Modulation of Cardiac Ca2+ Channel by Gq-activating Neurotransmitters Reconstituted in Xenopus Oocytes

L-type dihydropyridine-sensitive voltage dependent Ca2+ channels (L-VDCCs; α1C) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via Gq, such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on α1C isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking Gq coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of Gq, PLC, and PKC. The inhibitory modulation, present in both α1C isoforms, was Gq- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, as only basal levels of Ca2+ were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations. L-type dihydropyridine-sensitive voltage dependent Ca2+ channels (L-VDCCs; α1C) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via Gq, such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on α1C isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking Gq coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of Gq, PLC, and PKC. The inhibitory modulation, present in both α1C isoforms, was Gq- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, as only basal levels of Ca2+ were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations. The cardiac voltage-dependent, dihydropyridine-sensitive L-type calcium channel (L-VDCC) 1The abbreviations used are: L-VDCC, L-type voltage dependent Ca2+ channels; aa, amino acid; ACh, acetylcholine; AngII, angiotensin II; AT1R, angiotensin II receptor type 1; Bis, bis-indolylmaleimide; m1R and m3R, muscarinic receptors 1 and 3; NT, N-terminus; PKC, protein kinase C; PLC, phospholipase C; WT, wild-type; PMA, 4β-phorbol-12-myristate 13-acetate; ANOVA, analysis of variance; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. is the main calcium channel in the heart, where it contributes to the plateau of the action potential and thereby promotes cardiac cell contraction (1Reuter H. Nature. 1983; 301: 569-574Crossref PubMed Scopus (863) Google Scholar). In smooth muscle cells, these channels regulate tonus and contraction (2Hughes A.D. J. Vasc. Res. 1995; 32: 353-370Crossref PubMed Scopus (86) Google Scholar, 3Xiong Z. Sperelakis N. J. Mol. Cell. Cardiol. 1995; 27: 75-91Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Different hormones and transmitters, such as angiotensin II (AngII), bradykinin, acetylcholine (ACh), and norepinephrine, modulate the function of L-VDCC via G-proteins and protein kinases, profoundly affecting the function of the corresponding tissues (4Trautwein W. Hescheler J. Annu. Rev. Physiol. 1990; 52: 257-274Crossref PubMed Scopus (318) Google Scholar). AngII and ACh activate Gq-coupled receptors and are involved in cardiovascular function, regulation of blood pressure, and renal function (5Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Crossref PubMed Scopus (284) Google Scholar, 6Beech D.J. Pharmacol. Ther. 1997; 73: 91-119Crossref PubMed Scopus (79) Google Scholar). In the heart, ACh inhibits L-VDCC via m2 muscarinic receptors and the subsequent activation of the Gi signaling cascade and inhibition of adenylyl cyclase (1Reuter H. Nature. 1983; 301: 569-574Crossref PubMed Scopus (863) Google Scholar). However, in the smooth muscle, both AngII and ACh are potent vasoconstrictors that both induce Ca2+ release from intracellular stores and elevate intracellular Ca2+ concentration (7Jackson E.K. Garrison J.C. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman 15: 114-119Abstract Full Text PDF PubMed Scopus (237) Google Scholar). In heart and smooth muscle, AngII enhances Ca2+ channel currents (9Takenaka T. Forster H. Epstein M. Circ. Res. 1993; 73: 743-750Crossref PubMed Scopus (45) Google Scholar, 10Scholz H. Kurtz A. Am. J. Physiol. 1990; 259: C421-C426Crossref PubMed Google Scholar, 11Purdy R.E. Weber M.A. Circ. Res. 1988; 63: 748-757Crossref PubMed Scopus (63) Google Scholar, 12Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar, 13Macrez-Lepretre N. Morel J.L. Mironneau J. J. Pharmacol. Exp. Ther. 1996; 278: 468-475PubMed Google Scholar). ACh has also been reported to increase L-type Ca2+ channel currents in smooth muscle, mainly via m3 muscarinic receptors, m3R (14Benham C.D. Bolton T.B. Lang R.J. Nature. 1985; 316: 345-347Crossref PubMed Scopus (195) Google Scholar, 15Shi X.Z. Sarna S.K. Am. J. Physiol. 2000; 278: G234-G242PubMed Google Scholar, 16Liu X. Rusch N.J. Striessnig J. Sarna S.K. Gastroenterology. 2001; 120: 480-489Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Despite the clinical and physiological importance of the regulation of L-type Ca2+ channels by AngII and ACh, the molecular mechanisms remain poorly understood. The mechanism of AngII effect on L-type Ca2+ channels has been extensively studied but remains unclear and even controversial. Protein kinase C (PKC) is the most obvious and important mediator of AngII and ACh/m3R action. PKC is activated in native cells following AngII and ACh binding to Gq-coupled receptors (AngII receptor type 1, AT1R, and muscarinic receptors m3R and m1R, respectively). In mammals, PKC inhibitors block AngII-induced vasoconstriction (17Bauer J. Dau C. Cavarape A. Schaefer F. Ehmke H. Parekh N. Am. J. Physiol. 1999; 277: H1-H7PubMed Google Scholar, 18Nagahama T. Hayashi K. Ozawa Y. Takenaka T. Saruta T. Kidney Int. 2000; 57: 215-223Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Lambert C. Br. J. Pharmacol. 1995; 115: 795-800Crossref PubMed Scopus (19) Google Scholar, 20Fan Q.I. Vanderpool K. Marsh J.D. Biochim. Biophys. Acta. 2002; 1577: 401-411Crossref PubMed Scopus (12) Google Scholar). In cardiomyocytes and smooth muscle cells, the PKC activators phorbol esters and diacylglycerol mimic the effect of AngII, increasing force of contraction and Ca2+ influx (10Scholz H. Kurtz A. Am. J. Physiol. 1990; 259: C421-C426Crossref PubMed Google Scholar, 21Kato H. Hayashi T. Koshino Y. Kutsumi Y. Nakai T. Miyabo S. Biochem. Biophys. Res. Commun. 1992; 188: 934-941Crossref PubMed Scopus (32) Google Scholar, 22Savignac M. Badou A. Moreau M. Leclerc C. Guery J.C. Paulet P. Druet P. Ragab-Thomas J. Pelletier L. FASEB J. 2001; 15: 1577-1579Crossref PubMed Scopus (51) Google Scholar), as well as Ca2+ currents via the L-type channel (12Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar, 23Fish R.D. Sperti G. Colucci W.S. Clapham D.E. Circ. Res. 1988; 62: 1049-1054Crossref PubMed Scopus (163) Google Scholar, 24Lacerda A.E. Rampe Nature. 1988; PubMed Scopus Google Scholar, Am. J. Physiol. PubMed Google Scholar, Biochem. Biophys. Res. Commun. 1993; PubMed Scopus Google Scholar, K. K. 1994; PubMed Scopus Google Scholar, T. FASEB J. 1996; PubMed Scopus Google Scholar, C. J. Physiol. 1995; PubMed Scopus Google Scholar, C. Am. J. Physiol. Google Scholar). enhancement is by a later in the current A.E. Rampe Nature. 1988; PubMed Scopus Google Scholar, Am. J. Physiol. PubMed Google Scholar, C. S. Y. L. Am. J. Physiol. 2000; 278: Google Scholar). In only inhibition of the current in to PKC activation has been reported L. N. M. Circ. Res. 1997; 80: PubMed Scopus Google Scholar). In to PKC, protein J. Pharmacol. Exp. Ther. Google Scholar, T. H. M. M. Sperelakis N. J. Physiol. 1999; PubMed Scopus Google and the via activation of Mironneau J. A. Mironneau C. N. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), have been as involved in the of AngII in the molecular mechanisms of AngII and ACh modulations was that these modulations not be in heterologous expression A. N.M. H. 1995; PubMed Scopus Google studied the modulation of human L-VDCC expressed in Xenopus oocytes and reported that following the of PMA, the dihydropyridine-sensitive was M. N.M. M. Mol. Pharmacol. PubMed Scopus Google reported a in Ca2+ current in oocytes α1C and following of AngII, to a effect was by Ca2+ by intracellular Ca2+ stores The to reproduce AngII-induced Ca2+ channel enhancement be to the of isoforms of α1C that are not by PKC. In the and N-terminal (NT) isoforms of α1C are of the W.J. Full Text PDF PubMed Scopus Google Scholar). and α1C initial of and amino W.J. Full Text PDF PubMed Scopus Google Scholar, A. K. T. T. Y. H. S. S. Nature. PubMed Scopus Google Scholar, M. P. W. F. 1990; PubMed Scopus (195) Google Scholar, W.J. A. J. 1990; Full Text PDF PubMed Google of the of the NT of α1C is in the long-NT of the molecular mechanism of PKC modulation of the long-NT expressed in Xenopus the as crucial for PKC modulation T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). was to the for PKC action T. Y. N. T. J. 1999; Full Text Full Text PDF PubMed Scopus (51) Google Scholar). N-terminal isoforms of α1C have also been in the human The of the human α1C a the of the of α1C human long-NT to the long-NT Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, N. C. C. P. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). The previously human the of the a of N.M. 1994; PubMed Scopus Google Scholar). used by A. N.M. H. 1995; PubMed Scopus Google and M. N.M. M. Mol. Pharmacol. PubMed Scopus Google Scholar), is not by PKC, it not the segment crucial for the enhancement of The long-NT isoform of human L-VDCC, the crucial is enhanced by Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). We that it be to the enhancing effect of AngII and ACh on L-VDCC in a heterologous expression the long-NT isoform of reconstitution of molecular mechanisms of L-VDCC modulation by we demonstrate the reconstitution of the enhancing effect of Gq-coupled receptors on L-VDCC in Xenopus oocytes. The of modulation are The initial segment of the is crucial for AngII- and ACh-induced enhancement of the Ca2+ channel The activation of Gq, and the subsequent activation of PKC, are The long human isoform of α1C is in a to the long the human isoform currents that are only by AngII and were and as N. in Scholar). were 1 of the of α1C mutants 1 of m1R of and for in 1 1 cell currents were the the voltage in a and to T. T. M. N. J. Physiol. 1995; Scopus Google Scholar). Ca2+ currents were in the but of of AngII and ACh were in and to the a concentration of 1 and for the AngII currents were by to from a potential of and were as H. M. A. M. Y. S. Mol. Pharmacol. 2001; PubMed Scopus Google Scholar, N. J. Physiol. 1997; PubMed Scopus Google Scholar). In was in and in were of and in for to were of and in for were in for In most oocytes were of were from and of and were as M. F. N. PubMed Scopus Google Scholar). The heart α1C mutants used were in as T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of human isoform N.M. A. H. J. 1995; Full Text Full Text PDF PubMed Scopus Google was as Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of human long-NT isoform was in as Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). m1R is in and are in J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The were a of the of the and of in the of the N. in Scholar). and are as of cells the of in current the were as in was to the basal of were and were for of of different in the were several was analysis of by the Reconstitution of of L-type Ca2+ in Xenopus have previously that the long-NT isoform of cardiac L-VDCC, expressed in Xenopus is by PKC activators as in cardiac and smooth muscle initial increase in current via the channels by a T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In to the modulation of channel by we expressed the receptors and m1R in the of cardiac α1C long-NT A. K. T. T. Y. H. S. S. Nature. PubMed Scopus Google Scholar), and also Xenopus oocytes were the and currents were the voltage The m1R was for it is to be a Gq-coupled and it to be well expressed in oocytes. The m3R effects not The expression of m1R and was by currents that following activation of the receptor The of is to the activation of the Gq signaling to release of Ca2+ from intracellular stores and the activation of channels found in the oocytes N. Rev. Biochem. PubMed Scopus Google Scholar). the of currents oocytes were of to current T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). We not in the effects of AngII ACh the and were was by to from a potential of the current to was for and of ACh AngII in oocytes that expressed the channel not in In in oocytes that expressed the channel and the both ACh and AngII increase in the of a The enhancement of by AngII was of 1 is to the in native tissues the of declined the several below the initial and also in the of the the has not been not The of not even long of and of AngII not increase The of the as well as on physiological The of the enhancement of the current on the type of receptor to in the of the of the in the channel was expressed in Currents of the and the of expressed as of initial in the cell of the are The increase in by both transmitters was and The was and the in a following the was also The of the is for by a Gq-coupled PKC activators the initial segment of the was to be crucial for the enhancement of the current T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. N. T. J. 1999; Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the effect of ACh and AngII is mediated by PKC, the initial segment of the also be crucial for different deletion mutants of α1C were used in the are from the and respectively). that expressed the the and the m1R, were to a of to In was the current enhanced as a of receptor activation by the only a was following activation and The of the was in the deletion mutants the that are crucial for of the channel by PKC are also for the enhancement by the m1R in In the in the current to be a of the of the of by PKC was to on the in the long-NT isoform of α1C by Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The is the initial of the in the isoform of α1C we the long-NT human and not the to be in a to the long-NT was in oocytes the isoform of human α1C and m1R, was a of from the initial we a increase in oocytes the human long-NT a in the current the long and the isoform and effects were in oocytes not receptor not of Gq to phospholipase C and the to Ca2+ release and PKC is involved in the modulation we have expressed a mutated receptor in the of the C were mutated to mutated receptor was to Gq J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). expressed the long-NT channel the and the receptor the mutated current to AngII was in oocytes in the of Ca2+ and the of of to and that activation of receptor not elevate Ca2+ in the oocytes. The enhancement in the current via L-type Ca2+ channel long-NT α1C) following AngII was present only in oocytes the and only a was AngII was enhanced by in oocytes the receptor but was by in oocytes the mutated a of was for the of PLC, we used the were both and to current of oocytes the channel long-NT α1C) and m1R in a of the enhancement as oocytes and The currents and was m1R and and and increase in oocytes a and in respectively). In the was not by it to be the enhancement was the in demonstrate the of Gq and the cascade in of the long-NT isoform of We also the effect of on oocytes the deletion in the in we only a and enhancement The was even in in in oocytes that enhancing effect of was present in the the of the enhancement the mechanism by ACh inhibits PKC in of the the of PKC in the modulation via the receptors, we used PKC is a of PKC. It inhibits PKC by the P. H. P. T. M. P. F. J. Full Text PDF PubMed Google Scholar, G. H. G. H. C. J. 1993; Full Text PDF PubMed Google Scholar). the enhancement of the current by AngII via ACh via m1R but not the and a protein kinase a of action that only PKC M. S. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. Y. M. F. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), the increase in the current by ACh activation of it also most of the in the current PKC inhibits only not the m1R modulation not of Ca2+ on AngII of the of M. N.M. M. Mol. Pharmacol. PubMed Scopus Google reported that AngII-induced inhibition of L-VDCC in Xenopus oocytes the human isoform of α1C was by the oocytes to the M. N.M. M. Mol. Pharmacol. PubMed Scopus Google to that the AngII-induced in was mediated via the release to be that Gq is not involved in the long-NT is that the mechanisms of the in are not in and present we it the and of effect are in both isoforms, and of Ca2+ modulation in both isoforms is a in M. N.M. M. Mol. Pharmacol. PubMed Scopus Google the of that we have used we expressed the isoform and the to the oocytes to current concentration of of and a concentration of The inhibition was the of was in to the of The AngII-induced inhibition of was in oocytes of both and in oocytes the concentration The of the AngII-induced inhibition was by the effects of AngII in oocytes the long-NT isoform of the channel and AT1R, was used as the In as the increase in the current in to was by a that was 1, C and of M. N.M. M. Mol. Pharmacol. PubMed Scopus Google and a Ca2+ of the inhibitory effect. However, not the that is not mediated by the increase in Ca2+ but by a different mechanism that the of basal levels of Ca2+ is by Ca2+ we expressed the isoform the mutated receptor Gq and not elevate and the and concentration The inhibition of the current was in oocytes we expressed the isoform of α1C and and the oocytes the concentration of Ca2+ used of the The in the current was only in oocytes the concentration but it was the the type the of in The by activation of in the long-NT isoform current was the concentration of demonstrate modulation of L-type Ca2+ channel by a and a (AngII) to activate Gq and L-type Ca2+ currents in heart and/or smooth m1R were expressed in Xenopus oocytes in the long-NT and of the in enhancement of the current that over and was by a below baseline The of the Ca2+ channel is in the of activation of Gq, as well as and PKC. The initial of the to be important for PKC is to be crucial for human long isoform of α1C to the long The enhancement of L-VDCC currents by AngII and ACh, has been in a heterologous expression for the a for of reconstitution of the of Gq was of PLC, of Gq the enhancement of the expression of a Gq coupling, not a current increase the of Gq in the of that the enhancement of Ca2+ channel current by the transmitters studied was mediated by PKC. of PKC the of of the different PKC inhibitors that we and have both the enhancing effect of AngII and The that the and AngII-induced increase in Ca2+ channel current is mediated by PKC is also by the of modulation in mutants lacking the initial of the NT, to be crucial for the PKC and in the these have been previously to the enhancing of modulation by a phorbol T. Y. N. T. J. 1999; Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism by PKC enhances the current is not PKC enhances and and channels are by PKC different in the and the inhibition of the channel by J. Nature. 1997; PubMed Scopus Google Scholar). In the mechanism is the crucial initial of NT the are by PKC T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. N. T. J. 1999; Full Text Full Text PDF PubMed Scopus (51) Google Scholar). modulation by PKC be activated PKC a segment of the channel that the PKC the of the channel to the to increase in the The present study the the modulation of L-VDCC by Gq- and Despite the enhancing action of AngII in cardiac and smooth muscle cells and of ACh in smooth muscle cells, effect not be in heterologous expression that the in the different modulation of isoforms of α1C by these the long-NT isoform of long in and but only in is by PKC in the oocytes Y. N. G. T. N. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google it is not that only a in the current following is the isoform used in the of PKC and AngII modulation of human L-type channels expressed in Xenopus oocytes A. N.M. H. 1995; PubMed Scopus Google Scholar, M. N.M. M. Mol. Pharmacol. PubMed Scopus Google Scholar). to study PKC modulation of L-type Ca2+ channels be the of heterologous expression systems lacking of the for the T. Sci. S. A. 2000; PubMed Scopus Google reported that activation of PKC only L-type Ca2+ current via channels on long-NT α1C expressed in that cells be lacking the PKC present we not PKC is involved in the of AngII- and ACh-induced enhancement in Xenopus it is a the increase is not by of intracellular Ca2+ and by of PKC 1997; PubMed Scopus Google Scholar). The Xenopus the well enhancing effect of the ACh and AngII, be the of for molecular of type of regulation of the L-type The inhibitory effect of AngII and ACh was not in the of as it is not it is physiologically such inhibition is receptors in cardiac and smooth muscle The molecular mechanism of effect is it is different from that of the The in was Gq- and PLC-independent it was by the AngII receptor and the not The of in in oocytes that of the type but the effects were to activate signaling in of J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The AngII-induced inhibition via such a current is also of PMA, PKC T. Y. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. F. N. 1992; PubMed Scopus Google Scholar). It is to these by that the proposed pathway to the activation of a PKC to is by the that a potent PKC the protein kinase be involved is a kinase M. S. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. Y. M. F. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), but not the inhibitory effect of the inhibition by and by AngII be mediated by different The in current via long-NT channels reported by T. Sci. S. A. 2000; PubMed Scopus Google in cells was by and and the that these are by PKC and that the T. Sci. S. A. 2000; PubMed Scopus Google Scholar). In in the and ACh-induced remains in lacking the that the It that in in cells and in oocytes are mediated by different molecular The inhibition of the current via the human α1C isoform by AngII was Ca2+-dependent, but it was not We that basal levels of intracellular Ca2+ are crucial for of the in the is that the inhibition on of the of C of the of basal Ca2+ levels R.D. A. H. H. R.W. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). are to the mechanism of the AngII- and ACh-induced inhibition and to it is in cardiac and smooth muscle We M. for of m1R, N. and for the of and for of the