FL 33101, U.S.A.. The contractile state of vascular smooth muscle is a function of the cytoplasmic concentration of free Cat+. The free cytoplasmic Cat+ can be ...
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Biochemical Approaches to the Control of Hypertension Pharmacological Biochemistry Group Colloquium organized by T. J. Franklin (Alderley Park) and I. F. Skidmore (Ware), and edited by T. J. Franklin
Localized Determinants of Vascular Smooth-Muscle Contractility
Regulation of Cat+ levels in smooth-muscle cells CYNTHIA CAUVIN and CORNELIS VAN BREEMEN Department of Pharmacology, University of Miami, Miami, FL 33101, U . S . A . The contractile state of vascular smooth muscle is a function of the cytoplasmic concentration of free Cat+. The free cytoplasmic Cat+ can be derived from two sources: extracellular and intracellular Cat+ pools. We will discuss the mobilization of these pools to ellicit contraction and the inhibition of these processes to cause relaxation. Intracellular Cat+ stores The sarcoplasmic reticulum, mitochondriaand theplasmalemma represent storage sites for Cat+. Activation of vascular smooth muscle by agonists involves the release of intracellular Cat+ from storage sites. The degree to which this release occurs depends upon the particular agonist and blood vessel studied (Loutzenhiser et al., 1983). In the rabbit aorta, for example, NA can ellicit a phasic contraction after IOmin incubation in Cat+-freesolution that is about 80% as large as the control contraction obtained in normal Caz+-containing physiological saline. In contrast, in the rabbit superior mesenteric arterial tree, such a contraction would be only 60% of control in the mesenteric artery itself, only 30% of control in the first branch, and only 10% of control in the fourth branch (Cauvin et al., 1983~).The release of the Cat+ pool which supports these contractions most likely involves Cat+-induced Cat+ release (Sadia & van Breemen, 1983, 1984~).This process has been found to be increased by cyclic AMP in rabbit mesenteric artery (Saida & van Breemen, 19846). Hence, NA activation of aladrenoceptors leads to release of intracellular Ca2+(Cauvin et al., 1982); the intracellular Ca2+released is most likely a membrane-bound pool, which upon release acts as a trigger to stimulate Ca2+-induced Cat+ release from the sarcoplasmic reticulum. The latter process is enhanced by NA activation of /I-adrenoceptors (Saida & van Breemen, 19846). The release of intracellular Cat+ from these storage sites not only provides the initial elevation of cytoplasmic [Cat+]for contraction, but also prevents these storage sites from acting to sequester entering Cat+ (Loutzenhiser & van Breemen, 1983~).Under such conditions, even when no Cat+ is entering the cells through excitable Cat+ channels, the Cat+ entering through the passive-leak pathway can Abbreviations used : NA, noradrenaline; PSC,potential-sensitive channel; ROC, receptor-operated channel; CAt, calcium antagonist. VOl. 12
support a tonic contraction (Loutzenhiser & van Breemen, 1983b). Ca2+entry from extracellular pools The tonic phase of agonist-induced contractions as well as the entire contraction due to membrane depolarization is the result of entry of extracellular Ca2+into the cytoplasm. In the case of agonist stimulation, some of this Ca2+derives from extracellularly bound Cat+, which is released upon stimulation, then rapidly enters the cytoplasm (Loutzenhiser & van Breemen, 1983~). The pathways through which Ca2+enters the cell during activation may include the following: (a) the passive Ca2+ leak, (6) a PSC, and (c) an ROC. Evidence to support the hypothesis that separate PSCs and ROCs exist in vascular smooth muscle involves selective activation and inhibition of these entities, which are discussed below. Selective activation of Ca2+channels A number of investigators have observed in several blood vessels that NA is capable of eliciting contraction in depolarized vascular smooth muscle (Su et al., 1964; Droogmans et al., 1977). This phenomenon was first described as pharmacomechanical coupling (Somylo & Somylo, 1968). We have found that NA has no effect on membrane potential in rabbit aorta (Cauvin et al., 1984). NA can activate the aorta which has been depolarized by 8 0 m ~ - K +and , stimulates virtually the same Cat+ influx in the depolarized as in normal polarized preparations (Meisheri et al., 1981). These results suggest that NA activates a Ca2+influx pathway distinct from the PSC in this tissue. Use of a new Cat+ channel agonist, Bay K8644, which appears to bind to open Ca2+ channels and keep them open, has led to the discovery that the Cat+-influx pathway opened by NA in the rabbit aorta is also distinct from that opened by Bay K8644. In contradistinction, Bay K8644 and 8 0 m ~ - K +appear to open the same channel, presumably the PSC (Yamamoto et al., 1984). In the rabbit mesenteric resistance vessels, NA ( 1 0 - 4 ~ ) has, in contrast to aorta, been found to depolarize the tissues to the same extent as does 8 0 m ~ - K +This . NA depolarization is completely inhibited by 10- M-diltiazem (Cauvin et al., 1984), which completely inhibits the NA-stimulated Cat+ entry in the vessels (Cauvin et al., 1983~).The diltiazem has no effect on the depolarization induced by 8 0 m ~ - K +Moreover, . the NA is able to produce virtually the same contraction and stimulation of Ca2+ influx in depolarized ( 8 0 m ~ - K + resistance ) vessels as in normal
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BIOCHEMICAL SOCIETY TRANSACTIONS
activation of rabbit aorta causes a change in CAt sensitivity (van Breemen et at., 1983). The higher the NA activation level, the less sensitive the NA-stimulated Ca2+influx is to inhibition by D600, diltiazem and nisoldipine. It is possible that using selective a-adrenoceptor agonists may produce varying levels of activation (due to their different efficacies), and that this may account for the different CAt potencies observed when selectivea-agonists are compared. The reason why different levels of NA activation of the Selective inhibition of Ca2+channels rabbit aorta are differently sensitive to CAts presents an (1) fi-adrenergic relaxation. It has been shown that fi- intriguing problem. We have postulated that the varying adrenoceptor occupation with isoproterenol depresses K+- degrees of release of intracellular CaZ+ by NA may be stimulated Ca2+ influx in rabbit aorta, but not that responsible for this observation. Our rationale for this hypostimulated by methoxamine (Meisheri & van Breemen, thesis includes the following: (1) in tissues where the release 1981) and histamine. These results imply that the intra- of intracellular Ca2+ by NA is minimal (e.g. the rabbit cellular messenger cyclic AMP acts to inhibit PSCs but not mesenteric resistance vessels and the dog coronary), the sensitivity of NA-stimulated CaZ+influx is very high; (2) at ROCs. (2) Caz+ antagonists. Inhibition by CAts of CaZ+influx low concentrations of NA, there is little release of intrastimulated by depolarization or by agonist activation has cellular CaZ+in rabbit aorta, and the CAt sensitivity is high, been shown to be selective (for review see Cauvin et al., whereas at high "A], intracellular CaZ+release is sub19836). Generally Ca2+influx and contractions induced by stantial and CAt sensitivity of the stimulated CaZ+influx is K+ depolarization are more sensitive to Ca2+ antagonists low; and (3) low and high levels of NA activation of the than are those stimulated by agonists. This generalization rabbit mesenteric resistance vessels, neither of which applies to D600,diltiazem, nisoldipine (Loutzenhiser & van involve significant release of intracellular Cat+, are equally Breemen, 1983b) and PY10844 (Sandoz) in the rabbit aorta. sensitive to diltiazem (Cauvin et al., 1984). We have hypoWe hypothesized that PSCs were more sensitive to CAts thesized, therefore, that the release of intracellular Ca2+ than ROCs. In rabbit mesenteric resistance vessels, may render the ROC less sensitive to CAts. Differences in resting membrane potentials of vascular however, the opposite conclusion can be reached. Diltiazem is approximately 20 times more potent in inhibiting CaZ+ smooth muscles or in the degree to which agonists influx stimulated by NA than that stimulated by depolarize vascular tissues may also provide a basis for CAt 8 0 m ~ - K +in these vessels (Cauvin el al., 1983~).In either selectivity. For example, NA depolarizes rabbit mesenteric case, it is apparent that the CAts demonstrate selective resistance vessels and these are highly sensitive to the inhibition of the two proposed types of CaZ+channels. inhibitory effects of diltiazefn. One could speculate that in Moreover, the sensitivity of the PSC to CAts appears to be this tissue, NA opens PSCs more than it does in the aorta. rather constant throughout vascular beds and is rather The problem with this argument is that in the resistance comparable among species, whereas the proposed ROC vessels, diltiazem is about 20 times more potent in varies widely in its sensitivity to CAts (for review see inhibiting NA-induced CaZ+influx than that induced by 8 0 m ~ - K + Hence, . it is difficult to conclude what role Cauvin et al., 19836). membrane depolarization by NA plays in conferring Theoretical basis for CAt selectiuity diltiazem selectivity for the resistance vessels. Finally, the development of dihydropyridine Ca2+chanAlthough competitive radioligand-binding studies have indicated that the order of binding affinities and the nel agonists suggests that some CAts may act as partial agonists. The possibility must be investigated that different pharmacological potencies of CAts correlate well within a given tissue (e.g. guinea pig ileum; Janis & Triggle, 1984), tissues would have different agonistic/antagonistic ratios for a given CAt and, hence, would demonstrate pharmacosuch studies have shed little light on the reasons for marked variation in CAt potencies among tissues. Binding affinities logical CAt potencies in spite of similar binding affinities. for [3H]nitrendipine show remarkable similarities in nerve, cardiac, secretory, skeletal-muscle and smooth-muscle tissues, while the pharmacological potency varies tremendously among these tissues (Janis & Triggle, 1984), smooth Cauvin, C., Loutzenhiser, R., Hwang, 0. & van Breemen, C. muscle being most sensitive. Even among vascular tissues, (1982) Eur. J . Phurmucol. 84, 233-235 as mentioned previously, sensitivity to CAts varies markedCauvin, C., Saida, K . & van Breemen, C. (1983~)Blood Vessels 21, ly, particularly when agonist activation is studied. It is 23-31 reasonable to speculate that differing mechanisms of acti- Cauvin, C., Loutzenhiser, R. &van Breemen, C. (19836) Ann. Reu. Phurmucol. Toxicol. 23, 373-396 vation of different vascular tissues may account, at least in Cauvin, C., Lukeman, S.,Cameron, J., Meisheri, K., Yamamoto, part, for such variation. H. & van Breemen, C. (1984) J . Curdiouusc. Phurmucol. in the It has been postulated, for example, that activation of alpress adrenoceptors is less sensitive to CAt inhibition than is that Drmgmans, G . , Raeynmakers, L. & Casteels, R. (1977) J . Gen. of az-adrenoceptors, perhaps because a1-adrenoceptors PhySioI. 70, 129-148 may release intracellular Ca2+while a2-adrenoceptorsmay Janis, R. A. & Triggle, D. J. (1984) J . Med. Chem: Perspect. Ser. in induce CaZ+influx (Van Meel et al., 1981). We have, on the the press other hand, found in both rabbit and rat aorta and Janssens, W. J., Beyens, G . G . & Verhaughe, R. H. (1983) Arch. Znt. Phurmucodyn. 263, 320-321 mesenteric resistance vessels that a,-adrenoceptors mediate NA-induced Cat+ influx and intracellular Ca2+ release Loutzenhiser, R. & van Breemen, C. (19830) Circ. Res. 52 (Suppl. I), 97-103 (Cauvin et al., 1982).Other investigators have found that aLoutzenhiser, R. & van Breemen, C. (19836) in Symposium on Cu2+ adrenoceptors as well produce both Ca2+influx and intraEntry Blockers. Adenosine and Neurohumors (Merrill, G . F., cellular CaZ+release (Janssens et al., 1983). Nonetheless, Weiss, H. R. & Scriabine, A., eds.), pp. 73-91, Urban and the finding that selective a-adrenoceptor activation is Schwarzenberg associated with more or less sensitivity to CAts requires Loutzenhiser, R. & van Breemen, C. (1983~)Blood Vessels 20,295explanation. We have observed that varying the level of NA 305 polarized tissues (Cauvin etal., 1984), and it does so without further depolarizing the 80m~-K+-treatedtissues. These results suggest that NA may again be activating Ca2+influx through a different pathway from the PSC in spite of the depolarization produced by NA alone. It is possible that the NA may open ROCs in these issues and that the entering Ca2+ ions depolarize the tissues. This hypothesis may explain the inhibition of NA depolarization by diltiazem.
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Loutzenhiser, R., Leyten, P.,Saida, K. & van Breemen, C. (1983) Somylo, A. P. & Somylo, A. V. (1968) Pharmacol. Rev. 20,197-272 in Calcium and Smooth Muscle Contractility (Daniel, E. E. & Su, C . , Bevan, J. A. & Ursillo, R. C. (1964) Circ. Res. 15, 2& Grover, A. K., eds.). Humana Press Inc., New Jersey, in the 27 press van Breemen, C., Lukeman, S., Loutenhizer, R., Saida, S. & Merisheri, K. & van Breemen, C. (1981) Fed. Proc. Fed. Am. SOC. Cauvin, C. (1983) in Calcium Antagonists: The Stateofthe Art & Exp. Biol. 40,624 (Abstract) Role in Cardiovascular Disease (Hofmann, B., ed.), pp. 13-26 Meisheri, K., Hwang, 0.&van Breemen, C. (1981)J. Membr. Biol. Van Meel, J. A. C., DeJonge, A., Kalkman, H. 0.. Wilffer, B., 59, 19-25 Timmermans, P. B. M. W. M. & Van Zeieten, P. A. (1981) Saida, K.& van Breemen, C. (1983) Pflugers Arch. 397, 166167 Naunyn-Schiedeberg’s Arch. Pharmucol. 316, 288 Saida, K. & van Breemen, C. (1984~)Blood Vessels 21, 43-52 Yamamoto, H., Hwang, 0. & van Breemen, C. (1984) Eur. J . Saida, K. & van Breemen, C. (19846) J . Gen. Physiol. in the press Pharmacol. in the press
The identification of cytoplasmic membrane calcium-entry channels by ligand-binding studies H. GLOSSMANN, A. GOLL, D. R. FERRYand M. ROMBUSCH Rudolf Buchheim-Institutfur Pharmakologie, Justus LiebigUniversitat, Giessen, 0 - 6 3 Giessen, Frankfurterstrasse 107, Federal Republic of Germany During the last 2 years rapid progress has been made with respect to the molecular pharmacology of the voltagedependent CaZ+channel (Glossmann et al., 1982, 1983a; Glossmann & Ferry, 1983~).This breakthrough was made possible by the availability of labelled drugs, which act on the CaZ+channel in a highly selective and specific manner. Many of these drugs block the channel. There is, however, an increasing number of man-made molecules which keep or force the channel open but’direct labelling studies with these channel activators are still in an early stage. We have concentrated our work on mainly three tissues, namely heart, brain and mammalian skeletal muscle (Glossmann et al., 1982,1983~.Glossmann &Ferry, 1983a). These tissues have the highest density of high-affinity binding sites for tritiated or iodinated 1,Cdihydropyridines. The t-tubules of the mammalian skeletal muscle can be termed the electric eel for the biochemist working on calcium channels, since 1 mg of membrane protein contains 60000fmol of 1,4-dihydropyridine-bindingsites (Ferry & Glossmann, 1982a; Glossmann et al., 19836). Approx. 0.52% of the t-tubule protein is Caz+-channel protein. The metalloprotein-nature of the channel labelled with 1,4dihydropyridines When brain calcium channels are labelled with nimodipine, the binding is inhibited dose-dependently by the chelator EDTA (Glossmann & Ferry, 1983a; Glossmann et al., 1983~).This inhibition is due to a conversion of a highaffinity state of the channel into a divalent cation-free state which has very low affinity for the calcium-channel blocker. The important conclusion is, that the channel, as identified by these radioligands, has tightly bound divalent cations. Since the equilibrium constants of the coupled equilibria are interdependent, it follows that the metal must be more tightly bound when the 1,4-dihydropyridine occupies the channel. This supports the concept of saturable binding sites for divalent cations within the channel and furthermore it follows that these channel blockers act by increasing the tightness of the binding for certain divalent cations. The channel can be depleted of divalent cations with EDTA and refilled with different divalent cations. Each divalent cation has a typical reconstitution isotherm which is characterized by a Hill slope, an intrinsic activity factor and an EC50 value. Barium and strontium exhibit Hill slopes significantly smaller than I , whereas calcium and manganese have slopes greater than 2. Cobalt, zinc and
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nickel have biphasic reconstitution isotherms. This is evidence that there are co-operative effects between more than one metal-binding site and the 1,Cdihydropyridinebinding sites (Glossmann & Ferry, 1983~). Subtypes of channels Tissue- but not species-specific binding constants for a given 1,6dihydropyridine label exist and in addition there are tissue-specific effects of the positive allosteric regulator d-cis-diltiazem at 37°C (Ferry & Glossmann, 1983). All brains, from different species, which we have examined, have a 0 . 6 n ~KD for nimodipine, all skeletal muscles (rat, guinea pig or rabbit) have a KDof about 1.5nM and all hearts (frog, rat, guinea pig, bovine and human) have a 0.2511~KD value at 37°C. These ‘isoreceptors’most likely correspond to channel subtypes. The channel subtypes are further differentiated by their pH profile, their sensitivity to sulphydryl reagents, their heparin sensitivity and by subtype-selective drugs (Glossmann et al., 1984). The 1,Cdihydropyridine drug receptor of the channel is a highly conserved domain which is a tissue-specific but not species-specific phenomenon. The three main subtypes, exemplified by brain, heart and skeletal muscle, have nevertheless the same molecular size and d-cis-diltiazem modification of the radiation-sensitive domain when probed in situ by irradiation inactivation (Go11 et al., 1983; Glossmann et al., 1984). Channel agonists and antagonists For skeletal muscle membranes at 37°C one finds that different radiolabelled 1,4-dihydropyridines label different numbers of channel binding sites. [3H]PN-200-110 (overt antagonist) labels 5 times more sites than [3H]nifedipine (partial agonist). [’HIBay K 8644, the 1,Cdihydropyridine channel agonist, hardly labels any sites at 37°C in the absence of diltiazem. The ability of a given 1,4-dihydropyridine to stabilize this high-affinity state at 37°C is related to its ability to either ‘block’ or ‘activate’ the CaZ+channel. It can be suggested that the fraction of channels stabilized in the low-affinity state corresponds to the ‘open’ state population whereas the fraction of channels stabilized in the high-affinity state corresponds to the ‘closed’ channel. Multiple drug receptor sites within the calcium channel There are three main, distinct drug receptor sites on the calcium channel which were named after the prototype of drug which was employed to characterize these sites either by direct labelling and indirectly by drug competition studies (Ferry & Glossmann, 19826; Glossmann & Ferry, 1983a; Glossmann et al., 1982, 1983~).Each of these drug receptor sites can exist in low- and high-affinity states, induced by other drugs, temperature, ions or detergents.
