Interactions between a “calcium channel agonist”, Bay K 8644, and calcium antagonists differentiate calcium antagonist subgroups in K+-depolarized smooth muscle

Naunyn-Schmiedeberg's Arch Pharmacol (1984) 328:69-75 Naunyn-Schrniedeberg's

Archivesof Pharmacology �9 Springer-Verlag 1984

Interactions between a "calcium channel agonist", Bay K 8644, and calcium antagonists differentiate calcium antagonist subgroups in K +- depolarized smooth muscle Michael Spedding and Christiane Berg Merrell Dow Research Institute, Strasbourg Center, 16 rue d'Ankara, F-67084 Strasbourg Cedex, France

Summary. 1. The proposal that calcium antagonists have different sites of action has been tested by attempting to reverse their inhibitory effects with a dihydropyridine which augments Ca 2+ entry into cells, Bay K 8644.

2. Bay K 8644 (1-1000 nmol/1) increased the sensitivity to Ca 2+ of K+-depolarized taenia preparations from the guinea-pig caecum. Thus Bay K 8644 augmented established submaximal Ca2+-induced contractions and also shifted cumulative concentration-response curves to Ca 2+ to the left, even in the presence of an optimal K +- depolarization.

3. The inhibitory effects of nifedipine (10nmol/1), verapamil (0.2 ~tmol/1), diltiazem (1 ~tmol/1) and diclofurime (1 ~tmol/1) on Ca 2+ - induced contractions were reversed by Bay K 8644 (1-100 nmol/1). In contrast, Bay K 8644 did not reverse the effects of cinnarizine (1 ~tmol/1), flunarizine (1 ~tmol/1), fendiline (3 ~tmol/1), prenylamine (3~tmol/1), pimozide (l~tmol/1), bepridil (3~tmol/1), perhexiline (10 ~tmol/1) or the calmodulin antagonist W-7 (200 ~tmol/1).

4. Bay K8644 (1-100nmol/1) was less effective at reversing the effects of nisoldipine (10 nmol/1), a slowly dissociating dihydropyridine, than the effects of nifedipine. However, preincubation with Bay K 8644 (1 ~tmol/1) protected the taenia from the inhibitory effects of nisoldipine (10 nmol/1). These findings are compatible with interactions of nisoldipine and Bay K 8644 at a common site.

5. Taenia preparations incubated with Bay K8644 (1 ~tmol/1) were protected from the inhibitory effects of nifedipine (10 nmol/1), nisoldipine (10 nmol/1) and to a lesser extent verapamil (0.2 ~tmol/1) and diltiazem (1 ~tmol/1). However, there was no protection against the inhibitory effects of cinnarizine (1 ~tmol/1), flunarizine (l~tmol/1), pimozide (1 ~tmol/1), fendiline (3~tmol/1), prenylamine (3 ~tmol/1) or W-7 (200 ~tmol/1). Thus, the lack of protection indicates that there is no interaction between the dihydropyridine site and the site for the latter drugs. These experiments show that Bay K 8644 defines subgroups of calcium antagonists which are the same as those previously reported from functional studies.

Key words: Dihydropyridines - Calcium - Calcium antagonists - Verapamil - Diltiazem

Send offprint requests to M. Spedding at the above address

Introduction

The diversity of the drugs classed as calcium antagonists is so great that this group may form the most chemically disparate class of therapeutic agents (Kazda et al. 1983). It is evident that a single molecular site could not accept such diverse compounds and therefore drugs inhibiting Ca ~+ slow channels may act at several site either in the channel or intracellularly. Several workers have proposed that different subclasses of calcium antagonist may exist, depending on their site of action (Ferry and Glossmann 1982; Glossmann et al. 1982) or on functional differences between the compounds (Fleckenstein 1981, Spedding 1981, 1982a, b; 1983a, b; 1984a and b; Rodeukirchen et al. 1982; Bou et al. 1983); the various subclassifications are listed in Table 1. Generally there appear to be three major classes of drugs: a) dihydropyridines which bind to a site in the channel, b) drugs such as diltiazem and verapamil which can positively or negatively regulate the dihydropyridine site by an allosteric mechanism and c) diphenylalkylamines which can be readily differentiated from the other agents in functional tests (Spedding 1984b).

However, a unitary model for calcium antagonists has recently been proposed, where the dihydropyridine binding site, associated with a selective cation binding site, is allosterically modified by a site which is common to verapamil, diltiazem and the diphenylalkylamines (Gould et al. 1983; Murphy et al. 1983). Thus in this model the drugs would only act at two sites and Murphy et al. (1983) demonstrated modulation of the inhibitory effects of diphenylalkylamines on dihydropyridine binding by diltiazem and by a verapamil derivative, D 600. However, these important experiments were performed in guinea-pig brain membranes: there is at present no functional correlate to this binding site and it cannot clearly be ascribed to the Ca ~+ channel (Triggle 1984).

Functional consequences of calcium antagonist interactions are not striking in smooth muscle (Spedding, 1983c), but the recent availability of a dihydropyridine which stimulates Ca 2+ entry via Ca 2+ channels and thus behaves as a "calcium channel agonist" (Bay K8644, Schramm et al. 1983 a and b) should allow more striking functional interactions. Bay K8644 has been shown to bind to the dihydropyridine binding site (Glossmann et al. 1983; Sarmiento et al. 1984) arid to reverse competitively the effects of the dihydropyridine calcium antagonist nife-

70

dipine (Schramm etat. 1983b; Schramm and Towart 1984).

This report describes reversal of the effects of some, but not all, calcium antagonists by Bay K 8644. The results support the hypothesis that there are distinct subgroups of calcium antagonists. K+-depolarized taenia preparations from the guinea-pig caecum were used as these preparations are very sensitive to Ca 2+ and competitive kinetics can be used to describe the interactions between antagonists and Ca 2+ (Riemer et al. 1974; Spedding 1982 a).

Methods

Strips of taenia ( 1 - 2 m m diameter, 1 .5-2cm relaxed length) from the caecum of male guinea-pigs (200-350 g) were set up in 10 ml isolated organ baths containing K +- Tyrode solution which was gassed with 95% 05:5% CO2 and maintained at 35 ~ The K+-Tyrode solution had the following composition (mmol/1): NaC1 97; KC140; NaHCO3 11.9; NaH2PO4 0.4; glucose 5.5. In some experiments the concentration of KC1 was changed with the appropriate change of NaC1 to keep the molarity constant. The preparations were washed in the Ca 2 +-free, 40 mmol/1 K+-Tyrode solution for 30 rain by which time they were fully relaxed. Contractile responses were measured under isotonic conditions (lg load) using Harvard or Bioscience isotonic transducers connected to Rikadenki potentio- metric recorders. Cumulative concentration-response curves to Ca 2+ (30-3000 ~tmol/1) were obtained by increasing the Ca 2+ concentration at 3-5 min intervals in logarithmic increments (Van Rossum 1963). When concentration-response curves were repeated the curves were obtained at 40 rain intervals. Antagonists were preincubated for 25 rain. Concentration ratios were calculated as the ratios of the concentrations of Ca 2+ which produced a 50% maximal response (EC50) in the presence and in the absence of the antagonists. The slopes were calculated from the concentrations producing 80% and 20% maximal

effects (Stephenson 1956). Student's t-test was used for comparisons of mean values and the Mann-Whitney U test for comparison of % changes. Concentration ratios were calculated, and tested for statistical differences, in logarithmic units and the antilog,arithms are quoted in the test. SEMs are illustrated on percentage changes for representational purposes only. All concentrations refer to final bath concentrations of drugs.

Drugs. The following drugs were used: Bay K 8644, nifedipine, nisoldipine (Bayer AG, Leverkusen, FRG), cinnarizine tartrate, flunarizine hydrochloride, pimozide tartrate (Janssen, Dtisseldorf FRG), bepridil (Organon, France), diltiazem hydrochloride (Synthelabo, France), fendiline hydrochloride (Dr. Thiemann GmbH), prenylamine (Segontin Hoechst AG), perhexiline hydrochloride (Men'ell Dow, USA), (__)-verapamil hydrochloride (Knoll AG, Ludwigshafen, FRG) N-(6-aminohexyl)-5-chloro- 1-naphthalenesulphon- amide (W-7) (Synthelabo, France). Dihydropyridines were dissolved in ethanol (1 retool/l), prior to diluting in distilled water. Stock solutions were kept at 0 ~ for up to 12 h and aqueous dilutions prepared immediately prior to use. All solutions were shielded from light. The other drugs were dissolved in distilled water.

Results

Effects of Bay K 8644

K+-depolarized taenia preparations contracted in response to Ca 2+ (0.1-3 mmol/1) and submaximal contractions were sustained for > 4 h. At maximum contraction the tissues shortened to 20-30% of their relaxed length in Ca2+-free solution. Bay K 8644 (1-1000 nmol/1) did not contract the tissues in Ca2+-free medium but increased the sensitivity of the preparations to Ca 2+ (Fig. 1). Bay K8644 augmented established submaximal Ca2+-induced contractions in a concentration-dependent fashion with the threshold concentration being 1 nmol/1 and maximal effect at 100nmol/1 (Fig. 2). Cumulative concentration-response

8

B

A C

Fig. 1. Contractions induced by Ca ~+ in a K + (40mmol/1)- depolarized taenia preparation from the guinea-pig caecum. Contractions induced by Ca 2+ (100 ~nol/1) before and in the presence of Bay K 8644 (1 gmol/1) are shown by curves A and B respectively. The effect of Ca ~+ (30 ~tmol/1) in the presence of Bay K8644 (1 Nnol/l) is also shown (C); this concentration of Ca 2+ did not contract the taenia in the absence of Bay K 8644. The vertical calibration represents the maximal response of the taenia to Ca 2+ (3 mmol/l)

z _o

I-- Z

' Ld

[00

5 0

I I 0 0 0 I0 I O0

B e y K 8 6 4 4 , nm01 /

Fig. 2. Augmentation by Bay K 8644 of estabhshed submaximal contractions to Ca 2+ of K + (40 mmol/1)-depolarized taenia preparations. 1he contractions induced by Ca 2+ (t00 ~tmol/1) were 40-60% of the maximal response of the tissue; percentage increases are calculated taking the maximal contraction as 100%. Note that Bay K 8644 increased the sensitivity of the tissues to Ca 2+ in a concentration-dependent fashion. Vertical bars represent SEM, n = 5-6

Table 1. Literature classifications of calcium antagonists

Di- Vera- Dil- hydro- pamil tiazem pyridines

Diphenyl- alkyl- ammes

Fleckenstein (1981) I I I II Glossmann et al (1982) IA II III IB Rodenkirchen et al (1982) II I I III a Murphy et al (1983) I II II II Spedding (1981, 1982) I II II III

a fendiline classed with verapamil, prenylamine with dihydropyridines

Table 2. Effects of increasing K+-depolarization on the sensitivity of taenia preparations from the guinea-pig caecum to Ca z+, in the absence or presence of Bay K 8644, 1 btmol/1

K + concentration EC 50 for Ca 2 + retool/1 btmol/1 (range SEM)

Control Bay K 8644 n

10 376 (271- 523) 197 (161-241)* 4 20 237(153- 369) 87 (74-102)* 4 40 126 (108- 145) 57 (51-66)** 21 70 535 (282-1,023) 49 (36- 67)** 4

137 286 (234- 349) N.T. 12

EC 50 values were calculated from cumulative concentration- response curves to Ca 2+. An initial cumulative concentration- response to Ca 2+ was obtained in 40 mmol/1 K + and the K + concentration changed to the concentration indicated (four changes of bath solution, 30 min incubation). In some of the preparations Bay K 8644 (1 ~tmol/1) was added for the last 25 min of the incubation period, *p<0.05, **p<0.001. The maximum effect was not reduced during these experiments

curves to Ca 2+ were displaced to the left by Bay K 8644 (1 btmol/1), the converse of the effects in this preparation of nifedipine, a dihydropyridine calcium antagonist (Spedding 1982a). This displacement occurred at all K + concentrations tested (Table 2). The smallest effect occurred with 10 m M K +, where the preparations would not be depolarized sufficiently to fully activate the Ca ~+ channels ( Inomato and Kao 1976).

Reversal experiments

Incubation of taenia preparations for 20 min with nifedipine (10 nmol/1) reduced the sensitivity to Ca 2+ so that Ca ~+ (3retool/I) produced only a small contraction (Fig. 3). Cumulative addition of Bay K8644 (1-100 nmol/1; Fig. 3) reversed the inhibitory effects of nifedipine.

The ability of Bay K 8644 to reverse the effects of a range of calcium antagonists is shown in Fig. 4. Bay K 8644 reversed the effects o f nifedipine, verapamil, diltiazem and diclofurime. Bay K 8644 (1 nmol/1) was more effective in reversing the effects of diltiazem (1 ~tmol/1) th.an those of the other antagonists (Fig. 4, p < 0.05). In contrast Bay K 8644 did not reverse the effects of cinnarizine (1 ~tmol/1), flunarizine (1 ~tmol/1), prenylamine

71

NIFEDIPINE 10 nM ~ o 100 nM

BAY K 8 6 4 4

./,o 1 0 I l a i n

A

Fig. 3. Reversal by Bay K 8644 (1-100 nmol/1) of the inhibitory effects of nifedipine (10 nmol/1, preincubated for 25 min) on a contraction induced by Ca 2+ (A, 3 mmol/1) in a K + (40mM)- depolarized taenia preparation. The vertical bar represents the isotonic contraction of the taenia to Ca 2+ (3 mmol/1) prior to exposure to nifedipine

/•l? VERAPAMIL

,oo F~, DILTIAZEM

n -

/ / . . " , ~ q.CINNARIZ]NE

o L I I

I IQ IQQ

Bey K 8 6 4 4 , n m o l / I

Fig. 4. Reversal by Bay K 8644 of the inhibitory effects of calcium antagonists in K+-depolarized taenia preparations. The experiments were performed as in Fig. 3 and the antagonists preincubated in a Ca2+-free media for 25 min prior to addition of Ca 2+ (3retool/i). When the contraction had stabilized, Bay K8644 (1-100nmol/1) was added cumulatively. The calcium antagonists were: e - o nifedipine 10nmol/1, n=5; v - , , verapamil 0.2 btmol/1, n=5; []-D diltfazem 1 btmol/l, n=6; A-A diclofurime 1 ~tmol/1, n=4; ~ - ~ nisoldipine 10nmol/1, n=5; A-A pimozide 3 ~tmol/1, n=7; v - v cinnarizine 1 ~mol/1, n=6; o - o flunarizine 1 ~mol/1, n=5; []-~ bepridil 3 ~tmol/1, n---5. In additional experiments (not shown), the effects of fendiline (3~tmol/1, n=4), prenylamine (3~tmol/l, n=5), perhexiline (10 gmol/1, n=4) and W-7 (200 ~mol/1, n =7) were not reversed by Bay K8644 (1-100 nmol/1). "A" indicates that the reversal of the effects of diltiazem by Bay K 8644 (1 nmol/1) was significantly (p < 0.05) greater than for the other antagonists

(3 pmol/1), fendiline (3 gmol/1), perhexiline (10 ~tmol/1), bepridil (3 Nnol/1) or the calmodulin antagonist W-7 (200 ~tmol/1). The effects of pimozide (3 ~tmol/1) were only reversed by Bay K8644 (100 nmol/1) in three of seven experiments (Fig. 4). However, the effects of nisoldipine (10 nmol/1), a dihydropyridine calcium antagonist which dissociates slowly from calcium channels (Kazda and

72

~100 I.-- (J IZ F- Z 0 50 L)

X <Z =E

o

�9 /

oJ/ I I I I I I I

0.1 I I0

Ca~ (mmol / I )

Fig. 5. Protection of the taenia from the inhibitory effects of nifedipine (10 nmol/1) by Bay K 8644 (1 ~tmol/1). Initial control curves (n = 10) to cumulative addition of Ca 2+ are represented by e - e . Nifedipine (10nmol/1 for 25 rain, �9169 n=5) shifted the curves to the right. Bay K 8644 (1 ~tmol/1 for 25 min, A-A, n=5) increased the sensitivity of the preparations to Ca 2§ shifting the curve to the left. Subsequent addition of nifedipine (10 nmol/1 for 25 min, zx-zx, n= 5) did not cause antagonistic effects, in the presence of Bay K 8644 (1 ~tmol/1). Vertical bars represent SEM

CINNARIZINE

~ / O B a y K 8644

._o Control r f

== O

- log mol/I ANTAGONIST

Fig. 6. Arunlakshana and Schild (1959) plot of the inhibitory effects of cinnarizine in the presence (o -o ) or absence ( e - e ) of Bay K 8644, 1 gmol/1. Cinnarizine was preincubated, in increasing concentrations, for 25 rain prior to cumulative addition of Ca 2+ (0.1-30 mmol/1). Vcrtical bars represent SEM, n = 5-6.

Towart 1982), were only reversed by high concentrations of Bay K 8644 (30-100 nmol/1). The failure to reverse the inhibitory effects of the diphenylalkylamines might therefore have been due to very slow dissociation of these agents from their site of action (Spedding 1982 a). In order to exclude this possibility, Bay K8644 (1 ~mol/1) was preincubated prior to exposure to the antagonists.

Protection experiments

Preincubation with calcium antagonists shifts Ca ~+ cumulative concentration response curves to the fight in parallel, in the taenia (Spedding 1982a). Thus nifedipine (10 nmol/1; Fig. 5), nisoldipine (10 nmol/1), verapamil (0.2 ~tmol/1), diltiazem (1 ~tmol/1), cinnarizine (1 ~tmol/1) and W-7 (200gmol/1) markedly antagonized Ca2+-in - duced contractions (Table 3). Bay K8644 (1 ~mol/1) in contrast shifted the Ca 2+ concentration-response curves to the left (Fig. 5). The shift was maximal and thus Bay K8644 (1 ~tmol/1) presumably interacted with all the available channels. Pretreatment with this maximal concentration of Bay K 8644 completely protected the taenia preparations from the inhibitory effects of nifedipine (10nmol/1; Fig. 5) and nisoldipine (10nmol/1) and markedly reduced the inhibitory effects of verapamil (0.2 ~mol/1) and diltiazem (1 gmol/1) (Table 3). In contrast, the inhibitory effects ofcinnarizine (1 gmol/1), flunarizine (1 ~mol/1), pimozide (3 ~mol/1), fendiline (3 ~tmol/1), prenylamine (3 ~zmol/1) and W-7 (200 gmol/1) were not attenuated by Bay K 8644 (1 gmol/1) (Table 3); the augmented Ca 2+ concentration ratios seen in some of these exeriments reflect the enhanced initial sensitivity of the preparations treated with Bay K 8644, because the EC50s in the presence of the antagonists and Bay K 8644 were not greater than the EC50s in the presence of the antagonists alone (Table 3).

The effects of cinnarizine were not reduced by Bay K 8644 even over a wide range of cinnarizine concentrations and the pA2 value for cinnarizine was not modified by the presence of Bay K 8644 (Fig. 6). Thus the concen-

tration of calcium antagonist was not critical, as regards the ability of Bay K 8644 to reverse its inhibitory effects.

Discussion

The K+-depolarized taenia preparation from the guinea- pig caecum is the most sensitive smooth muscle preparation to exogenous Ca ~+ yet described (Spedding 1982a), but Bay K 8644 further increased this sensitivity, shifting concentration-response curves to Ca 2+ to the left, in parallel. Thus in the presence of Bay K 8644, concentrations of Ca 2+ as low as 30 9mol/1 produced large contractions. Such sensitivity approaches that of skinned smooth muscle (Sparrow etal. 1981; Spedding 1983a). Bay K 8644 reduces the stimulation threshold for depolarizing responses in the taenia (Godfraind etal. 1984), but its mode of action on Ca 2+ channels must be different from that of depolarization because the effects of the compound were imposed on sensitivity which was already optimal for K+-depolarization. These findings are consistent with a site of action of Bay K8644 on the dihydropyridine binding site of the Ca 2+ channel to increase Ca 2+ entry through activated channels (Schramm et al. 1983 a and b). Nevertheless, the marked increase in sensitivity to Ca 2+ in the presence of Bay K 8644 leads to the conclusion that Ca 2+ channels are normally inhibited, even in this sensitive preparation.

A site of action of Bay K 8644 on the dihydropyridine binding site would also be indicated by interactions with the structurally related dihydropyridine, nifedipine. Thus Bay K 8644 both reversed the inhibitory effects of nifedipine and prevented their onset; the data are compatible with a direct interaction between the compounds, as has previously been suggested (Glossmann et al. 1983; Schramm et al. 1983 a and b; Albus et al. 1984; Towart and Schramm 1984; Sarmiento et al. 1984).

In dihydropyridine binding studies verapamil and diltiazem have been shown to allosterically modify (3H) nitrendipine or (3H)nimodipine binding but with opposite

73

Table 3. Effects of preincubation with Bay K 8644 (1 gmol/1) on the inhibitory effects of calcium antagonists in K+-depolarized taenia preparations from the guinea-pig caecum. The experimental protocol was as in Fig. 5. Ca 2 + EC50s in the presence or absence of antagonist are expressed in Nnol/1 (range SEM). The Ca 2+ concentration ratio (C.R.) represents the EC50 ratio in the presence and absence of antagonist (range SEM)

Control BAY K 8644

Initial Antagonist Ca 2 + C.R. Initial Antagonist Ca 2 + C.R EC50 EC50 EC50 EC50

Nifedipine 106 8,028 76 50 74 1.7 * 10 nmol/1 (68-123) (6,325-10,170) (57-100) (38-63) (59-95) (1.4-2.0) Nisoldipine 268 > 30,000 > 100 59 138 2.3* 10 nmol/1 (156-464) (39-90) (61-294) (1.3-4.0) Verapamil 184 4,169 23 49 179 3.6* 0.2 ~tmol/l (138-248) (2,984-5,808) (16-32) (41-58) (152-212) (2.7-4.8) Diltiazem 216 6,710 31 48 460 9.2* 1 ~tmol/1 (143-323) (475-9,484) (23-42) (39-60) (305-696) (6.4-13.5) Cinnarizine 119 1,520 13 74 2,812 38" 1 ~mol/l (101-138) (1,072-2,150) (8-18) (54-101) (1,600-4,510) (22-67) Flunarizine 103 1,126 11 43 352 8.2 1 ~tmol/1 (59-184) (760-1,678) (7-18) (35-52) (190-675) (5.1-15) W-7 194 5,535 36 69 8,318 94 200 ~tmol/1 (157-241) (3,356-9,136) (19-66) (62-78) (4,148-14,450) (58-153) Pimozide 115 5,500 46 74 5,418 69 3 ~mol/l (82-162) (3,805-7,930) (23-88) (65-82) (2,489-11,178) (34-141) Fendiline 111 3,860 43 64 4,303 66 3 ~xmol/1 (88-140) (2,939-5,064) (31-59) (44-91) (3,515-5,269) (47-93) Prenylamine 167 8,320 50 110 4,235 38 3 ~tmol/1 (104-272) (6,738-10,290) (37-67) (79-144) (1,823-9,750) (17-85)

* p < 0.05 compound with control Ca 2+ concentration ratio

effects, verapamil reducing binding (Ehlert etal. 1982; Ferry and Glossmann 1982; Marangos et al. 1982; Wil- liams and Tremble 1982; Bellemann et al. 1983; Gould et al. 1983; Murphy et al. 1983) and diltiazem enhancing it (DePover et al. 1982; Ferry and Glossmann 1982; Yama- mura et al. 1982). The present functional study shows that the converse can take place and that a dihydropyfidine can reduce the effectiveness, and presumably the binding, of verapamil. Bay K 8644 reversed the inhibitory effects of verapamil and diltiazem and prevented their onset. Again, these findings extend the work of Schramm et al. (1983 b), who reported non-competitive interactions between Bay K 8644 and verapamil and diltiazem. In the present work verapamil and diltiazem produced greater antagonistic effects than nifedipine in the presence of Bay K 8644 (Table 3) and further kinetic studies indicate that this is due to a complex non-competitive interaction whereas the interaction of Bay K 8644 and nifedipine is apparently competitive (Spedding, in preparation). The only other drug to be reversed by Bay K 8644 was diclofurime which resembles verapamil and diltiazem in a variety of functional tests (Gittos and Spedding 1984).

Verapamil and diltiazem seemed similar even though they have opposite effects on dihydropyridine binding; this presumably reflects the fact that the drugs must block calcium channels in order to influence dihydropyridine binding and hence the functional consequences of such interactions will be small (Spedding 1983c; 1984b; Humphrey and Robertson 1983). Nevertheless the finding that Bay K 8644 (1 nmol/1) was more effective in reversing

the effects of diltiazem than equivalent responses to the other antagonists (Fig. 6) is probably a functional correlate of the increased dihydropyfidine binding seen in the presence of dilfiazem (DePover etal. 1982; Ferry and Glossmann 1982; Yamamura etal. 1982). Thus these functional studies can be considered to be in broad agreement with some of the binding studies (Glossmann et al. 1982), but indicate that the allosteric effects between the site(s) for verapamil and diltiazem and the dihydropyridines are bidirectional and that the dihydropyfidines can reverse the effects, and presumably the binding, of verapamil and dilfiazem.

The present findings are difficult to reconcile with the preliminary report of Reynolds et al. (1983), who did not observe changes in (3H)verapamil binding to guinea-pig cerebral cortex membranes after occupation of the dihydropyridine binding site. However, the reversal of the effects of verapamil by Bay K 8644 in the taenia was presumably a direct effect, because it could not be due to increased Ca a+ entry displacing intracellular verapamil (Reynolds et al. 1983) since the concentration ratio technique uses equivalent contractility states, and hence presumably equivalent Ca 2+ entry, to define antagonistic potency.

In complete contrast to the above effects Bay K 8644 did not reverse the inhibitory effects of cinnarizine, flunarizine, fendiline, prenylamine, pimozide, perhexiline, bepridil or the calmodulin inhibitor (Karaki etal. 1982) W-7. The failure of Bay K8644 to reverse these drugs did not appear to be due to kinetic differences

74

between these agents and nifedipine, because Bay K 8644 did not protect the taenia from the inhibitory effects of these drugs even when pre-incubated in very high concentrations. Kinetic differences explain the failure of Bay K 8644 to reverse the inhibitory effects of nisoldipine. Nisoldipine dissociates very slowly from its site of action (over 10 h; Kazda and Towart 1982) and thus there are presumably few dihydropyridine sites available for Bay K 8644 to interact with. In contrast, when Bay K 8644 is ,preincubated with the tissue prior to addition of nisoldipine it can protect the taenia from the antagonistic effects of nisoldipine because both drugs presumably interact with the dihydropyridine site. Thus the failure of Bay K 8644 to protect the taenia from the inhibitory effects of cinnarizine and the other diphenylalkylamines means that the site of action of these drugs is independent of the dihydropyridine site, and consequently also independent of the site(s) for verapamil and diltiazem, the latter site(s) being allosterically linked to the dihydropyridine site.

These experiments can be considered as confirmatory evidence that calcium antagonist subgroups exist. The drugs which were not reversed by Bay K 8644 form a distinct subgroup of calcium antagonists: all the drugs are very lipophilic and most have a diphenylalkylamine moiety (Spedding 1984b). Their effects can be readily distinguished from the dihydropyridines, verapamil and diltiazem in that they are much less selective for calcium channels (Fleckenstein 1981; Bayer etal. 1982; Roden- kirchen etal. 1982; Anno etal. 1984), have time- and calcium-dependent effects in smooth muscle (Spedding 1982a; 1984b; Wadsworth and Moss 1983) and, like calmodulin inhibitors, can interact with the contractile proteins in high concentrations (Spedding 1983 a). Bepri- dil, flunarizine (Lugnier et al. 1984), pimozide (Levin and Weiss 1979), fendiline and prenylamine (Johnson and Fugman 1983) bind directly to calmodulin in concentrations similar to, or only slightly higher than, those used in this study. Furthermore, changing surface charge with salicylate increases the effectiveness of this group of antagonists in the taenia whereas the effectiveness of verapamil and diltiazem is reduced and that of nifedipine unchanged (Spedding 1983b and 1984a). There is thus considerable evidence that these very lipophilic calcium antagonists behave differently from the dihydropyridines, and from verapamil and diltiazem.

The results from these functional experiments cannot be reconciled with a unitary mechanism of action for calcium antagonists, proposed by Gould et al. (1983) and Murphy et al. (1983). These workers claim that verapamil, diltiazem and the diphenylalkylamines interact at a common site which modifies the dihydropyridine binding site. The functional consequences of this model would be that cinnarizine should resemble verapamil. Why is this not the case? Cinnarizine and the diphenylalkylamines at the relevant pharmacological concentrations have certainly been shown to reduce dihydropyridine binding in membranes from all tissues studied, including smooth muscle (Bolger et al. 1983). However, the relevance of this phe- nomenon is questionable, because this effect is directly paralleled by the ability to bind to calmodulin (Sarmiento et al. 1983), which in turn parallels non-specific mem- branal interactions (Landry et al. 1981). Thus the effect of these lipophilic drugs on (3H)nitrendipine binding most likely reflects non-specific effects (see Spedding 1984b).

The results with Bay K 8644 in the present study indicate that the effects of cinnarizine would be independent of whether the dihydropyridine site was occupied or not. An effect of cinnarizine on events subsequent to Ca 2+ entry, similar to those demonstrated for W-7 (Karaki et al. 1982), cannot be ruled out.

In conclusion, differential reversal of the effects of calcium antagonists by Bay K 8644 confirm the proposal that the subgroups of calcium antagonists act at different sites (Spedding 1981 and 1982a; 1983 a and c; 1984a and b). Bay K 8644 reverses the effects of dihydropyridines such as nifedipine competitively (Schramm and Towart 1984), whereas the interaction of Bay K 8644 with verapamil and diltiazem is non-competitive (Schramm and Towart, 1984). Bay K 8644 does not reverse the effects of the lipophilic diphenylalkylamines. Actions at different sites would account for the disparate profiles of the subgroups in in vivo experimental models (Spedding 1982 b; Hof 1984) and in man (Opie 1980).

Acknowledgements. We are very grateful to Dr. M. Schramm (Bayer AG) for the gift of Bay K 8644 and to Janssen Pharma- ceutica, Synthelabo, Hoechst, Dr. Thiemann GmbH for gifts of drugs.

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Received May 7, 1984/Accepted July 24, 1984

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Interactions between a “calcium channel agonist”, Bay K 8644, and calcium antagonists differentiate calcium antagonist subgroups in K+-depolarized smooth muscle