Dynamic Targeting of the Agonist-stimulated m2 Muscarinic Acetylcholine Receptor to Caveolae in Cardiac Myocytes

In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand 3Hquinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that 3HQNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this 3HQNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the 3HQNB binding detected in the low density fractions. Moreover, the 3HQNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells. In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand 3Hquinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that 3HQNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this 3HQNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the 3HQNB binding detected in the low density fractions. Moreover, the 3HQNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells. The activation of a muscarinic acetylcholine receptor (mAChR) 1The abbreviations used are: mAchR, muscarinic acetylcholine receptor(s); GPR, G protein-coupled receptor; β-AR, β-adrenergic receptor; eNOS, endothelial isoform of nitric-oxide synthase; NO, nitric oxide; ARVM, adult rat ventricular myocytes; QNB, 1-quinuclidinyl benzilate; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; MBS, Mes-buffered saline; PVDF, polyvinylidene difluoride; TBST, Tris-buffered saline with Tween 20; PAGE, polyacrylamide gel electrophoresis. triggers a number of signal transduction pathways that, in the heart, may elicit both positively and negatively inotropic and chronotropic effects (1Korth M. Kühlkamp V. Pfluegers Arch. Eur. J. Physiol. 1985; 403: 266-272Crossref PubMed Scopus (47) Google Scholar, 2Eglen R.M. Montgomery W.W. Whiting R.L. J. Pharmacol. Exp. Ther. 1988; 247: 911-917PubMed Google Scholar). Recent studies have shown that, of the five mAchR subtypes identified to date, only the m1 and m2 subtypes are expressed in adult mammalian cardiac tissues (3Gallo M.P. Alloatti G. Eva C. Oberto A. Levi R.C. J. Physiol. 1993; 471: 41-60Crossref PubMed Scopus (73) Google Scholar, 4Sharma V.K. Colecraft H.M. Wang D.X. Levey A.I. Grigorenko E.V. Yeh H.H. Sheu S.-S. Circ. Res. 1996; 79: 86-93Crossref PubMed Scopus (67) Google Scholar). According to these reports, the m2 mAchR, which is expressed at a much higher level than the m1 mAchR, triggers the inhibitory response while m1 receptor activation elicits, when stimulated by higher concentrations of agonist, a compensatory excitatory effect on heart function. Therefore, distinct downstream signaling cascades must be involved following m1 and m2 mAchR activation. Both m1 and m2 receptor subtypes also have been reported to undergo translocation into specific subcompartments derived from the plasma membrane (5Harden T.K. Petch L.A. Traynelis S.F. Waldo G.L. J. Biol. Chem. 1985; 260: 13060-13066Abstract Full Text PDF PubMed Google Scholar, 6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar, 7Ho A.K.S. Zhang Y.-J. Duffield R. Zheng G.-M. Cell. Signalling. 1991; 3: 587-598Crossref PubMed Scopus (6) Google Scholar, 8Svoboda P. Milligan G. Eur. J. Biochem. 1994; 224: 455-462Crossref PubMed Scopus (38) Google Scholar, 9Goldman P.S. Schlador M.L. Shapiro R.A. Nathanson N.M. J. Biol. Chem. 1996; 271: 4215-4222Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), a characteristic of many G protein-coupled receptors (GPR) following agonist binding. To date, two major pathways for GPR clustering and sequestration have been reported, which involve plasma membrane modifications that lead to the formation of either clathrin-coated or non-coated vesicles (11Sandvig K. van Deurs B. Trends Cell Biol. 1994; 4: 275-277Abstract Full Text PDF PubMed Scopus (73) Google Scholar). While the human muscarinic cholinergic receptor Hm1 has been shown to internalize via clathrin-coated vesicles (10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), mAchR have also been shown to be internalized through non-clathrin-coated vesicles in human fibroblasts, although the identity of these vesicular structures has not been defined (6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar). Recently, a clathrin-independent sequestration pathway has received attention with the characterization of a population of plasmalemmal vesicles termed caveolae. Caveolae are small flask-shaped invaginations of the plasma membrane characterized by high levels of cholesterol and glycosphingolipids (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar), the principal scaffolding protein of which are the caveolins, 20–24 kDa integral membrane proteins that undergo homo-oligomerization (13Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T.V. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (401) Google Scholar). These specialized lipid microdomains have been shown to play a role in the compartmentation of a number of plasma membrane-linked signal transduction pathways, including those mediated by receptor tyrosine kinases (14Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 15Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). In addition, a recent report by Parton et al. (16Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (299) Google Scholar) provides additional evidence that coalescence and fission of caveolae may be essential for the development of the T-tubular system that is essential for normal intracellular calcium homeostasis and excitation-contraction coupling in cardiac and skeletal muscle. The specific mechanisms involved in receptor sequestration may differ among distinct cellular phenotypes. For example, several reports have proposed the involvement of clathrin-coated pits in the mechanism of internalization of β-adrenergic receptors (β-AR) (17Muntz K.H. Trends Cell Biol. 1994; 6: 356Google Scholar), and yet a recent report indicated that in epidermoid A431 cells, β-AR are clustered within caveolae in response to agonist stimulation (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar). The recent development of antibodies directed against different tissue-specific isoforms of caveolin has permitted a better characterization of caveolar microdomains. Using these antibodies in immunoprecipitation experiments, we have recently shown that eNOS, the constitutively expressed isoform of nitric-oxide synthase in cardiac myocytes, is targeted to sarcolemmal caveolae in cardiac myocytes and endothelial cells (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Interestingly, reports from our laboratory and by others have shown that the generation of nitric oxide (NO) is an obligate intermediate step in the signal transduction cascade involved in the m2 mAchR-mediated inhibitory responses of the heart, particularly following β-adrenergic stimulation (20Balligand J.-L. Kelly R.A. Marsden P.A. Smith T.W. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 347-351Crossref PubMed Scopus (632) Google Scholar, 21Balligand J.-L. Kobzik L. Han X. Kaye D.M. Belhassen L. O'Hara D.S. Kelly R.A. Smith T.W. Michel T. J. Biol. Chem. 1995; 270: 14582-14586Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 22Han X. Shimoni Y. Giles W.R. J. Gen. Physiol. 1995; 106: 45-65Crossref PubMed Scopus (144) Google Scholar, 23Han X. Kobzik L. Balligand J.-L. Kelly R.A. Smith T.W. Circ. Res. 1996; 78: 998-1008Crossref PubMed Scopus (116) Google Scholar). Caveolae may, therefore, constitute the structural framework within which this signaling cascade operates. Thus, the dynamic targeting of agonist-stimulated muscarinic cholinergic receptors to caveolae in cardiac myocytes could facilitate the activation of eNOS, which we have shown to be quantitatively and specifically associated with caveolin-3, the muscle-specific isoform of caveolin (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Way M. Parton R.G. FEBS Lett. 1996; 378: 108-112Crossref PubMed Scopus (59) Google Scholar, 25Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The in caveolae of this isoform with proteins to including a and proteins T. J. Cell Biol. 1993; PubMed Scopus Google Scholar), as well as with G proteins (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), that these plasmalemmal microdomains may constitute a for the and of the signaling proteins involved in the muscarinic cholinergic pathway in heart muscle. In this report, we to the that m2 mAchR are targeted to plasmalemmal caveolae agonist stimulation in adult rat ventricular Using a detergent-free method for caveolae isolation by isopycnic centrifugation, we evidence that the m2 mAchR, after agonist with caveolin-3 and eNOS. Furthermore, we that the m2 mAchR can be specifically immunoprecipitated from these caveolin-enriched fractions using antibodies directed against adult rat ventricular myocyte primary cultures were on and for in a defined as reported previously (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). were either with or carbachol and on in and myocytes were in the of or carbachol treatment. cells were with saline to of was by the of in specific 3Hquinuclidinyl binding levels in of ARVM, or not with a muscarinic agonist or were in a of and by using a to a method from Song et al. K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The was to by of a in Mes, and at the of a gradient containing for an The gradient was in fractions to concentrations and and the intermediate was with proteins were and on and to a membrane with in Tris-buffered saline with Tween were with the primary antibody for in containing the were for with a antibody at a in containing five additional the were in TBST, with a to the and exposed to was determined by with to facilitate the in as previously P.A. J. Cell Biol. 1996; PubMed Scopus (116) Google Scholar). at for by with was at using a binding was determined as O. M. T. J. Pharmacol. PubMed Scopus Google binding was in the of were on with and the was determined in a and binding are expressed as of of and of specific The gradient fractions at were to and and of the different fractions were with 3HQNB at for binding was determined in the of were in and by filtration on or by an immunoprecipitation from those in J. Mol. Pharmacol. 1988; Google and A.I. J. 1991; PubMed Google Scholar). For these immunoprecipitation experiments, the binding also and 3HQNB binding was determined by the of the immunoprecipitation in the of of the receptors with an antibody directed against the m2 mAchR and were by centrifugation, with containing and CHAPS, and in was used for the immunoprecipitation with the caveolin-3 antibody that binding and did not contain The isoform and of of the caveolin (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Way M. Parton R.G. 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Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, E.J. Ying C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), as well as in of and proteins into caveolae T.V. E. P. Trends Cell Biol. 1995; Full Text PDF PubMed Scopus Google Scholar). Therefore, for caveolae from cardiac myocytes, we have a detergent-free method on the to of caveolin by and on the of cellular membrane by (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, after of in a the was to and at the of a gradient for an of the different fractions were by and with or The in that the of caveolin-3 and eNOS in ventricular myocytes in fractions and which to the of eNOS and caveolin-3 is in with our on the of these two proteins from cardiac myocyte (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar) and on the of eNOS and in endothelial cells Smart E.J. Z. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The gradient fractions were also for protein as well as for the of as a P.A. J. Cell Biol. 1996; PubMed Scopus (116) Google Scholar), and for the level of specific binding O. M. T. J. Pharmacol. PubMed Scopus Google Scholar), as a specific of a at the sarcolemmal of cardiac shown by the of of these the gradient the of cellular protein that at the high density to and sarcolemmal The small of caveolin-3 and eNOS associated with these high density fractions is to of both proteins with the L. O. Kaye D.M. Michel T. Smith T.W. Kelly R.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) or to to density gradient the effects of a muscarinic cholinergic agonist, on the of mAchR using the to a in receptor was by agonist binding. The following were on primary cultures of exposed to carbachol for myocytes were and to isopycnic on a of the different fractions were with a muscarinic at for In a of experiments, were on shown in in from myocytes, the binding of 3HQNB is only detected in the fractions. In following carbachol of the 3HQNB binding can be in the fractions and which to the caveolin-enriched The of the 3HQNB binding in fractions and binding to sarcolemmal muscarinic translocation of muscarinic receptors in cardiac The of muscarinic receptors in is determined by the of specific 3HQNB binding detected by on or by immunoprecipitation with antibodies control and carbachol are by and In the in of carbachol was by a with or a with For binding was determined in the of The are expressed as the of specific 3HQNB binding and are of those in to In a of experiments, we used a to the in 3HQNB binding. The different fractions after isopycnic were immunoprecipitated using an m2 mAchR and the of specific 3HQNB binding in was shown in the of of m2 mAchR is to that from the 3HQNB binding to a of 3HQNB m2 mAchR the low density fractions when myocytes have been exposed to carbachol. of the 3HQNB binding is detected in the caveolar fractions and Importantly, when are with carbachol the of the m2 mAchR in fractions and is indicating the of the clustering Interestingly, in a Raposo et al. (6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar) reported that of human fibroblasts, either with a muscarinic cholinergic agonist or with the muscarinic cholinergic atropine, the of the Hm1 mAchR into specific of the plasma caveolae, and that only with the agonist lead to the receptor Furthermore, and Lameh (10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) using that the Hm1 mAchR, after agonist are internalized via clathrin-coated vesicles in cells with the Hm1 with the reported these results that the and the of receptor compartmentation in response to agonist stimulation may be by both the receptor subtype and the in which is In our is that clustering of the m2 mAchR into pits can the in mAchR into density evidence from the indicates that the density of clathrin-coated pits is higher than that of caveolae M.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and not the of of muscarinic receptors in and B. Furthermore, when myocytes are with a to K. S. Deurs J. Cell Biol. 1987; PubMed Scopus Google Scholar), a of m2 mAchR into caveolin-enriched fractions is detected To the dynamic targeting of muscarinic receptors to caveolae in cardiac myocytes, we used a caveolin-3 antibody to caveolar and the m2 mAchR by radioligand binding In these cardiac myocytes either with or carbachol were and on and the fractions to caveolae were and with with either an antibody or a IgG1 antibody and were by centrifugation, and was determined in a in in the of carbachol was immunoprecipitation of 3HQNB binding by caveolin-3 antibodies the level of 3HQNB binding was to that when using the IgG1 for the immunoprecipitation. In following agonist a of specific 3HQNB binding can be immunoprecipitated by antibodies in caveolin-3 was after carbachol In of the 3HQNB binding in fractions and by direct filtration on could be after immunoprecipitation. on fractions which to the of plasma membrane of protein when did not specific 3HQNB binding in the caveolin-3 in with the low of caveolin-3 in these fractions Importantly, in myocytes with carbachol in the of the muscarinic atropine, the 3HQNB binding immunoprecipitated by antibodies at the level detected in a control immunoprecipitation with a is in with the shown in in which binding was detected in the mAchR from caveolar fractions of myocytes with carbachol in the of these that the m2 mAchR to plasmalemmal caveolae of cardiac myocytes following agonist binding. The dynamic targeting of the m2 mAchR to caveolae has for muscarinic receptor as well as for the of eNOS activation. several have reported evidence for the translocation to low density gradient fractions of the muscarinic receptors agonist stimulation (5Harden T.K. Petch L.A. Traynelis S.F. Waldo G.L. J. Biol. Chem. 1985; 260: 13060-13066Abstract Full Text PDF PubMed Google Scholar, 6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar, 7Ho A.K.S. Zhang Y.-J. Duffield R. Zheng G.-M. Cell. Signalling. 1991; 3: 587-598Crossref PubMed Scopus (6) Google Scholar), to our in the that the specific of these The and and also O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google and Smart E.J. Z. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) of eNOS, and the agonist-stimulated m2 mAchR in isopycnic which than of the of that caveolae are the structural for these with that, in A431 cells, β-AR are within caveolae in response to agonist stimulation (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar), our that clathrin-coated formation can be as the pathway for clustering G protein-coupled receptors within specialized plasmalemmal microdomains. The of caveolar β-AR and mAchR is is not caveolae from the plasma membrane and lead to this is the that pathways of receptor internalization may in cells. While studies the sequestration of G protein-coupled receptors after agonist stimulation as a for a of C. E. E. J. Pharmacol. Exp. Google Scholar), the in this the that, following stimulation by agonist, cardiac m2 mAchR translocation to caveolae may be to initiate specific downstream signaling Interestingly, several recent studies have shown that internalization of the m2 and mAchR is mediated by mechanisms distinct from the by the G protein-coupled receptor to lead to receptor R. Y. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, K.H. van Mol. Pharmacol. 1996; Google Scholar). The translocation of muscarinic receptors within caveolae with the G protein to be within these plasmalemmal microdomains (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and after of and intermediate to the activation of eNOS, a caveolar protein in cardiac of caveolin-enriched fractions to additional signaling involved in the muscarinic cholinergic stimulation of the pathway in cardiac myocytes is in our The caveolar compartmentation for the muscarinic cholinergic pathway may as a for G protein signaling cascades that are targeted to caveolae.