Experimental Gerontology, Vol. 25, pp. 369-373, 1990
0531-5565/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
Printed in the USA. All rights reserved.
RECEPTOR SITES INVOLVED IN THE ACTION OF CALCIUM BLOCKING AGENTS
J.-C.
STOCLET,P. SCHAEFFERand G. JtrLou-ScHAEFFER
Laboratoire de Pharmacologie Cellulaire et Mol6culaire (U.R.A. CNRS 600), Universit6 Louis Pasteur de Strasbourg, BP 24, 67401, lllkirch Cedex, France
Abstract -- The affinities of calcium blocking agents (CBAs) for membrane binding sites
and for calmodulin were compared to their potencies at inhibiting contraction of various isolated arteries. Two allosterically linked binding sites, the dihydropyridine (DHPS)and benzothiazepine-phenylakylamine (BS) one, were characterized. In spite of a correlation between the affinities of a number of hydrophobic CBAs and calmodulin antagonists for DHPS and for calmodulin, these drugs displayed higher affinities for BS. Furthermore, their potency at inhibiting calcium-induced contraction in depolarized rat aorta rings was correlated to their affinity for the latter site. These results suggest that binding to membrane sites was the basis of the inhibition of depolarization-elicited contraction by all CBAs, including calmodulin antagonists. Differences in sensitivity to CBAs depending on the artery and whether they were depolarized or stimulated by noradrenaline were shown in further experiments on rat cerebral artery and resistance arterioles. These differences in sensitivity did not correspond to differences in the apparent affinity o f the drugs. This suggests that the receptors of CBAs (and therefore the associated calcium channels) involved in the responses to depolarization and to the agohist were identical. Key Words: calcium blocking agents, calmodulin antagonists, vascular smooth muscle, resistance arteries
INTRODUCTION TrmRE ARE various chemical classes of calcium blocking agents (CBAs) with relatively selective effects on different organs (Godfraind et al., 1986). These drugs are of the utmost importance in the treatment of cardiovascular disease associated with aging. All CBAs have the common property of being able to antagonize the effects of calcium in the response of cardiac and vascular smooth muscle cells to various stimuli. Most of them inhibit calcium entry into stimulated cells, but some can also interfere with calcium transport into, and release from intracellular organelles, or intracellular calcium receptor proteins such as calmodulin. Indeed, hydrophobic CBAs are calmodulin antagonists (Stoclet et al., 1987), and it has been suggested that this and other intracellular mechanisms could be involved in the tissue selectivity of CBAs.
Correspondence to: J.-C. Stoclet.
369
370
3.-c. STOCLET et al.
One possible approach towards elucidating the respective participation of these mechanisms would be to compare the affinities of CBAs for different target sites with their potencies in altering tissue functions. In the present paper, recent work based on this approach will be reviewed with respect to the effects of CBAs on different arteries. INTERACTIONS OF CALMODULIN AND CALCIUM ANTAGONISTS WITH MEMBRANE BINDING SITES AND WITH CALMODULIN As reviewed by Godfraind et al. (1986) at least 2, perhaps more. distinct high affinity binding sites for CBAs have been characterized in a large number of studies on crude membranes or plasma membrane enriched fractions from many tissues. One of these sites (DHPS) is highly specific for dihydropyridines. Following recent detailed studies, the DHPS can perhaps be divided in 2 closely associated subsites, one for agonists and the other for antagonists. The other site (BS) has high affinities for other CBAs, including the benzothiazepine dittiazem and an heterogeneous group of arylalkylamines (phenylalkylamines such as verapamil and derivatives, and diphenylalkylamines such as bepridil, flunarizine and other hydrophobic compounds). Detailed binding studies suggest that the site can also be divided in 2 subsites, respectively, binding diltiazem and verapamil. All these sites are linked by allosteric interactions. Since identical binding sites have all been found on the purified L-type calcium channel from skeletal muscle (Galizzi et al.. 1986), it is reasonable to hypothesize that membrane high affinity binding sites for CBAs are located on this channel. In view of structural similarities between calmodulin antagonists, such as naphthalene sulfonamide W7 or the phenothiazine derivatives, and diphenylalkylamine CBAs, interactions between all these drugs and both calmodulin and CBAs binding sites have been investigated (Lugnier et al.. 1984; Schaeffer et al., 1988). Contrary to the observation that dihydropyridines bound to membranes at much lower concentrations than those necessary to inhibit catmodulin, the affinity of both diphenylalkylamine CBAs and: calmodulin antagonists on the DHPS site were comparable to those inhibiting calmodulin (Table 1). However, the apparent affinity of these drugs for the BS site was generally higher, suggesting that their pharmacological effect, as those of dihydropyridine derivatives, might rather be due to binding to membrane sites associated with calcium channels than to inhibition of calmodulin. In addition, these results support the view that a calmodulin-like component is associated with DHPS. INHIBITION OF VASCULAR SMOOTH MUSCLE CONTRACTION ELICITED BY DEPOLARIZATION In vascular smooth muscle, depolarization induces an increase in calcium permeability through T- and L-type calcium channels, and this increased permeability is dose-dependently decreased by dihydropyridines (Bolton et al., 1988). Experiments with 45Ca have also shown that not only other CBAs, but also calmodulin antagonists decrease calcium influx and subsequent contraction in stimulated vascular smooth muscle cells (Flaim et al., 1985). Therefore, the question has been raised as to whether or not this could be produced by calmodulin inhibition. In addition to producing calcium entry, depolarization increases the affinity of plasma membrane L-type calcium channels for CBAs, which probably bind to an inactivated state of the channel (Bean, 1984). Since isolated membranes are depolarized, one expects a correlation
RECEPTORSOF CALCIUMBLOCKINGAGENTS
371
TABLE I. COMPARISON OF INHIBITORYCONCENTRATIONS OFCALCIUMANDCALMODULINANTAGONISTSON [3H]d-cis
DtLTIAZEM (BS), AND [3H]NITR~NDIP~ (DHPS)~BINDIN~ TO RATBRAINM~MBRANES(--iog Ki), ON CALMODULIN-INDUCED ACTIVATIONOFCYCLICNUCLEOTIDEPHOSPHODIESTERASE ( - iog IC5o), ANDONCaCI2-INDUCED
CONTRACTIONOF K+-DEPOLARIZEDRAT AORTA(x) OR GUINEAPIG TAENIACOLI(xx) (--log KB OR pAz) BS
Calcium entry blockers Nitrendipine Nifedipine Nicardipine d-cis diltiazem Verapamil Bepridil Prenylamine
0531-5565/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
Printed in the USA. All rights reserved.
RECEPTOR SITES INVOLVED IN THE ACTION OF CALCIUM BLOCKING AGENTS
J.-C.
STOCLET,P. SCHAEFFERand G. JtrLou-ScHAEFFER
Laboratoire de Pharmacologie Cellulaire et Mol6culaire (U.R.A. CNRS 600), Universit6 Louis Pasteur de Strasbourg, BP 24, 67401, lllkirch Cedex, France
Abstract -- The affinities of calcium blocking agents (CBAs) for membrane binding sites
and for calmodulin were compared to their potencies at inhibiting contraction of various isolated arteries. Two allosterically linked binding sites, the dihydropyridine (DHPS)and benzothiazepine-phenylakylamine (BS) one, were characterized. In spite of a correlation between the affinities of a number of hydrophobic CBAs and calmodulin antagonists for DHPS and for calmodulin, these drugs displayed higher affinities for BS. Furthermore, their potency at inhibiting calcium-induced contraction in depolarized rat aorta rings was correlated to their affinity for the latter site. These results suggest that binding to membrane sites was the basis of the inhibition of depolarization-elicited contraction by all CBAs, including calmodulin antagonists. Differences in sensitivity to CBAs depending on the artery and whether they were depolarized or stimulated by noradrenaline were shown in further experiments on rat cerebral artery and resistance arterioles. These differences in sensitivity did not correspond to differences in the apparent affinity o f the drugs. This suggests that the receptors of CBAs (and therefore the associated calcium channels) involved in the responses to depolarization and to the agohist were identical. Key Words: calcium blocking agents, calmodulin antagonists, vascular smooth muscle, resistance arteries
INTRODUCTION TrmRE ARE various chemical classes of calcium blocking agents (CBAs) with relatively selective effects on different organs (Godfraind et al., 1986). These drugs are of the utmost importance in the treatment of cardiovascular disease associated with aging. All CBAs have the common property of being able to antagonize the effects of calcium in the response of cardiac and vascular smooth muscle cells to various stimuli. Most of them inhibit calcium entry into stimulated cells, but some can also interfere with calcium transport into, and release from intracellular organelles, or intracellular calcium receptor proteins such as calmodulin. Indeed, hydrophobic CBAs are calmodulin antagonists (Stoclet et al., 1987), and it has been suggested that this and other intracellular mechanisms could be involved in the tissue selectivity of CBAs.
Correspondence to: J.-C. Stoclet.
369
370
3.-c. STOCLET et al.
One possible approach towards elucidating the respective participation of these mechanisms would be to compare the affinities of CBAs for different target sites with their potencies in altering tissue functions. In the present paper, recent work based on this approach will be reviewed with respect to the effects of CBAs on different arteries. INTERACTIONS OF CALMODULIN AND CALCIUM ANTAGONISTS WITH MEMBRANE BINDING SITES AND WITH CALMODULIN As reviewed by Godfraind et al. (1986) at least 2, perhaps more. distinct high affinity binding sites for CBAs have been characterized in a large number of studies on crude membranes or plasma membrane enriched fractions from many tissues. One of these sites (DHPS) is highly specific for dihydropyridines. Following recent detailed studies, the DHPS can perhaps be divided in 2 closely associated subsites, one for agonists and the other for antagonists. The other site (BS) has high affinities for other CBAs, including the benzothiazepine dittiazem and an heterogeneous group of arylalkylamines (phenylalkylamines such as verapamil and derivatives, and diphenylalkylamines such as bepridil, flunarizine and other hydrophobic compounds). Detailed binding studies suggest that the site can also be divided in 2 subsites, respectively, binding diltiazem and verapamil. All these sites are linked by allosteric interactions. Since identical binding sites have all been found on the purified L-type calcium channel from skeletal muscle (Galizzi et al.. 1986), it is reasonable to hypothesize that membrane high affinity binding sites for CBAs are located on this channel. In view of structural similarities between calmodulin antagonists, such as naphthalene sulfonamide W7 or the phenothiazine derivatives, and diphenylalkylamine CBAs, interactions between all these drugs and both calmodulin and CBAs binding sites have been investigated (Lugnier et al.. 1984; Schaeffer et al., 1988). Contrary to the observation that dihydropyridines bound to membranes at much lower concentrations than those necessary to inhibit catmodulin, the affinity of both diphenylalkylamine CBAs and: calmodulin antagonists on the DHPS site were comparable to those inhibiting calmodulin (Table 1). However, the apparent affinity of these drugs for the BS site was generally higher, suggesting that their pharmacological effect, as those of dihydropyridine derivatives, might rather be due to binding to membrane sites associated with calcium channels than to inhibition of calmodulin. In addition, these results support the view that a calmodulin-like component is associated with DHPS. INHIBITION OF VASCULAR SMOOTH MUSCLE CONTRACTION ELICITED BY DEPOLARIZATION In vascular smooth muscle, depolarization induces an increase in calcium permeability through T- and L-type calcium channels, and this increased permeability is dose-dependently decreased by dihydropyridines (Bolton et al., 1988). Experiments with 45Ca have also shown that not only other CBAs, but also calmodulin antagonists decrease calcium influx and subsequent contraction in stimulated vascular smooth muscle cells (Flaim et al., 1985). Therefore, the question has been raised as to whether or not this could be produced by calmodulin inhibition. In addition to producing calcium entry, depolarization increases the affinity of plasma membrane L-type calcium channels for CBAs, which probably bind to an inactivated state of the channel (Bean, 1984). Since isolated membranes are depolarized, one expects a correlation
RECEPTORSOF CALCIUMBLOCKINGAGENTS
371
TABLE I. COMPARISON OF INHIBITORYCONCENTRATIONS OFCALCIUMANDCALMODULINANTAGONISTSON [3H]d-cis
DtLTIAZEM (BS), AND [3H]NITR~NDIP~ (DHPS)~BINDIN~ TO RATBRAINM~MBRANES(--iog Ki), ON CALMODULIN-INDUCED ACTIVATIONOFCYCLICNUCLEOTIDEPHOSPHODIESTERASE ( - iog IC5o), ANDONCaCI2-INDUCED
CONTRACTIONOF K+-DEPOLARIZEDRAT AORTA(x) OR GUINEAPIG TAENIACOLI(xx) (--log KB OR pAz) BS
Calcium entry blockers Nitrendipine Nifedipine Nicardipine d-cis diltiazem Verapamil Bepridil Prenylamine
