Studies on mechanisms of calcium channel modulators

J Mol Cell Cardiol 19, (Supplement II) 49-62 (1987) STUDIES ON MECHANISMS OF CALCIUM CHANNEL MODULATORS ARNOLDSCHWARTZ

Department of Pharmacologyand Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575 USA INTRODUCTION The extensive studies of Albrecht Fleckenstein and his colleagues, beginning with verapamil and D-600, in 1964, culminated in the establishment by Fleckenstein of a new therapeutic modality he termed CALCIUM ANTAGONISM [15,16].

The organic calcium

antagonists, also referred to as calcium channel inhibitors, are currently enjoying unprecedented popularity in the treatment of coronary artery disease. These drugs, however, appear to have perhaps the widest spectrum of potential beneficial c l i n i c a l effects than any other type of drug. Someof the areas in which these drugs are being used or investigated include coronary artery disease, hypertension, peripheral vascular disease, arrhythmias, migraine, cerebral ischemia, esophogealspasm, asthma, atherosclerosis, epilepsy, ischemic heart disease, cardiomyopathy,and myocardial infarction.

While the calcium antagonists have been referred to as a class of drugs,

i t is clear that there are perhaps three, even four, distinct types or subtypes. In fact, a recent World Health Organization Committeehas decided to subdivide the antagonists into at least three distinct classess DIHYDROPYRIDINEor N type, PHENYLALKYLAMINE or V type, and BENZOTHIAZEPINE or D type.

The N, V, and D refer to

nifedipine, verapamil, and diltiazem, respectively (Table !).

Someof the chemical

structures and space-filling models (Fig. 1 and 2) reveal three completely different organic molecules, which strongly suggest the existence of multiple receptors. TABLE i.

These

Pharmacological and Structural Classification of Ca Channel Modulators I.

1,4-DIHYDROPYRIDINES A. ANTAGONISTS (+)Bay k 8644 (-)202-791 Nitrendipine Nifedipine PN200-110

II. PHENYLALKYLAMINES Verapamil Desmethoxyverapmil (D-888) D-600 I I I . BENZOTHIAZEPINES Diltiazem TA 3090

B. AGONISTS (-)Bay k 8644 (+)202-791 CGP 28392 YC-170

0022-2828/87/$20049 + 14 $03.00/0

9 1987 Academic Press Inc. (London) Limited

50

A. S c h w a r t z

DILTIAZEM S

.~

NIFEDIPINE

OCH3

OC~o~CC'CH3 [~[~!H2_CH2_N/ CH3 ~CH3

NO2

H3COOC- ~ COOCH3 H3C-~N..~- CH3 / VERAPAMIL

H3C\/CH3

CH CH3 I I CH30~"~'~'-G-CH2-CH2-CH2-N-CH2-CH2--f~OCH3 CH30~ ] C--N ~ ' - OCH3 Figure I

Figure 2 receptors, of course, could be located on a single, large protein.

In fact, despite

the heterogeneity, and we probably should include bepridil, (1-N-benzylanilino-2pyrolidino-3-isobutoxypropane) as a potential fourth type ~6] of these drugs, they do share some important pharmacological effects [13,15,18,26,28,29,40,42,5~, chiefly, vasodilation, a negative inotropic action, and in the case of some, inhibition of excitation of sinoatrial and atrioventricular nodes [29].

In recent years, a

Calcium ChannelModulators

51

large number of laboratory animal studies have provided support for the exciting possibility that some calcium antagonists may successfully retard or prevent myocardial ischemia [39]. In a very recent multi-institutional clinical study, Roberts and his colleagues have clearly demonstrated that diltiazem in a daily dose of 360 mg significantly prevented the development of reinfarction in patients who had suffered non-Q-wave infarction [33,19]. Withinthepast two years, a most interesting aspect of the calcium antagonist "field" has emerged, ViZo, the existence of enantiomers of the dJhydropyridines that have the opposite effects of the antagonists, and hence are referred to as calcium channel agonists [14,38,45-4~.

Further, there is evidence that even those drugs

known as calcium antagonists exhibit dual effects, i.e., agonism and antagonism, and hence may in fact be termed "partial agonists '~ ~0].

We prefer, therefore, to refer

to the organic calcium channel drugs as "modulators." Our approach to the mechanism(s) of action of these important agents is programmatic in that it includes pharmacology of cardiac and vascular smooth muscle, electrophysiology (skeletal and cardiac), biochemistry and molecular biology. This review is not intended to be comprehensive, but rather a summary of the important and interesting findings and problems. THE CALCIUM CHANNEL It is thought that a voltage-dependent calcium channel, similar to the description for the sodium channel, exists in three "states": resting, open, and inactivated.

A highly schematic depiction of such a channel with its "gates" is shown

in Figure 3 and represents a compilation of electrophysiological studies from a number of groups and is based upon the Hille-Katzung modulated receptor concept ~,3~.

An alternative concept for calcium channel gating characteristics, based

upon the action of dihydropyridine calcium agonists and antagonists, favors the existence of different "modes" ~3,24].

The calcium antagonist, or "partial

a g o n i s t , " would i n h i b i t the entry of calcium by promoting mode 0 of the channels. Whether or not the "mode concept" or " i n a c t i v a t e d hypothesis" i s h e l p f u l in e x p l a i n i n g the behavior of the calcium channel modulators, i t i s apparent that the channel p r o t e i n ( s ) are probably of large molecular weight and subject to conformational changes, which are associated with channel a c t i v i t y and might confer speci-

A. S c h w a r t z

$2

(R) resting

(0) activated

([) inactivated |

Figure 5 ficity of drug action.

We will not understand the molecular mechanisms for channel

activity or drug action until the protein(s) is/are purified and reconstituted.

To

compound the problems of isolation, the density of calcium channels in heart is low compared to other channels and pumps. The calcium channel, however, is probably efficient, such that one calcium channel per square micron of membrane admits about 400 calcium ions in one millisecond.

Furthermore, it is apparent that multiple chan-

nel types (isochannels) exist; in fact, at least two or three voltage-dependent calcium channels have been described, one "long-lasting" and the other "transient" with the former being sensitive and the latterbeing insensitive to organic calcium channel modulators.

These have been found in heart [4,3~, smooth [17,54], skeletal

muscle [6], and neuronal tissue [P,51]. CARDIAC PHARMACOLOGY We and others have reported a number of complex actions of the calcium modulators. Stated simply, these drugs exert dual and concentration-dependent positive and negative inotropic effects. Incidentally, this is also true at least for pig coronary artery rings. A summary of these actions are shown in Table 2 and Figure 4.

C a l c i u m Channel Modulators TABLE 2.

53

Positive and Negative Inotropic Effects of Bay k 8644

Rat Langendorff Atria Ventricles Guinea pig atria Ventricles Dog atria Ventricles Purkinje

Positive inotropie effect Control to maximum

Negative inotropic effect. Maximum to control

N

ICso (~M)

ED50 (nM)

7 5 13 6 6 8 8 2

35 • 74 • 91.6t 104 t 120 • 30.5t 29.5t 67.5

15 45 21.7 28 34 9.7 8.0

6.8• 12.4• 9.9• 4 t 1.7 6.6• 3.71• 1.48 4.65• 2.31 7.5

200

% E

Bay K 8644

150

100

~6 50

10 9

10-8

10 7

10-6

10-5

[M] OihydropyFidine

Figure 4. Concentration-dependent effect of Bay k 8644 (closed circles) and nimodipine (open circles) on contractility (dP/dt) of isolated, retrogradely perfused rat heart preparations. The drugs were added in cumulative manner. Eash point represents the mean response • S.E.M. of 7 or 8 ex4~eriments. [Taken from A. Schwartz, et al. BBRC 125:387-394 (1984).J These effects are not due to "calcium overload," in the case of the agonists, since addition of calcium restores rather than deteriorates contractility [50].

We do not

know whether this is due to multiple receptor sites or multiple "states" of a single protein or site. With respect to the interesting discrepancy between "low affinity" pharmacology of the dihydropyridine antagonists on intact cardiac preparations, and "high affinity" binding of these drugs to isolated membrane preparations (150 - 600 nM vs. KD - O.1 to 0.3 nM), a reasonable explanation has been put forth by Sanguinetti and Kass [36] and by Bean [ 3 ] .

The l a t t e r author, using i s o l a t e d dog v e n t r i c u l a r myo-

54

A. S c h w a r t z

cytes, found an apparent affinity for nitrendipine of 6.36 nM when the cells were held at a depolarizing voltage (-20 mV) and about 750 nM when polarized (-80 mV). These values are very close to our pharmacological and binding data, using the same tissue.

In a combined binding, pharmacological, and electrophysiological single cell

study ~3], using an optically pure dihydropyridine enantiomer, (-)R202-791, we found similar results to the above that are consistent with the concept of "state" or conformation-dependent affinity of the dihydropyridine calcium antagonists dependent upon the membrane potential.

Thus, we found a K i for (-)R202-791 on

[3H]-nitrendipine binding of 1.6 nM consistent with an inhibition of peak inward calcium current at an IC50 of i nM when the cell was held at -30 mV, and an IC50 of 200 nM at -90 mV.

The ICso for (-)R202-791 on inhibition of contractile force of the

same tissue, however, was 320 nM.

In contrast, the agonist enantiomer, (§

produced a positive inotropic effect with an EDso of i17 nM, which is in the same range as inhibition of [3FO-nitrendipine binding (Ki=230 nM) and augmentation of peak inward calcium current (EDso=80 nM~ less dependent upon membrane potential than the antagonist).

In other words, the "discrepancy" between pharmacology and binding that

exists with the antagonist does not appear to be the case with the agonist.

To

further complicate the issue, neither diltiazem nor the phenylalkylamines (verapamil, desmethoxyverapamil (0888), or D-600), exhibit such discrepancies, yet these drugs, like the dihydropyridines, are state and use-dependent [unpublished data~ 49]. we dealing with multiple "receptor" sites?

Are

Is the putative calcium channel protein

highly complex with multiple membrane-potential conformational states?

Again, we

won't unravel this confusion until the structure of the channel is known, reconstituted and re-studied with these and other drugs. Another curious effect of the calcium antagonists is the pharmacological modification of dihydropyridine effects by diltiazem.

We have shown that pretreatment of

heart tissue with very low concentrations of d-cis-diltiazem (not the less active lcis-isomer) potentiates the negative inotropy and possibly coronary blood flow produced by nifedipine, nitrendipine (unpublished data) and nimodipine [9]. This synergism appears to be consistent with the acute "up-regulation" of dihydropyridine receptors by d-cis-diltiazem in both heart and coronary artery membranes [i0]. Preliminary clinical information regarding "combination therapy" with diltiazem and

C a l c i u m Channel Modulators

55

nifedipine is consistent with these experimental findings (Drs. William Frishman, Richard Conti, and Nyron Weisfeldt, personal communications). VASCULAR SMOOTH MUSCLE AND POSSI~E I~NOLVEHENT OF CALCIUM CHANNELS IN THE ENDOTHELIUN In our studies, we have employed pig coronary rings because of stability, ease of acquisition with minimal cost, and their presumed similarity to the human coronary vasculature. The presence of a threshold concentration of KCI (15 mM) is needed in most studies using isolated rat and rabbit aorta to demonstrate contractile effects [44,55], but there is no need for "partial depolarization" in pig coronary arteries Ill,12]. This could be due to a difference in the resting membrane potential of different blood vessels, with the isolated pig coronaryring perhaps being already partially depolarized, although we do not know for certain.

We observed a very complex

interaction between the activator and inhibitor 1,4-dihydropyrldines in our pig coronary artery ring preparations [ll,12~. In thissystem, pretreatmentwith calcium channel inhibitors resulted in a paradoxical potentiation of the subsequent activator-induced contraction (Figure 5).

The mechanism of this potentiation may

10 10-8M(-)202-791 Pretre~ (1) I--

r

(.9

T 0

J

10 -8

~

Control I

I

10-7

10-6

A

10 -5

Mer)202-7g1 5.

Figure Effect of (-)202-791 pretreatment on (+)202-79l doseresponse curves in tissues with intact endothelium. Coronary artery Lest rings were pretreated for 60 minutes with tO nM (-)202-791. At the same time point, paired control rings in a separate bath received the corresponding volume of solvent. Following this pretreatment period, the resting tensions which had been altered by the vasodilator or the solvent were restored to the original 5g baseline tension by mechanical adjustment of the rings. (+)202-791 was then added to both the test and control rings in a cumulative fashion. Each point represents the mean response • SEN for 4-6 rings.

56

A. Schwartz

involve multiple binding sites for dihydropyridine calcium channel modulators [44,50]. A recently discovered effect of the calcium channel activators on endothelium cells possibly explains the potentiation phenomenon.

We have shown that calcium

channel activators, such as (-)Bay k 8644 and (+)$202-791, stimulate the release of endothelium-derived relaxing factor (EDRF) from dog femoral arteries [35]. The EDRF release was inhibited by nitrendipine and by (-)R202-791 which strongly suggests that a "dihydropyridine

receptor" and/or a potential-dependent calcium channel may exist

in endothelial cells.

We speculate that Ca2+ entering through these putative

agonist-stimulated endothelial calcium channels somehow triggers the release of EDRF which in turn induces relaxation of vascular smooth muscle.

If this is the case,

then the response to a calcium channel activator would be a balance between its action on vascular smooth muscle and endothelial cells. The overall contractile effect would thus be smaller than when the drug was acting on smooth muscle denuded of endothelium.

Our experimental data show that thecontractile response to

(+)$202-791 is indeed higher in coronary arteries without endothelium than with endothelium (Figure 6). "potentiates"

This suggests that the mechanical removal of endothelium

the contractile effect of (+)$202-791 in pig coronary arteries in a

manner similar to that when tissues are pretreated with a calcium channel inhibitor. 10

8

W i t h o u ~

.-

~

4

2

0

I

10-8

I

I

10-7 10-6 M (+)202-791

i

10-5

Figure 6. Dose-response curves to (+)202-791 in tissues with and without endothelium. The agonist was added to the musclebath in a cumulative fashion! when equilibrium was obtained, the next dose was added. Each point represents the mean response ~ SEM for 3-15 rings.

C a l c i u m Channel Modulators

57

SKELETAL MUSCLE

Here we have a situation that is even more fascinating than cardiac, smooth, or neuronal tissues.

There is no question that the number of dihydropyridine binding

sites in the T-system is much higher than in any other tissue thus far examined [8,20-22,40,43].

Yet despite the evidence of the presence of a large number of

"calcium channels," the physiological function remains elusive. Based primarily on recent experiments showing that charge movementcould be reduced in the presence of nifedlpine, i t has been suggested that part or a l l of charge movement is associated with calcium channels in the T-system [25,27,32]. Voltage-dependent charge movementwas f i r s t reported by Schneider and Chandler [37] in frog skeletal muscle who proposed a link betweenexcitation of the T-tubular membranes with release of calcium from the sarcoplasmic reticulum.

A problem with

the mechanismis illustrated by the study of Schwartz et al [4~.

Using frog sar-

torlus muscle and [3~PN200-110 to measure binding to the intact muscle, these investigators found that calcium current was blocked by concentrations 100-1000 times higher than that required for the saturation of dihydropyridine sites.

The authors'

conclusion is that only a few percent of the binding sites in skeletal muscle represent functional caiciEm channels, which is contrary to general opinion.

This major

discrepancy seems not to be the case when "myoball" preparations (primary cultures) of neonatal rat skeletal muscle were used and comparedto binding of the dihydropyridine to membranesfrom adult rat T-tubules and using a whole cell patch clamp technique on the "myoballs" in culture.

In this study, the KO.5 value for inhibiting

calcium current (0.15 nM) was close to the binding constant for [3F~PN200-110 to membranes (KD = 0.22 nM) [7]. In our studies, led by Dr. Kenneth B. Walsh, a number of i n t e r e s t i n g paradoxical r e s u l t s have emerged, summarized as f o l l o w s , 1.

o L c i s - d i l t i a z e m produces a concentration-dependent, s t e r e o s e l e c t i v e , augmen-

t a t i o n o f twitch amplitude and a decrease in mechanical threshold in mouse and r a t

skeletal muscle fibers [5~. 2.

Calcium current in rabbit muscle is blocked by cadmium, diltiazem, and vera-

pamil. The concentrations of diltiazem and verapamil required were, however, high (IC50=63 and lO ~M, respectively).

Nitrendipine, however, even in concentrations of

58

A. S c h w a r t z

I-i0 ~M, had no blocking action even after 20 minutes of exposure ~2]. 3.

Diltiazem blocks calcium current butenhances charge movement in frog fibers

while nitrendipine even at high concentrations could not block inward calcium current, but greatly reduced charge movement (K.B. Walsh, Ph.D. Dissertation, 1986). These and other results reinforce our paucity of knowledge regarding calcium channels in skeletal muscle and their function.

Since most investigators, including

ourselves, are using skeletal muscle as the source for purification [1,5,8], it is imperative that detailed studies of the role of the T-tubule calcium channel be emphasized. UPDATE ON RECEPTOR ACTIVITY AND ONPURIFICATION Professor Glossmann, who was the first to demonstrate stereoselective binding of a calcium channel modulator to membranes, will cover the details of mechanisms involved in receptor binding and provide information regarding purification of the putative calcium channel protein(s) from guinea pig skeletal muscle. Our studies regarding binding can be summarized in Figure 7, which illustrates the complex allosteric effects of verapamil and diltiazem using cardiac muscle membranes.

I | [

(-) NICARDIPINE (Ki=2.5 nM)

9

50 --

i" ~

~o

(-) 2 0 2 - 7 9 1 ~ ~ K i = 2 . 1 nM) 9

\

9

~ . ~.,OZVERAPAMIL

(+) NICARDIPINE ~e V'\ (Ki=0,2 nM) "~e \~.~o o

"'-%~-~:~o-o

10-11

d-CIS-DILTIAZEM

%\~

\.\\

9

'~ :o

~m /

~ /

\,

~-o..~__0

\. . (+) 202-791 A & ~ (Ki=200 nM)

-~._,

I

I

I

i

i

i

i

l

10-10

10-9

10-8

10 -7

10-s

10-5

10 -4

10-3

Figure 7. Effects of 202-791 and nicardipine stereoisomers, d-cisdiltiazem and verapamil on (-)[5F~202-791 binding to canine cardiac membranes. (Vaghy et al. unpublished results.)

Calcium C h a n n e l M o d u l a t o r s

59

In terms of our progress in purifying the protein(s) from rabbit skeletal muscle led by Or. Natsuki Nakayama, we have purified a membrane preparation, which reveals a major 150 kdalton protein on SOS-PAGE, silver stained; this purified preparation exhibits the same characteristics described by Professor Glossmann.

There may be

lower molecular weight bands present, but their significance at this writing is unknown.

We have been successful thus far in obtaining the N-terminal sequence of

both our rabbit muscle preparation and Professor Glossmann's guinea pig protein, purified by a different method, and the two are identical.

In preliminary experi-

ments, these preparations have been reconstituted in lipid bilayers and demonstrate voltage-dependent calcium channel activity.

Similar reconstitutlon experiments using

cardiac SL vesicles and skeletal T-tubular membranes, have been recently reported

[1,34. ACKNOWLEDGMENTS The original studies referred to in this paper were supported by grants from the N.I.H. POI HL22 619 and the American Heart Association, Southwestern Chapter.

I

should l i k e to express my sincerest appreciation to my colleagues who have contributed in a major way to the studies in my laboratory. They are:

Drs. Yung Baik,

Joseph Balwierczak, Alain DePover, Gregory Dube, Gunter Grupp, Ingrid Grupp, Kunihisa Miwa, Natsuki Nakayama, Pal Vaghy, and Judith Williams.

My special thanks to

Dr. Williams for reading and critiquing the manuscript, to Mrs. Pamela Frohman and Mrs. Kathleen Smidebush for typing and proofreading, and to Ms. Gwen Kraft for her contributions on the figures and tables.

Support and some of the drugs were

generously supplied by Marion Laboratories, Miles Laboratories, and Sandoz Pharmaceutical Company. REFERENCES 1. 2. 3. 4. 5.

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34. 35. 36. 37. 38. 39.

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