Naunyn-Schmiedeberg's

Naunyn-Schmiedeberg's Arch Pharmacol (1990) 341 : 577- 585

Archivesof Pharmacology © Springer-Verlag1990

Antagonism of novel inotropic agents at A1 adenosine receptors and m-cholinoceptors in human myocardium* Martin Ungerer, Michael Biihm, Robert H. G. Schwinger, and Erland Erdmann Medizinische Klinik I der Universit/it Mfinchen, Klinikum Gro6hadern, Marchioninistrasse 15, D-8000 Mfinchen 70, Federal Republic of Germany Received August 2, 1989/Accepted February 12, 1990

Summary. The effects of the new inotropic agents saterinone, sulmazole, UD-CG 212.C1 and milrinone at A1 adenosine receptors and m-cholinoceptors were evaluated in human myocardium from patients with heart failure. At A1 adenosine receptors, all compounds inhibited aH-DPCPX-binding to ventricular membrane preparations at micromolar concentrations. As judged from the Ki-values, the rank order of potency was saterinone > sulmazole > UD-CG 212.C1 > milrinone. The new inotropic agents also displaced the binding of 3H-QNB at m-cholinoceptors. Except for saterinone, the concentration ranges of mean Ki-values were considerably higher at m-cholinoceptors than at A1 adenosine receptors. The rank order of potency was saterinone > sulmazole > UD-CG 212.C1 > milrinone. Competition of the A1 adenosine receptor agonist R-PIA to 3HDPCPX-binding showed a biphasic curve with a shallow slope (Hill coefficient nH= 0.63) and revealed two affinity states of the A1 adenosine receptor. In the presence of guanine nucleotides [Gpp(NH)p], the competition curve showed one low affinity class of binding sites and was shifted to the right. In contrast, the competition curves of the new inotropic agents were characterized by a monophasic, steeper slope (mean Hill coefficient nn = 0.98). Guanine nucleotides had no effect. Similar results were obtained with saterinone and carbachol at mcholinoceptors. Competition with carbachol revealed three affinity states of the m-cholinoceptor, the superhigh affinity binding was reversed by Gpp(NH)p. Competition with saterinone revealed one class of binding sites which was not influenced by Gpp(NH)p. Accordingly, in isolated, electrically driven human atrial trabeculae, the negative inotropic effect of adenosine was antagonized concentration-dependently by saterinone, sulmazole and UD-CG 212.C1. Similarly the negative inotropic effect ofcarbachol was antagonized concentration-dependently by saterinone. It is concluded that the new inotropic agents bind to A1 adenosine receptors and that their Send offprint requests to M. B6hm at the above address

* Supported by the Deutsche Forschungsgemeinschaft

interaction is of antagonist nature. This mechanism might contribute to their capacity to enhance force of contraction by stimulation of cAMP-formation in addition to phosphodiesterase inhibition. The effects of saterinone may be partially due to antagonism at m-cholinoceptors. This is presumably not the case with the other inotropic agents studied given their low affinity for this receptor.

Key words: Heart failure - AI adenosine receptors m-Cholinoceptors - Phosphodiesterase inhibitors Human myocardium - Positive inotropic agents

Introduction In recent years, research efforts have been made to develop new positive inotropic agents for the therapy of cardiac failure which should overcome the problems encountered with digitalis glycosides, especially their small therapeutic range. A new approach was offered by new classes of substances which were classified as inhibitors of the low Km cAMP-specific phosphodiesterase II! (PDE III; Weishaar et al. 1986; Brunkhorst et al. 1989) and some of which were also found to exert a calcium-sensitizing effect on myocardial contractile elements (Herzig et al. 1981). Moreover, an effect of A1 adenosine receptors was suggested (Earl et al. 1986; Parsons et al. 1988). The existence of A x adenosine receptors in human myocardium has recently been demonstrated by radioligand binding studies (B6hm et al. 1989a, b) showing that At adenosine receptors could be directly characterized with the radiolabeled antagonist 3HDPCPX, and that the adenosine receptor is identical in human atria and ventricles (B6hm et al. 1989a). Adenosine exerts a "direct" negative inotropic effect on the atrial myocardium which can be observed in electrically stimulated auricular trabeculae in a variety of species (Chiba and Himori 1975; Rockoff and Dobson 1980) including man (B6hm et al. 1989a). In ventricular

578 papillary muscles, this effect c a n n o t be demonstrated. A negative inotropic response is only observed if the preparations are prestimulated with a c A M P - d e p e n d e n t positive inotropic substance ("indirect" negative ino t r o p i c effect; Schrader et al. 1977a). Similar effects are exerted by cholinergic substances acting via muscarinic cholinoceptors in the heart ( B r o w n 1979). In the ventricle, b o t h receptors play a role in the regulation o f c A M P f o r m a t i o n (Schrader et al. 1977a) or directly affect slow the Ca 2" inward current ( B 6 h m et al. 1984). I n the auricle, however, adenosine receptors and m - c h o l i n o c e p t o r s seem to be coupled to K + - c h a n n e l s (Belardinelli and Isenberg 1983; J o c h e m a n d N a w r a t h 1983; Ransnfis et al. 1986) and to a m i n o r extent to adenylate cyclase ( E n d o h et al. 1983). Effects o f the new inotropic agents milrinone, sulmazole and U D - C G 212.C1 on radioligand binding to A1 adenosine receptors have been d e m o n s t r a t e d in rat adipocytes (Parsons et al. 1988). In the heart, radioligand binding has only been studied for milrinone in rat atria (Earl et al. 1986). Therefore, the question arose whether an effect o f the new inotropic c o m p o u n d s can also be observed in h u m a n m y o c a r d i u m and whether functional a n t a g o n i s m is detected in c o n t r a c t i o n experiments o f hum a n heart tissue. The effect on m - c h o l i n o c e p t o r s was studied for comparison. The effects o f saterinone, sulmazole ( A R - L 115 BS), U D - C G 212.C1 a n d milrinone on radioligand binding at A1 adenosine receptors a n d at m - c h o l i n o c e p t o r s were examined in ventricular m y o c a r d i u m o f patients with cardiac failure. 3 H - D P C P X and 3 H - Q N B were used to label A1 adenosine receptors and m-cholinoceptors, respectively. Moreover, h u m a n atrial tissue was used to evaluate the influence o f the new inotropic agents on the negative inotropic effect o f adenosine a n d c a r b a c h o l in c o n t r a c t i o n experiments o f isolated, electrically driven muscle strips. Auricular tissue was chosen because adenosine a n d c a r b a c h o l p r o d u c e a "direct" negative inotropic effect only in atrial m y o c a r d i u m . The a n t a g o n istic properties o f the new inotropic c o m p o u n d s can be evaluated w i t h o u t the presence o f a third, c A M P - i n c r e a s ing substance (e.g. isoprenaline). Moreover, the atrial receptors seem to be p r e d o m i n a n t l y coupled to K---channels (Belardinelli and Isenberg 1983; G o y a l 1989) so that elevated intracellular cAMP-levels due to p h o s p h o d i esterase inhibition should n o t influence the effect o f adenosine and o f carbachol.

Materials and methods Patients and methods. Radioligand binding experiments

were performed on myocardial membrane preparations from the left ventricles of 5 patients with cardiomyopathy. The mean age was 46 _+6 years (x + SD). Atrial preparations were from 10 patients who underwent operation because of coronary heart disease without evidence for heart failure. The mean age was 51 _+4 years (x _+SD). Medical therapy consisted of nitrates, cardiac glycosides, diuretics and enalapril in all cases. Patients receiving catecholamines were withdrawn from the study. All patients gave written informed consent prior to operation. After excision, the tissue was placed immediately in ice-cold cardioplegic solution and delivered to the laboratory within 10 rain.

For radioligand binding experiments, the tissue was minced with scissors in ice-cold homogenization buffer (10 mmol/1 TrisHC1, 1 mmol/1 EDTA, 1 mmol/1 dithiothreitol, pH 7.4) and prepared with a motor-driven glass-teflon Elvehjem-potter. The homogenate was spinned with 2000 rpm (Beckman rotor A-641). The supernatant was diluted with an equal volume of 1 mol/1 KC1 and stored on ice for 10 min. This suspension was pelleted twice with 100000 g for 45 rain. The pellet was resuspended and used for binding experiments. The incubation buffer consisted of 50 retool/1 Tris-HCl and 0.2 gg/ml adenosine deaminase at pH 7.4 in the case of adenosine binding experiments and of 50 mmol/1 TRIS-HC1 and i mmol/1 MgC12 in the case of m-cholinoceptor binding experiments. Membranes were incubated with 0.05-12.0 nmol/1 3HDPCPX for 120 min or with 0.02-10 nmol/13H-QNB for 100 rain. All assays were carried out in a total volume of 250 gl at 22° C. The chosen conditions allowed complete equilibration of the receptors with the radioligand. The reaction was terminated by vacuum filtration and washing of the filters were ice-cold incubation buffer. Before use, filters were presoaked in 0.1% Chaps {3-[(3chelamidopropyl)-dimethylammonio]l-propanesulfonate} in order to reduce nonspecific binding of 3H-DPCPX to the glass fibers. Nonspecific binding was determined with 1 mmol/1 theophylline and amounted to 35.2 _+5.8% of total binding at KD in the case of A1 receptor binding experiments. Total binding at Kd was approximately 520 cpm with a nonspecific binding of approximately 170 cpm; only experiments in which the amount of specific binding exceeded 300 cpm were used. At m-cholinoceptors, it was determined with 0.1 mmoI/1 atropine and amounted to 5 _+1% of total binding at Km Total binding was approximately 1500 cpm with a nonspecific binding of approx. 80 cpm. The maximal density (Bmax) and apparent affinity (KD) were obtained in individual experiments by linear regression analysis. The mean protein concentration was 246 + 11 gg/tube. Force of contraction was measured in contraction-responseexperiments performed on electrically driven (1 Hz) human atrial trabeculae. The atrial preparations of uniform size (diameter < 1.0 ram, length 3 - 6 mm) were dissected in aerated bathing solution (composition see below) at room temperature. The preparations were attached to a bipolar platinum stimulating electrode and suspended individually in 75 ml glass tissue chambers for recording isometric contractions. The bathing solution was a modified Tyrode solution containing (retool/l) NaC1 119.8, KC1 5.4, CaClz 1.8, MgCI2 1.05, NaHzPO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, glucose 5.0. It was continuously gassed with 95 % 02 and 5% COz and maintained at 35°C; the pH was 7.4. The force of contraction was measured with an inductive force transducer (W. Fleck, Mainz, FRG) attached to a Hellige Helco Scriptor or Gould recorder. Each muscle was stretched to the length at which force of contraction was maximal. The resting force (approximately 5 raN) was kept constant throughout the experiment. The preparations were electrically paced at 1 Hz with rectangular pulses of 5 ms duration (Grass stimulator SD 9), the voltage was about 20% above threshold. All preparations were allowed to equilibrate in drugfree bathing solution until complete mechanical stabilization. All compounds were freshly dissolved in prewarmed and preaerated bathing solution. Muscle strips were allowed to equilibrate for 5 rain after addition of drugs before force of contraction was measured. Stock solutions of saterinone and UD-CG 212.C1 were prepared in 100 % dimethylsulfoxide (DMSO). Concentration-dependent effects were obtained in the following manner: adenosine and carbachol were applied cumulatively. For experiments with new inotropic agents, the preparations were prestimulated with these substances and concentration-response curves of adenosine or carbachol were measured under maintenance of the concentration of the prestimulating agent. Materials. R-(-)-N6-phenylisopropyladenosine (R-PIA), adenosine

and guanylylimidodiphosphate [Gpp(NH)p] were purchased from Boehringer (Mannheim, FRG). Theophylline and atropine were from Serva (Heidelberg, FRG). Carbachol was from Sigma (Deisenhofen, FRG). 3H-DPCPX (1,3-dipropyl-8-cyclopentyl-

579 A 13

A

COMPETITION AT A t - R E C E P T O R S IN HUMAN

o

VENTRICULAR MYOCARDIUM

Table 1. Inhibition constants (Ki) and Hill coefficients (nil) of saterinone, sulmazole, UD-CG 212.C1 and milrinone at A1 adenosine receptors (A) and m-cholinoceptors (B)

100

,a z

E _E

\

E o

•

SATERINONE

O

SULMAZOLE

(A) Al-adenosine receptors

•

UD-CG212,CL

Saterinone

.

ML..o.E

Sulmazole (AR-L 115 BS) UD-CO 212.C1

50

Milrinone

i-o

0.1

1

10 Concentration

B

100

1OOO

1 0 0OO

COMPETITION AT M-CHOMNOCEPTORS IN HUMAN VENTRICULAR MYOCARDIUM

| ?

nH

8.3 (0.45 - 135) 22 (7.7-63) 46 (25-83) 292 ( 9 0 - 946)

0.96 _+0.04

3.5 (1.9-4.0) 2000 (1460-2750) 530 (370-770) > 10000

0.92 +_0.05

1.02 + 0.03 0.99 _+0.05 0.93 _ 0.06

(B) m-cholinoceptors Saterinone

(pmol/I)

Ki (gmol/1)

Sulmazole (AR-L 115 BS) UD-CG 2:12.C1

100

Milrinone

0.87 _+0.07 0.95 + 0.05 -

For Ki-values geometric means are shown with 95% confidence limits in parenthesis. Hill coefficients are given as means _+ SEM

E

[ o

50

values were calculated according to Cheng and Prusoff (1973). Agonist competition curves were analysed with the SCTFIT-program according to DeLean et al. (1982). Curve fits assuming one, two and three affinity states were compared with an F-test. The significantly (p < 0.005) best fit was accepted. For contraction experiments, a value ofp < 0.05 was considered to be significant.

i 0

O.1

1

10

100

1OOO

10 O00

Concentration ( p m o l / I )

Fig. 1. Competition of saterinone ( e ) , sulmazole (AR-L 115 BS; ©), UD-CG 212.C1 ( I ) and milrinone (A) at A1 adenosine receptors (A; 3H-DPCPX-binding) and m-cholinoceptors (B; 3H-QNBbinding) in human ventricular myocardium. The concentration of 3H-DPCPX was 2 nmol/1 and that of 3H-QNB was 0.5 nmol/1. Data are means of triplicate determinations in 5 different tissues. Ordinates." specific binding of radioligand in % of the maximum radioligand bound. Abscissae: concentration of compounds, expressed in gmol/1. On an average, 232 _+ 13 gg protein per tube were used in the assays xanthine) and 3H-QNB (quinuclidyl benzilate) was from Amersham-Buchler (Braunschweig, FRG). Saterinone [BDF 8634; 1-[4-(cyano-l,2-dihydro-6-methyl-2-oxopyrinidyl-5)-phenoxy]-3[4-(2-methoxyphenyl)-piperazinyl-t]-propanol} was from Beiersdorf AG, Hamburg, FRG. Milrinone [1,2-dihydro-6-methyl-oxo-5(4-pyridyl)nicotinonitrile] was from Sterling-Winthrop GmbH, Hamburg, FRG. Sulmazole [AR-L t 15 BS; 2-methoxy-(4-methylsulfinyl)phenyl-lH-imidoazol(4,5-b)pyridine] and UD-CG 212.C1 [4,5 - dihydro- 6- 2- (4- methoxyphenyl)- 1H-benzimidazole- 5 - yl- 5methyl-3(2H)-pyridazinone) were donated by Dr. Karl Thomae GmbH, Biberach, FRG. For radioligand binding experiments, sulmazote was diluted in the incubation buffer, UD-CG 212.C1 and R-PIA were diluted in incubation buffer and 0.2% dimethylsulfoxide (DMSO), milrinone and saterin0ne were diluted in incubation buffer and 2% DMSO. The final concentration of 2% DMSO decreased specific binding by 10%. It did not affect the pHvalue in the incubation medium. Statistics. ICso-values in competition experiments were estimated graphically as half maximal inhibition of radioligand binding. Ki-

Results 3H-DPCPX concentration-dependently labeled adenosine r e c e p t o r s in h u m a n v e n t r i c u l a r m e m b r a n e s w i t h a Bmax v a l u e o f 18.3 -+_ 1.7 f m o l / m g p r o t e i n . T h e m e a n K w value was 2.1 ( 1 . 3 - 3 ) nmol/1 (n = 5). m - C h o t i n o c e p t o r s were d e t e c t e d w i t h 3 H - Q N B in the s a m e h e a r t s w i t h a Bmax-value o f 251 + 33 f m o l / m g p r o t e i n (n = 5) a n d a KD o f 0.4 ( 0 . 1 8 - - 0 . 7 ) nmol/1 (n = 5). I n b o t h cases, b i n d i n g was m o n o p h a s i c a n d S c a t c h a r d r e g r e s s i o n analysis r e v e a l e d one class o f b i n d i n g sites ( n o t shown). S a t e r i n o n e , s u l m a z o l e , U D - C G 212.C1 a n d m i l r i n o n e c o m p e t i t i v e l y i n h i b i t e d 3 H - D P C P X - b i n d i n g to c a r d i a c A1 a d e n o s i n e receptors. C o m p e t i t i o n curves a r e s h o w n in Fig. 1 A. T h e curves are m o n o p h a s i c a n d s h o w Hill coefficients close to 1. A l l K i - v a l u e s were in the m i c r o m o l a r range. T h e y are listed in Table 1 A. Besides, it was f o u n d t h a t the n e w i n o t r o p i c a g e n t s b i n d to mc h o l i n o c e p t o r s . F i g u r e 1 B shows t h a t b i n d i n g o f 3HQ N B is also i n h i b i t e d b y the new i n o t r o p i c agents. T h e affinity o f s a t e r i n o n e for m - c h o l i n o c e p t o r s was similar to t h a t for A1 a d e n o s i n e r e c e p t o r s w h e r e a s s u l m a z o l e a n d U D - C G 212.C1 d i s p l a y e d a c o n s i d e r a b l y lower affinity for m - c h o l i n o c e p t o r s t h a n for A1 a d e n o s i n e receptors. M i l r i n o n e d i s p l a c e d 3 H - Q N B - b i n d i n g o n l y slightly at m i l l i m o l a r c o n c e n t r a t i o n s . T a b l e I s u m m a r i z e s the Kivalues a n d the Hill coefficients (nil) o f the new i n o t r o p i c

580

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-

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.

Carbachol 1oo

,-G°o,N.,o

'~i 2 U so

.

,0

g,

o

u

0.01 o

//, 0.1

1

10 Con©entratlon

100

1000

10 000

0.1

1 10 100 Concentration (pmol/I)

F~

(nmol/l)

B

~,,aterinone

S A I

|1o.

1000

m

¸

£

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e

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e, = 100 0 .O m

z 0 m

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t

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:

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1000

Concentration (pmoi/I)

o

q/ t

0.1

1

10

100

1000

10000

CoeceantJon (t~oO/t)

Fig, 2, Displacement of 3H-DPCPX-binding to human cardiac membranes by R-PIA (A) and saterinone (B). The competition curves were detected in absence ( e ; A) and in presence (©; A) of 100 gmol/1 Gpp(NH)p. The concentration of 3H-DPCPX was 2 nmol/1. Data are means of triplicate determinations in 5 different tissues. Ordinates: specific binding in % of maximum 3H-DPCPX bound. Abscissae: Concentration of R-PIA in nmol/1 (A) or concentration of saterinone in I.tmol/1(B)

compounds at A1 adenosine receptors and m-cholinoceptors, In order to analyse the agonist or antagonist nature of receptor interaction, competition experiments in the presence of 100 gmol/1 Gpp(NH)p were carried out. The competition curves of receptor agonists are usually steepened and shifted to the right in the presence of nonhydrolysable guanine nucleotides whereas competition curves of antagonists are usually not. Agonist competition is demonstrated with the A1 adenosine receptor agonist R-PIA in Fig. 2A. R-PIA competed for 3HDPCPX-binding with a shallow curve (nil = 0.63) in the absence of Gpp(NH)p. A steeper curve (nn --- 0.95) was detected in the presence of Gpp(NH)p. Competition without Gpp(NH)p revealed a high and a low affinity state

Fig. 3. Displacement of 3H-QNB-binding to human cardiac membranes by carbachol (A) and saterinone (B). The competition curves were detected in absence ( I ; • ) and in presence ([3; &) of 100gmol/1 Gpp(NH)p. The concentration of 3H-QNB was 0.5 nmol/1. Data are means of triplicate determinations in 4 different tissues. Ordinates: specific binding in % of maximum 3H-QNB bound. Abscissae: Concentration of carbachol (A) or of saterinone (B) in gmol/1

of the At adenosine receptor whereas with Gpp(NH)p only one low affinity state was detected. Figure 2 B shows that this was not the case with saterinone. 100 ~tmol/1 Gpp(NH)p had no effect on the competition of saterinone at one class of low affinity At adenosine receptors. Similar results were obtained with the other agents (not shown). Similarly, guanine nucleotides produced no change in the shape or affinity of the competition curves of saterinone to 3H-QNB-binding (Fig. 3). Figure 3A shows that the m-cholinoceptor agonist carbachol competed for 3H-QNB-binding with a shallow curve (nil = 0.55) displaying three affinity states in the absence of Gpp(NH)p. Competition in the presence of Gpp(NH)p steepened the curve (nil = 0.91) and two affinity states were detected. In contrast, Fig. 3 B shows that the competition of saterinone could not be influenced by guanine nucleotides. With and without 100 ~tmol/1 Gpp(NH)p, one class of low affinity m-cholinoceptors was detected. Accordingly, the antagonism of saterinone, sulmazole and UD-CG 212.C1 on the negative inotropic effect of

581

A

B

HUMAN ATRIUM

HUMAN ATRIUM

1toNI bathing solution __

, bathing solution

J

Ad i

L

Carb

"J

2.5 mN [

2.5 mN [

,

bathing solution

1bathing solution

iIso

i Iso

i

=Ad

•

Carb

2.5 m N [

2.5 mN [

= bathing solution

bathing solution

,Sat =Ad

oi

I Sat

|

5|

= i Carb

i 10 min i Ad 1 0 0 0 prnol/I

0 b

=

5 i

!

1 0 rain m Carb 10 pmol/I

Iso 0.03 pmol/I

Iso 0.03 pmol/I

Sat 1 0 0 JJmol/I

Sat 1 0 0 pmol/I

adenosine were evaluated in isolated, electrically driven atrial preparations; and the antagonism of saterinone on the negative inotropic effect of carbachol was measured similarly. Given their low affinity for the receptor in radioligand binding studies, the antagonist potencies of milrinone at adenosine receptors and that of sulmazole, UD-CG 212.C1 and milrinone at m-cholinoceptors were not measured in contraction experiments. The increase in force of contraction achieved by saterinone was 320 _+ 31% of the basal value. The ECsovalue was 2 (0.8-5) gmol/1 and maximum stimulation of force of contraction was obtained at 30 gmol/1 (not shown). Sulmazole increased contractile force to 346 -t- 41% of basal value at a concentration of 300 gmol/1 [ECso-value: 60 (30-105) gmol/1]. UD-CG 212.C1 had a positive inotropic effect of 172 -t- 26% of basal force at a concentration of 0.3 gmol/1 [ECso-value: 0.03 (0.0150.06) gmol/1] (not shown). The original tracings of the antagonism by saterinone on the effect of adenosine (A) and of carbachol (B) are shown in Fig. 4. The influence of isoprenaline is illustrated for comparison. The original tracing in the upper panel of Fig. 4A shows the negative inotropic effect of 1000 gmol/1 adenosine. The same effect is observed after prestimulation with 0.03 gmol/1 isoprenaline (below). In contrast, adenosine had only a small negative inotropic effect after prestimulation with 100 gmol/1 saterinone (bottom). The original tracing in the upper panel of Fig. 4B shows the negative inotropic effect of 10 lamol/1 carbachol. The same effect is observed after prestimulation with 0.03 gmol/1 isoprenaline (middle tracing).

Fig. 4. Original tracings illustrating force of contraction in isoiated, electricallydriven human atrial trabeculae. A upper panel: effectof 1000 gmol/l adenosine (Ad) alone, middle panel." effectof 1000 lamol/1 adenosine after prestimulation with 0.03 gmol/1isoprenaline (Iso), lowerpaneI: effectof 1000 gmol/1adenosineafterprestimulation with 100 gmol/1 saterinone (Sat). B upper panel: effect of 10 gmol/1carbachol (Carb) alone, middle panel: effect of 10 grnol/1carbachol after prestimulation with 0.03 gmol/1 isoprenaline (Iso), lower panel: effect of 10 lamol/1carbachol after prestimulationwith 100 grnol/1saterinone(Sat)

Again, 100 lamol/1 saterinone abolished the negative inotropic effect of carbachol almost completely (bottom). Similarly, suhnazole and UD-CG 212.C1 at a concentration of 1000 gmol/1 attenuated the negative inotropic response to adenosine (not shown). Concentration-response curves of the negative inotropic effect of adenosine and carbachol were measured and the effects of saterinone, sulmazole and UD-CG 212.C1 were evaluated. Exemplarily, Fig. 5A shows the effect of 30 gmol/1 and 300 ~tmol/1saterinone on the negative inotropy exerted by adenosine. Adenosine alone concentration-dependently reduced force of contraction with a maximal effect at 1000 gmol/1. The effect amounted to 28% of the value before application of adenosine. In the presence of saterinone, this negative inotropic effect is diminished or almost abolished and the concentrationresponse curve of adenosine is shifted to the right. The respective IC2s-values are given in Table 2. Accordingly, Fig. 5B shows the effect of 2 lamol/1 and 10 gmol/1 saterinone on the negative inotropy exerted by carbachol. Carbachol alone reduced force of contraction to 0% of the value before application of carbachol. It was maximal at a concentration of 10 gmol/1. At concentrations of 2 gmol/1 and 10 gmol/1, saterinone inhibited this effect and shifted the curve to the right. Table 2 summarizes all inhibition constants. As the negative inotropy of adenosine and carbachol in the presence of the new inotropic compounds did not reach the same level as it did in their absence, pAz-values were not calculated.

582 Table 2. ICe s-values of the negative inotropic effect of adenosine (A Ad, 0.1 - 1000 Ixmol/1)or ofcarbachol (B Carb, 0.0001 - 10 Ixmol/1), alone or in the presence of different concentrations of new inotropic agents in isolated, electrically driven preparations from human atrial myocardium

A lOO ¢O

IC25 (gmol/1)

O

(A) Adenosine

,50 O

Ad Ad + Ad + Ad + Ad + Ad + Ad +

O

"8 o

+ 300 Lumol/ISATERINONE

O tl.

o

I0.1 1

i

i

i

I

i

I

I

I

1 10 100 1000 Concentration OJrnol/I)

30 lamol/1saterinone 300 gmol/1 saterinone 100 gmol/1 sulmazole 1000 gmol/1 sulmazole 100 gmol/1UD-CG 212.C1 1000 btmol/1UD-CG 212.C1

10 (8.6-11.5) 61 (20-- 188)* 520 (288--950)* 55 (25-- 138)* 380 (105-- 1200)* 35 (7-185) 910 (395-2340)*

(B) Carbachol

B

Carb Carb + 2 gmol/1 saterinone Carb + 10 gmol/1 saterinone

0.035 (0.016-0.075) 0.85 (0.5--1.44)* 2.65 (0.8-8.9)*

Data give the mean with 95% confidence limits. * p < 0.05 vs. Ad or Carb

e-

.o 0

"~ O 50 I c ARBACHOL ~_ o ,3 pmol/I SATERtNONE~

O) 0 u_ o

'~

~ e c ARBACHOL +10 pmol/I SATERINONE ~ . . . . . ? -~,,~.._~ 0

0.0001

0.001

0.01

Concentration

• "~

0.1

1

-

~

0 10

OJmol/I)

Fig. 5. A Cumulative concentration-response curves for the effect of adenosine alone (©, n = 7), in presence of 30 gmol/1 (11, n = 7) and 300 gmol/1 (0, n = 7) saterinone. B Cumulative concentrationresponse curve of the effect of carbachol alone (O, n = 7), in presence of 2 gmol/1 (11, n = 6) and 10 gmol/1 (0, n = 6) saterinone. Ordinates: force of contraction in % of the value before addition of adenosine (A) and carbachol (B). Abscissae: Concentration of adenosine (A) and of carbachol (B). Basal force of contraction was 2.2 _+0.4 mN (n = 47). 2 gmol/1 Saterinone increased force of contraction to 201 _+ 15% (n = 7) of predrug value, 10 gmol/1 to 290 + 35% (n = 7), 30 gmol/1 to 320 _+31% (n = 7) and 300 gmol/1 to 281 -_t-_8.5% (n = 7). Note that saterinone antagonized the negative inotropic effect of both adenosine and carbachol

Discussion

The new inotropic agents examined inhibited aHDPCPX-binding at A1 adenosine receptors in h u m a n cardiac m e m b r a n e s with the following rank order of potency: saterinone > sulmazole > U D - C G 212.C1 > milrinone. In contrast to their effect on the binding of the A1 adenosine receptor agonist R-PIA, G p p ( N H ) p did not change the slope or the affinity of the competition curves of the compounds. In electrically driven, isolated h u m a n atrial trabeculae, saterinone, sulmazole and U D C G 212.C1 antagonized the negative inotropic effect of adenosine and shifted its concentration-response curve to the right. At m-cholinoceptors in the h u m a n heart, all c o m p o u n d s also inhibited radioligand binding, but except for saterinone, the Ki-values were considerably higher. G p p ( N H ) p did not change the slope or affinity of

the competition curves of saterinone. Saterinone accordingly antagonized the negative inotropic effect of carbachol and shifted its concentration-response curve to the right. J a k o b et al. (1989) had reported that a "direct" negative inotropic effect of R - P I A and o f c a r b a c h o l in h u m a n atria could only be observed for a few seconds and was very instable. In contrast, we found a m a r k e d and stable "direct" negative inotropic effect of both c o m p o u n d s in h u m a n atria which is in accordance with several reports from animal atria (e.g. Chiba and Himori 1975; Sorota et al. 1986). The apparent divergence of results cannot be explained; it might be due to different experimental conditions. It has been shown that an increased a m o u n t of adenosine is released during cardiac hypoxia (Berne 1963; Schrader et al. 1977b), cardiac ischemia (Fox et al. 1974) or excessive stimulation with catecholamines (Schrader et al. 1977 a). Although adenosine receptor agonists failed to exert an antiadrenergic effect in dogs in vivo (Seitelberger et al. 1984; Schipke et al. 1987), adenosine has been suggested to be an important feed back inhibitor protecting the heart from overstimulation with catecholamines (Schrader et al. 1977a). Moreover it was shown that adenosine is also released in heart failure (Newman et al. 1984). Consequently, substances which antagonize adenosine receptors might be useful at increasing force of contraction in the therapy of severe heart failure. The relevance of these findings is supported by the fact that adenosine receptors were detected in h u m a n cardiac tissue by functional studies (B6hm et al. 1985) and by radioligand binding techniques (B6hm et al. 1989a, b). They are coupled to intracellular effectors via a pertussis-toxin sensitive G-protein in rat m y o c a r d i u m (Hazeki and Ui 1981) and in the h u m a n heart (B6hm et al. 1989b). As the a m o u n t of inhibitory G-proteins (Gi) is increased by a b o u t 40% in the failing h u m a n m y o c a r d i u m (Feldman

583 et al. 1988), the A1 adenosine receptor-Gi complex might even be more important in heart failure. The new inotropic agents inhibit phosphodiesterase III activity (Weishaar et al. 1986; Ahn et al. 1988). Consequently, most compounds were shown to stimulate intracellular cAMP-formation (Honeljfiger et al. 1981; Parsons et al. 1988). Moreover, amrinone has been demonstrated to functionally antagonize A 1 adenosine receptors in guinea-pig atria (Dorigo and Maragno 1986). Milrinon displaced radioligand binding to these receptors in rat atria (Earl et al. 1986). Other new inotropic agents were demonstrated to bind to A1 adenosine receptors at rat fat cells (Parsons et al. 1988). The possible relevance of the latter findings are substantiated by functional studies which showed that phosphodiesterase inhibition alone is unlikely to account for the whole inotropic effect of sulmazole (Ahn et al. 1986). This was also supported by the findings of Parsons et al. (1988) who demonstrated that it stimulates cAMP beyond the level which is attainable with maximal PDE inhibition by papaverine. Inconsistencies between the data for PDE III-inhibition and those for inotropic action also exist for other novel inotropic agents such as milrinone (Brunkhorst et al. 1989). Consequently, additional mechanisms might contribute to their action. A direct effect of novel inotropic substances on Gi was postulated by Parsons et al. (1988). Inhibition of Gi function was observed in rat fat cells but millimolar concentrations were needed to observe this effect. Since the interpretation of these data is still subject to controversial discussion (e.g. Linden 1989), it was not investigated in the present study. Furthermore, this mechanism might be not relevant in the heart because of the high concentrations required. Of the four compounds studied, saterinone was shown to be both an A1 adenosine receptor antagonist and an m-cholinoceptor antagonist at micromolar concentrations. In these concentrations, it had an effect on the concentration-response curves of adenosine and carbachol, shifting them to the right. These antagonist properties might be explained partly by its bipyridine-like molecular structure which is presumably responsible for A1 adenosine receptor antagonism and by a tertiary amine followed by three carbohydrates, an oxygene and another carbohydrate which might explain m-cholinoceptor antagonism (Fig. 6). Saterinone was also reported to be a potent inhibitor of crude cAMP phosphodiesterase in human myocardium with an ICso-value of 25 gmol/1 (Pieske et al. 1988) and of PDE III in guinea pig myocardium at an ICso-value of 0.02 gmol/1 (von der Leyen et al. 1988). The positive inotropic effects of saterinone were observed at an ECso-value of 2 gmol/1. Taken together, all effects compared are exerted at similar concentration ranges and could, therefore, equally contribute to the action of saterinone. Suhnazole and UD-CG 212.C1, which both show structural similarities to methylxanthines, inhibited radioligand binding to A~ adenosine receptors at micromolar concentrations and antagonized the negative inotropic effect of adenosine in the same range. At considerably higher concentrations they also displaced 3HQNB-binding from m-cholinoceptors. Their potency to

O

C,~r't

ATROPINE

Iz---x

/ IH ~

]

SATERINOI~E

/

SULMAZOLE (AR-L 118 H

~ N..

I

I

I

I

I I I

u .co ,,,c, \ \

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MILRINONE Fig. 6. Molecular structures of the new inotropic agents in comparison to the A1 adenosinereceptor antagonisttheophylline(left panel

in frames) and the m-cholinoceptorantagonistatropine (right panel in frames). Arrows indicate decreasingaffinityto the A1 adenosine receptor. Relevantstructures are shownin bold lines. All substances studied display either bipyridine- or methylxanthine-likestructure which is similar to the two-ring structure of theophylline. In addition, the central chain of saterinone shows similarities to the structures whichdetermineparasympatholyticpotency of atropine

inhibit PDE III in guinea pigs was reported to be 225 gmol/1 for sulmazole (Ahn et al. 1986) and 0.19 gmol/ 1 for UD-CG 212.C1 (Brunkhorst et al. 1989). Hence, sulmazole is five times more potent in antagonising A1 adenosine receptors than in inhibiting PDE III whereas UD-CG 212.C1 is a much more potent inhibitor of PDE III. Besides, a direct action of sulmazole on contractile elements sensitizing them to Ca 2 ÷ was shown in cat hearts (van Meel et al. 1988) with an ICso-value of about 50 gmol/1. Serum levels at which the positive inotropic effects of sulmazole were reported in patients were 10 gmol/1 (Renard et al. 1983). Sulmazole had a positive inotropic effect in human myocardium at an ECso-value of 60 gmol/1. UD-CG 212.C1 showed positive inotropic effects already at an EC5o-value of 0.03 gmol/1. If we base our judgement on a comparison of these concentration ranges, adenosine antagonism might contribute in vivo to the cAMP-stimulation caused by sulmazole whereas it is presumably less important for the action of UD-CG 212.C1. Although sulmazole and UD-CG 212.C1 show

584

strong structural similarities, their mechanism of action might be quite different. The bipyridine milrinone was shown to be a less potent adenosine antagonist. Similar results were reported for rat myocardium (Earl et al. 1986). Its effect on mcholinoceptors was negligible. Phosphodiesterase III inhibition by milrinone in guinea pig myocardium was reported at ICso-values of 1.5 gmol/1 (Brunkhorst et al. 1989) and 45 gmol/l (Ahn ct al. 1986); thus it was observed at lower concentrations than adenosine antagonism. Besides, milrinone was reported to show inotropic effects in muscle strips in human N Y H A IV myocardium at an ICso-value of 386 gmol/1 and, if added to isoprenaline, at an 1C5o-value of 4 gmol/1 (B6hm et al. 1988). Therefore, it has to be assumed that - in contrast to the findings with sulmazole - PDE III-inhibition matches the inotropic and in vivo effects of milrinone rather than adenosine antagonism. However, Brunkhorst et al. (1989) found evidence that PDE III inhibition could not account as the only mechanism for the positive inotropic effect of milrinone in guinea pigs since its concentration-response curve in contraction experiments was biphasic showing 24% of the inotropic effect at an IC50value of 0.2 ~tmol/1 and a second phase ofinotropic effect at an ICs0-value of 686 gmol/1. Consequently, the second, low affinity phase of its action could depend on a mechanism different from PDE III-inhibition. Therefore, also in the case of milrinone a partial importance of Aa adenosine receptor antagonism cannot be completely ruled out. In order to explain the affinity of the new inotropic substances to purinergic and cholinergic receptors, Fig. 6 shows the molecular structures of the studied compounds listed according to their potency at An adenosine receptors. The structures of theophylline and atropine are given for comparison. Arrows symbolize decreasing affinity. All relevant molecular structures are shown in bold lines. Dashed lines drawn between the molecules further underline the homogeneities between the adenosine antagonist theophylline and the novel inotropic agents studied and between atropine and saterinone. As shown, saterinone and milrinone show a bipyridine-like structure. Sulmazole and UD-CG 212.C1 have structural similarities to methylxanthines. In comparison, the adenosine antagonist theophylline (in frames at left top) also displays a two-ring structure containing nitrogene common to all substances studied. Moreover, the central molecular chain of saterinone which contains a tertiary amine followed by three carbohydrates, an oxygene and another carbohydrate can be regulated to structural elements of the parasympatholytic drug atropine (right, at top). In summary, all new inotropes studied were shown to be A 1 adenosine receptor antagonists in human ventricular myocardium. Depending on their structural properties, this mechanism might be of variable importance compared to other intracellular actions such as phosphodiesterase III inhibition or Ca2+-sensitation. mCholinoceptor antagonism could also contribute to the action of saterinone. It is probably of minor or no importance for the action of the other compounds studied.

Acknowledgement. We thank Heidrun Fluch and Karl La Ros6e for their excellent technical help. We are indepted to all collegues of the Herzchirurgische Klinik der Universit/it M/inchen (Director: Prof. Dr. B. Reichart) for providing us with human myocardial samples.

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