, pertussis toxin (which activates inhibitory G-proteins) failed to modify cromakalim-induced vasorelaxation or enhancement of =Rb+ efflux in the rat aorta and portal vein (Ref 123; Coldwell, unpublished). Cholera toxin (which activates stimulatory G-proteins) did not influence cromakalim-enhanced 86Rb efflux in rabbit mesenteric artery cultured smooth muscle cells (Howlett and Longman, unpublished). These results tend to rule out any involvement of PCAs with G-proteins, with the exception of an action through pertussis or cholera toxin-insensitive G-proteins. The putative effects of PCA drugs upon second messengers such as protein kinase C have also been investigated. Although phorbol esters inhibited cromakalim-induced increase in &Rb+ efflux, no evidence exists for a direct influence of such drugs upon this en~yrne.'",'~ Similarly, PCA drugs +

+

I ( +)

Zromakalim ( - )

Low-conductance Apamin Caz+-activated 0.1 pM; K + channel 0.15 mg/kg i.v.

I

Cromakalim Pinacidil Diazoxide Minoxidil Sulph.

Cromakalim Pinacidil Nicorandil Diazoxide Minoxidil Sulph.

Nicorandil

Nicorandil

Cromakalim Pinacidil Nicorandil Minoxidil Sulph. Diazoxide NIP 121

I

Vasorelaxation

Cromakalim

Cromakalim (+)

Cromakalim

Electrophysiology (hyperpolarization; K+ conductance)

Effects on PCA

Cromakalim

( )

Cromakalim Pinacidil (+) Vicorandil

Procaine 1-5 mM

Nicorandil Minoxidil Sulph.

Pinacidil

Cromakalim

+

Rb +/K efflux

4-AP 6.3 pM-10 mM

Channel

Concentration (where reoorted)

+

Blocking agent

(+)

Cromakalim

( -)

Haemodynamic profile ( .1 blood pressure)

Table I1 Influence of K+ Channel Blocking Agents upon the Vascular Effects of PCA Drugs”

+

31,42,112,113

40,70,112

40,43

34,40,42,43,69, 70,77,112

Refs.

m

m


-

89

+

h

h

I

Y

Y

+

h

J

4

w

h h

I +

w w

+

h

h

Y

Y

w

+

-.-E 4E

8 h

I

w

E

4

2

E

8

+

h

+

h

w

90


have shown no effect upon phosphatidyl inositol (PI) turnover in brain tissue.42 Thus, since extensive studies failed to demonstrate an effect of PCA drugs upon membrane receptors and second messenger systems or to identify specific recognition sites for these drugs,42further work has attempted to characterize the nature of the K + channel opened by drugs such as cromakalim and has examined the sensitivity of PCAs to a wide range of K+ channel blocking agents (Table 11). The conclusion drawn from this is that it is probable that a common mechanism must underlie the vasorelaxant, ion flux, and electrophysiologcal properties of PCAs as indicated by their sensitivity to TEA, procaine, 4-AP,and Ba2+.The consistent inability of apamin to modify responses to PCAs has inferred that these drugs do not influence or interact with a low-conductance Ca2+-activated K+ channel. The situation is more complex when large conductance Ca2+-activatedK+ channels are considered. Studies in vascular smooth muscle cells from rabbit aorta or rat azygous vein have demonstrated that cromakalim (0.1-1 p M ) or pinacidil (10 pM) increased the "opening time" of large conductance Ca2+that were blocked by scorpion toxin or charactivated K + channel-ffects y b d o t o ~ i n . ~ ~Similarly, ,"~ in the rat portal vein, a high concentration of cromakalim (30 pM) enhanced the conductance of Ca2+-activated K+ channels (and inhibited a Ca2+ inward current).lZ6An increase in the "opening time" of large-conductance Ca2+-activated K+ channels (which had been incorporated into planar lipid bilayers) has been reported for cromakalim (0.05-0.5 pM).lZ7However, other reports have indicated that the vasorelaxation and the increases in 86Rb+ efflux and K+ current evoked by PCA drugs are not the result of an action upon large-conductance Ca2+-activatedK+ channels, as judged by insensitivity to charybdotoxin and the nature of the increased K f c o n d ~ c t a n c e . In ~ ~a~study ~ ~ ~ '(using ~ ~ whole cell or patch clamp techniques) conducted in vascular smooth muscle cells derived from rabbit portal vein, the rank order of potency of inhibitors indicated that the cromakalimsensitive K f current may be carried by channels which underlie the delayed rectifier current (IK) rather than the Ca2+-dependentK + c ~ r r e n t . ~ ~ , ~ ~ Further evidence to reject any involvement of Ca2+-dependentK+ channels with the K + current evoked by PCAs has come from the inability of blockade of Ca2+influx (with either La3+ or nifedipine) to inhibit the hyperpolarization or increase in 86Rb+efflux evoked by cromakalim (see above). In addition, cromakalim was able to enhance %Rb+efflux in rabbit mesenteric artery bathed in a Ca2+-freebuffer (containingEGTA) suggesting that the K+ channel opened by this drug is not dependent upon the influx of Ca2+, although Ca2+ is apparently required to close the cromakalim-sensitive K+ channel.85 K + channels that are inhibited by i[ATP] and sensitive to blockade by sulphonylureas such as glibenclamide have been described in heart, airway smooth muscle, pancreatic @-cells,skeletal muscle and neurones (see respective sections). Thus the consistent reversal by glibenclamide (albeit at higher concentrations than those effective in the pancreas) of PCA-induced inhibition of spontaneous or spasmogen-induced contractions of blood vessels, in addition to inhibition of both hypotension and enhanced %Rb+ eff l ~ ~ , ~ , ~has, suggested ~ ~ , ~ that ~ ~PCA , ~drugs ~ ~ may , ~act~ by~ opening ATPsensitive K + channels. These channels have been identified for the first time


91

in vascular smooth muscle.128Furthermore, the ability of glibenclamide to inhibit cromakalim (1 FM) evoked opening of such channels in rat and rabbit isolated mesenteric artery smooth muscle cells confirms an interaction between cromakalim and vascular ATP-sensitive K+ channelss (see Fig. 5). Interestingly, these authors reported that the hyperpolarizing actions of other smooth muscle relaxants [Ach, vasoactive intestinal polypeptide (VIP), and calcitonin-gene-related peptide (CGRP)] are sensitive to blockade by glibenclamide, suggesting that hyperpolarizing agents may act through a common mechanism by opening ATP-sensitive K + channel^.^^"^^'^^ Support for these findings has been derived from studies conducted in rabbit mesenteric artery cultured smooth muscle cells where cromakalim-enhanced s6Rb efflux was associated with modest decreases in the concentration of i[ATP].I3' Further studies demonstrated that a relationship may exist between cromakalim-induced K + channel opening and i[ATP]-the effects upon i[ATP] being stereospecific in nature and also sensitive to glibenclamide.I 3 l The single-channel conductance (described by Standen and co-workers) for the ATP-sensitive K + channel in rabbit mesenteric artery smooth muscle cells was 135 pS. This is greater in magnitude than the ATP-sensitive K + channel conductance in many other tissues. The properties of single-channel conductances for ATP-sensitive K + channels in a variety of tissues are shown in Table IV. Interestingly, cromakalim, pinacidil, and nicorandil have been reported to increase the open-time of a low-conductance (10-11 ps) ATP-sensitive K + channel in porcine coronary artery and rat portal vein smooth muscle The affinity of a given ATP-sensitive K+ channel for i[ATP], coupled with the density of such channels in the membranes of different tissues (see Ref. 132), may influence resting membrane potential and thus the action of drugs such as PCAs. Differences in the potency of sulphonylureas to block ATP-sensitive K + channels in vascular tissue (half-maximal inhibition of cromakalim-evoked 86Rb efflux was achieved with glibenclamide 0.3 pM'17) relative to other tissues [glibenclamide (60 pM) produced half-maximal inhibition of s6Rb+efflux following lowered i[ATP] in rat insulinoma cells133] has indicated that there may exist distinct physiological differences between ATP-sensitive K + channels of varying origin. In this respect, it is interesting to note that cromakalim and diazoxide, which both open K + channels in vascular tissue, show diverse activity in pancreatic p-cells (see Sec. VII). In contrast to diazoxide, cromakalim is more selective for K+ channels in vascular smooth muscle than in the pancreas5' (see Fig. 14). An initial study in rabbit vascular smooth muscle cells has suggested that cromakalim may enhance K+ current via an action upon low-conductance (7.5 pS) K + channels, insensitive to i[ATP] but sensitive to glibenclamide.25 This highlights the need for further characterization of the large number of K + channels and their functional role in vascular smooth muscle. Although substantial data exist to demonstrate an interaction between PCA drugs and sulphonylureas, no evidence of a direct interaction with a 3Hglibenclamide binding site has been reported in any tissue. To date, a recognition site for 3H-glibenclamide has not been described in vascular tissue. Thus, although initial reports suggest that PCA drugs may act at ATP-sensitive K+ channels, further evidence of the existence of these channels in vascular tissue and the exact nature of the antagonism by sulphonylureas are both +

+

7

3

2

1

0 1

4

3

2

PA

1

0

1

PA

O

4

3

2

1

0

1

PA

O

5 4

3

PA

2

1

0

1

Figure 5. Effect of cromakalim and glibenclamide on a membrane patch from rabbit mesenteric artery at a holding potential of 0 mV. Singlechannel recordings and amplitude histograms are shown: (a) in the absence of ATP in the solution bathing the cytoplasmic face of the patch; (b) in the presence of ATP (1 mM); (c) in the presence of ATP (1 mM) and cromakalim (1 pM); (d) and in the presence of ATP (1 mM), cromakalim (1 pM), and glibenclamide (20 pM). The single-channel recordings are continuous (left to right, top to bottom) and the amplitude histograms were made from longer records; 302.9 s in (a), 77.25 s in (b), 197.7 s in (c) and 196.4 s in (d). The mean unitary currents were -2.36 pA in (a), - 2.09 pA in (c) and -2.08 pA in (d) as determined from Gaussian fits to the histograms. The single-channel currents were inward at 0 mV because the bathing solution contained 6 mM K + instead of 120 mM (with Na+ substituted for K+). The data were filtered at 1 kHz. The closed level is indicated by arrows. (Reproduced from N.B. Standen, J.M. Quayle, N.W. Davies, J.E. Brayden, Y. Huang, and M.T. Nelson, Science, 245,177-180, with permission. 0 1989 American Association for the Advancement of Science.)

O 4

O

W

> 1 W

> 2

15

> 10

L 5 5

E

W 1

a

25

30

m 2o

X

.0

m

a,

4

E 2

cn

X

-3

0

6

10

:

c

m 15

X

-

m0 25 20

Fa, 3

v)

6 6 0 - 5 x 4

3

8z

$ d

z

6


93

issues that need to be addressed in order to broaden our understanding of the mechanism underlying the relaxant activity of PCA drugs.

B. Vascular Smooth Muscle-In

Vivo Studies

The effects of acute oral or intravenous administration with PCA drugs have been characterized by dose-related falls in systemic blood pressure in conscious normotensive animals, including the dog, cat, and rhesus monkey. 120,134-141 Studies have also been conducted using a wide variety of animal models of hypertension, including the DOCA-salt hypertensive and spontaneously hypertensive rat (SHR),36,51,119,120,134,141-145 the renal hypertensive dog,120,134,142 and renal hypertensive at.^^,^^^,'^^ An initial report of the bloodpressure-lowering ability of the PCA/Ca2+ antagonist KRN 2391 has been described in conscious or anaesthetized SHR5' and anaesthetized dog.146 Although PCAs have been shown to evoke reproducible hypotensive responses over a chronic period of daily oral dosing,119~135,136~138,141-143 an isolated incidence of cardiovascular tolerance has been reported in ovariectomized female rats following repeated intravenous administration of a high dose of cromakalim (1 mg/kg) over a 24-h period.147This observation remains unexplained at the present time. Early studies demonstrated that the development of DOCA-salt hypertension in rats could be prevented by diazoxide.lMLongterm pinacidil therapy has been shown to normalize the blood pressure in young SHRs treated from 1 month until 12 months of age.'& However, in a later study, once daily oral doses of cromakalim or SR 44866 failed to prevent the development of genetic hypertension in SHRs dosed from 1 until 5 months of age.149 In common with in vitro studies, the in vim activity of PCA drugs appears to be stereospecific in nature, as demonstrated for pinacidil and cromakalim.136,142,150 In some studies, these drugs have displayed greater potency than Ca2 antagonists as blood-pressure-lowering agents in various models of hypertension. For instance, cromakalim (at oral doses of 0.01-0.5 mg/kg) was 10-30 times more potent than nifedipine in SHR, renal hypertensive dogs, and renal hypertensive and normotensive at^.^^^,^^^ Differences between PCAs in terms of potency/duration of antihypertensive response in different animal models may reflect differing pharmacokinetic profile of such drugs between species. An appropriate example is the apparent lack of activity of cromakalim compared with SDZ PCO 400 in the rhesus monkey, contrasting with efficacy of both drugs in SHR (oral dose range for SDZ PCO 400, 0.1-0.3 mg/kg; cromakalim 0.3-1 rng/kg).l4l The fall in blood pressure following administration with PCA drugs is normally associated with concomitant tachycardia, although in the conscious renal hypertensive cat, low doses of cromakalim (0.007-0.01 mg/kg orally) have been reported to lower blood pressure without influencing heart rate.39 In view of the number of animal models employed, the relative ability of different PCAs to evoke rises in heart rate is difficult to assess. In the renal hypertensive dog, comparing equihypotensive doses of EMD 52692 with cromakalim, the tachycardia evoked by EMD 52692 was less pronounced than following cromakalirn.lu However, in the renal hypertensive or normotensive cat, the heart rate increases following cromakalim were consistently less than +

94


achieved with equihypotensive doses of pinacidil or nicorandil.39,140 In the latter models, cromakalim-inducedtachycardia was also less pronounced than witnessed following equihypotensive doses of the Ca2+ antagonist nifedipine. 140,142 Reports that the elevation in heart rate evoked by PCA drugs may be blunted in anaesthetized animals compared with conscious animal^^^^,'^ is consistent with the notion that such increases in heart rate are reflexly mediated. This is substantiated by the ability of p-blockers such as propranolol or bopindolol to prevent the tachycardia induced by drugs such as pinacidil, nicorandil, cromakalim, lemakalim, NIP 121, and KRPd 2391.113,119,120,137,142,146,151 Furthermore, the ability of high concentrations of cromakalim or EMD 52692 (in excess of 100 times the concentration required to inhibit spontaneous myogenic tone in portal vein) to have only minimal influence upon the spontaneous rate of beating of rat or rabbit isolated right atria37,39,152 has suggested the absence of a direct cardiac stimulatory effect of PCA drugs. More detailed study of the systemic haemodynamic effects of PCAs has indicated that the fall in blood pressure induced by these drugs is due to a fall in total peripheral resistance (TPR). The reflex tachycardia associated with these drugs probably constitutes haemodynamic counterregulation to the reduction in TPR and consequent fall in blood pressure. In some models, such as the conscious or anaesthetized normotensive cat or rabbit120,139,'50 and the anaesthetized or conscious normotensive dog,120,135,136 the fall in TPR evoked by cromakalim, lemakalim, minoxidil or pinacidil has been accompanied by increased cardiac output (probably by increased heart rate and stroke volume). This increase in heart rate and cardiac output may arise as a result of baroreceptor mediated increase in sympathetic nerve activity and concomitant decrease in parasympathetic nerve activity. However, other studies employing cromakalim in the anaesthetized normotensive dog or rat, 118,15334 pinacidil in the conscious normotensive dog,151and EMD 52692 or Ro 31-6930 in the anaesthetized normotensive d ~ g ' ~have ~ , demonstrated ' ~ variable effects upon heart rate, with little change in cardiac output in the face of a fall in TPR. Such studies serve to highlight the variation in haemodynamic profile witnessed following administration of a PCA in different animal models. As stated earlier, the systemic effects of these drugs may differ from those evoked by a Ca2+antagonist such as n i t r e n d i ~ i n e or '~~ the vasodilator hydra1a~ine.l~~ The increases in heart rate or cardiac output evoked by the latter drugs were exaggerated compared with responses to pinacidil or cromakalim. Interestingly, in one study conducted in anaesthetized SHR, cromakalim produced a greater rise in heart rate than pinacidil, although enhancement of sympathetic nervous activity (as measured in renal and splenic nerves) was most pronounced for ~ i n a c i d i 1 . Such l ~ ~ observations remain unexplained at the present time. Investigation of the regional haemodynamic effects of PCAs has produced variations in profile that may reflect differences in selectivity of these drugs for different vascular beds and also between the species and experimental conditions employed. Drugs such as diazoxide, nicorandil, pinacidil, EMD 52692, cromakalim, lemakalim, WAY 120491, and KRN 2391 evoked increases in coronary blood f l o ~ , ~ ~ ~suggesting , ~ ~ that , ~ these ~ drugs ~ , may ~ ~ ~ , ~ be useful as anti-ischaemic agents. In order to prove useful, PCAs would be


95

required to increase blood flow to the ischaemic subendocardial layers of the myocardium. Although PCAs may enhance coronary blood flow in nonischaemic muscle, their effects are somewhat more variable in ischaemia. The coronary vasodilator effect of cromakalim in anaesthetised rabbits (in nonischaemic muscle) was more pronounced in the outer ventricular wall (subepicardium) than subendocardium. 150 In conscious dogs, pinacidil increased coronary blood flow in nonischaemic tissue, although, unlike sodium nitroprusside, pinacidil was ineffective in ischaemic muscle.'58 In other studies in conscious dogs, pinacidil has been shown to induce a situation analogous to coronary steal, reducing blood flow to the subendocardium during ischaemia induced by acute coronary artery occlusion and coexisting coronary stenosis.165,166 In contrast, in anaesthetized dogs, EMD 52692 selectively diverted blood to ischaemic collateral-dependent myocardium,167 although flow was equally distributed between subepicardium and subendocardium. 15' Encouraging information regarding the anti-ischaemicpotential of PCA drugs has come from the ability of cromakalim, pinacidil, and RP 52891 to produce considerable improvement in the reperfusion function of globally ischaemic rat hearts or ischaemic myocardium in anaesthetized dog^.'^'^' Cromakalim may have direct myocardial protective effects, as evidenced by improved contractile function (during reperfusion) in the anaesthetized dog following intracoronary administration of drug.169In this study cromakalim preferentially enhanced subepicardial flow, but coronary steal was not observed. Interestingly, the coronary dilator effects of cromakalim in guinea pig isolated hearts16' and cardioprotective effects of cromakalim, EMD 56431, or RP 52891 in ischaemic rat hearts or canine myocardium are glibenclamide sensitive, suggesting a direct involvement with K channel ~ p e n i n g . ~ ~ KRN ,~~ 2391 ~ , enhanced '~~ the recovery of myocardial mechanical function and depressed depletion of high-energy phosphates during reperfusion following global ischaemia in rat isolated perfused hearts. 173Glibenclamide failed to influence these myocardial protective properties of KRN 2391, although increased coronary blood flow (witnessed during ischaemia) was glibenclamide sensitive. Thus the nature of the cardioprotectiveeffects of KRN 2391 appear complex and possibly reflect the mixed properties of this drug. The coronary vasodilator properties of nicorandil have been researched more widely than for other PCA drugs. Despite producing variable (often transient) falls in systemic blood p r e s ~ u r e , ~ ' , 'nicorandil ~ ~ ' ~ ~ has been shown to relax agonist-induced coronary vasoconstriction1p~'78and to enhance coronary blood flow in a variety of nonischaemic models. 137,156*174-177~179Comparison of the effects of nicorandil with those of glyceryl trinitrate'75,1p,180,181 have demonstrated that a broad similarity exists between the in vivo effects of these drugs. In nonischaemic models (conscious dog and anaesthetized pig) nicorandil evoked marked increases in myocardial blood flow, most notably in the outer layers of the heart (subepicardium and mid-myocardi~m).'~~,~@-' In one study, conducted in the anaesthetized dog, although intravenous nicorandil did not influence collateral blood flow, an improvement in postischaemic myocardial contractile function was 0 b ~ e r v e d . When l ~ ~ administered into the ischaemic coronary artery the drug proved ineffective, suggesting that the beneficial action of nicorandil may be related to its systemic +

96


effects (reduced preload and afterload) rather than any direct effect upon the myocardium. Although nicorandil failed to have a beneficial effect on the porcine ischaemic myocardium, la2other studies employing ischaemic models (usually anaesthetized dogs) have demonstrated that nicorandil may enhance collateral blood flow predominantly to the deeper subendocardium.181,183 These latter observations, coupled with the ability of nicorandil to reduce large coronary artery resistance, indicate that nicorandil may prove to be an effective antianginal agent, diverting blood flow to the areas most susceptible to ischaemic damage. Additionally, nicorandil has been shown to antagonize the pathophysiological events produced by Ca2 overload, thus conferring an additional cardioprotective characteristic on the drug. PCA drugs appear to possess vaned properties in terms of their ability to influence skeletal muscle blood flow. Whilst an increase in femoral or hindquarter blood flow (or decrease in femoral vascular resistance) has been reported in the anaesthetized dog, rat, or cat following diazoxide, cromakalim, SDZ PCO 400, or Ro 31-6930,"8~134~141~143~1a5~186 other studies in anaesthetized dogs, cats, or rabbits have failed to show any effect of pinacidil or cromakalim upon skeletal muscle blood f l o ~ . ~ However, ~ ~ , ~increases ~ ~ , in~ femoral ~ ~ , ~ ~ ~ blood flow or decreased femoral vascular resistance have been observed with nicorandil and EMD 52692 in the anaesthetized dog or mini pig.144,175-177 More striking effects of PCAs were seen in models of ischaemic skeletal muscle disease such as the rat chronically ischaemic gastrocnemius muscle. Whereas cromakalim, pinacidil, and nicorandil (unlike Ca2+ antagonists) had little influence during normal perfusion, they produced marked increases in blood flow and oxygen availability in an ischaemic preparation (Ca'+ antagonists and other vasodilators were ineffective in this situation).lS7These studies highlight differences between PCAs and other vasorelaxants such as Ca2+ antagonists, suggesting that PCA drugs may dilate large arteries (including collateral vessels), which would explain the increased blood flow to hypoxic muscle. Increased cerebral blood flow has been reported in anaesthetized rabbits or pigs and conscious normotensive rats following intravenously administered cromakalim or EMD 52692.113,144~150,160Although no change in carotid blood flow was observed with pinacidil in the anaesthetized dog, 157 carotid artery blood flow was enhanced by intravenously administered cromakalim or SDZ PCO 400 in the anaesthetized normotensive cat or SHR.141*1" Similarly, increases in blood flow to the stomach and small intestine have been reported following an intravenous injection of cromakalim to anaesthetized rabbit^."^,'^^ This is substantiated by reports of increased mesenteric blood flow or decreased mesenteric vascular resistance following intravenous administration of nicorandil, cromakalim, pinacidil, EMD 52692, SDZ PCO 400, Ro 31-6930, or KRN 2391 to the anaesthetized normotensive dog, rat, cat, or pig, the conscious normotensive rat or anaesthetized SHR39,141,143.144,16,1~,175,176,185(Fig. 6). Studies examining the influence of PCA drugs upon renal perfusion and function have reported wide diversity between the actions of these drugs. Increases in renal blood flow or decreases in renal vascular resistance in the anaesthetized dog, normotensive rat, and SHR have been reported following intravenous administration with nicorandil, pinacidil, cromakalim, EMD 52692, +


+20 r

97

(a)

(b)

(4

HR

Mesenteric vascular resistance

Renal vascular resistance

r

-5 -10 -15-20-25

-5 -10 -15 -20-25

D~~~~~~~in DBP (rnrnHg)

-10

1 * *

-50

L

*

*

Figure 6. Percentage change in (a) heart rate (beatdmin)(HR), (b) mesenteric vascular resistance, and (c) renal vascular resistance during intravenous infusions of cromakalim, 2.5 pg/kg/min (0); pinacidil, 10 pg/kg/min (0); nicorandil, 50 pg/kg/min (A);or vehicle (0) in anaesthetized cats. Values are mean f SEM from 4-5 animals. Statistical significance from preinfusion values; *p < 0.05 and **p < 0.001 (Dunnett's test). (Reproducedfrom S.D. Longman, J.C.Clapham, C. Wilson and T.C. Hamilton, I. Curdiovusc. Phrmucol., 12, 535 (1988), with permission.)

SDZ PCO 400, and Ro 31-6930.'36,141~143~144,153,175,176,185 In anaesthetized or conscious normotensive cats, intravenous or oral administration of cromakalim produced increases in renal blood flow (Fig. 6),39,139,1a,'42,188 although this was not seen in anaesthetized rabbits following intravenous administrat i ~ n . ~ 'In ~ ,anaesthetized '~~ or conscious cats, equihypotensive doses of pinacidil or nicorandil failed to enhance renal blood flow (Fig. 6),39,'a,'s8 indicating that the latter drugs possess profiles of activity characteristic of nifedipine in these models Although increases in renal blood flow have been reported in conscious or anaesthetized dogs following low doses of PCA,'36,157,'89other studies have shown decreased renal blood flow or function [decreased renal clearance, glomerular filtration rate (GFR), diuresis, and natriuresis] with increasing doses of PCA drugs such as diazoxide and p i n a ~ i d i l , ' ~ , but ' ~ ~not , ~ ~cro~ makalim in anaesthetized SHR.153These reductions in renal parameters were observed during a more pronounced hypotensive phase, indicating that the effects of these drugs on renal haemodynamics are dependent upon the level of hypotension attained. A direct effect upon tubular reabsorption may explain minoxidil-induced increases in sodium and water retention in spite of maintained GFR in the conscious dog (see Ref. 190). Overall, the decline in renal function with a wide variety of PCAs may result from markedly reduced renal perfusion pressure. Support for this theory has come from studies with cromakalim in anaesthetized rats, where kidney function and regional blood flow were maintained (presumably by a system of autoregulation) for modest decreases in blood p r e ~ s u r e . ' ~ ' ~ ' ~ ~ Reports of enhanced hypotensive effects following nephrectomy or administration with an ACE inhibitor suggest that the renin-angiotensin system (RAS) may also be stimulated as a consequence of the hypotensive response

98


to a PCA drug.'93,'94In a variety of animal models, the reduction in blood pressure produced by PCAs such an minoxidil, pinacidil, cromakalim, and SDZ PCO 400 have been associated with increases in plasma renin activity (PRA).136,140,141,188,189,195,196 In the conscious renal hypertensive cat cromakalim-induced falls in blood pressure were not significantly enhanced by the ACE inhibitor e n a l a ~ r i l , 'and ~ ~ increases in PRA evoked by cromakalim in the normotensive cat were less marked than those witnessed following equihypotensive doses of pinacidil or the Ca2 antagonist nifedipine. 140,188 Evidence that PCAs may act directly upon K + channels in juxtaglomerular cells to evoke renin release has come from the ability to demonstrate cromakalim stimulation of renin production in primary cultures of rat juxtaglomerular cells.198 Hyperpolarization of juxtaglomerular cells by cromakalim (resulting in decreased i[Ca2'I) may explain these results, since renin release from juxtaglomerular cells may involve lowered i[Ca'+]. 199 Work within our own laboratories has failed to confirm a direct effect of cromakalim and lemakalim upon renin release in rat juxtaglomerular cells (Watson, unpublished). Raised levels of A11 and aldosterone may be expected as a consequence of renin release in vivo. Thus the ability of minoxidil or cromakalim to increase plasma levels of aldosterone in conscious animal r n ~ d e l s ' ~may ~ ~be ' ~a~consequence of increased levels of angiotensin I1 due to enhanced sympathetic nervous activity and renin release. This is supported by the ability of a pblocker or ACE inhibitor to inhibit any rise in plasma aldosterone evoked by cromakalim in the conscious renal hypertensive cat. 197 Studies in bovine or rat isolated adrenal zona glomerulosa cells have attempted to examine a possible direct effect of cromakalim upon aldosterone secretion. Although some discrepancy exists between the concentrations used, cromakalim (1-40 pM) and pinacidil (1-100 FM) evoked inhibition of basal, K + or agonist (A11 or ACTH) induced aldosterone release in these preparations.200-202 The association of the inhibitory effects of cromakalim with enhanced K + efflux,201 suggests that agonist-evoked aldosterone release may be a consequence of depolarization of the adrenocortical cell membrane. Thus, despite reports of increases in PRA or aldosterone, the ability of PCAs to produce hypotension over a chronic period of dosing (reported earlier in this section) suggests that stimulation of the RAS in animals does not lead to a diminuition in hypotensive response to PCAs. Major differences between the antivasoconstrictor effects of PCA drugs and Ca2+ antagonists have been observed in anaesthetized rat^.^^^,^^^ Whereas cromakalim inhibited pressor responses to NA and phenylephrine, Ca2 antagonists inhibited responses to these agonists in addition to methoxamine, vasopressin, and AII.'03 Interestingly, cromakalim also inhibited endothelininduced vasoconstriction in conscious dogs.205In addition, drugs such as pinacidil and cromakalim inhibited pressor responses to 01'- or a2-adrenoceptor or electrical s t i m ~ l a t i o nin~ pithed ~ ~ , ~ normotensive ~~ rats or SHR. The ability of vasopressin infusions to reverse the inhibition evoked by pinacidil or cromakalim (but not Ca2+ antagonist^)^^^,^^^ suggests that the effects of PCAs may be influenced by the prevailing level of blood pressure. This theory may be supported by reports of enhanced hypotensive potency of cromakalim but not nifedipine, in SHR compared with normotensive rats, +

+


99

(a)

w 80

40-

t 1

1

0

20

40 60 Time (min)

80

0

20

40 60 Time (rnin)

80

Figure 7. Interaction between intravenously administeredglibenclamideand the blood-pressurelowering activity of lemakalim and nifedipine in anaesthetized female SHR. In panel (a) glibenclamide, 20 mg/kg i.v. (H)or vehicle 5 ml/kg i.v. (0)was administered 15 min prior to (first arrow) lemakalim 0.05 mg/kg i.v. (second arrow). In panel (b) glibenclamide, 20 mg/kg i.v. (0) or vehicle 5 mYkg i.v. (0) was administered 15 min prior to (first arrow) nifedipine at 0.1 mg/kg i.v. (second arrow). Values are mean f SEM for 7 rats. *Indicatessignificance (p < 0.05)between groups (Dunnett‘s test). (Reproduced from Ref. 120 with permission.)

implying that the activity of cromakalim may be dependent upon intrinsic vascular tone. 154 Evidence for lack of a direct sympathoinhibitory effect of PCA drugs has come from studies in the pithed normotensive rat, where cromakalim did not influence levels of circulating catcholamines (Buckingham, unpublished). Thus, although PCAs may inhibit responses to endogenous and exogenous substances, their ability to lower blood pressure in pithed rats193,203,207 suggests that these drugs predominantly relax vascular tissue via a direct action. In vivo evidence that the vasorelaxant effects of PCA drugs may be mediated via opening ATP-dependent K + channels has come from studies in the anaesthetized rat that have shown the ability of the sulphonylureas, glibenclamide or glipizide, to inhibit the hypotensive responses to cromakalim, diazoxide, SDZ PCO 400, or lemakalim (Fig. 7),35,51,117,119,120 but not Ca2+ antagonist^.^','^^ The ability of the sulphonylureas to inhibit the in vivo responses to PCAs is seen only with high intravenous doses of drug (20-30 mg/kg), with no inhibitory effect of glibenclamide being observed when administered by the oral route.208Thus the inhibitory effects of glibenclamide are witnessed at doses in considerable excess of those required to cause hypoglycaemia in rats (0.1-3 pmoVkg or 0.05-1.5 mg/kg i.v.).’09 The inability of apamin to influence the blood-pressure-lowering activity of cromakalim in anaesthetized rabbits (see Table 11) excludes any involvement of the drug with low-conductance Ca2+-activatedK + channel^."^ As demonstrated in in vitro studies, the in vivo vasorelaxant effects of PCAs such as pinacidil or cromakalim do not appear to be mediated by interaction with receptors such as aand p-adrenoceptors, muscarinic or histaminergic. receptors, or substances such as prostaglandins and platelet activating factor (PAF).118r157,194r210

C. Cardiac Many different K + currents have been identified in the heart and the properties of these currents have been extensively reviewed (see Ref. 211). The

100


currents that have been most widely studied are the inwardly rectifying current ( I K I )(permitting long depolarizing responses such as the plateau phase of the cardiac action potential), the delayed outward rectifier current (IK) (responsible for initiating the termination of the action potential) and the transient early outward current (produces an early repolarization phase or "notch" in the action potential). Another K + current characterized in cardiac tissue is a current activated by ACh and adenosine [IK(ACh)], with muscarinic ACh and adenosine receptors being linked via the a-subunit of a GTP-binding The opening of K+ channels resulting in protein to a K + channe1.29*212*213 enhanced IK(ACh) conductance is probably the mechanism underlying the ability of vagally released ACh to slow the heart (see Ref. 214). In the myocardium the resting membrane potential is high with mean values of approximately - 87 mV and - 70 mV being reported in human ventricular and atrial muscle respecti~ely.~'~ As a consequence of myocardial ischaemia, oxygen-derived free radicals may be formed and may mediate some of the events associated with ischaemia or postischaemic reperfusion, namely, loss of cellular K + together with shortening of the cardiac action potential duration (APD) and loss of high-energy phosphates.216The loss of cellular K + is considered to involve an ATP-sensitive K + channel,13 channel opening being induced by conditions known to lower i[ATP] such as ischaemia or metabolic inhibition (see Ref. 217). ATPsensitive K + channel activity has been recorded throughout the heart, unlike other cardiac K + currents such as IKI (not present in nodal pacemaker cells) and IK(ACh) (not present in ventricular fibers) (see Ref. 217). Another K + current that may play a functional role in pathological conditions such as ischaemia Although the function of this is the Na+-dependent K + current [IK(Na+)]. current is not clear, it may be activated by a marked rise of i[Na+]. Activation of IK(Na+) may cause repolarization of the cell and improve Ca2+ extrusion via the Na+/Ca2+exchanger (see Ref. 211). Inhibition of IKI and IK appears to underlie the antiarrhythmic property of class I11 agents such as sotalol, bretylium, bethanidine, and c l o f i l i ~ m . ~ ~ ~ , ~ ~ Thus modulation of ATP-sensitive K+ conductance [IK(An)] may also have profound effects upon electrical and mechanical events in the heart. Sulphonylureas have been shown to inhibit IK(ATP), as evidenced by their ability to inhibit channel opening in guinea pig or rat isolated ventricular cells"0-222 and reverse the shortening of APD induced by hypoxia or lowered i[ATP] in ferret and guinea pig papillary muscle or guinea pig ventricular Radioligand studies have demonstrated specific binding of sulphonylureas in cardiac tissue."' In such studies there was a close association between the rank order of potency of the sulphonylureas in cardiac cells and in insulinoma cells, although glibenclamide has been reported to display less affinity at the sulphonylurea receptor in the heart (cardiac cells Kd 2 nM, insulinoma cells Kd 0.3 nM).221 Similarly, in patch clamp studies, tolbutamide has also displayed less sensitivity in cardiac tissue compared with pancreatic p-cells."O Although glibenclamide evoked minimal effect upon ventricular fibrillation induced by coronary artery occlusion in the anaesthetized rat or rat isolated perfused heart,"4 in other studies, sulphonylureas demonstrated antiarrhythmic


101

- -

Cromakalim

Cromakalim

1OpM

30pM

/I\

; ;

>

Glibenclamide 0.3pM

200

a

d b

50 0

I

Time 40 (min) C

0

(b)

O1 -90

l\i. a

b

80

120

i d

C

200 msec Figure 8. Glibenclamide reverses the effect of cromakalim on action potential duration (APD) measured at 90% repolarization (APD 90) and effective refractory period (ERP) in guinea pig papillary muscle. (a) APD 90 and ERP recorded during a single microelectrode impalement. The top of the graph shows the order in which the drugs were added. Letters a 4 correspond to times when action potentials shown in (b) were recorded. (Reproduced from Ref. 223 with permission.)

activity, abolishing ventricular fibrillation in the globally ischaemic rat heart .225-z7 Thus it becomes apparent that if PCAs were to influence K + conductance in the heart this may have profound effects upon impulse conduction and mechanical activity. Early studies in canine a t i a demonstrated that nicorandil produced a hyperpolarization and decrease in Al'D.228 Subsequent studies in Purkinje fibers or ventricular myocytes from a variety of species have shown that n i c ~ r a n d i l , p~ i~ n- ~a ~~ i d iand l ~ ~~ r~o~m~ a~k a l i m ' ~all ~ shortened ~"~~~~~~ the duration of the fast or slow action potentials, without affecting the upstroke phase of the action potential (see Fig. 8). Cardiac effective refractory period (ERP) may also be reduced by PCAs as seen in ferret or guinea pig papillary musclez3 and in the normal or ischaemic myocardium of the conscious or anaesthetized d0g.242-244 Reduction in APD by PCAs appears to be highly stereoselective as judged by the activity of

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lemakalim.u7 In addition, cromakalim (or hypoxia) may reverse the prolongation of APD evoked by the antiarrhythmic agents ICS 205-930 and sotalo1.237,245 Although evidence for an influence upon resting membrane potential is equivocal, PCAs, have been shown to have a repolarizing effect upon depolarized cardiac t i s s ~ e . ~ ~ ~ , ~ ~ ~ , ~ ~ ~ In addition to shortening APD, nicorandil, cromakalim, and pinacidil have displayed negative inotropic activity in isolated cardiac tissue39~234,238,246,248-E1 (see Table I) or perfused whole heartsIE2 and inhibition of the positive inotropic effects of agents such as ~henylephrine.’~~ Indirect evidence of decreases in both the influx of Ca2+ and in i[Ca’+] have also been reported for nicorandil in frog atrial muscle.E0 In isolated working heart models, cromakalim has demonstrated marked species- and age-dependent negative inotropic effects.254In this study cromakalim (10 pM) depressed mechanical activity to a greater extent in hearts from older (20 month) than younger rabbits (2 month) whilst the drug was ineffective at this concentration in SHR hearts. Such observations remain unexplained at the current time. The ability of K + channel blockers such as TEA to inhibit such negative inotropic confirms that an enhancement of K+ current(s) underlies the conduction and mechanical responses evoked by PCA drugs. In addition, PCAs have displayed some antiarrhythmic activity, as judged by their ability to decrease spontaneous activity and enhanced automaticity (evoked by a variety of arrhythmogenic stimuli) in isolated cardiac tissues.uo,234,2246 In anaesthetised rabbits, intravenous infusion of lemakalim was associated with a markedly reduced incidence and duration of fibrillation during acute myocardial ischaemia and also during r e p e r f u s i ~ nEvidence . ~ ~ ~ such as this may confer a beneficial profile of activity on PCAs, particularly since K + conductance may be decreased in canine subendocardial Purkinje fibers 24 hours after coronary artery ligation.257In vivo, pinacidil has also shown marked antiarrhythmic activity in conscious dogs at 22-24 h after coronary artery ligation, although these events were observed at high doses that produced significant falls in blood pressure.E8 In general, it must be noted that the effects of PCAs in cardiac tissue are observed at considerably higher concentrations than required for effective vasorelaxation (see Table I). Although nicorandil may not display marked selectivity, other drugs such as cromakalim and pinacidil have demonstrated selectivity ratios in favor of vascular t i s s ~ e . ~In~canine , ~ ~ Purkinje , ~ fibers and cephalic vein, pinacidil and cromakalim displayed selectivity for vascular rather than cardiac tissue.234In this study pinacidil displayed greatest selectivity and was about 6-10 times more potent in relaxing venous tissue than in decreasing APD and contractility. However, both drugs displayed a greater degree of vascular selectivity in rabbit tissues39(see Table I). A close correlation between the cardiac electrophysiological effects and antihypertensive properties in SHR has been reported for pinacidil and a series of structurally related pyridylcyanoguanidines, supporting the concept that a common mechanism may underlie both phenomena.233 Studies examining the nature of PCA-enhanced K + conductance have suggested an alteration in the properties of a variety of K+ currents i n c l ~ d i n g ” ~ , ~ ~ ~ IK 01;128,231,239,241,260-263 lKI.In guinea pig ventricular myocytes, recent patchclamp techniques (with ATP present in the pipette solution) have demon-


103

strated that cromakalim, pinacidil, nicorandil, and RP 49356 may act predominantly by opening ATP-sensitive K channels. 14,223232,w263-267 Interestingly, in inside-out patches of cardiac myocytes, both cromakalim2&and RP 49356266 are capable of reactivating ATP-sensitive K+ channels that have experienced channel "run-down" following a prolonged recording period in an ATP-free solution. The significance of this phenomenon remains unexplained at the present time. In general, increases in IK(Am) evoked by PCAs in cardiac myocytes were observed at high concentrations (10-300 pM). This highlights possible differences in the functional properties of ATP-sensitive K+ channels in different tissues and confirms the relative selectivity of drugs such as cromakalim for the channels in vascular tissue* (see Table IV). Sulphonylureas have been shown to block the increase in IK(ATp) and shortening of APD or ERP evoked by PCA drugs (Fig. 8).14,w,235,2~,2~,2~,269 Unlike other PCA drugs, diazoxide (500 pM) did not evoke opening of ATP-sensitive channels in rat ventricular myocytes, but like tolbutamide, inhibited IK(ATp) and prolonged APD in the ventricular epicardium of perfused intact rat heartsw This interesting "discrepancy" with diazoxide requires further investigation. The enhancement of ZK(ATp) by cromakalim, pinacidil, and RP 49356 has been reported to show temperature dependence, with little increase in channel activity being evident at room temperature."3,235,265,269 Although this phenomenon is not understood it may indicate some enzymatic regulation of ATP-sensitiveK channel activity, possibly involving CAMP-dependent phosphorylation.269 Thus, although the antiarrhythmic properties of K + channel blockers (including sulphonylureas) have been described, the question arises as to the therapeutic potential of PCA drugs in the heart. Evidence of enhanced ventricular fibrillation has been reported following extremely high doses of nicorandil (21 mg/min intra-arterially) in canine isolated papillary muscle.270 During ischaemic injury, high doses of nicorandil (50, 100 p M ) reduced the incidence of ventricular f i b r i l l a t i ~ nwhilst , ~ ~ ~ pinacidil(30 pM) or cromakalim (10 pM) showed arrhythmogenic activity by their ability to shorten APD, together with enhancement of ventricular fibrillation during global ischaemia in rat isolated heart^."^ Similarly, in conscious dogs, pinacidil(O.3 mg/kg i.v. given as a single injection or infused over 6 h) proved arrhythmogenic in models of sudden death.242,243 In the anaesthetized dog, incidences of atrial arrhythmias (during electrical pacing) have been reported following intravenous doses of cromakalim (0.25,0.5 mg/kg) or pinacidil (1,2, mg/kg), which produced excessive (240%)decreases in mean arterial pressure.244However, the clinical implications for such drugs are unknown in situations of diseased tissue (and also ischaemia) where cardiac tissue may become partially depolarized and produce arrhythmia^^^^,^^^ and where myocardial hypertrophy may be associated with enhanced nucleotide sensitivity of ATP-sensitive K+ channels.273 It has been postulated that shortening APD (as witnessed with PCA drugs), relative to ERP, may be effective in conditions of depressed conduction such as re-entrant-type arrhythmias.274The lack of effect of nicorandil upon ERP in canine cardiac tissue, together with its ability to inhibit enhanced automaticity at low concentrations (10 pM)230close to therapeutic concentrations of the drug,275suggests that the drug may possess a useful profile in the +

+

104


heart. Maintenance of normal automaticity and conduction in the rabbit AV node276indicates a possible beneficial influence of nicorandil in ischaemic heart disease with manifested abnormal AV nodal functions. Further evidence that PCAs may possess antiarrhythmic potential in ischaemia has been shown by the inhibitory effects of cromakalim (5pM) on abnormal pacemaker activity in canine Purkinje fibers and reduction in oscillatory potentials due to Ca2+ overload.246Evidence that PCA drugs may preserve levels of ATP during ischaemia is largely equivocal. A beneficial profile of these drugs may be inferred by improved reperfusion function following cromakalim (7 pM) and RP 52891 (3 pM) in globally ischaemic rat heart^,^^,'^^ and minimal effect of a similar concentration of cromakalim upon high-energy phosphates (despite suppression of mechanical activity)in a rabbit isolated working heart In addition, nicorandil (100 pM) also preserved levels of high-energy phosphates during hypoxia in rat cultured myocytes and rat isolated perfused heart^,^^,^^' although this was not observed in a rat perfused working heart Nicorandil(l0 mg/kg orally) has demonstrated beneficial activity in a model of myocardial injury induced by isoprenaline in rats.280In such a study, oral administration of nicorandil was associated with decreased incidence of myocardial infarction and an improved survival rate of isoprenalinetreated animals. Differences in the electrophysiological characteristics of endocardial and epicardial cells have been observed in pathological conditions such as ischaemia (prolongation of conduction and refractoriness being more prominent in epicardial than endocardial tissue) .'" These differences have been highlighted by evidence that ATP-sensitive K+ channels in myocytes from the feline epicardium and endocardium display different sensitivity to ATP.282 Since PCAs may alter the distribution of blood flow between these layers (see Sec. II.B), this highlights the necessity of thorough investigation of the clinical potential of these drugs as antiarrhythmic agents.

D. Clinical Since extensive studies have demonstrated that PCAs relax vascular tissue from a variety of animals, in vivo techniques have been employed to examine the selectivity of these drugs for different vascular beds in man. In human volunteers and hypertensive patients, PCA drugs have demonstrated arterioselective vasodilation, as judged by the ability of a single oral dose or intravenous infusion of minoxidil, diazoxide, pinacidil, or cromakalim to produce increases in forearm blood flow or decreased forearm vascular r e s i ~ t a n c e ~ ' ~ - ~ ~ ' with no change in venous capacitance or ~ e n o d i l a t i o n . ~ These ~ , ~ ~results ,~'~ have been confirmed in patients with hypertension or ischaemic heart disease where minoxidil, pinacidil, and cromakalim evoked marked reductions in systemic, but not pulmonary, vascular r e ~ i s t a n c e , ~ ' ~indicating -~~' that PCA drugs act as vasodilators with an arterial, rather than venous, site of action. Evidence of a reduction in left ventricular afterload and improvement in cardiac performance have been demonstrated following intravenous diazoxide administration to hypertensive patients during exercise,292and following intravenous administration with cromakalim in patients with angina p e ~ t o r i s . ~ ~ ~


105

These studies highlight the necessity of evaluating such drugs for the longterm management of ischaemic heart disease. Although shown to lower blood pressure in hypertensive patients,294nicorandil appears to offer most potential in the treatment of angina,295demonstrating beneficial effects upon cardiovascularhaemodynamics and left ventricular function in patients with coronary artery disease.296Nicorandil has been shown to produce arterial and venous dilation, but as outlined earlier, this drug possesses "hybrid" characteristics of a nitrovasodilator in addition to an ability to open K+ channels (see Ref. 297). In addition to PCA drugs vasodilators, such as Ca2+ antagonists, also display arterioselectivity in their The ability of PCAs to produce selective arteriolar vasodilation with a consequent reduction in afterload indicate that they should be of value in the treatment of arterial hypertension where the primary haemodynamic abnormality is increased TPR (see Ref. 299). Wide clinical experience has been gained with PCA drugs for the treatment of hypertension. Extensive studies in normotensive subjects and hypertensive patients have demonstrated that diazoxide and minoxidil lower blood pressure (as a consequence of reduced TPR) in man (see Refs. 190 and 300). Despite its antihypertensive efficacy, the incidence of severe side effects (predominantly hyperglycaemia, sodium and water retention) has precluded the use of diazoxide as a long-term blood-pressure-lowering agent. The hyperglycaemic effects of diazoxide are probably a consequence of its influence upon pancreatic p-cells and also at extrapancreatic sites via catecholamine release and adrenergic receptors. Attempts to combine chronic administration of diazoxide with the antidiabetic sulphonylureas (in an attempt to stabilize blood glucose) have proved unsuccessful due to the incidence of pronounced hypoglycaemia.wl Similarly, combined therapy of diazoxide with a diuretic enhanced impairment of carbohydrate metabolism (see Ref. 300). Thus the use of diazoxide has been confined to some incidences of acute hypertensive crises. Despite isolated reports that minoxidil may alter glucose metabolism (the development of latent diabetes in severe hypertension),m,m it has proved effective in the treatment of severe, essential and renal hypertension, being a potent, long-acting, orally effective antihypertensive agent following acute or chronic administration (see Ref. 190). The lack of correlation between the onset and duration of hypotension (up to 24 h), and plasma levels of parent drug (half-life of approximately 4 h), suggested that an active metabolite may be responsible for the pharmacological activity of m i n ~ x i d i l .In~vitro , ~ ~studies have suggested that the active metabolite is minoxidil sulphate,% although confirmatory evidence has not been reported demonstrating the existence of this metabolite in man. Pinacidil has been studied extensively as an antihypertensive agent in man (see Ref. 307). Evidence that acute or chronic orally administered pinacidil may be short acting as a blood-pressure-lowering agent in volunteersw8 and hypertensive patients,= together with knowledge that the elimination halflife of the drug is approximately 2-3 h,310,311led to the development of a "slow-release" formulation of the drug. Although an improvement on the previous formulation, the inability of slow release pinacidil 20-40 mg b.i.d. to provide a sustained reduction in blood pressure over 24 h, in addition to

106


high incidence of fluid retention (see below), cast doubts upon the suitability of pinacidil as monotherapy. Administration of PCA drugs may stimulate several counter-regulatory reflex mechanisms including activation of the sympathetic nervous system (increased heart rate, cardiac output, and stroke volume) and the RAS in response to a drug-induced reduction in TPR. As a consequence, reports of sodium and water retention, palpitations, and vasodilator headache have been well documented for minoxidil, diazoxide, and pinacidil (see Refs. 300, 307, and 312). Uncontrolled, the activation of counter-regulatory reflexes may lead to the tendency of such drugs to become less effective as blood-pressurelowering agents during prolonged treatment. As seen with vasodilator drugs such as hydralazine, co-administration with a P-blocker and/or a diuretic has proved effective in reducing the incidence of tachycardia, headache, and oedema following acute or chronic administration of minoxidil,283,313,314 diaoxide,^^^ or p i n a ~ i d i l ~to~hypertensive ,~'~ patients. Following such combined therapy, the incidence of raised levels of plasma catecholamines following pinacidil has also been a t t e n t ~ a t e d . ~ 'Despite ~ , ~ ' ~ increases in PRA, plasma aldosterone has generally not increased following minoxidil or pinacidil therapy.317,318In addition to a possible direct effect of PCAs upon aldosterone secretion (see previous section), increased metabolic clearance resulting from enhanced hepatic blood flow may explain the inability of drugs such as minoxidil to raise plasma aldosterone levels.317The effect of PCA drugs upon hepatic blood flow has not been widely reported, although nicorandil has produced reductions in hepatic blood flow in a study conducted in normal volunteers.319 By virtue of their recent discovery, there are no published reports of clinical trials in man with drugs such as lemakalim, RP 49356, EMD 5269~,Ro 316930, EMD 56431, NIP 121, and KFW 2391. Clinical experience with cromakalim is less extensive than for PCA drugs synthesized prior to cromakalim. In human volunteers, acute or chronic oral administration of cromakalim (doses up to 2 mg) had little influence upon resting blood p r e ~ ~ ~ r e . ' ~ ~ , ~ ~ ~ However, in patients with mild to moderate essential hypertension, cromakalim has been reported to lower blood pressure following single oral doses of 0.5-1.5 or following chronic once daily administration.324327Antihypertensive activity was maintained during repeat dosing with no signs of tolerance developing during a six week open trial with c r ~ m a k a l i mAs . ~with ~~ other PCA drugs, the hypotension produced by cromakalim was normally accompanied by small increases in heart rate; some incidence of a dose-related headache has been reported. Antihypertensive efficacy has been observed in hypertensive patients up to 8 h after a single oral dose of cromakalim (1.5 mg).323Following oral administration of cromakalim in healthy volunteers, the plasma half-life of the active (-)-enantiomer (lemakalim) in man is 1824 h.328If this prolonged pharmacokinetic half-life is matched by an extended pharmacodynamic effect, lemakalim should provide adequate reduction in blood pressure over a 24-h period using a once daily dosing regimen. Increases in PRA have been reported following acute or repeated oral administration of cromakalim to human volunteer^'^^,^^^,^^^ and acute administration to hypertensive patients.321However, in patients, repeated single oral daily doses of cromakalim at effective antihypertensive doses failed to


107

produce increased PRA326,327 and no evidence of oedema or weight gain have been r e p ~ r t e d . ~As ’ ~with , ~ ~pinacidil or minoxidil, there has been no evidence of raised levels of plasma or urinary aldosterone following acute or repeated administration of cromakalim in patients or normal volunteer^.^^**^'^,^^^ The lack of oedema witnessed with cromakalim in patients may reflect the differences in haemodynamic profile between cromakalim and other drugs such as pinacidil in animal models (see Sec. 1I.B). In spite of incidences of reduced K+ content in red blood cells and leucocytes in volunteer^,^'^ cromakalim has generally been reported to have little influence upon plasma or urinary electrolytes in normal volunteers or hypertensive patients. 198,321,325,327,329 Although renal haemodynamics were preserved during an initial short-term study in hypertensive patients,327more extensive long-term studies will be required in order to establish any putative benefit that cromakalim may possess regarding water and electrolyte balance over existing PCA drugs. The limited clinical experience with cromakalim has suggested that the drug is well tolerated and appears to be free of adverse side effects. In addition to causing fluid retention, another side effect of pinacidil is h y p e r t r i c h o s i ~ , ~ ~ although its incidence is less than reported for min~xidil.~~’ Changes in ECG pattern have been reported for minoxidi1330and to a lesser extent with pinacidiI.=l The changes, which were transient (rarely occurring after 3 months therapy), primarily consisted of T-wave flattening or inversion. Such repolarization changes appeared to be independent of alterations in blood pressure or heart rate but may be due to changes (shortening) in APD. To date, no change in ECG pattern has been detected during repeated oral administration (up to 8 days) with cromakalim in hypertensive patients.332 Since there is evidence that the hypertensive state may be associated with enhanced responsiveness to pressor investigators have attempted to examine the influence of PCA drugs upon pressor responses in human blood vessels. Using a myograph technique, pinacidil has been shown to produce concentration-related inhibition of NA contractions (2.5-5 pM) with little effect upon contractions to high concentrations of KC1 (>120 mM) in small (internal diameter 200 pm) human arteries (vessels with relevance for control of TPR).%In this study, the effective concentration of pinacidil was similar to that reported in human crural veinsm (0.2-1 pM) and comparable to a therapeutic plasma concentration of pinacidil(~50ng/ml) in clinical studies.335Despite evidence of nonspecific relaxant effect of cromakalim in human crural veins,336in human mesenteric artery, cromakalim selectively inhibited contractions to NA (EC50 0.1 p.M) in preference to high KC1.337In human volunteers cromakalim has evoked either an inhibition3%or had no effecPZ9 upon pressor responses to infusions of NA or AII. However, interpretation of such studies is complicated by constant infusion rates of agonist in the face of cromakalim induced changing haemodynamics. Another feature of essential hypertension is believed to be an impairment of baroreceptor (heart) reflexes.339Thus the apparent ability of cromakalim to enhance baroreceptor sensitivity to pressor challenge in the anaesthetized c a p 0 may confer a possible advantage over other PCAs such as pinacidil. A useful additional property of PCAs would be the ability to modify hypertrophy of the left ventricle, a feature that is often inherent in hypertension. Pinacidil has not been reported to influence cardiovascular hypertrophy in hyperten-


108

Left ventricular mass (9) Pinacidil

Nifedipine

700

7

, 600 0°:

-

\

500400-

600

500

400 0

200

-

Change -75.1f 47.3 (-22.7%) p < 0.001

300200

-

05~

Change -26.3f 34.7(-8.6%) p = 0.04

Figure 9. Left ventricular mass before and after 6 months of treatment with either pinaadil or nifedipine in 22 patients with mild to moderate hypertension pretreated with bendrofluazide 5 mg daily. -0represents mean value. During the first 6 weeks of combination therapy, vasodilator dosage was increased if the diastolic blood pressure remained above 90 mmHg. The following dose-steps of pinacidil were used: 12.5, 25, 37.5, and 50 mg twice daily; nifedipine dose-steps were: 10, 20, 30, and 40 mg twice daily. Combination therapy was maintained for 6 months. (Reproduced from Ref. 341 with permission.)

sive rats (SHR).148However, an initial clinical study demonstrated that during co-administration with a diuretic, pinacidil possessed greater efficacy than nifedipine in evoking regression of left ventricular hypertrophy during longterm treatment (6 months) in patients with mild to moderate hypertensionM1 (see Fig. 9). Evidence from in vitro studies that cholesterol enrichment may inhibit the enhancement of 86Rb+efflux evoked by cromakalim in rabbit vascular smooth muscleM2highlights the need for clinical studies examining the influence of PCA drugs upon plasma lipids. Limited work with pinacidil has shown the drug to lower plasma cholesterol and triglycerides or evoke increases in highdensity lipoprotein (HDL) cholesterol during repeated monotherapy or coadministration with diuretic or p-blockeP-’ suggesting that PCA drugs may possess a favorable profile of activity on plasma lipids (Table 111). An initial four-week study in hypertensive patients with cromakalim has demonstrated small reductions in total plasma cholesterol and low-density lipoprotein (LDL) cholesterolrM8highlighting the need for more detailed trials of this nature to identdy any putative antiatherosclerotic potential for PCA drugs. Interestingly, minoxidil and nicorandil have been shown to decrease the proliferation of vascular smooth muscle cells in ~ l i f r 0indicating , ~ ~ ~ ~that PCA drugs may prevent the hyperproliferation of vascular smooth muscle cells that may be witnessed in the vascular walls of hypertensive patients.


109

Table I11 Serum Lipid and Lipoprotein Concentrations During Pinacidil Treatment in Hypertensive Patientsa 6 Weeks

Baseline (mgdl) Total patients (n = 22) Cholesterol Total VLDL LDL HDL HDLi HDL3 Trig1yceride Total VLDL

220 39.4 137 44.2 26.9 19.7

39 24.8 f 43 f 12.0 f 11.4 f 8.1

142 78.2

f f

f f

78 56.5

mgdl

% Change

12 Weeks

mg/dl

211 + 28 -4.1 25.1 f 18.4+ -36.2 138 f 27 0.7 49.5 f 9.0* 11.8 29.3 f 12.3 8.9 21.7 f 9.2 9.9

208 30.0 126 47.3 28.0 21.3

105 f 3 7 59.4 2 31.0

115 f 56* 62.3 f 36.5

-26.1 -24.1

f 37+ 2 32.5 f 45 f 12.4 f 12.9

f 11.4

% Change

-5.8 -23.9 -7.8 7.0 4.0 8.3 -19.1 -20.4

"VLDL = very-low-densitylipoproteins;LDL = low-density lipoproteins;HDL = high-density lipoproteins. Values are mean f SD. Statistical significance: *p < 0.05; 'p < 0.10. Extracted, with permission, from K. Saku, H. Ying, and K. Arakawa, Clin. Ther. 12(2), 132-138 (1990).

In in vitro studies, the ability of cromakalim to reduce resting tone and antagonize the vasospastic activity of 5-HT in rat and rabbit basilar, coronary, or cerebral arteries49,50,351 and human intracranial a r t e P 2 suggests that like some Ca2+ antagonists, PCA drugs may offer therapeutic potential in the treatment of vasospastic disorders such as unstable angina, subarachnoid haemorrhage, and migraine (see Refs. 353 and 354). The PCA/Ca2+antagonist KRN 2391 may have potential as an antianginal agent as judged by inhibition of rhythmic contractions to the K+ channel blocker, 3,4-diaminopyridine (DAP) in canine isolated coronary arteries355and also by its ability to demonstrate a protective effect in an anginal model (induced by 5-HT treatment) in the anaesthetized normotensive rat.356Interestingly, nicorandil has previously demonstrated an ability to reduce spasm and spontaneous electrical activity of canine basilar arteries following subarachnoid haemorrhage (induced by cisternal injection of autologous blood).357 Evidence of improved skeletal muscle blood flow and oxygen availability in animals with hypoxic skeletal muscle (see Sec. I1.B) has indicated that PCAs may prove effective in the treatment of occlusive vascular diseases such as Raynaud's disease and intermittent claudication. Although consistent increases in calf blood flow have been reported following oral administration of pinacidil(25 mg twice daily for 1 week) to healthy volunteers,358more extensive studies are required to evaluate the use of PCAs in patients with peripheral vascular disease (see Sec. 1I.B). 111. AIRWAYS SMOOTH MUSCLE

In airways, the direct relaxant properties of PCA drugs such as cromakalim have been assessed predominantly in guinea pig isolated trachealis muscle. This tissue has also been used for ion flux studies and for intracellular recording of change in membrane potential. More recently, smooth muscle from bovine trachea has been employed by some workers, this tissue being a richer source of smooth muscle.

110


Spontaneous (prostaglandin-dependent) tone in guinea pig isolated trachealis is reduced by cromakalim, lemakalim, pinacidil, nicorandil, and Ro 31-693031,249,359361 with the following relative order of potency: Ro 31-6930 > lemakalim > cromakalim > pinacidil > nicorandil. In human isolated bronchial smooth muscle, cromakalim (IC500.35 pM) was about 3 times more potent as a relaxant of spontaneous tone than in the guinea pig t r a c h e a l i ~ . ~ ~ ~ Examination of the ability of PCAs to inhibit or reverse contractions produced by a variety of agonists in different species and tissues has revealed some interesting differences. For example, although cromakalim and pinacidil are weak inhibitors of contractions induced by cholinomimetics (ACh and cromakalim is approxicarbachol) in the guinea pig mately 50 times more potent as an inhibitor of carbachol-induced tone in human bronchial smooth muscle.365The relaxant effect of lemakalim in the human preparation has been confirmed in preparations precontracted by carb a c h 0 1 . ~Overall, ~~ these findings presumably reflect a difference in the mechanism of action of carbachol between species and show that in this instance, the guinea pig is not a good predictor of the inhibitory response of PCA drugs in man. However, in the guinea pig, cromakalim and RP 49356 produced greater inhibitions of contractions induced by low, rather than high, concentrations of carbach01.~~~ In contrast, these drugs inhibited contractions induced by a wide range of concentrations of histamine367although others3&found that cromakalim was more effective against contractions produced by low, rather than high, concentrations of histamine. An earlier report stated that cromakalim produced only a minor shift in the concentration effect curves of histamine and ACh.359 In addition to inhibitory activity against histamine and carbachol, PCAs relax tissues precontracted by PGE2,361LTD4 and 5-HT,365,U46619 and betahi~tine,~",e n d ~ t h e l i n neurokinin ,~~~ Arx6, PGF2a,363and phorbol dibutyrate.368Most commonly, KCl has been employed as the spasmogen and, predictably, contractions to low (15-30 mM), but not high (40-120 mM), concentrations were inhibited by drugs such as cromakalim, nicorandil, RP 49356, pinacidil, and diazoxide in guinea pig tracheal preparat i o n ~ , ~ ~ and , ~ by ~ cromakalim, ~ , ~ ~ * pinacidil, ~ ~ ~ , and ~ ~ diazoxide ~ in bovine tracheal smooth muscle.371These results support the proposal that the major inhibitory mechanism of action of these compounds is due to the opening of K + channels. The relatively low potency of cromakalim as an inhibitor of contractions produced by exogenous cholinomimeticsin guinea pig isolated trachea prompted a comparison with the influence of cromakalim on responses mediated by vagal stimulation. In an innervated, tubular preparation of guinea pig isolated trachea, cromakalim, in concentrations up to 10 p M , inhibited increases in intraluminal pressure due to preganglionic stimulation of the vagus, but not those mediated by exogenous ACh362,372 or electrical field stimulation (EFS) (postganglionic).362 These findings suggest that cromakalim probably does not interfere with ACh release from postganglionic cholinergicnerve terminals (since EFS is not inhibited) but acts by another, prejunctional, mechanism to inhibit cholinergic neuroeffector transmission, for example, in ganglion cells. As yet, other PCAs have not been investigated in this model and the effects of such drugs upon 13H]-AChrelease from parasympathetic nerve endings


111

have not been reported. It should also be noted that in a study involving stimulation of perivascular nerves in rabbit mesenteric small arteries, no evidence was obtained that cromakalim had a prejunctional inhibitory effect (on e.j.p.s.).u Cromakalim (1 and 10 pM) and nicorandil (1 mM) have been shown to stimulate 86Rb+efflux from guinea pig tracheal smooth m u s ~ l e .Subse~~~,~~ quently lemakalim (5 pM) also stimulated 43'42K+ efflux in this preparation.373 Cromakalim (1-30 mM) and pinacidil (3-30 mM), but not diazoxide (1 mM), increased 86Rb+efflux in bovine tracheal smooth muscle.371This suggests that diazoxide relaxes this muscle by a mechanism unrelated to the opening of Rb +-permeable K+ channels. However, in the latter preparation, cromakalim (1and 10 pM) caused greater increases in QK+ than =Rb+ In contrast to other workers, diazoxide (100 and 300 pM) evoked similar increases in the efflux of both ions.375The reason for the disparity in the diazoxide results is ~ n c l e a r . ~In ~ guinea ' , ~ ~ ~pig trachealis, cromakalim (10 pM) caused a greater stimulation of 43'42K+ than of 86Rb+efflux in single-label studies. However, in dual-label experiments, the magnitude of cromakalim-induced efflux of 42/43K+ and 86Rb+was the same, but that of =Rb' efflux was similar to that in the single-label experiment,376suggesting that extracellular Rb may reduce the permeability of the cromakalim-activated K channel. Additionally, cromakalim-induced relaxation of spontaneous tone was transformed into a transient response when Rb+ replaced extracellular K +.377 Taken together, these latter findings suggest that cromakalim may open Rb -permeable (transient relaxation) and Rb+-impermeable(prolonged effect) K+ channels in the guinea pig trachealis. In guinea and tracheal smooth muscle cells, cromakalim (1-10 pM) produced concentration-dependent hyperpolarization. In the latter preparation, diazoxide (100 pM),375lemakalim (1-10 pM),380and RP 49356379were also effective. In earlier nicorandil (50 pM) evoked hyperpolarization in canine isolated tracheal cells, and nicorandil (10 pM-1 mM) produced a similar effect in the guinea pig trachealis.360Thus, although only a limited number of studies have demonstrated hyperpolarization produced by PCAs in airway smooth muscle cells, the evidence obtained supports K + channel opening as being involved in the tracheal relaxant activity of these drugs. This conclusion is consolidated by the following data from experiments using a variety of K+ channel blockers. (5 mM) inhibited both the In guinea pig isolated t r a ~ h e a l i s ,procaine ~~~ relaxant activity and hyperpolarization induced by cromakalim, whereas TEA (8 mM) was more effective as an inhibitor of hyperpolarization than of relaxation. Nicorandil-induced hyperpolarization was antagonized by TEA in guinea pig and canine t r a ~ h e a . Apamin ~ ~ , ~ ~ (0.1 pM) was ineffective against ~romakalim~~' and n i c ~ r a n d i induced l~~ relaxations in guinea pig trachealis, as was charybdotoxin (up to 180 nM) against relaxations of carbachol-induced tone by cromakalim and p i n a ~ i d i l However, .~~~ charybdotoxin antagonized relaxation induced by the p-agonists, isoprenaline, and salbutamol. The latter findings suggest that cromakalim and pinacidil, unlike p-agonists, do not modulate the opening of the large-conductance Ca2+-activated K+ channel. This conclusion is supported by the failure of cromakalim and RP 49356 to affect the open state probability of this channel in inside-out membrane patches +

+

+

112


from bovine trachealis cells.379Interestingly, procaine was found to block nicorandil-induced hyperpolarization, but not relaxation, providing further evidence that nicorandil possesses additional properties as a smooth muscle relaxant.360 In guinea-pig trachealis precontracted by histamine,383or by low concentrations of KCl,384glibenclamide (1 pM) antagonized cromakalim-, pinacidil-, and RP 49356-induced relaxation. Similarly, in bovine tracheal smooth muscle contracted by 20 or 25 mM KC1,371,374 glibenclamide (1 pM) inhibited relaxation produced by cromakalim, pinacidil, and diazoxide. Reductions in spontaneous tone in guinea pig trachealis by cromakalim were inhibited by 1-pM g l i b e n ~ l a m i d e , there ~ ~ ~ ,being ~ ~ ~ some evidence for a competitivelike effect. These workers also reported that phentolamine antagonized, in a noncompetitive manner, cromakalim-induced relaxation. In human isolated bronchial smooth muscle, lemakalim-induced relaxation of agonist-induced contractions was inhibited by glibenclamide (0.1 and 1 pM).366Glibenclamide (1-10 pM) also reversed cromakalim-induced hyperpolarization in bovine tracheal smooth muscle.374In guinea pig tracheal preparations, glibenclamide (1 pM) and phentolamine (100 pM) reversed cromakAs previously described for vascular alim-induced (10 pM) hyperpolarizati~n.~~~ preparations, the blocking action of phentolamine is apparently not due to its a-adrenoceptor blocking properties since neither prazosin nor yohimbine modified cromakalim-induced relaxation.378 In bovine tracheal smooth muscle, glibenclamide (1pM) inhibited increases in =Rb+ efflux induced by cromakalim and p i n a ~ i d i l ~and ~ l ,to ~ ~a ~lesser degree inhibited 42K+ efflux induced by cromakalim. In guinea pig tracheal tissue, BRL 31660 (a putative K + channel blocker/antiarrhythmic drug with Class I and IV properties385)antagonized, in a noncompetitive manner, relaxation, and stimulation of 42’43K+ efflux produced by cromakalim and lemakalim.373Thus, utilizing a variety of K+ channel blockers, data support the notion that Ca2+-activatedK + channels are not involved in the relaxant effects of PCAs in airways smooth muscle. Recent evidence has demonstrated that lemakalim (1pM) prolonged the opening of an ATP-sensitive K+ channel in rabbit and bovine airways smooth muscle cells12(see Table IV). As reported for vascular smooth muscle (Sec. II.A), evidence has been obtained that PCAs may affect intracellular Ca2+ stores in airways.386Thus, in rabbit airway cultured smooth muscle cells, lemakalim (10 pM) reduced the Ca2+ content of the Ca2+ store and reduced inositol 1,4,5-triphosphate (IP,) (10 pM) induced Ca2+ release (but not that produced by guanosine-5’0-(3-thio) triphosphate (100 pM). Thus a contribution to the relaxant effects of lemakalim in airways may stem from this influence on Ca2+ stores and be initiated by an action of the drug on K+ channels in the SR. The results of further studies in this area, including those using K+ channel blocking agents, are awaited with interest. Moreover, the reported lack of effect of cromakalim and RP 49356 (up to 7.5 pM) on the content of cyclic nucleotides in guinea pig isolated trachealis muscle379has emphasized the different intracellular actions of PCAs from other bronchodilators such as theophylline. Other findings have excluded an effect of cromakalim and RP 49356 on Na+/Ca2+exchange as a mechanism mediating the bronchodilator response.387 In vivo investigations of the respiratory actions of PCAs have, to a large


113

extent, been limited to studies in guinea pigs. The antibronchoconstrictor activity of cromakalim has been demonstrated following oral, intraduodenal, and intravenous routes of administration.361In the anaesthetized guinea pig (Konzett-Rossler model), cromakalim (EDm0.084 mg/kg i.v.) inhibited 5-HTinduced bronchoconstriction and was about 7 times more potent than pinacidil. By contrast, low doses of the Ca2+channel antagonist nifedipine reduced blood pressure with no effect on airways.361In conscious guinea pigs, cromakalim, in oral doses >1 mg/kg, protected animals from histamine-induced bronchospasm and, in ovalbumin-sensitized animals, afforded protection from antigen challenge. In both situations, pinacidil was approximately 5 times less potent than c r ~ r n a k a l i mRo . ~ 31-6930 ~~ was also effective in the histamine challenge model.= Comparison of Ro 31-6930 and cromakalim against 5-HT-induced bronchospasm in anaesthetized guinea pigs showed Ro 31-6930 (EDm 0.012 mg/kg i.v.) to be about 10 times more potent than ~ r o m a k a l i mFollowing .~~ intravenous administration, cromakalim was more potent than RP 49356 and pinacidil as an inhibitor of 5-HT-induced increases in lung resistance and decreases in dynamic ~ o m p l i a n c eIn . ~this ~ study, each drug was slightly more potent against resistance than compliance changes produced by 5-HT. Similarly, in the anaesthetized cromakalim inhibited 5-HT-induced changes in resistance and compliance (respective IDms 0.032 and 0.07 mg/kg i.v.). Additionally, in the anaesthetized dog, intra-arterially administered nicorandil (3-300 pg) reduced basal intraluminal pressure390and tone elevated by ne~stigmine.~~~ cromakalim and lemakalim prolonged time to colBy the inhaled lapse induced by aerosolized histamine in conscious guinea pigs. In anaesthetized animals, lemakalim (250 pg/ml) caused similar inhibitions of histamine-induced changes in resistance and compliance whilst salbutamol was more effective against resistance than compliance. By this route, lemakalim had no effect on blood pressure at effective bronchodilator doses (Fig. 10). Thus administration of PCAs by the inhaled route would be expected to be effective at rapidly reversing an acute bronchoconstriction during an asthmatic attack. In contrast, by the i.v. route, cromakalim, RP 49356, and pinacidil lowered blood pressure at bronchodilator dose^.^^,^^ Thus administration of these drugs by the inhaled route should provide a means of avoiding unwanted cardiovascular effects observed following systemic administration. However, studies in man using oral cromakalim have shown a lack of cardiovascular changes at effective bronchodilator doses (see below). Evidence suggesting that K + channels in the airways may differ from those in the vasculature has come from two independent studies using K + channel blockers. Firstly, although glibenclamide(20 mg/kg i.v.) antagonized the bronchodilator and hypotensive effects of lemakalim (50 pg/kg i.v.) in anaesthetized guinea pigs, there were temporal differences in the blocking action of glibenclamide on these two parameters .3y3 It has also been reported394that cromakalim (0.4 mg/kg i.v.) inhibited bronchoconstriction evoked by vagal stimulation in anaesthetized guinea pigs pretreated with atropine and propranolol. In this study, glibenclamide (20 mg/kg i.v.) prevented the hypotensive response to cromakalim and its inhibitory action on the nonadrenergic, noncholinergic (NANCe)nerve mediated bronchoconstriction, whereas phen-

I

I

15 pMI5

Rat neonatal

Pancreatic P-cell 5415

-100 pM (100%inhibition at 500 uM)I3

8013

Guinea pig, rabbit

Cardiac ventricle

2mM

0.1 In isolated detrusor muscle from humans and rats, and in muscle from rat hypertrophied bladders, pinacidil(r1 pM) inhibited spontaneous contractile activity.411.412 In addition to their inhibitory effects on myogenic activity, cromakalim and pinacidil also inhibited, to varying degrees, contractions in detrusor muscle produced by a number of agonists. With the exception of the guinea pig detrusor muscle, cromakalim reduced carbachol-induced contractions in norIn mal or unstable bladder preparations from a number of guinea pig and human detrusor muscle, cromakalim (10 pM) and pinacidil (130 pM) inhibited contractionsto low, but not high, concentrationsof KCl,409,413 whilst in some other studies, these drugs also reduced contractions to high concentrations of KCl.410-412 In rat isolated detrusor muscle, lemakalim, Ro 31-6930, S 0121 (an analog of BRL 38226), pinacidil, P1060 (an analog of pinacidil), and RP 49356 produced concentration-dependent relaxation of tissue precontracted by 20 mM KCl, the relative order of potency being Ro 31-6930 > P1060 r lemakalim > pinacidil > RP 49356 > S O121.414Of these agents, only pinacidil relaxed contractions due to 80 mM KCl. In this study, lemaka h , pinacidil, and S 0121 were about 8 times more potent as relaxants of spontaneous tone in rat isolated portal vein than in detrusor muscle.


120

Although data are limited, pinacidil(1-30 p M )reduced contractions evoked by frequency-dependent electrical stimulation in normal and, to a greater extent, in rat hypertrophied bladders.411Similar findings were reported for c r ~ m a k a l i mHowever, .~~~ in other studies using unstable bladders from minipigs and humans, cromakalim did not significantly inhibit contractions induced by transmural nerve s t i m ~ l a t i o n .Moreover, ~ , ~ ~ ~ cromakalim antagonized responses to carbachol, but not those evoked by electrical stimulation of nerves.410 Overall, the ability of PCAs to inhibit electrically induced contractions appears to differbetween tissues and laboratories. These results contrast with the predictable inhibitory actions of these drugs against myogenic responses (electrical and mechanical) and may be related to the degree of depolarization produced by neuronal stimulation in different tissues. In comparison with the number of efflux studies undertaken in other smooth muscles, limited investigations have been performed in the detrusor muscle. Initial observation^^^,^^^ revealed that in guinea pig detrusor muscle, cromakalitn (10 pM) did not affect 86Rb+efflux, although concentrations in the range 5 to 100 pM increased 43K+ efflux. However, in rat bladder, a number of PCAs including lemakalim, pinacidil, and RP 49356 stimulated both &Rb+ and QK+ efflux.414In the mini-pig, muscle from unstable bladders was more sensitive to stimulation of 43K+ efflux by cromakalim than muscle from normal bladder^.^" However, in the rat, normal and hypertrophied muscles were equisensitive to 86Rb+ stimulation by pinacidil (1-30 PM).~"Pinacidil (3-30 pM) also stimulated 86Rb+efflux in normal human bladdeF3 (Fig. 13).Thus, in contrast to the guinea pig detrusor muscle, &Rb+-permeableK+ channels are present in detrusor muscle from rat and man. Muscle from the guinea pig, as well as that from the rat, mini-pig, and man, is able to efflux 43K+ ions, although, as found in other smooth muscles, the concentrations of drug required are higher than those inhibiting myogenic activity. Intracellular recording techniques, and the double sucrose gap method, have been employed to evaluate the effects of cromakalim on the electrical properties of detrusor muscle cell membranes. In guinea pig bladder muscle cromakalim (0.1 and 0.5 pM) abolished electrical spiking and, at higher concentrations (1 to 10 pM), evoked concentration-dependent hyperpolarizat i ~ n . ~Thus , ~ cromakalim ' ~ raised the membrane potential from about - 60 to about -82 mV, a value approaching the K + equilibrium potential. Supportive evidence for the hyperpolarizing action of the drug via K + channels was obtained by its reversal towards the resting membrane potential following the addition of 20.2 mM KCl. Also, using the double sucrose gap m e t h ~ d , ~ , ~ ~ ~ cromakalim (1 to 10 pM) reduced the size of the electronic potential, hyperpolarized the membrane, and produced relaxation in muscle from normal guinea pigs or Landrace pigs with bladder instability. In the guinea pig bladder, cromakalim-induced 43K+ efflux was unaffected by apamin (10 pM), whilst data obtained using procaine (5 mM) and TEA (10 mM) were equivocal due to stimulation of basal K+ efflux by these blockers.416 Moreover, in the presence of both nifedipine and apamin, cromakalim still stimulated 43K efflux. In this report, hyperpolarization produced by cromakalim (5 pM) was antagonized by procaine (5 mM) and quinidine (50 pM), although TEA, apamin, and 4-AP were ineffective. The effects of glibenclam+


121

"g +n

[r

P

0 1

3

10

30

Pinacidil concentration (pM)

Figure 13. Increments in the rate of =Rb+ efflux induced by pinacidil in human bladder muscle. Results are expressed as percentage of average of 5 samples obtained before applying drug and represent peak efflux rate produced upon drug application. Each value is presented as mean with SEM from number of experiments shown in parentheses. Statistical significance 'p < 0.05, **p < 0.01 (Student's t-test). (Reproduced from M. Fovaeus, K-E. Anderson, and H. Hedlund, I. Urol. 141, 637-620, with permission. 0 1989 American Urological Association, Inc.)

ide upon cromakalim-induced hyperpolarization have not been reported. Glibenclamide, however, has been shown to antagonize the relaxant effect of cromakalim416and, in rat detrusor muscle,414both the relaxant activity and enhancement of 42K+and =Rb+ efflux by PCAs were antagonized by glibenclamide. Evaluation of the effects of PCAs on bladder contractility in vivo is limited to the mini-pig410and rat.411,417 In mini-pigs with chronic urethral obstruction, cromakalim (0.3 mg/kg i.v.) abolished contractile activity, but not the ability of the animals to void. The dose of cromakalim used was about 10 times higher than the threshold dose for reducing blood pressure (see Sec. 1I.B); lower doses of cromakalim were not examined in the mini-pig model of bladder obstruction. Given orally, cromakalim and pinacidil(1 and 5 mg/kg) had little or no effect upon spontaneous contractile activity or bladder capacity in normal rat bladders. However, in a model of bladder hypertrophy induced by urethral obstruction, cromakalim and pinacidil abolished spontaneous contractions and pinacidil reduced micturition pressure, although an ability to void was retained.

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Since these drugs selectively inhibit spontaneous contractile activity in detrusor muscle without affecting the micturition reflex, PCAs may offer a novel treatment for detrusor muscle instability secondary to bladder outflow obstruction. However, initial data following a study with pinacidil in patients with bladder hyperactivity and bladder outflow obstruction (secondary to prostatic hyperplasia) failed to demonstrate any clear improvement in bladder As described above, S 0121 is a relaxant in rat bladder and portal vein. S 0121, like cromakalim, is also a relaxant in isolated ureter from rabbit and humans,419although S 0121 produced a smaller hyperpolarization than cromakalim in rabbit tissue. On the basis of these data, PCAs may be useful in the treatment of kidney stones by aiding their passage along the ureter.420 V. GASTROINTESTINAL SMOOTH MUSCLE In guinea pig isolated taenia caeci, cromakalim (0.4 to 10 pM), nicorandil ( 2 3 pM) and pinacidil (21 pM) reduced basal Ro 31-6930 was about 8 times more potent than cromakalim on basal tone in this Although pinacidil relaxed spontaneous tone in guinea pig taenia caeci and rat fundal strips, no effect was detected upon baseline tension in guinea pig isolated ileum, despite both carbachol- and histamine-induced contractions being inhibited (IC504 and 10 pM, respe~tively)."~In mouse distal colon, spontaneous contractile activity was inhibited by pinacidil and diazoxide, respective IC50 values being 6 and 100 pM.425In guinea pig taenia caeci, cromakalim (0.3-3 pM) relaxed contractions induced by 10 mM KCl or carba~hol.~' Similar findings were obtained with pinacidil and nicorandil against 30 mM KCl contractions and, interestingly, both drugs fully relaxed contractions due to 100 mM KCl.421When caffeine was employed to contract the tissue, presumably by release of intracellular Ca2 from stores, pinacidil and nicorandil had an inhibitory effect. After saponin treatment to permeabilize the cell membrane in taenia caeci, Ca2+-induced contractions were not inhibited by pinacidil (300 pM), although trifluoperazine and W-7 (protein kinase C inhibitors) were active.84 Measurement of peristaltic reflexes (induced by increasing intraluminal pressure) in guinea pig isolated ileum showed that cromakalim was about 10 times more potent as an inhibitor of contractions in longitudinal (IC50approximately 1 pM) than circular muscle.426This difference may be related to different numbers, and types, of K+ channels in these muscle layers. In guinea (1 pM) pig longitudinal muscle myenteric plexus p r e p a r a t i ~ n , ~ cromakalim '~ abolished phasic contractions but higher concentrations (100 pM) had no effect on basal tone. In this preparation, cromakalim (10 pM) inhibited contractions to electrical field stimulation and inhibited agonist-induced contractions by varying degrees (5-HT > nicotine > pilocarpine) but had no effect on 3HACh release evoked by these stimuli. However, cromakalim, 1 to 100 pM, inhibited release of 3H-AChcaused by stimulation of muscarinic (MI)receptors by pilocarpine. In rat oesophageal tunica muscularis mucosae, cromakalim and ( - )pinacidil suppressed contractions evoked by electrical field stimulation by KC1 (10-50 mM) and relaxed tonic contractions produced by the muscarinic agonist cis-dioxolane.428 +


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In guinea pig stomach muscle, cromakalim (3 pM) reduced mechanical activity and evoked a small hyperpolarization, while slow-wave activity per~ i s t e d . ~Cromakalim ’~ (1-10 pM) evoked relaxation and hyperpolarization in the guinea pig anal sphincter preparation-relaxation was always associated with hyperpolarizati~n.~~ Cromakalim (L 1 pM) also hyperpolarized the membrane potential of circular muscle in canine colon.431In an earlier nicorandil (10 pM-0.3 mM) evoked hyperpolarization in guinea pig terminal ileum by a mechanism insensitive to apamin, but inhibited by local anaesthetics. Moreover, the nicorandil-evoked hyperpolarization persisted in the absence of Ca’+ in the bathing fluid, suggesting that nicorandil was acting upon a Ca’+ -independent K + channel in this tissue. A limited number of investigations have been performed on the effects of PCAs upon K + efflux in gastrointestinal smooth muscle. Cromakalim and nicorandil stimulated =Rb+ efflux in guinea pig taenia ~aeci.~’’Subsequently, cromakalim was shown to stimulate 42K+ efflux and to hyperpolarize this preparation.433The effects of cromakalim on tension, 86Rb+efflux, and membrane potential were unaffected by apamin.a*m*4z4*4m Cromakalim-induced relaxation was not antagonized by haemoglobin (an inhibitor of guanylate cyclase).48This result is corroborated by the lack of effect of cromakalim on cGMP levels in this although CAMPlevels were increased by cromakalim in guinea pig taenia caeci. Such observations remain unexplained at the present time. Glibenclamide (0.1 pM) has been found to antagonize, in a competitive manner, the relaxant properties of cromakalim and Ro 31-6930 in guinea pig taenia ~ a e c i . ~Cromakalim-induced ’~ hyperpolarization was inhibited by glibenclamide in guinea pig taenia ~ a e c i ~ and ’ ~canine Also, in mouse distal colon, glibenclamide antagonized the relaxant effects of pinacidil and diazoxide, but not that due to nifedi~ine.~‘~ Interestingly, in this preparation, phentolamine (1 pM) was ineffective as an antagonist. Overall these data support findings in other smooth muscles that PCAs produce their inhibitory effects in gastrointestinal smooth muscle by a glibenclamide-sensitive mechanism suggesting an effect on ATP-sensitive K+ channels. In the rat, cromakalim given subcutaneously inhibited gastric motility as assessed by gastric emptying (Cooper and McRitchie, 1985, unpublished observations) and also had an inhibitory action on transit time for a charcoal meal in the mouse.426 The significance of these inhibitory properties of PCAs in the gastrointestinal tract is unexplained at the present time. The evaluation of such drugs in other clinical conditions has not revealed an incidence of adverse side effects such as constipation. However, the animal data suggest that PCAs may have utility in conditions associated with disturbances in gastrointestinal motility such as irritable bowel syndrome.

VI. UTERINE SMOOTH MUSCLE In isolated uterus from the term pregnant rat, cromakalim (0.04-1.3 pM) inhibited spontaneous phasic contractile activity and contractions evoked by low (40 mM) concentrations of KCl, actions sug-

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gesting an effect of cromakalim on K + channels.434The phasic component of oxytocin-induced contractions was inhibited by cromakalim (10 pM) in this preparation4%and by pinacidil, cromakalim, and RP 49356 in oestradiol-primed rat uterus.249,435 A number of K+ channel blocking drugs (procaine, quinine, quinidine, TEA, and 4-AP) inhibited the relaxant effect of cromakalim upon oxytocinenhanced phasic spasm^.^^,^^ Cromakalim, RP 49356, pinacidil (1-10 pM), and minoxidil sulphate (100 pM) relaxed 20-mM KC1-induced contractions and oxytocin-induced phasic spasms in uteri from oestradiol-treated rats.435 Glibenclamide (10 pM) inhibited the relaxant properties of cromakalim, RP 49356, and pinacidil. However, the relaxant effect of higher concentrations (100 pM) of these PCAs against contractions to high (80 mM) KCl was glibenclamide insensitive. In addition to its uterine relaxant properties, cromakalim (10 pM) inhibited the electrical spike activity associated with oxytocin but only a small hyperpolarization (5 mV) was recorded in myometrial strips.434In single cells, cromakalim had no effect upon action potentials, Ca2+ inward current, or K + outward current. Additionally, ion flux studies showed that cromakalim (10 pM) did not stimulate the efflux of eithei'34 86Rb+or 436 &K+, although oxytocin did stimulate the efflux of both ions. Thus, although channels permeable to Rb+ and K + exist in the uterus, cromakalim does not stimulate ion efflux in this tissue, despite evidence supporting an action of PCAs on uterine K + channels. Evidence for the existence of at least two K + currents has been reported in rat ~ t e r u ~These . ~ include ~ ~ , a~fast ~ outward ~ K + current (reversal potential -80 mV) and a slow K + current (reversal potential -65 mV). The small hyperpolarizing effect of cromakalim may be related to the enhancement of this slow K + current, which may be involved in the generation of pacemaker a ~ t i v i t yNo . ~ evidence ~ appears to exist at present to support the notion that such K + channels may be ATP dependent. The uterine relaxant activity of cromakalim in isolated uteri has been confirmed following i.v. administration (0.1 and 1 mg/kg) to conscious nonpregnant and Day 18 pregnant rats439;similarly, RP 49356 was active in nonpregnant rats.435The inhibitory effects of these drugs in uterine smooth muscle were immediate and complete. At 0.1 mg/kg, both drugs reduced blood pressure (by about 50%)over a similar time course as their uterine relaxant activity. Glibenclamide (20 mg/kg i.v.) inhibited the actions of both drugs on the uterus and v a ~ c u l a t u r e . ~ ~ ~ Interestingly, tolerance developed to the uterine relaxant effects of a large i.v. dose (1 mg/kg) of cromakalim, given at 8-12-h intervalsm9and subsequently was also shown to apply to the hypotensive effect of the drug (i.v.) in ovariectomized ratsw The induction of tolerance in the uterus was more marked in pregnant than nonpregnant rats and contrasts with a lack of tolerance to daily oral doses of cromakalim in cardiovascular studies using SHR.lQr3" The reason for the development of tolerance to the uterine inhibitory action of cromakalim is unclear.439However, the mechanism appears to be related to K channel opening, since cross-tolerance developed between cromakalim and RP 49356 but not between cromakalim and relaxin (a uterineselective polypeptide relaxant).441 +


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The clinical potential of PCAs in the uterus requires investigation, since such smooth muscle relaxants may have utility in events such as premature labor and dysmenorrhoea. VII. PANCREAS Glucose is the most important physiological stimulus for the release of insulin from p-cells in the pancreas. When pancreatic p-cells are stimulated by glucose, a depolarization of the cell membrane occurs, initiating a cyclical which invokes insulin secretion pattern of electrical activity and influx of Ca2+, by exocytosis (see Ref. 442). Early studies in rat or mouse pancreatic islets and latterly in membrane patches demonstrated that the depolarization evoked by glucose or carbohydrate metabolism was associated with decreased K + permeability and closure of a K C ~ h a n n e l . This ~ ~ -channel ~~ was subsequently identified as an ATP-regulated K + channel, being closed by intracellular ATP, thus forming a link between carbohydrate metabolism and the ionic events involved in insulin ~ecreti0n.l~ Further studies led to the identification of ATP-sensitive K + channels in pancreatic p-cells from animals or man and also in insulin-secreting cell lines (see Ref. 132). The properties of this ATP-sensitive K + channel and the mechanism involved in its regulation are currently under intensive investigation. It has been suggested that channel regulation may involve phosphorylation (via stimulation of protein kinase C).446-449 The inhibitory properties of nonhydrolyzable analogs of ATP suggest that the inhibitory effect of ATP itself does not involve pho~phorylation,'~ although some hydrolysis of ATP (possibly via channel protein phosphorylation) may be necessary to maintain the channel in a functional state.450The ability of much lower concentrations of ATP to inhibit channel activity in excised inside-out patches, rather than in whole cells, led to the suggestion that other intracellular modulators of channel function may exist in intact cells. Modulatory factors that have subsequently been identified are ADP (highlightingthe importance of the cytosolic ATP/ADP ratio),451-453 intracellular GDP and GTP,454and the pyridine nucleotides NADP and NAD.455 Sulphonylureas such as tolbutamide and glibenclamide have been shown to be specific blockers of ATP-sensitive K + channels in pancreatic p-cells (respective ICm values 7-17 pM and 27 nM, see Refs. 456 and 457), indicating their ability to increase insulin release and thus act as antidiabetic agents. Radioligand binding studies in rat pancreatic islets with 3H-glibenclamide have demonstrated reversible binding to saturable, high-affinity recognition sites for sulphonylureas. 133,458 Compared with other sulphonylureas, glibenclamide displayed greatest potency in these radioligand binding studies correlating with its potency in enhancing insulin release from pancreatic islets.459 Although high-affinity binding sites for 3H-glibenclamidehave been identified in brain membranes458and cardiac cells,221some disparity exists between the conductance and properties of ATP-sensitive K channels in these different tissues (see Ref. 132 and Table IV). In addition to the initial identification, purification, and affinity labeling of the subunit structure of the ATP-sensitive K+ channel from pig brain or rat p - ~ e l l s ~and ~ , ~the ' functional expression (in Xenopus laevis oocytes) of mRNA coding for the ATP-sensitive K + channel +

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in an insulin-secreting cell line,462further studies are essential to extend current knowledge of the function and regulation of such channels. Since glibenclamide inhibits the hyperpolarizing and vasorelaxant properties of PCAs (Sec. II.A, Table II), the question arises as to whether PCAs may modulate ATP-sensitive K+ channels in the pancreas and thus influence insulin secretion and plasma glucose. The hyperglycaemic effects of diazoxide in man have been well documented (see Ref. 463). Following initial reports that diazoxide could decrease glucose-evoked insulin release in pancreatic islet^,^,^^^ diazoxide has been shown to increase K+ efflux (using %Rb+) from p-cells resulting in a hyperpolarization.466In contrast, the sulphonylurea tolbutamide was shown to inhibit %Rb+efflux and cause depolarization. Thus diazoxide appeared to open a K+ channel which influenced the resting membrane potential of pancreatic p-cells. Following characterization of an ATPsensitive K + channel in the pancreas (see above), work in pancreatic p-cells or insulin-secreting cell lines (using membrane patches or whole cells) has confirmed that diazoxide increased IK(ATP) currents (evoking hyperpolarization) and reversed the inhibition of IK(ATP) produced by tolbutamide or glucose.17,448,456,457,467-470 Depending upon experimental conditions, these effects of diazoxide were observed over a wide range of concentrations (up to 600 pM), although IC50values of 20 and 102 pM were reported in the presence of 0.3 and 1 mM ATP (in the recording pipette) re~pective1y.l~ Although electrophysiological studies have indicated that the presence of ATP (at the intracellular surface) and/or inhibition of the ATP-sensitive K+ channel with sulphonylureas appears to be a prerequisite for diazoxide to enhance 17,457 the precise mechanism is under current investigation. Evidence obtained, using an insulin-secreting cell line, that Mg2+ was not obligatory and that high levels of ATP (5mM) inhibited diazoxide-induced increases in IK(ATp)I suggested that diazoxide may interfere with the ability of A T P to inhibit ATP-sensitive K+ However, this theory is confounded by evidence that diazoxide only increased IK(ATp) in the presence of Mg-ATP and was dependent upon the presence of a source of hydrolyzable ATP (the activity of diazoxide was lost when ATP was replaced with the nonhydrolyzable analog AMP-PNP). This suggests that channel phosphorylation may be implicated in the activity of diazoxide.448Interestingly, in isolated membrane patches, diazoxide displays greater potency at enhancing IK(ATp) when applied to the internal rather than the external side of the membrane, suggesting that the specific recognition site for diazoxide may be accessed more readily from the intracellular side of the membrane.448These authors also reported that in the absence of ATP and Mg2+ (i.e., not under normal physiological conditions), diazoxide may inhibit ZK(ATp), possibly in a manner similar to sulphonylureas. An explanation for the inhibitory action of diazoxide may be that it enhances the rate of channel run down (decline in channel activity if ATP is not present). An initial study conducted in the p-cell line HIT T15 has indicated that diazoxide and sulphonylureas may interact at different recognition sites as judged by the inability of diazoxide (200 pM, a concentration producing marked stimulation of %Rb+efflux), to modify 3H-glibenclamidebinding.471 Potentiation of glucose-induced insulin release and decreased 86Rb efflux has been demonstrated with a high concentration (500 pM) of minoxidil sul+


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phate in mouse pancreatic p-cells, suggesting closure of ATP-sensitive K + channels in the pancreas.%' In contrast, earlier clinical studies have indicated that minoxidil may increase plasma glucose in patient^,^^,^^^ although such effects may be partly explained by increased levels of circulating catecholamines and stimulation of glycolysis. Thus the influence of minoxidil upon IK(ATp) and pancreatic function remains unclear at the present time. Using whole cells or outside-out membrane patches, nicorandil had no effect upon IK(ATP) in mouse pancreatic p - ~ e l l salthough ,~~ at high concentrations (200-500 pM) increases in current were observed using the insulinoma cell line RIN,5F.474 Similarly, RP 49356 (200-500 pM) evoked increases in IK(AT~), although like nicorandil, such increases in current were weak compared to the effects of diazoxide. Evidence that pinacidil may open ATPsensitive Kf channels in the pancreas has come from studies demonstrating enhanced %Rb+efflux from mouse or rat pancreatic islets (in the presence of glucose).469,475,476 High concentrations of pinacidil (200-500 pM), like nicorandil and RP 49356, are associated with increased IK(ATp).469r474 Initial work has suggested that unlike other drugs of its class, SDZ PCO 400 (at concentrations as low as 50 p M ) appears to act as a K+ channel blocker (Dunne, unpublished observations). This interesting discrepancy requires further investigation. Cromakalim (-500 pM) was found to have a minimal influence upon %Rb efflux in rat pancreatic islets475and did not enhance IK(ATp) in mouse pancreatic p - ~ e l l sAlthough .~~~ cromakalim (100 pM) was ineffective in the insulin-secreting cell line, CRI-G1,448,468 ATP-sensitive K + channels were opened in RIN,5F cells following exposure to cromakalim (100-200 pM).16 In addition, both pinacidil and cromakalim hyperpolarize the cell membrane and have been shown to inhibit glucose evoked d e p ~ l a r i z a t i o n ' ~and , ~ ~rises in intracellular Ca2+.16* 475,476 In vitro studies in rat or mouse pancreatic islets have demonstrated that pinacidil(500 pM) or cromakalim (250-500 pM) may inhibit insulin s e ~ r e t i o n ,although ~ ~ , ~ ~other ~ studies have demonstrated cromakalim and nicorandil to be without e f f e ~ t . ~ ~ ~ , ~ " Despite indirect evidence in vivo that intravenously administered cromakalim may inhibit insulin release,478plasma levels of insulin or glucose were largely unaffected by oral or intravenous administration of blood-pressurelowering doses of cromakalim to rats51,477 (see Fig. 14). The ability of other PCA drugs such as nicorandil, pinacidil, cromakalim, and RP 49356 to exert effects within the pancreas was observed only at high concentrations (up to 1000 times greater than those necessary to relax vascular or tracheal smooth muscle). This suggests that hyperglycemia will not be a problem associated with these drugs in the clinic and to date no adverse effects have been reported in terms of blood glucose homeostasis. In healthy volunteers chronic administration (up to 2 weeks) with clinically relevant doses of pinacidil has failed to demonstrate an effect upon glucose-stimulated insulin secretion or oral glucose tolerance.479Long-term studies in hypertensive patients are therefore awaited with interest. In general, studies in insulin-secreting cell lines or mouse pancreatic islets have indicated that a good correlation exists between the concentrations of diazoxide or sulphonylureas required to produce either a respective half maximal increase in IK(ATp) (diazoxide 20-100 pM),17 or an inhibition of IK(A*p) +

(b) Diazoxide

Figure 14. Effects of i.v. administration of (a) cromakalim (0.1 mg/kg, A and open columns; 0.3 mg/kg, A and spotted columns), and (b) diazoxide (10 mg/kg, and open columns; 30 mg/kg; 0and spotted columns) on blood pressure (BP), heart rate (HR), and plasma glucose levels in conscious spontaneously hypertensive rats (SHR). Control animals (0 and filled columns) received the vehicle of cromakalim or diazoxide. Values represent mean with SEM from 46/group. Basal values of BP ranged from 180-207 mmHg and HR from 349-385 bts/min and did not vary significantlybetween the groups. (Reproduced from U. Quast and N.S. Cook, 1. Pharmacol. Exp. Ther., 250 261-271, with permission. 0 1989 American Association for Pharmacology and Experimental Therapeutics.)

(a) Cromakalim


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(glibenclamide 4-27 nM,17,457and the concentrations of each drug required to modify insulin secretion.17*457,459 Since such concentrations are comparable to the therapeutic plasma concentrations of the free drugs480,481 this suggests that an ATP-sensitive K + channel is the primary site of action for mediating the effects of diazoxide and sulphonylureas upon insulin release. In addition, the influence of diazoxide upon insulin release and vasorelaxation occurs within the same concentration range, highlighting the fact that diazoxide is unsuitable for the treatment of hypertension owing to its ability to alter glucose homeostasis. Interestingly, in anaesthetized dogs, nicorandil has demonstrated a direct influence on pancreatic exocrine secretion, evoking increases in pancreatic fluid secretion with increased concentration of bicarbonate and amylase.482Such effects may be attributed to the nitrate moiety, since pinacidil was without effect in this study. Other substances shown to influence ATP-sensitive K + channels in the pancreas and inhibit insulin release in a variety of experimental conditions include galanin, somatostatin, and Galanin has been shown to increase IK(ATP) and hyperpolarize the cell membrane of pancreatic p-cells or RINm5F However, it is unlikely that galanin or somatostatin interact with the ATP-sensitive K+ channel in exactly the same manner as PCA drugs, since they are thought to open ATP-sensitive K+ channels via a pertussis-toxin-sensitive G - p r ~ t e i n , ~a' ~property ,~~ not possessed by drugs such as cromakalim. VIII. CENTRAL NEURONES

The diversity of K+ channels found in the central nervous system (CNS) has recently been reviewed (see Ref. 491). The channel types described include voltage-activated K+ channels (delayed rectifier, transient K+ current, slowly inactivating outward current, inward rectifier, and the M current), Ca2+activated K+ channels (slow, medium, and fast Ca2+-activated currents), a Na+-activated K+ conductance, and an agonist-activated K+ conductance. ATP-sensitive K + channels have been described in cortical and hypothalamic neurones21,22and in substantia nigra= (Table IV). The conductance of these K+ channels is in the range 53-150 pS and their low sensitivity to ATP suggests these are low-affinity ATP-inhibited K+ channels. The role of these channels in nerve cells has not yet been fully elucidated but it seems likely that they would be activated by glucose, neurotransmitters, and hormonal peptides. A study in the substantia nigra showing a link between decreased glucose concentration, opening of ATP-inhibited K+ channels, and decreased GABA release suggests a functional role in seizure control for this type of K+ In addition, other findings suggest a modulating function for this channel in ischaemic and/or anoxic conditions, which lead to a decrease in i[ATP] and hence to channel opening.493Glibenclamide blocks the anoxic response (hyperpolarization) of CA3 hippocampal n e u r o n e ~ supporting ,~~ the involvement of ATP-sensitive K+ channels in this response. High-affinity sulphonylurea binding sites have been described in several brain regions456*m,4954w and were most dense in areas such as the substantia nigra, in contrast to the hypothalamus, spinal cord, and medulla oblongata. These neuronal binding sites may be identical to those found on insulin-

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secreting cell membranes and in heart cells. This contention is supported by the finding that sulphonylurea binding protein solubilized from pig brain membranes has a similar molecular weight to that from rat @-celltumour membrane^.^^,^^^ However, it is not yet known if the sulphonylurea receptor actually constitutes the K+-ATP channel. To date, no evidence exists for a direct interaction of PCA drugs with the sulphonylurea receptor, as shown by the inability of pinacidil to displace ['251]-iodoglibenclamidefrom rat brain tissue.499 Limited studies have reported the effects of PCAs on electrical discharges or ion efflux in neurons. In rat whole brain, cortical and hippocampal synaptosomes, cromakalim (up to 100 pM) and high concentrations of pinacidil and nicorandil did not affect 86Rb+efflux.500In cerebellar slices, inhibition of the release of glutamate by 60-mM KC1 or by the K+ channel blocker tetrapentylammonium required a high concentration (1 mM) of cromakalim.50' The excitability of hippocampal pyramidal cells was depressed by 100-pM c r ~ m a k a l i mThese . ~ ~ ~ findings were extended to embryonic rat cultured hippocampal neurons where cromakalim (50-500 pM) augmented an outward voltage-dependent K+ current, which may be similar to the delayed rectifier In similar preparations (exposed to Mg? +-free, glycine-supK+ plemented solutions to induce fluctuations of i[Ca2+]and excitatory synaptic activity), high concentrations of cromakalim (100 pM) and diazoxide (500 pM) decreased these effects by a glibenclamide-sensitive mechanism.504These findings suggest that PCAs are able to inhibit excitation associated with synaptic activation of NMDA (N-methyl-D-aspartate) receptor^.^^ In slices of rat substantia nigra, lemakalim, cromakalim, pinacidil, and nicorandil stimulated %Rb+ efflux and inhibited [3H]-GABA release by a sulphonylurea-sensitivemechanism% (Fig. 15).Surprisingly, lemakalim was about 50 times more potent than cromakalim. An excellent correlation existed for the ability of these drugs to inhibit [3H]GABA release and to stimulate 86Rb+ efflux. Thus it appears that PCAs, by their ability to open ATP-sensitive K+ channels, can inhibit neurotransmitter release. Hence, by virtue of their ability to diminish neuronal excitability, PCAs could have therapeutic utility in conditions such as epilepsy. Data obtained from some animal models of epilepsy support this view. In genetically epileptic rats lemakalim (10 nmol intracerebroventricularly, i.c.v.) reduced seizures.506In addition, seizures in mice induced by the K + channel blocker mast cell degranulating peptide (MCD, a bee venom neurotoxin) were inhibited by prior i.c.v. administration of lemakalim (1-100 nmol) and RP 49356 (10 nmol), but not by BRL 38226.507However, lemakalim and RP 49356 were inactive, even at higher doses, if administered after MCDinduced seizures were established. In addition, neither lemakalim nor RP 49356 affected seizures induced by two other K + channel blockers, dendrotoxin I (DTX) and 4-AP, suggesting that MCD blocks a different voltagedependent K+ channel. However, despite the specificity of the inhibitory action of PCAs for MCD-induced seizures, the PCAs (up to 100 pM) did not affect the binding of either '=I-MCD or lZ5I-DTXto their respective receptors in rat synaptic membranes.508 Thus lemakalim has shown anticonvulsant activity in two rodent models. These findings provide encouragement that lipophilic PCAs with the ability


-

131

log [lernakalirn], M

0

s? I00

a a

m

E

50

I

"0 -10

-8

-6

-4

log [PCA], M Figure 15. Inhibition of [3H]-GABA-evokedrelease from substantia nigra (SN) slices by increasing concentrations of lemakalim. Release was evoked either by 15-mM K+ (0), by 20-mM glucose (O),or by 1WnM gliquidone (m). The basal release of [3H]-GABA in 3.5-mM K + medium was not affected by 100-nMlemakalim. (Inset) Activation by different effectors of [3H]-GABArelease. Columns: 1, basal release in 3.5-mM K+ containing medium; 2, 15-mM K+; 3, 20-mM glucose; 4,100-nM gliquidone. Time of [3H]-GABA-evokedrelease was 5 min. Ordinate shows [3H]-GABA release in fractional rate ( n = 4). (b) Inhibition by different PCAs of [3H]-GABA release evoked by 100-nM gliquidone. This release was inhibited by increasing concentrations of lemakalim (0) (half-maximal effects, K0.5 = 10 2 2 nM), nicorandil(0) (G.5= 120 2 5 nM), cromakalim (H) (K0.5 = 300 15 nM), pinacidil(0) (G.5 = 400 2 20 nM) and P1075 (A)(&.5 = 500 & 20 nM) (n = 4). (Reproduced from Ref. 505 with permission.)

*

to penetrate the blood-brain barrier and possessing specificity for neuronal K+ channels, may have potential as treatment in some convulsive states. In an in vivo study examining the behavioral properties of PCAs, intraperitoneal administration of cromakalim (1-10 mg/kg) reduced pilocarpine-induced chewing movements in mice, whilst i.c.v. administration of cromakalim also had this inhibitory effect (EDm about 0.1 pg).m However, cromakalim did not displace the binding of either [3H]N-methylscopolamineor [3H]-o~otremorine and had no effect on basal- or carbachol-stimulated PI hydrolysis in rat cerebral cortex. Thus the ability of cromakalim to inhibit a behavioral response to the muscarinic agonist pilocarpine is not due to a direct interaction with muscarinic or monoamingergic neuronal receptors but may be achieved by opening of neuronal K+ channels causing a decrease in cholinergic nerve function. Further work is required to elucidate the type(s) of K+ channel involved and the significance of these findings.

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Finally, novel series of amidochromanols510and of benzopyrans5" have been reported to have potential in the treatment of depression based upon their ability to improve swimming performance in mice. Interestingly, the latter compounds lacked blood-pressure-lowering activity in SHR and therefore appear to be selective for the CNS. However, despite their structural similarity to cromakalim, it is uncertain if these compounds act by virtue of being PCAs in neurones. IX. SKELETAL MUSCLE Subsequent to the discovery of ATP-sensitive K+ channels in the heart13 (see also Sec. II.B), similarly gated K + channels were found to be widely distributed on the surface membrane in skeletal muscle.18-z0,512514 Such channels are inhibited by the presence of i[ATP] at the inner surface of the membrane and it appears that sulphydryl groups near the ATP-binding site are functionally important in the modulation of these K+ channels.514Although i[ATP] is normally well buffered, the concentration may fall in conditions of skeletal muscle rigor, leading to a high K+ permeability and a beneficial decrease in the excitability of diseased skeletal muscle. It has recently been shown that falls in intracellular pH reduce the inhibitory effect of ATP on excised patches of frog skeletal muscle515and this mechanism may be operative as one means of offsetting skeletal muscle rigor. A limited number of studies have examined the effects of PCAs in skeletal muscle preparations. Relatively high concentrations of cromakalim (2100 pM) increased %Rb efflux, whilst a small hyperpolarization was demonstrated in frog sartorius muscle following cromakalim (200 pM).516Glibenclamide antagonized both effects of cromakalim, indicating an effect on an ATP-sensitive K+ channel. Diazoxide (600 pM) did not affect %Rb+ efflux in this preparation. Using inside-out patches from mouse skeletal muscle,z0in the presence of 0.1 mM i[ATP], internally applied cromakalim (0.2-0.8 mM), pinacidil (0.4 mM) and RP 49356 (0.4 mM), but not diazoxide (0.4 mM), increased opening time of the ATP-sensitive K+ channel. The effects of cromakalim were found to be temperature dependent, the drug being more active at 30 "C than at 20 "C. Another PCA, SR 44866, also opened K + channels, an effect that was inhibited by increasing i[ATP], or by 10-pM gliben~lamide.~~' However, in this study, high concentrations of SR 44866 also inhibited the Na+ inward current. In addition, SR 44866 inhibited electrically evoked twitches in mouse isolated skeletal muscle by a mechanism sensitive to both temperature and gliben~lamide.~'~ However, the potency of SR 44866 as an inhibitor of skeletal muscle twitch is much less than that as a smooth muscle relaxant. It was notedz0that, in the absence of ATP, RP 49356, but not cromakalim, pinacidil, or diazoxide, could reactivate channel opening following channel run down. This phenomenon has also been reported for this drug in pulmonary artery and cardiac myocytes. A similar effect was reported for cromakalim in cardiac myocytes.z65It has been suggestedz0that RP 49356, but not cromakalim or pinacidil, is able to recruit new K+ channels which are normally inactive. Other K+ channels exist in skeletal muscle, the delayed rectifier K+ channel +


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(TEA sensitive), and both small- and large-conductance Ca2+-activated K+ channels have been d e s ~ r i b e d . ~No l ~ -evidence ~~ has been obtained for an action of PCAs on the large conductance Ca2+-activatedK + channel.20 In biopsy specimens from human skeletal muscle, cromakalim, pinacidil, and RP 49356 (each at 100 pM) produced hyperpolarization sensitive to blockade by tolbutamide, glibenclamide, or Ba2 .524526 Larger hyperpolarizations were observed in specimens from diseased muscle (such as in patients with myotonic dystrophy or hypokalaemic periodic paralysis). This difference is probably related to the less negative membrane potential in such tissue compared to that from normal muscle. Concomitantly, a small decrease in intracellular [K+J occurred.526Cromakalim (10-100 pM) and EMD 52692 (1-10 pM) were also shown to suppress after contractions and spontaneous mechanical activity in isolated fiber bundles from myotonic human skeletal muscle.527 In a more recent report from a study employing the patch clamp technique in human skeletal muscle cell membranes, EMD 52692, RP 49356, and cromakalim (100 pM) increased the open probability of two types of K + channels, one being ATP sensitive and the other ATP independent." Interestingly, glibenclamide (5 pM) antagonized the actions of the PCAs on both types of K + channel. This study therefore provides information that PCAs are able to open both ATP-sensitive and ATP-insensitive K+ channels in skeletal muscle and that glibenclamide is not a specific blocker at ATP-sensitive K+ channels in this tissue. Since changes in ion permeability (Na+, C1-, K+) may contribute to membrane depolarization and muscle fatigue, selective PCAs may find an application in some disorders manifested as skeletal muscle rigidity, although these drugs are more potent as PCAs in smooth muscle. In addition, other studies in human muscle have implicated the small-conductance (apamin-sensitive) Ca2+-activatedK+ channel in the condition, myotonic muscular dystrophy, which is characterized by muscle stiffness.528It has been shown that PCAs like cromakalim do not activate this apamin-sensitive K + channel in smooth muscle (see Sec. 1I.A) and therefore these drugs are unlikely to act upon this particular K + channel in skeletal muscle. +

X. OTHER EFFECTS OF PCAs

In the treatment of impotence, vasodilators such as papaverine, injected locally, are commonly used. The established smooth muscle relaxant properties of PCAs in the vasculature suggest that these drugs may offer an alternative to current treatment. Moreover, recent data from rabbit isolated cavernosal tissue have shown that cromakalim (0.1 pM) and pinacidil(1 pM) are more potent than papaverine as inhibitors of spontaneous contractile activity, electrically induced contractions and those produced by NA.529Both cromakalim and pinacidil, but not papaverine, increased %Rb+ efflux from the tissue, suggesting that their inhibitory effects against contractile responses are due to K+ channel opening. Similar results were obtained for pinacidil in human isolated cavernosum.530 The occurrence of hypertrichosis during antihypertensive treatment with minoxidil (see Ref. 531) has led to the subsequent evaluation of the drug (applied topically) to enhance hair regrowth in areas of baldness. 532 Hyper-

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trichosis has also been reported in pinacidil-treated patients,307although its incidence is far less marked than witnessed with minoxidil. This evidence, coupled with the ability of topically applied minoxidil to promote hair regrowth in states of alopecia with differing aetiology, suggests that this effect may be mediated by a common mechanism on K+ channels. Indeed, the active metabolite of minoxidil, minoxidil sulphate, has been found to be more potent than the parent drug in stimulating hair growth in neonatal mouse cultured hair follicles.533Although it is unknown whether other PCAs induce hair growth, their potential in the treatment of baldness requires more extensive investigation. Another area where PCAs could offer clinical potential may be via an action upon immunoresponsive cells. These cells possess a number of ion channels including several types of K + channels (see Ref. 534). Although the functional importance of such K+ channels is unknown, it is possible that modulation of K+ flux (with PCAs) may influence various aspects of immune function such as cellular proliferation. Recent evidence in rabbits demonstrating that repeated topical application of pinacidil, its ( - )-enantiomer, cromakalim and nicorandil lower intraocular pressure has served to highlight a potentially beneficial influence of PCAs in disorders such as glaucoma.535Whether these effects in the eye may be attributed to enhanced K+ ion movement and consequent relaxation of smooth muscle or the result of K + channel modulation in epithelial cells requires further elucidation. XI. CONCLUDING REMARKS

In this article we have attempted to review the vast literature generated over the last few years in the field of PCAs. Since the K + channel opening property of cromakalim in smooth muscle was elucidated, there has been rapid and increasing interest in the role of K + channels in a variety of tissues. This is reflected in numerous reports of the biological properties of a number of structural analogs of cromakalim, nicorandil, pinacidil, and RP 49356. Marketed PCA drugs such as nicorandil, pinacidil, and diazoxide represent a variety of structural types distinct from the benzopyrans. It is anticipated that a number of novel PCA drugs may currently be under development in one or more therapeutic areas including hypertension, asthma, and angina. However, if tissue-selective PCA drugs are discovered, it is likely that these drugs may prove useful for the treatment of some other disorders, for example, detrusor muscle instability, irritable bowel syndrome, and epilepsy. Drugs for such indications should lack the cardiovascular effects associated with current PCAs and, by their selective action on K + channels in the target tissue, offer a novel therapy or an alternative to existing treatment. Although current PCAs are apparently nonselective in their inhibitory effects on a variety of normal smooth muscles, recent patents have suggested selectivity of S 0121 for smooth muscle in the ureter. Also, the CNS properties of some other benzopyrans, which are devoid of peripheral actions, suggest a different profile of selectivity. "Use" selectivity may be achievable with current PCAs, since such drugs will be used in patients where some type of abnormality


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prevails in a particular tissue. In this instance it is likely that the PCA drug may act at lower doses in this tissue to restore normal K+ channel function. Following the discovery of PCA drugs, further characterization of some K+ channels has been achieved by electrophysiological, pharmacological, and biochemical means and has been complemented by sequence data from molecular biology studies. It is only through increasing our knowledge of the factors implicated in K+ channel function, including changes that may prevail in certain disease states, that the clinical application of drugs that modulate K+ channel function may be realized. This knowledge is vital not only to exploration of novel areas for the therapeutic application of tissue-selective drugs that open K + channels, but also to areas where K+ channel blockade may prove useful. For example, in the heart, a more complete understanding of K + channel type and function should aid the search for effective antiarrhythmic agents. The mechanism(s)by which PCA drugs interact with K+ channels in smooth muscle, and in other tissues, is not fully understood. A number of different K+ currents, including the delayed rectifier (IK) and high-conductance Ca2+activated K+ currents, have been implicated in the actions of PCA drugs in tissues such as vascular smooth muscle and cardiac muscle. However, most evidence.suggests that drugs such as cromakalim may interact with an ATPsensitive K + channel. The ability of sulphonylurea drugs such as glibenclamide to antagonize the effects of PCAs on membrane potential, muscle tension, and K + efflux supports this contention. However, the concentrations of glibenclamide required are higher than those blocking such channels in pancreatic P-cells (Table IV). Moreover, glibenclamide has also been shown to block an ATP-independent K + channel in some tissues. Thus, it seems clear that PCAs activate both an ATP-sensitive K + channel, which is one of a family of such channels in various tissues, and another glibenclamide-sensitive, but ATP-independent, K+ channel. Additionally, in a given tissue there may be discrepancies between the K + modulating properties of PCA drugs. For example, diazoxide and SDZ PCO 400 block ATP-sensitive K + channels in the heart and pancreas, respectively, unlike other PCAs that are known to open K+ channels in these tissues. Recent evidence of an association between GABA release and modulation of ATP-sensitive K + channels in the substantia nigra has encouraged speculation that PCA drugs may have therapeutic application in the CNS. Thus drugs exhibiting specificity for neuronal K+ channels may prove beneficial in disorders such as epilepsy. Despite evidence of an association between the action of PCAs and ATPsensitive K+ channels, there is no published evidence for the existence of any specific recognition site for these drugs. Results from other investigations infer the existence of stereospecific recognition site(s). The possibility that PCAs may bind to an intra- or extracellular region within the ATP-sensitive K + channel requires investigation. It must be noted that although the target site for sulphonylureas appears to be the ATP-sensitive K+ channel, radioligand binding studies to date have shown that pinacidil does not interact at sulphonylurea receptor sites. The impact and success of PCAs as new therapeutic agents will depend

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upon demonstration that they possess additional properties over existing drugs and exhibit fewer side effects. For currently known PCAs it will therefore be important to identify properties beyond smooth muscle relaxation. In the treatment of hypertension, useful additional properties of PCAs may be their ability to have beneficial effects on plasma lipids, left ventricular hypertrophy and/or coronary heart disease, in addition to reducing blood pressure. Encouraging evidence, from animal experiments, that PCAs inhibit neuronally mediated bronchoconstriction suggests that these drugs may attenuate airways inflammation in addition to relaxing smooth muscle directly. However, in any particular therapeutic area, the results of extensive clinical trials, including long-term administration of these drugs, are eagerly awaited before their full potential can be assessed. The authors wish to acknowledge the helpful comments from a number of colleagues: Antoine Bril, Robin Buckingham, Mike Cawthorne, John Clapham, Colin Campbell, Diana Good, David Howlett, Brian King, Steve Taylor (SB Pharmaceuticals), and from Mike Hollingsworth (University of Manchester). We also thank Karen Hayes and Ilana Toegg for their diligence in obtaining literature references and Helen Robey for her skill and patience in typing this manuscript.

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