Pharmac. Ther. Vol. 47, pp. 329-345, 1990 Printed in Great Britain. All rights reserved

0163-7258/90 $0.00 + 0.50 © 1990 Pergamon Press plc

Associate Editor: M. J. LEWIS

CYCLIC AMP A N D MECHANISMS OF VASODILATION KENNETH J. MURRAY Department of Cellular Pharmacology, Smith Kline & French Research Ltd, The Frythe, Welwyn, Herts AL6 9AR, U.K.

Abstract--Cyclic AMP and the mechanism of vasodilation have been reviewed by first discussing the enzymes involved (adenyl cyclase, cyclic nucleotide phosphodiesterases, cyclic AMP-dependent protein kinase) and then agents that increase cAMP in smooth muscle. Two mechanisms of vasodilation are described: (i) effects on contractile proteins; (ii) effects on Ca 2÷ levels. Evidence for compartments of cAMP is also presented.

CONTENTS 1. Introduction 2. Enzymes Involved in cAMP Metabolism 2.1. Adenyl cyclase 2.2. Cyclic nueleotide phosphodiesterases 2.2.1. Ca2+/calmodulin-dependent phosphodiesterases 2.2.2. cGMP-specific phosphodiesterase 2.2.3. cGMP-stimulated phosphodiesterase 2.2.4. cGMP-inhibited phosphodiesterase 2.2.5. Rolipram-sensitive phosphodiesterase 2.3. cAMP-dependent protein kinase 3. Agents that Increase cAMP in Smooth Muscle 3.1. Receptor agonists (including forskolin) 3.2. Phosphodiesterase inhibitors 3.2.1. Nonselective inhibitors 3.2.2. Inhibitors of the rolipram-sensitive phosphodiesterase 3.2.3. cGMP-inhibited phosphodiesterase inhibitors 3,3. cAMP derivatives 4. Mechanisms of Vasodilation 4.1. Activation of cAMP-dependent protein kinase 4.2. Protein phosphorylation 4.3. Effects on the contractile apparatus 4.4. Effects on Ca `'+ levels 4.4.1. Studies on whole tissues and cells 4.4.2. Inhibition of Ca:÷-influx 4.4.3. Stimulation of Ca2+-efflux 4.4.4. Uptake of Ca `'+ by internal stores 4.4.5. Membrane byperpolarization 5. Compartments of cAMP 6. Conclusions Acknowledgements References

1. I N T R O D U C T I O N The role of cyclic nucleotides in regulating smooth muscle contractility has undergone what might be termed a yin-yang transformation in recent years. Reviews written over 10 years ago generally highlighted the importance of c A M P in mediating relaxation whereas c G M P appeared to be more associated with contraction (e.g. Bar, 1974). Recent reviews concentrate on the role of c G M P as a vasodilator (Lincoln, 1989) and it has been proposed that c A M P plays a minor role in the relaxation process (Francis et al., 1988). Undoubtedly, the discovery that relax-

329 330 .330 330 330 331 331 331 331 331 331 332 333 333 334 334 334 335 335 335 336 337 337 338 338 339 339 339 340 341 341

ants such as endothelium-derived relaxing factor, the atrial natriuretic peptides and nitrovasodilators all increase vascular smooth muscle c G M P levels has shifted interest away from cAMP. It seems an appropriate time, therefore, to review the role of c A M P as a vasodilator and to determine whether there is a role for both cyclic nucleotides in the regulation of smooth muscle contraction. It is obvious that vascular smooth muscle can relax by mechanisms that do not involve changes in c A M P levels so that, the question to be asked is: " W h e n vasodilators increase cAMP, is the relaxation due to this increase?" To attempt to answer this, first the 329

330

K.J. MURRAY

enzymes involved in vascular smooth muscle cAMP metabolism will be discussed and then proposed biochemical mechanisms by which cAMP causes relaxation reviewed. Finally, the evidence for compartments of cAMP and/or associated enzymes in vascular smooth muscle will be discussed.

2. ENZYMES INVOLVED IN cAMP METABOLISM 2.1. ADENYLCYCLASE The mechanism by which hormones modulate the activity of adenyl cyclase has been the subject of much recent research and reviews (Freissmuth et al., 1989; Johnson and Dhanasekaran, 1989; Gilman, 1987) and so will not be discussed at length in this article. Present evidence suggests that the smooth muscle adenyl cyclase system is composed of the same proteins as have been described for other tissues (Krall et al., 1983). Briefly, as shown in Fig. 1, the specificity of hormone binding is determined by the receptor; those coupled to the stimulatory guanine regulatory protein (G~) activate the catalytic subunit of adenyl cyclase, resulting in an increased production of cAMP. Those coupled to the inhibitory guanine regulatory protein (Gi) have the opposite effect due to inhibition of the adenyl cyclase catalytic subunit. Various agents can be used to increase the activity of adenyl cyclase and therefore the tissue content of cAMP. Forskolin activates the adenyl cyclase catalytic subunit directly; the same end effect is obtained with cholera toxin which acts through ADP-ribosylation of G~. These agents and the hor-

mones of physiological and pharmacological significance in vascular smooth muscle are described in Section 3.

22. CYCLICNUCLEOTIDEPHOSPHODIESTERASES It is now well established that tissues, including vascular smooth muscle, contain multiple forms (isoenzymes) of cyclic nucleotide phosphodiesterase (PDE) (reviewed in Beavo, 1988). The original practice of naming PDEs by the order in which they elute from anion exchange chromatography should be superseded as it is now realized that the number of peaks and the order of elution is highly dependent on tissue type and chromatography conditions. The nomenclature used in this review will follow that proposed by Beavo (1988) which is largely based on selectivity to substrates and regulators, and the kinetic properties of the PDE. Another, important, feature of the nomenclature is the use of isoenzyme selective inhibitors and it is the use of such inhibitors that has allowed the delineation of multiple forms of PDE. Finally, it must be remembered that the properties of the isolated PDE must be extrapolated with caution when determining the physiological relevance of that PDE in regulating cyclic nucleotide hydrolysis in intact tissue. For example, the activities of the cyclic nucleotide generating systems and the physical location of the PDE must be considered. The properties of the forms of PDE known to be present in vascular smooth muscle will now be discussed with emphasis on those thought to be primarily responsible for cAMP hydrolysis. It should be pointed out that these reports concentrate on soluble PDEs and little work has been carried out on the membrane bound enzyme(s).

cAMP derivatives

i/ R e c e p t o r Agonists

j/

," (~hoqera Forskolin /oxln

=

I R I--

/

/

/

/

PDE Inhibitors i

/ /

//I

cAMP

(~

(~ AMP

cAMP-dependent Protein K i n a s e R2C2 ~ R2cAMP4 (inactive) +2C ( a c t i v e )

PROTEIN

Phosphodiester-

ases

I

G,A R

ATP

2.2. I. C a : ~ / C a l m o d u l & - D e p e n d e n t

PHOSPHOPROTEIN

~ u n ction altered)

Phosphoprotein Phosphatase

FIG. 1. cAMP metabolism in smooth muscle and agents used to modify it. The pathway of cAMP 'metabolism' is shown in bold lines, the site of action of agents that may be used to modify it are shown by broken lines. See text for further details. Abbreviations: R, hormone receptor; Gs,G~,the stimulatory and inhibitory guanine regulatory proteins; ACase, adenyl cyclase; PDE, cyclic nucleotide phospbodiesterase.

The Ca2+/calmodulin-dependent phosphodiesterases (Ca2+/CaM-PDE) are a family of at least three closely related isoenzymes (Beavo, 1988). Ca 2+/CAMPDE has been found in a number of vascular smooth muscles (Prigent et al., 1988; Silver et al., 1988a; Weishaar et al., 1986) including human aorta (Lugnier et al., 1986). Reported kinetic parameters for the hydrolysis o f c G M P are very similar with a K m in the range of 1 3 #M (Lugnier et al., 1986; Weishaar et aL, 1986). However, this is a common characteristic of Ca:+/CaM-PDE and differences in the hydrolysis of cAMP have been observed (Silver et al., 1988a) suggesting the presence of different Ca2+/CaM-PDEs in different vascular smooth muscles. Vascular smooth muscle Cae+/CaM PDE(s) are inhibited nonselectively by calmodulin antagonists and 3-isobutyl-l-methylxanthine (IBMX) (Hidaka et al., 1984; Prigent et al., 1988) and more selectively by l-methyl-3-isobutyl-8-methoxymethylxanthine and vinpocetine; use of these latter inhibitors indicates that Ca-'+/CaM-PDE is primarily responsible for cGMP hydrolysis in intact tissue (Lorenz and Wells, 1983; Hagiwara et al., 1984). A method for estimating the association of calmodulin with CaZ+/CaM-PDE in coronary arteries has been devised (Miller and Wells, 1988).

Cyclic AMP 2.2.2. eGMP-Specific Phosphodiesterase A PDE that elutes early on anion-exchange chromatography, that is not activated by calmodulin and is specific for the hydrolysis of cGMP has been identified in aorta (Lugnier et al., 1986). This enzyme is inhibited by M&B 22948 (Zaprinast) and dipyridamole with a degree of selectivity, and also by the non-selective inhibitors IBMX and papaverine (Prigent et al., 1988; Beavo, 1988). 2.2.3. c G M P - S t i m u l a t e d Phosphodiesterase The hydrolysis of cAMP by the cGMP-stimulated PDE is increased dramatically by micromolar concentrations of cGMP, and it is a major form of PDE in many tissues including heart (Beavo, 1988). In contrast, this PDE does not appear to be present in vascular smooth muscle (Weishaar et al., 1986; Prigent et al., 1988). 2.2.4. cG M P-Inhibited Phosphodiesterase The realization that the new generation of cardiotonic agents inhibit the cGMP-inhibited PDE (cGIPDE) has meant that this enzyme has received recent, intensive study (for review see Silver, 1989). The cGI-PDE (loosely referred to as PDE III) has been most extensively studied in heart and platelet (reviewed in Beavo, 1988). The enzyme has a low K m for cAMP and cGMP (approximately 0.1 pM), is inhibited by cGMP (Ki=0.1 #M), and by the socalled 'PDE-III inhibitors'/cardiotonic agents exemplified by milrinone (K~ = 0.3 #M). cGI-PDE has a native molecular weight of 230,000 and is subject to phosphorylation and activation by cAMP-protein kinase; phosphorylation of cGI-PDE in intact platelets has also been observed (MacPhee et al., 1988). cGI-PDE has been described in a number of vascular smooth muscles including human and bovine aorta (Lugnier et al., 1986; Silver et al., 1988a, b) and bovine coronary artery (Weishaar et al., 1986). All present reports indicate that vascular smooth muscle cGI-PDE is very similar, in both its kinetic properties and its sensitivity to inhibition by pharmacological agents, to the more studied cardiac and platelet enzyme (Silver, 1989; Weishaar et al., 1986; Prigent et al., 1988); at present no studies on the phosphorylation of smooth muscle cGI-PDE have been reported. The effects of cGI-PDE inhibition on intact vascular smooth muscle have shown that the new cardiotonic agents, in addition to being inotropes, are also potent vasodilators; this is discussed in Section 3.2. 2.2.5. Rolipram-Sensitive Phosphodiesterase It is now apparent that early preparations of 'PDE-III' contained two PDEs; the cGI-PDE described above and another form inhibited by rolipram (ZK 62711) and Ro 20-1724, but not by agents such as milrinone (Reeves et al., 1987). A rolipram-sensitive PDE that is not inhibited by cGMP has been identified in bovine aorta (Prigent et al., 1988). In general, it appears that the 'low K m cAMP-PDE' observed in vascular smooth muscle is

331

a mixture of cGI-PDE and rolipram-sensitive PDE (Ahn et al., 1989). 2.3. cAMP-DEPENDENT PROTEIN KINASE The cyclic AMP-dependent protein kinase (cAMPPrK) appears to be ubiquitously distributed among mammalian tissues. It is composed of two subunits; the regulatory (R) subunits which bind cAMP and the catalytic (C) subunits responsible for the phosphotransferase reaction. The holoenzyme form R2C 2 is inactive; binding of 2 cAMP molecules to each R-subunit results in dissociation to give the free, active C-subunits (for review see Beebe and Corbin, 1986). cAMP-derivatives are also able to dissociate the holoenzyme directly and have been used to activate cAMP-PrK in intact vascular smooth muscle (see Section 3.3). Early studies showed that cAMPPrK existed as two isoenzymes, named type I and II after their order of elution from anion-exchange chromatography and that the differences between the two types were due to their R-subunits. More recent studies, using molecular genetic techniques, have shown that there are at least four forms of R-subunit and at least two forms of C-subunit. However, only the s-form is expressed in all tissues, the ]~-form is expressed mainly in brain (reviewed in McKnight et al., 1988). From the information on vascular smooth muscle cAMP-PrK there is no reason to assume that the enzyme is different from the widely studied striated muscle cAMP-PrK, as to date no significant tissue variability of cAMP-PrK has been reported, and, indeed, monoclonal antibodies to heart type lI R-subunits recognize those from trachea (Scott and Mumby, 1985). An approximately equal distribution of type I and II cAMP-PrK has been reported for rat aorta, caudal and femoral arteries and for bovine coronary artery (Silver, 1987; Silver et al., 1985). The two isoenzymes isolated from pig coronary arteries showed activation by a number of cAMP-derivatives similar to the corresponding isoenzymes from striated muscle (Francis et al., 1988). As with cardiac muscle (Corbin and Keely, 1977) and uterus (Krall et al., 1978), cAMP-PrK in vascular smooth muscle has been found in both soluble and particulate fractions (Silver, 1985, 1987). However, as is the case with the isoenzyme distribution, the physiological significance of this is unknown. In contrast to most other tissues, where cAMP-PrK is present in excess, vascular smooth muscle contains almost equal amounts of cAMP-PrK and cyclic GMP-dependent protein kinase (cGMP-PrK) (Francis et al., 1988).

3. AGENTS THAT INCREASE cAMP IN SMOOTH MUSCLE This section describes some of the agents that are known to raise cAMP levels in vascular smooth muscle. The categories are loosely defined, the first part contains agents, e.g. forskolin, that are not strictly receptor agonists and the agents in the last section, cAMP-derivatives, do not raise cAMP levels. The main purpose is not to give an exhaustive list of all the agents that raise cAMP in all types of vascular

332

K.J. MURRAY

smooth muscles. It is intended to point out the range of different agents that may be used, and to highlight some of the problems encountered in trying to establish whether the relaxant effects of the agent are mediated through cAMP. While realizing that this was not always the aim of some of the work described here, points that could be used for future studies are: (1) measuring the effects on cAMP levels and relaxation on the same sample (in a number of reports, described below, a PDE inhibitor was included only when the tissues were being used for cAMP measurements); (2) measuring both the cAMP and cGMP levels (some agents, especially PDE inhibitors (Section 3.2) elevate the levels of both cyclic nucleotides); (3) determining the endothelial dependence of relaxation and changes in cyclic nucleotide content. 3.1. RECEPTORAGONISTS(INCLUDING FORSKOLIN) The role of cAMP as a mediator of the relaxant actions of fl-adrenoceptor agonists has been recently reviewed (Bulbring and Tomita, 1987) and is described in more detail in subsequent sections. Although it is clear that occupation of fl-adrenoceptors results in activation of adenyl cyclase and elevated tissue levels of cAMP, there is increasing evidence that this is not the only result of receptor occupation, as the direct linking of fl-adrenoceptors to Ca2+-channels has been demonstrated in a number of tissues, including smooth muscle (see Section 4.4). The numerous studies of the effect of fl-agonists on smooth muscle range from instances where the increases in cAMP are tightly coupled to the degree of relaxation (e.g. Silver et al., 1982) to those where the two effects are completely dissociated (Fermum et al., 1984). The latter authors reported that diisopropyl-fluorophosphate inhibited the ability of isoprenaline to increase cAMP levels in coronary artery while having no effect on its ability to relax the tissue. The diterpene, forskolin, is a potent activator of the catalytic subunit of adenyl cyclase (Seamon and Daly, 1981; see Fig. l) and has also been widely used in the study of smooth muscle relaxation; again more detail is given in subsequent sections. Forskolininduced relaxation and rises of cAMP in guinea pig aorta were highly correlated, although it was noted that other agents produced equal relaxation with smaller changes in cAMP (Silver et al., 1988b). As with the fl-agonists, the effects of forskolin on vasodilation and cAMP content have been dissociated, but in the opposite manner, as it has been reported that forskolin can raise cAMP without causing relaxation (Vegesna and Diamond, 1983, 1984). Adenosine is usually considered as a physiologically important regulator of blood flow in a number of organs (Olsson, 1981). Cell surface receptors for adenosine may be positively (A, receptors) or negatively (A~ receptors) linked to adenylate cyclase (Ramkumar et al., 1989). It has been proposed that the vasodilator actions of adenosine are due to occupation of A 2 receptors resulting in increased smooth muscle cAMP content; adenosine analogs have been reported to activate adenylate cyclase in membranes prepared from cultured rat aorta cells (Anand-Srivastava et al., 1982). In contrast, working

with membranes prepared directly from pig aorta, Diocee and Souness (1987) found an effect of forskolin but not of the adenosine analogs. The effect of adenosine on intact tissue cAMP levels is not clear cut (see Kurtz, 1987, for discussion). In contrast, Kurtz (1987) has proposed that the vasodilator actions of adenosine are mediated through Al receptors, occupation of which stimulates a particulate guanyl cyclase resulting in increases in cGMP. This conclusion was based on the observations that, in cultured rat aorta cells, adenosine and its analogs stimulated guanyl cyclase activity in membranes and increased cGMP levels in intact cells. The rises in cGMP were small (60%) although they could be potentiated by the inhibitor of the cGMP specific phosphodiesterase, zaprinast (M&B 22948). These observations again highlight the importance of measuring both cAMP and cGMP levels when studying smooth muscle function. A 5-hydroxytryptamine receptor has been described in neonatal porcine vena cava that mediates both relaxation and elevation of cAMP (Sumner et al., 1989) and the two events show a temporal correlation (Trevethick et al., 1984). However, IBMX was included in the study of cAMP levels but not for the tension measurements, therefore this may not be a true comparison. There are postsynaptic dopamine receptors (DA~) present in vascular smooth muscle that are distinct to the presynaptic receptors found mainly within sympathetic terminals (DA2) (Niznik, 1987). Occupation of DA~-receptors is known to stimulate adenyl cyclase in, and cause relaxation of, renal vascular smooth muscle (Stoof and Kebabian, 1984; Goldberg and Rajfer, 1985). Dopamine and fenoldopam (a DA~-agonist) caused modest ( > 5 0 % ) increases in cAMP that were blocked by a selective DAi-antagonist (SCH-23390) in homogenates of renal arteries (Alkadhi et al., 1986). Dopamine relaxed rabbit afferent arterioles (Edwards, 1986) and increased cAMP content of the corresponding canine tissue (Tamaki et al., 1989). A more detailed relationship between these two events does not appear to have been investigated. Glucagon relaxes some, but not all, vascular preparations including renal artery, and there is indirect evidence that the relaxation was mediated by increases in cAMP (reviewed in Farah, 1983). More recently, Okamura et al. (1986) have shown that glucagon increases cAMP in canine renal, but not coronary, arteries and that glucagon can only relax the former. The vasodilator effects of acetate have been known for a long time (Bauer and Richards, 1928) and have been ascribed to metabolic effects by increased A M P or adenosine levels (Liang and Lowenstein, 1978). Acetate caused an endothelium-independent increase in the cAMP, but not the cGMP, content of rat caudal artery. Temporal and dose-dependent correlations between increases in cAMP and relaxation were observed; however, IBMX was included in studies of the former, but not the latter (Daugirdas et al., 1988). Parathyroid hormone (PTH) has been known for some time to have vasodilatot effects (Charbon, 1968) and there is evidence that this is due to increased

Cyclic AMP tissue cAMP content. Membranes from rat aorta contain a PTH sensitive adenyl cyclase (Nickols et al., 1986) and PTH causes a 2-10-fold increase in the cAMP content of various vascular tissues and cells (Nickols, 1985; Pang et al., 1986; Bergmann et al., 1987). The vasodilator and cAMP-increasing effects of PTH are potentiated by nonselective PDE inhibitors (Nickols, 1985; Pang et al., 1986). There is reasonable evidence, then, that the vasodilator actions of PTH are mediated by increases in cAMP, although it has been reported that PTH decreases cAMP in cultured bovine aorta cells (Stanton et al., 1985). The 28 amino acid, vasoactive intestinal polypeptide (VIP) shows a range of biological effects including vasodilation (Said and Mutt, 1970a, b). VIP stimulates adenyl cyclase in various smooth muscle cells and membranes (Ganz et al., 1986; Amenta et al., 1988). The effects of VIP on tissue preparations are not clear cut as both endothelium-dependent (Davies and Williams, 1984) and independent relaxation (Schoeffter and Stoclet, 1985) have been reported. In addition, Schoeffter and Stoclet (1985) reported that VIP was a less effective relaxant than isoprenaline in rat aorta despite the fact that it was more effective in raising cAMP levels. In contrast, VIP-induced relaxation and increase in cAMP showed the same dose-dependent relationship in rabbit mesentery (Ganz et al., 1986). Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide encoded in the calcitonin gene, and widely distributed in both the central and peripheral nervous systems (for review see Goodman and Iversen, 1986). CGRP is a potent vasodilator (Brain et al., 1985) and this property may be linked to the formation of cAMP in vascular smooth muscle. In rat aorta smooth muscle cells, CGRP increases cAMP, but not cGMP, levels, although these were measured in the presence of IBMX (Kubota et al., 1985; Hirata et al., 1988). However,

333

the physiological significance of this is unclear as CGRP relaxation of rat aorta is reported to be highly endothelial dependent (Brain et al., 1985; Kubota et al., 1985). In contrast, CGRP raised the level of cAMP in, and relaxed endothelial stripped porcine coronary and feline cerebral arteries (Edvinsson et al., 1985; Shoji et al., 1987). Therefore, CGRP may relax different vascular smooth muscles by different mechanisms as CGRP receptors have been located on both smooth muscle and endothelial cells (Kubota et al., 1985). 3.2. PHOSPHODIESTERASEINHIBITORS 3.2.1. Nonselective Inhibitors

There are a number of problems in the use of nonselective PDE inhibitors (Wells and Kramer, 1981) and the difficulties are particularly acute in smooth muscle as both cAMP and cGMP are proposed to mediate relaxation. Therefore it is important to measure the levels of both cyclic nucleotides after treatment with nonselective inhibitors. The problem that many of these nonselective inhibitors are also nonspecific must be considered. For example, IBMX can interact with adenosine receptors (Fredholm, 1980); and also block Gi (Parsons et al., 1988); both of these effects could also increase cAMP levels. As would be expected from their inhibition of the isolated PDEs, IBMX and papaverine increase both cAMP and cGMP levels in intact vascular smooth muscle (e.g. Silver et al., 1988b; Schoeffter et al., 1987) (see Table 1) confirming their limited use if one wishes to specifically study cAMP-dependent mechanisms of vasodilation. There are, however, ways of using these inhibitors to provide evidence that the increases in cAMP play a role in mediating their relaxant effects. (1) The inhibitors can be used to potentiate the effects of receptor agonists that

TABLE 1. The Effect o f Phosphodiesterase Inhibitors on Vascular Smooth Muscle Cyclic Nucleotide Levels Inhibitor Non-selective IBMX IBMX IBMX Papaverine Rolipram sensitive Rolipram Ro 20-1724 cGI-PDE Cilostamide Trequinsin Milrinone

LY 195115

Tissue (agonist) Rat aorta (5-HT) Bovine coronary (K) Pig coronary (K) Pig coronary (K) Rat aorta (5-HT) Rat aorta (5-HT) Rat aorta (5-HT) Rat aorta (5-HT) Rat aorta (NA) Guinea pig aorta (Phen) Guinea pig aorta (Phen) Rat aorta (5-HT) Rat aorta (5-HT)

[Inhibitor] (/AM)

Relaxation (%)

10 15 30 30

50 nd 72 69

180" 147 124 124

146 185 180 170

Schoeffter et Lorenzaand Kramer and Kramer and

150 250

50 50

166' 217"

132 135

Schoeffter et al. (1987) Schoeffter et al. (1987)

50 50 50 50

203* 158" 210 120

119 136" nd 100

Schoeffter et al. (1987) Schoeffter et al. (1987) Linz et al. (1988) Silveret al. (1988b)

300

100

180"

164" Silveret al. (1988b)

100 I00

100 100

240* 140"

350* Kauffman et al. (1987) 410' Kauffman et al. (1987)

35 0.3 0.9 2

cAMP cGMP (% of control)

Reference al., (1987)

Wells (1983) Wells (1979) Wells (1979)

The table shows the effect of various phosphodiesterase inhibitors on some vascular smooth muscle preparations. For the given inhibitor concentration, the % changes in tension and cAMP and cGMP levels are shown. Abbreviations: 5-HT, 5-hydroxytryptamine; K, K+-depolarization; NA, noradrenaline; Phen, phenylephrine; nd, not determined; *statistically significant change in cAMP or cGMP levels (determined by the authors).

334

K.J. MURRAY

stimulate the production of cAMP (Lorenz and Wells, 1983; Schoeffter et al., 1987). (2) Their effects on activation of cAMP-PrK can be ascertained (Silver et al., 1988b). Also a comparison of selective and nonselective inhibitors may provide some insight into compartmentation of cyclic nucleotide action (Schoeffter et al., 1987). 3.2.2. Inhibitors o f the R o l i p r a m - S e n s i t i v e

Phospho-

been overcome by the use of inhibitors of cGI-PDE as this enzyme hydrolyzes both cyclic nucleotides, and a functional role for cGMP in the vasodilation caused by these agents has been proposed (Kauffman et al., 1987). Further work with inhibitors of the rolipram-sensitive PDE may be fruitful as this enzyme is highly specific for cAMP; however, the high concentration required for these agents to cause vasodilation has already been noted.

diesterase

The two selective inhibitors presently available are rolipram itself and Ro 20-1724. Both of these compounds, albeit at high concentration, have been shown to relax rat aorta with significant increase in cAMP but not cGMP (Shoeffter et al., 1987; Table 1). 3.2.3. c G M P - I n h i b i t e d

Phosphodiesterase

Inhibitors

The effects of cGI-PDE inhibitors on vascular smooth muscle are shown in Table 1. This class of agent has potent vasodilator activity and there is growing evidence that this is due to increases in cAMP. (1) There is a correlation between a compound's ability to inhibit cGI-PDE and its potency as a vasodilator (Kauffman et al., 1987; Silver et al., 1988b). (2) Increases in cAMP in intact tissue are observed, but it should be noted that similar rises in cGMP are also found (see Table 1). (3) cAMP-PrK is activated in intact vascular smooth muscle (Silver et al., 1988b). (4) Relaxation is agonist independent (Table 1) and does not require the presence of endothelium (Kauffman et al., 1987), although it has been reported that cyclic nucleotide changes do not occur in endothelium stripped aorta (Linz et al., 1988). Some selectivity for the coronary vasculature has been reported for two of these inhibitors, amrinone and milrinone (Harris et al., 1989). To conclude, PDE inhibitors are effective relaxants of vascular smooth muscle, but only produce small increases in tissue cAMP content and this is often accompanied by similar increases in the level of cGMP. Therefore, the use of these inhibitors to show a role of cAMP in vasodilation is not as straight forward as could be hoped. This problem has not

3.3. cAMP DERIVATIVES Although, strictly speaking, they do not increase intracellular cAMP, cAMP-derivatives (also known as analogs) have been used in many tissues, including vascular smooth muscle, to investigate the effect of cAMP. In general, cAMP is derivatized at either the C-8 or N-6 position of the adenosine moiety to produce compounds that are more lipophilic than cAMP which itself is poorly, if at all, cell penetrant. When they enter the cell the derivatives mimic the action of cAMP by activating cAMP-PrK (Fig. 1) although, as discussed below, this is not their only potential mechanism of action. The commonly used cAMP derivatives have potencies similar to cAMP for activation of cAMP-PrK. However, a notable exception is N 6 , 0 2 ~ - d i b u t y r y l cAMP (DB-cAMP) which is a very poor activator of cAMP-PrK (Ogreid et al., 1985); this is due to the requirement of the T-OH group for activation, so DB-cAMP requires hydrolysis to N6-monobutryl cAMP or cAMP itself in the cell for it to be effective. The results of a number of studies that have investigated the effects of cAMP-derivatives on vascular smooth muscle are summarized in Table 2. 0.3mM-l.0mM DB-cAMP has been shown to partially relax rabbit mesenteric artery, and rat and rabbit aorta (Itoh et al., 1985; Meisheri and van Breemen, 1982; Bresnahan et al., 1975; Hwang and van Breeman, 1987; Lincoln, 1983); complete relaxation of rat aorta was achieved after 30 min by the addition of 0.1 mM DB-cAMP (McMahon and Paul, 1986). However, in the same tissue Lincoln (1983) reported 0.5 m~ 8-BrcAMP to be totally without effect; the same result was obtained by Napoli et al.

TABLE 2. Relaxation o/ Vascular Smooth Muscle by cAMP-Derivatives Tissue Rat aorta

Agonist Adrenaline

cAMP derivative

Dibutyryl N6-butyryl O:-butyryl Noradrenaline Dibutyryl Noradrenaline Dibutyryl 8-bromo K + or acetylcholine Dibutyryl 8-bromo K" 8-CPT N%benzoyl 8-AHA 8-SCH~ 8-SCHzCH~ 8-bromo

Concentration (raM) 1.0 1.0 1.0 0.1 0.5 0.5 0.1 0.1 0.04 0.6 0.6 1.2 0.5 1.7

Relaxation (%) 48 33 7 100 43 0 0 0 50 >50 > 50 50 50 50

Reference

Bresnahan et al. (1975) Bresnahan et al. (1975) Bresnahanet al. (1975) McMahon and Paul (1986) Lincoln (1983) Lincoln (1983) Bovine coronary Napoli et al. (1980) Napoli et al. (1980) Pig coronary Francis et al. (1988) Francis et al. (1988) Francis et al. (1988) Francis et al. (1988) Francis et al. (1988) Francis et al. (1988) The table shows the effect of various cAMP derivatives on some vascular smooth muscle preparations. For the given derivative concentration, the % change in lension is shown. Abbreviations: K ~. K+-depolarization: 8-CPT, 8-(4-chlorophenylthio); 8-AHA, 8-(6-aminohexyl)amino.

Cyclic AMP (1980) working with bovine coronary artery, they also reported DB-cAMP to be ineffective. The concentrations of derivatives used show that vascular smooth muscle relaxation is not very sensitive to their addition, however, the same is true of known cAMP-dependent events, e.g. lipolysis in fat cells (Beebe et al., 1988). The use of high concentrations does increase the possibility that the derivatives could be acting by nonspecific mechanisms, e.g. interaction with adenosine receptors. The mechanism of action of cyclic nucleotide derivatives has been recently studied in detail by Francis et al. (1988). Working with K ÷ contracted pig coronary arteries, these authors found relaxation by a number of cAMP derivatives, although by far the most potent of these was 8-(4-chlorophenylthio)cAMP (CPTcAMP) (see Table 2). With this exception, cGMP derivatives were more effective relaxants than cAMP derivatives in this tissue; e.g. compare 8-BrGMP (ECs0= 0.05 mM) with 8-BrcAMP (ECs0 = 1.7mM). Similar results were obtained in carbamylcholine contracted guinea pig trachealis, although all derivatives were more potent in this preparation. The authors investigated a number of reasons for the lower potency of the cAMP derivatives and found it was not due to (i) lower cell penetration (as measured by partition coefficient) (ii) metabolism of the derivatives by phosphodiesterases or (iii) failure of the derivatives to activate cAMPPrK from the same tissue. Indeed, the authors conclude that the cAMP derivatives that relax the smooth muscle preparations do so by their ability to activate cGMP-PrK, as they found a correlation between the potency of derivatives to activate cGMPPrK and relax the tracheal strips. These results do not completely exclude the role of cAMP-PrK in mediating relaxation as all the cAMP derivatives employed were far more potent activators of cAMPPrK than cGMP-PrK. Therefore, if the derivatives reached an intracellular concentration sufficient to activate cGMP-PrK then cAMP-PrK should have already been activated. The selectivity of cyclic nucleotide derivatives obviously should be considered by any future studies.

4. MECHANISMS OF VASODILATION 4.1. ACTIVATION OF cAMP-DEPENDENT PROTEIN KINASE

The vast majority of the effects of cAMP in tissues are assumed to be mediated through activation of cAMP-PrK and subsequent increases in protein phosphorylation. Therefore to show that cAMP is responsible for vasodilation it is necessary to show activation of cAMP-PrK in intact vascular smooth muscle. The method used to assess this is the cAMPPrK activity ratio assay (Corbin, 1983), although the results are somewhat dependent on the actual assay conditions used (Murray et al., 1990). Significant temporal and concentration-dependent correlations between the degree of relaxation and the cAMP-PrK activity ratio have been shown in bovine coronary artery using isoprenaline (Silver et al., 1982) and adenosine (Silver et al., 1984) as relaxants: in rat

335

aorta and mesenteric artery with forskolin and isoprenaline (Silver et al., 1985; Deisher et al., 1989) and in canine saphenous vein with isoprenaline (Kikkawa et al., 1986). In contrast, Vegesna and Diamond (1984) have reported that 0.1 #ra forskolin increases the activity ratio in bovine coronary artery without causing relaxation. However, higher concentrations of forskolin and isoprenaline caused both relaxation and an increased activity ratio. Similarly, forskolin produced a maximal increase in activity ratio after 3 min, but required 15 min to completely relax rat aorta (Lincoln and Fischer-Simpson, 1983). Kikkawa et al. (1986) reported that isoprenaline caused a similar increase in activity ratio in both canine saphenous and portal veins while causing a 90% relaxation in the former compared with 15% in the latter and the authors suggest that the two events are 'dissociated' in portal vein. The majority of studies show that, with a number of different agonists and vascular smooth muscles, an increase in the cAMP-PrK activity ratio is accompanied by relaxation and, in general, there is both a temporal and concentration-dependent correlation between the two. Despite the somewhat qualitative nature of the activity ratio assay, these results provide the required evidence for activation of cAMP-PrK in intact vascular smooth muscle. Interestingly, with the exception of one study (Deisher et al., 1989) the reported basal activity ratio is approximately 0.5 (showing that 50% of the cAMP-PrK is active). This is higher than the control activity ratio reported for most other tissues (0.14).3) and suggests that the activity ratio assay may require further investigation in vascular smooth muscle along the lines indicated by Murray et al. (1990). 4.2. PROTEIN PHOSPHORYLATION The result of activation of cAMP-PrK, described above, is the increased phosphorylation of its specific substrate proteins. To understand their physiological significance, it is necessary to study protein phosphorylation in intact tissue preparations. This section describes work in which smooth muscle strips or cells have been incubated with 32p~, to generate intracellular [7-32P]-ATP, and the resulting phosphoproteins analyzed by gel electrophoresis. Consequently, this method gives information regarding the Mr of proteins whose phosphorylation states are altered by a particular agent, and not much more. More detailed analysis requires the identification of the phosphoprotein and, for example, analysis of P-light chain phosphorylation in intact vascular smooth muscle has been made by this method (e.g. Murray and England, 1981). However, there are few studies that have looked specifically at cAMP-dependent phosphorylation in intact preparations. One reason is that there is no phosphoprotein that has been conclusively linked to the relaxation process. It should be noted that, if the phosphoprotein is present in small quantities in the tissue, it may be hard, or impossible, to identify it by this method. The effects of DB-cAMP and isoprenaline were studied in rat aorta by Rapoport et al. (1982), the two agents caused similar, but not identical, changes in the phosphorylation of a number of proteins. Again,

336

KI J. MURRAY

some, but not all, of these proteins were phosphorylated in response to 8-BrcGMP, showing that there are specific substrates for cAMP-PrK. 4.3. EFFECTSON THE CONTRACTILE APPARATUS

Force is generated in smooth muscle by the same mechanism as in its striated counterpart i.e. by the cyclic interaction of actin and myosin. In the latter, contraction is regulated by Ca :+ binding to troponin; however, this mechanism does not operate in smooth muscle. Various mechanisms have been proposed for the regulation of smooth muscle contraction, although the majority of experimental evidence supports the hypothesis that regulation is by phosphorylation of myosin light chains. This hypothesis is outlined in Fig. 2. Ca 2+ binds to its ubiquitous binding protein, calmodulin and this complex activates the enzyme, myosin light chain kinase (MLCK). MLCK phosphorylates a particular 20,000 MW light chain of smooth muscle myosin (P-light chain), and it is only myosin containing phosphorylated P-light chain that can interact with actin. This process is reversed by the action of a specific phosphoprotein phosphatase which removes the phosphate from the P-light chain. There are numerous studies in intact smooth muscle, including vascular, that show a relationship between cytoplasmic free Ca 2+, P-light chain phosphorylation and tension over short periods of contraction (reviewed in Kamm and Stull, 1985; Hai and Murphy, 1989). However, under some circumstances sustained contraction of smooth muscle is achieved even though Ca t+ levels and the degree of phosphorylation of the P-light chain return to basal or near basal. This particular contractile state has been termed the latch state. Although a hypothesis has been proposed that explains this state solely in terms of P-light chain phosphorylation (Hai cAMP-PrK

Ca

MLCK-@.

)P',a,e

~"

MLC K~/-~'-~

~MLLCK Ca"M r''2+/ ,.,=4~

,~MvosfN.,~S 1

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k

~

.j.

MYOSIN-~ +

.~

=' ACTOMYOSIN-@

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contracted

FIG. 2. Proposed mechanism for relaxation due to cAMPdependent phosphorylation of myosin light chain kinase. Phosphorylation of myosin is required for its interaction with actin and, therefore, contraction. It is proposed that cAMP-dependent phosphorylation of myosin light chain kinase (MLCK) decreases its activity by lowering its affinity for calmodulin (CAM), resulting in decreased myosin phosphorylation and, consequently, relaxation. Processes leading to relaxation are shown by broken lines, those leading to contraction by solid lines. See text for full details.

and Murphy, 1989; Rembold and Murphy, 1988), these, and other, observations have led to proposals that there is a second regulatory system, linked to the thin filament, operating in smooth muscle (Marston and Smith, 1985). At present, interest is focused on the protein caldesmon which has binding sites for actin, myosin, tropomyosin and the calmodulin Ca 2 + complex (Clark et aL, 1986; Adam et al., 1989). Obviously, the lack of definition of the regulatory system makes its potential regulation by cAMP hard to study. The experimental approaches taken have been to study the effects of phosphorylation by cAMP-PrK on individual proteins. In this respect the studies have been limited to MLCK, or to various preparations of the contractile apparatus e.g. demembranated muscle, myofibrils or actomyosin. The most commonly reported mechanism for vasodilation by cAMP is the cAMP-PrK catalyzed phosphorylation of MLCK (see Fig. 2). The initial observations showed that gizzard MLCK could be phosphorylated at two sites by cAMP-PrK, and that this phosphorylation dramatically reduced the affinity of MLCK for the calmodulin-Ca 2+ complex. Ultimately, this would result in a lower degree of P-light chain phosphorylation and relaxation of the tissue (Sellers and Adelstein, 1987). MLCK isolated from various vascular smooth muscles are also substrates for cAMP-PrK with similar effects on its interaction with Ca 2+-calmodulin (Vallet et al., 1981; Bhalla et al., 1982; Hathaway et al., 1985). However, subsequent studies have shown that the MLCKCa2+-calmodulin complex is only phosphorylated at one site and this does not alter the affinity of MLCK for Ca:+-calmodulin (Sellers and Adelstein, 1987). This suggests that, if as predicted, MLCK is present in contracted smooth muscle as the MLCKCa2+-calmodulin complex, then phosphorylation by cAMP-PrK at the effective site would not occur and, therefore, would not have a relaxant effect. This is supported by the observations of Miller et al. (1983) who reported that isoprenaline, although it relaxed precontracted tracheal strips, did not alter the kinetic properties of MLCK, suggesting that no phosphorylation had taken place. In contrast, forskolin treatment of the same tissue was shown directly to increase the phosphorylation of MLCK, however, the effects of this on its activation by calmodulin were not investigated (de Lanerolle et al., 1984). So at present, there is no direct evidence from studies of intact smooth muscle to support this hypothesis. The results of Gerthoffer et al. (1984) showed that forskolin could increase cAMP and relax carotid artery without changing P-light chain phosphorylation which had, however, already fallen to basal levels before the challenge with forskolin, Studies measuring both the phosphorylation state and the kinetic properties of MLCK are required in intact vascular smooth muscle to test the hypothesis outlined in Fig. 2. The effect of cAMP-PrK on various in citro preparations has also been studied. When bovine aortic, or pig carotid artery actomyosin was incubated with cAMP-PrK there was a decrease in both P-light chain phosphorylation and the actomyosin ATPase, due to a rightward shift in the pCa :+ relationship. These decreases were overcome by the addition of

Cyclic AMP calmodulin. There was also increased phosphorylation of a protein of Mr = 100,000, assumed to be M L C K (Silver and DiSalvo, 1979; Silver et al., 1981; Mrwa et al., 1979). cAMP-PrK has also been found to depress the contraction caused by submaximal Ca 2+ in chemically skinned preparations from carotid fibers (Ruegg and Paul, 1982), mesenteric artery (Itoh et al., 1985) and coronary arteries (Pfitzer et al., 1985); similar results have been reported for other, nonvascular smooth muscles (e.g. Sparrow et al., 1984; Kerrick and Hoar, 1981). In contrast, cAMPPrK had no effect on the tension in skinned rat aorta (McMahon and Paul, 1986). Together, these results indicate that it is possible for cAMP-PrK-dependent phosphorylation to cause relaxation by decreasing the affinity of the contractile apparatus for Ca 2+, although interpretation is hampered as some skinned preparations do retain a C a 2+ uptake system. However, by their very nature all these preparations result in the loss of soluble proteins so these results must be extrapolated to the intact tissue with caution. For example, effects of cAMP-PrK seen on actomyosin preparations are overcome by adding back calmodulin to concentrations which probably more accurately reflect the conditions found in intact tissue (Aksoy and Murphy, 1983). Similarly, Pfitzer et al. (1985) reported that cAMP-PrK relaxed skinned coronary arteries, but that this could be antagonized by Ca 2+ and calmodulin. Therefore, the hypothesis that cAMP-PrK phosphorylation of contractile proteins is responsible for smooth muscle relaxation, although theoretically possible based on the actomyosin studies, requires more evidence. A suitable system may be that recently reported by Nishimura and van Breemen (1989). They have investigated rat mesenteric artery permeabilized with Staphylococcal a-toxin, which results in a preparation permeable to cyclic nucleotides, that retains its intracellular proteins. The authors showed that both cAMP and cGMP could relax this preparation at a fixed Ca 2÷ concentration. The effect was on the Ca2+-sensitivity of the contractile apparatus, as ionomycin was included to discharge any internal stores. Similarly, work using intact vascular smooth muscle preparations (Abe and Karaki, 1989; DeFeo and Morgan, 1989) also suggests a change in Ca 2+ sensitivity of the contractile proteins (see Karaki, 1989; Section 4.4). There is very limited information on cAMP-PrK effects on contractile proteins other than MLCK. Caldesmon is phosphorylated in intact carotid arteries (Adam et al., 1989) and is a substrate for several protein kinases but not for cAMP-PrK (Ngai et al., 1984). Another actin-binding protein, fodrin can stimulate smooth muscle actomyosin ATPase and, interestingly, this stimulation is blocked when fodrin is phosphorylated by cAMP-PrK (Wang et al., 1987). Obviously, elucidation of the thin filament regulatory systems themselves is required before the effects of cAMP-PrK can be fully assessed. 4.4. EFFECTSON CA2+ LEVELS 4.4.1. S t u d i e s on W h o l e Tissues a n d Cells The use of the bioluminescent protein aequorin and the fluorescent indicators quin-2 and fura-2 have

337

allowed the direct measurement of cytoplasmic free Ca 2+ concentration ([Ca 2+ ]i) in smooth muscle cells and, more recently, in intact strips. This approach should provide an unequivocal answer as to whether cAMP causes relaxation by reducing [Ca 2+ ]i- However, the indications from the studies published so far are that, as with so many aspects of smooth muscle contraction, a consensus view is going to be hard to come by. There are some technical problems and differences that must be taken into consideration, in particular digital analysis of fura-2 fluorescence in arterial cells showed a spatially unequal Ca~+-distribution (Goldman et al., 1989), suggesting the possibility that there are compartments of Ca 2+ (Hai and Phair, 1989). Differences between the indicators quin-2 and aequorin have also been reported (DeFeo and Morgan, 1986). Forskolin decreased [Ca 2+ ]i and caused relaxation of K+-depolarized rat and ferret aortic strips; in the rat, forskolin was more effective in reducing [Ca 2+ ]i when noradrenaline was the contractant (Abe and Karaki, 1989; DeFeo and Morgan, 1989). The results from both these studies suggest that the relaxant effects of forskokin cannot solely be explained by the reduced [Ca 2+ ]i indicating that there is a change in the Ca2+-sensitivity of the contractile proteins. Studies treating bovine tracheal cells with isoprenaline have yielded unexpected results. Despite its known relaxant effects, when added by itself isoprenaline caused an increase in [Ca2+]i due to a stimulation of influx. Isoprenaline could, however, reverse the carbachol-induced [Ca 2÷ ]~rise (Takuwa et al., 1988; Felbel et al., 1988). It has recently been shown that isoprenaline can stimulate Ca2+-influx into cardiac cells directly via a G-protein coupled channel (Rosenthal et al., 1988), and the same now appears true for smooth muscle (Felbel et al., 1988). This effect of isoprenaline, in addition to increasing cAMP levels, could explain differences obtained with /~-agonists and cAMP-derivatives. In general, cAMP-derivatives are far less potent than their cGMP counterparts in lowering smooth muscle [Ca2+]i although a full systematic study has not been reported. Dibutyryl-cAMP only partially reduced the [Ca 2+ ]i increased by K + depolarization in rat aortic smooth muscle cells, whereas 8-BrcGMP caused a complete and more rapid reduction (Kai et al., 1987). Injection of cGMP-PrK, but not cAMPPrK, lowered the carbachol dependent-[Ca 2+ ]i increase in bovine tracheal smooth muscle cells and, similarly, 8-BrcGMP was more effective than 8-BrcAMP in reducing [Ca 2+ ]i (Felbel et al., 1988). There is growing evidence that cGMP could cause vasodilation by reducing [Ca2+]i (but see Yanagisawa et al., 1989); the best evidence that cAMP operates through this mechanism comes from studies using forskolin. However, the possibility that forskolin activates cGMP-PrK cannot be discounted (Lincoln et al., 1989; see Section 6). There are a number of potential points at which the [Ca2÷ ]i may be regulated (see Fig. 3). Ca 2+ may enter the smooth muscle cell either through voltage- or receptor-operated channels and leave either by active transport or in exchange for Na ÷. Additionally, vascular smooth muscle contains internal Ca 2+-stores in the form of sarcoplasmic (or endoplasmic) reticu-

338

K.J. MURRAY

Plasma MembraneRocJ~

VOC[][

I[

Sarcoplasmic" Reticulum FIG. 3. Ca2+ transport mechanisms of smooth muscle. The figure shows some of the ways a smooth muscle cell may modulate [Ca2+]i; processes leading to lowered [Ca2+]i and therefore relaxation, are shown by broken lines. The text describes how these processes may be modulated by cAMP. Abbreviations: ROC, receptor operated channel; VOC, voltage operated channel; IP3, inositol 1,4,5-triphosphate; PL, phospholamban.

lum. Release from this store is through an inositol, i,4,5-triphosphate (IP3)-sensitive mechanism and there is an ATP-driven CaZ+-pump for uptake. Reduction of [Ca2+]~ can therefore be achieved by inhibiting Ca 2+ influx (or release) or by stimulating its uptake or extrusion; mechanisms for the regulation of all these pathways by cAMP have been proposed. The importance of each may well vary with the vascular smooth muscle and the particular agonist and relaxant used, so generalization is not always possible. 4.4.2. Inhibition o f Ca2+-Influx The requirement for infux of extracellular Ca 2+ for contraction of vascular smooth muscle is both tissue and agonist dependent, e.g. K+-depolarization is dependent on its presence; whereas aorta will contract, in response to noradrenaline, in its absence (see van Breeman and Saida, 1989; Loutzenhiser et al., 1985 for reviews). Isoprenaline and dibutyrylcAMP were shown to inhibit 145 mM K+-stimulated 45CaZ+-influx into rabbit aorta and in this study, there was a temporal correlation between the inhibition of influx and relaxation (Meisheri and van Breeman, 1982). In a subsequent report it was found that, when external K + was varied, dibutyryl-cAMP and, more markedly, forskolin caused a shift to the right in the force versus Ca2+-influx relationship. The greater effect of these agents on inhibiting contraction relative to 45Ca2+ influx suggests other mechanisms (e.g. sequestration by internal stores) are more important (Hwang and van Breeman, 1987). There is obviously a difficulty in studying this process in preparations of vesicles as the agonist and cAMPPrK need to be on opposite sides of the membrane, and patch-clamp studies of cells could well be of use. 4.4.3. Stimulation o f Ca2+-Efftux Ca 2+ may leave the smooth muscle cell either by active transport or in exchange for Na+; the relative contribution of each is a matter of debate and may

well be tissue dependent (Blaustein, 1988). Smooth muscle plasma membranes appear to contain an erythrocyte-type Ca2+-calmodulin stimulated Ca 2+ATPase (reviewed in Eggermont et al., 1988a, b). The regulation of its activity by protein kinases has been studied in microsomal fractions and on the purified protein. There is growing evidence that the activity of this Ca2+-ATPase may be increased by phosphorylation by cGMP-PrK although this is not a direct effect on the ATPase itself (Lincoln, 1989; Baltensperger et al., 1988). The effects of protein kinases on rat aortic microsomes containing a calmodulin-stimulated Ca2+-ATPase were studied by Rashatwar et al. (1987). Incubation of the microsomes with cAMP-PrK did not alter the activity of the ATPase, whereas an increase was found following treatment with cGMP-PrK. In contrast, both kinases stimulated Ca 2+ uptake into pig aortic sarcolemmal vesicles, cAMP-PrK phosphorylated proteins of molecular weight 22,000 and 28,000 and a cGMP-PrK one of 35,000 (Suematsu et al., 1984). Using cultured rat aortic smooth muscle cells, Furukawa et al. (1988) observed Ca 2+ efflux due to the activity of both the Ca2+-ATPase and Na+-Ca :~ exchange although the authors concluded that the former was more important for maintaining resting [Ca2+]i; 8-BrcGMP caused a stimulation of Na + independent Ca 2+ efflux, whereas two cyclic AMP derivatives and forskolin were ineffective. This result is consistent with the observations that cAMP-PrK does not alter the activity of the Ca2+-ATPase. However, in toad stomach smooth muscle cells, isoprenaline and dibutyryl-cAMP have been reported to stimulate Ca 2+ efflux. In this case it is proposed that cAMP-PrK activates Na+/Ca 2+ exchange (Scheid and Fay, 1984; see Fig. 3). Therefore, whether Ca 2~ movements are observed in smooth muscle cells or microsomes is dependent on the exact ionic conditions used and the smooth muscle studied. The observations that cAMP-PrK and cGMP-PrK can phosphorylate distinct proteins in vascular smooth muscle sarcolemma allows the possibility that each regulates Ca 2+ extrusion from the cell via a different

Cyclic AMP mechanism. At present, the majority of evidence indicates that the plasma membrane Ca2+-ATPase is regulated by cGMP and not by cAMP. 4.4.4. Uptake o f Ca 2+ by Internal Stores Vascular smooth muscle contains a network of sarcoplasmic (or endoplasmic) reticulum (SR) that by taking up or releasing Ca 2÷ is capable of regulating [Ca2+]i (Somlyo and Himpens, 1989). In smooth muscle there is good evidence that release of Ca 2+ from this store is mediated through the action of IP 3 (Saida el al., 1988; van Breemen and Saida, 1989). Recent studies have indicated that the regulation of Ca 2+ uptake by the SR in smooth muscle is very similar to that occurring in cardiac tissue. In the latter, the SR Ca2+-ATPase is regulated by the phosphorylation of an associated protein, phospholamban. Phosphorylation by cAMP-PrK of phospholamban in cardiac SR vesicles results in increased Ca2+-ATPase and Ca 2+ uptake (Tada and Katz, 1982). In addition, phospholamban has been identified in SR from slow skeletal muscle (Kirchberger and Tada, 1976) and more recently in several smooth muscles. Antibodies to canine cardiac phospholamban cross react with proteins in pig stomach, dog and rabbit aorta and, as in the heart, smooth muscle phospholamban was located exclusively in the sarcoplasmic reticulum. Interestingly, no phospholamban was found in pig aorta (Raeymaekers and Jones, 1986). In the above study, cAMP-PrK catalyzed the phosphorylation of smooth muscle phospholamban with 32p being incorporated into proteins of M r = 22,000 and 11,000. Watras (1988) reported that incubation of bovine aortic sarcoplasmic reticulum vesicles with cAMP-PrK resulted in both phosphorylation of a protein of M r = 11,000 (that comigrated with cardiac phospholamban on electrophoresis) and stimulation of Ca 2+ uptake. In stark contrast, Chiesi et al. (1984) found no phospholamban or cAMP-PrK stimulated Ca 2+ uptake in the same tissue. Phospholamban has also been identified, by its characteristic change in M r o n boiling, in bovine and ovine pulmonary artery microsomes (Raeymaekers et al., 1988; Huggins et al., 1989). These studies also report that cardiac and smooth muscle phospholamban are substrates for both cAMP-PrK and cGMP-PrK. Studies with isolated microsomes have shown that there is a qualitative similarity between cardiac and smooth muscle sarcoplasmic reticulum. Both membranes contain equal amounts of phospholamban and the Ca-'+-ATPase, although these proteins are more abundant in cardiac membranes (Raeymaekers and Jones, 1986; Watras, 1988). In general, smooth muscle microsomes show a relatively low rate of Ca :+ uptake which is rather poorly stimulated by cAMP-PrK, although, due to the technical problems involved, this need not mean that this is not of physiological significance. Indeed, cAMP-dependent stimulation of Ca 2+ uptake into the sarcoplasmic reticulum has been demonstrated in intact and skinned vascular smooth muscle preparations (Saida and van Breemen, 1984; Hwang and van Breemen, 1987; Twort and van Breemen, 1988). cAMP was reported to enhance Ca :+ storage in portal vein cells

339

studied by whole-cell patch clamp (Komori and Bolton, 1989). Although not conclusive, the results described in this section suggest that cAMP could cause relaxation of vascular smooth muscle by cAMP-PrK catalyzed phosphorylation of phospholamban which stimulates Ca 2÷ uptake into the sarcoplasmic reticulum. However, this mechanism appears to be tissue and/or species dependent and also not unique to cAMP as phosphorylation of phospholamban is also catalyzed by cGMP-PrK. Phosphorylation of phospholamban has been shown in perfused heart (Wegener et al., 1989) but remains to be demonstrated in an intact smooth muscle preparation. Huggins et al. (1989) did not observe any phosphorylation of phospholamban in untreated rabbit aorta, or tissue treated with agents known to elevate cGMP. These authors did not investigate the effects of increasing cAMP, and this study is obviously required to determine the physiological significance of cAMP-dependent phospholamban phosphorylation. 4.4.5. Membrane Hyperpolarization The relaxant effects of isoprenaline are associated with membrane hyperpolarization in some vascular smooth muscles (Bulbring and Tomita, 1987). Recent studies, using the patch-clamp technique, suggest that this is due to cAMP-dependent phosphorylation of a Ca2+-dependent K + channel. Injection of cAM P-PrK mimicked the external application of isoprenaline; both resulted in an increased open-state probability of the channel (Sadoshima et al., 1988; Kume et al., 1989).

5. COMPARTMENTS OF cAMP It is obviously incorrect to regard an intact tissue or even a single cell, as being homogenous, therefore assays of metabolites or enzymes that, necessarily require disruption of the tissue will record only an average value. This could allow for discrepancies between measured levels of cAMP and vasodilation to be accounted for by the existence of compartments of cAMP and/or its associated enzymes. In the heart, the observations that PGE~ and rolipram could increase cAMP and activate cAMP-PrK without the expected increases in contractility have suggested the existence of compartments of cAMP in this tissue (Buxton and Brunton, 1983; Murray et al., 1989). However, it should be noted that the role of cAMP as an inotrope is rather better defined than its role as a vasodilator. In the studies on heart, it has been shown, that, although PGEI increases cAMP and activates cAMP-PrK, it does not lead to increased myocardial protein phosphorylation (Hayes et al., 1982). This study therefore provides a rational explanation for the lack of inotropic effects of PGE~ and also highlights the need to measure cAMP, cAMP-PrK activity, protein phosphorylation and physiological response at the same time when studying compartments. At present, as there are no well defined substrates for cAMP-PrK in vascular smooth muscle, such a study is not possible. However, there are a number of reports that indicate that there are compartments of cAMP in vascular smooth muscle.

340

K.J. MURRAY

Working with rabbit aorta, Vegesna and Diamond (1986) reported that isoprenaline, forskolin and PGE~ all increased cAMP levels. Whereas the first two agents relaxed phenylephine-contracted tissue, PGE~ caused contraction both in the presence or absence of phenylephrine. The data presented showed that the PGE~-induced contractions were very resistant to cAMP-mediated relaxation. For example, PGE 1 caused a contraction in forskolintreated aorta in which the cAMP content had been increased over 30-fold; in contrast isoprenaline could relax these preparations without a further increase in cAMP. A similar increase in cAMP with no relaxant effect has been reported for forskolin (Vegesna and Diamond, 1983) and arachidonic acid (Rubanyi et al., 1986) treatment of bovine coronary arteries. In guinea pig aorta, Silver et al. (1988b) found from their concentration and temporal studies that there was a significant correlation between cAMP levels (and activation of cAMP-PrK) and vasodilation induced by milrinone, papaverine or forskolin. However, when the two PDE inhibitors were compared with forskolin, it was found that the latter caused larger increases in cAMP and activation of cAMPPrK for equal levels of relaxation. Similar 'overproduction' of cAMP relative to its contractile effects has been reported in heart (England and Shahid, 1987). Reports have also suggested the presence of compartments of cAMP in uterine smooth muscle (reviewed in Krall et al., 1983). The present observations cannot be said to provide conclusive evidence for the compartmentation of cAMP in vascular smooth muscle, and further work in this area requires more knowledge about the mechanism(s) by which cAMP causes vasodilation.

6. CONCLUSIONS Sutherland et al. (1968) proposed the original set of criteria to determine if cAMP was involved in a particular physiological response and, if it is assumed that all actions of cAMP are through activation of cAMP-PrK, these criteria can then be linked to those of Krebs and Beavo (1979) for the involvement of protein phosphorylation. The criteria may be stated with regard to vasodilation as: (1) Receptor agonists should stimulate the formation of cAMP in intact tisue and membrane preparations. (2) Phosphodiesterase inhibitors should mimic or potentiate the physiological action of the receptor agonist and have similar effects on the cAMP levels. Inhibition of the phosphodiesterase in a tissue extract should be demonstrated. (3) cAMP-derivatives should cause relaxation. (4) Activation of cAMP-PrK in the intact tissue preparation in response to relaxant agents should be demonstrated. (5) Increased phosphorylation of specific protein(s) should be observed in intact preparations. The degree of phosphorylation should correlate temporally and in a concentration-dependent manner with the relaxation.

(6) The isolated protein should be a substrate for cAMP-PrK, be phosphorylated stoichiometrically and at a significant rate. The functional properties should be altered by, and correlated with its phosphorylation. The first two criteria have been satisfied to some extent with respect to vasodilation. Appropriate receptor agonists (Section 3.1) and PDE inhibitors (Section 3.2) cause relaxation that is associated with increased cAMP levels. It should be noted that the ultimate event in the cAMP pathway is phosphorylation of proteins (Fig. I) so that relaxation should correlate with the degree of protein phosphorylation and not necessarily with the preceding events. Nevertheless, there is evidence that relaxation in response to some agents does proceed in a temporal or concentration-dependent basis with increased cAMP levels. Again the third and fourth criteria have also been observed in intact preparations (Section 4.1). Contrasting observations have also been made, with receptor agonists having been postulated to relax in a cAMP-independent manner or to cause an increase in cAMP without relaxation, and cAMP derivatives have failed to cause relaxation. There is growing evidence to suggest that some of the effects of cAMP are mediated through cGMP-PrK rather than cAMP-PrK. This has been suggested by Francis et al. (1988) based on their observations with cAMP derivatives although, at present, there is no direct proof that cGMP-PrK was activated in the tissue. However, an elegant system to test this has recently been devised. Lincoln et al. (1989) observed that primary cultures of rat aortic smooth muscle contained both cAMP-PrK and cGMP-PrK; whereas cGMP-PrK was selectively depleted in passaged cells. Forskolin and isoprenaline decreased [Ca 2+ ]i in primary cultures but increased it in passaged cells. Pinocytic addition of cGMP-PrK to passaged cells restored the ability of isoprenaline and forskolin to decrease [Ca2+]i. These results indicate that (1) to lower [Ca2+]~, cGMP-PrK must be activated and forskolin and isoprenaline are capable of doing this and (2) activation of cAMP-PrK by itself raises [Ca 2~ ].. Although cGMP-PrK, especially in its autophosphorylated form, is capable of being activated by cAMP (Landgraf et al., 1986) these are the first reports to suggest that this may be physiologically relevant. The last two criteria remain to be satisfied. Although phosphorylation of specific proteins in response to increased cAMP has been observed in intact vascular smooth muscle (Rapoport et al., 1982), the function of these proteins is unknown. As there are unique substrates for both cAMP-PrK and cGMP-PrK in vascular smooth muscle membranes (Section 4.3), study of their phosphorylation in intact tissue might help elucidate the kinase(s) activated by agents such as isoprenaline, forskolin and CPTcAMP. However, the major problem remains the identification of the protein, or more probably, proteins responsible for relaxation. With regard to the contractile proteins, MLCK activity is regulated in an appropriate manner by cAMP-PrK: further work is required to show this occurs in intact vascular

Cyclic AMP smooth muscle and is accompanied by decreased P-light chain phosphorylation. Any effects of c A M P on the postulated second regulatory mechanism requires elucidation of that system, although it appears direct effects of c A M P - P r K on caldesmon can be excluded (Section 4.3). Similarly, the effects of c A M P on [Ca 2+ ]~ require further study on the exact mechanism, although evidence is increasing that uptake by the sarcoplasmic reticulum and phospholamban phosphorylation may be the cause (Section 4.3). Once again, the differences between species, tissues and the agents used both for relaxation and contraction must be emphasized. The differences between the regulation of contraction of cardiac and smooth muscles are usually emphasized (e.g. England, 1988). In heart, c A M P elevating agents act as inotropes (i.e. increase the force of contraction) but also increase the rate of relaxation, c A M P - P r K regulates [Ca 2+ ]i in cardiac cells by phosphorylation of the voltage sensitive calcium channel and phospholamban. There is also evidence that the contractile proteins are desensitized to Ca :+ by phosphorylation of the inhibitory subunit of troponin (England, 1983; Murray et al., 1989). There is increasing evidence that similar mechanisms operate in smooth muscle; isoprenaline and forskolin have been reported to increase the probability of opening of the voltage-sensitive calcium channels, phospholamban has been found in smooth muscle (Section 4.4) and the contractile apparatus is desensitized to Ca 2+ (Section 4.3). There are many agents that elevate vascular smooth muscle c A M P levels by diverse mechanisms, and cause vasodilation. Therefore, it seems improbable that c A M P has no role as a vasodilator. At present, it appears that relaxation may be caused both by desensitization of the contractile elements and by lowering [Ca 2+ ]~, mainly by promoting uptake by the sarcoplasmic reticulum. Acknowledgements--I am grateful for the assistance of

Dr David Owler and Ms Tracy Beadle in the preparation of this manuscript, and also to Dr A. J. Kaumann and Dr P. J. England for their useful comments.

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