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• W2022992564 abstract "The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A1 receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 ± 5% of adenosine A1receptors were isolated with caveolae. However, incubation with the adenosine A1 receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A1 receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A2areceptor agonist did not alter the localization of A1receptors to caveolae. These data suggest that the translocation of A1 receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A1 receptors are associated with few direct effects. The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A1 receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 ± 5% of adenosine A1receptors were isolated with caveolae. However, incubation with the adenosine A1 receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A1 receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A2areceptor agonist did not alter the localization of A1receptors to caveolae. These data suggest that the translocation of A1 receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A1 receptors are associated with few direct effects. 2-chloro-N 6-cyclopentyladenosine polyacrylamide gel electrophoresis endothelial nitric-oxide synthase protein kinase C 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine 8-cyclopentyl-1,3-dipropylxanthine Adenosine has multiple and profound effects on cardiac function. Adenosine increases coronary blood flow and exerts direct negative chronotropic and dromotropic effects (1.Belardinelli L. Shryock J.C. Song Y. Wang D. Srinivas M. FASEB J. 1995; 9: 359-365Crossref PubMed Scopus (249) Google Scholar). There is also significant evidence that adenosine reduces both reversible and irreversible myocardial ischemic-reperfusion injury (2.Lasley R.D. Pelleg A. Belardinelli L. Effects of Extracellular Adenosine and ATP on Cardiomyocytes. R. G. Landes Co., Austin, TX1998: 133-172Google Scholar). The cardiac effects of adenosine are mediated by the activation of specific extracellular adenosine receptor subtypes, A1, A2a, and A3, located on vascular smooth muscle, coronary endothelial cells, and cardiac myocytes (1.Belardinelli L. Shryock J.C. Song Y. Wang D. Srinivas M. FASEB J. 1995; 9: 359-365Crossref PubMed Scopus (249) Google Scholar). The adenosine A1 receptor subtype is localized primarily on cardiac myocytes (1.Belardinelli L. Shryock J.C. Song Y. Wang D. Srinivas M. FASEB J. 1995; 9: 359-365Crossref PubMed Scopus (249) Google Scholar). The adenosine A1 receptor is a 36-kDa, seven-transmembrane domain protein localized to the sarcolemma (3.Linden J. FASEB J. 1991; 5: 2668-2676Crossref PubMed Scopus (218) Google Scholar). Agonist binding induces a conformational change in the receptor, which facilitates its interaction with an inhibitory guanine nucleotide-binding protein (Gi) (3.Linden J. FASEB J. 1991; 5: 2668-2676Crossref PubMed Scopus (218) Google Scholar). With the exception of the ferret, adenosine A1 receptor occupation does not significantly alter contractility, intracellular calcium, or cAMP concentration in normal mammalian ventricular myocardium (4.Song Y. Belardinelli L. Am. J. Physiol. 1996; 271: C1233-C1243Crossref PubMed Google Scholar). However, A1 receptor occupation does reduce β-adrenergic receptor-induced increases in all of the above parameters. Although the anti-adrenergic effect of adenosine is well studied, evidence of other signal transduction mechanisms mediated by adenosine A1receptor occupation in ventricular myocardium is lacking. The fact that other receptors in ventricular myocytes, which presumably couple to the same Gi protein, produce direct effects (5.Brown J.H. Buxton I.L. Brunton L.L. Circ. Res. 1985; 57: 532-537Crossref PubMed Google Scholar, 6.Minshall R.D. Nakamura F. Becker R.P. Rabito S.F. Circ. Res. 1995; 76: 773-780Crossref PubMed Scopus (82) Google Scholar, 7.Barry W.H. Matsui H. Bridge J.H. Spitzer K.W. Adv. Exp. Med. Biol. 1995; 382: 31-39Crossref PubMed Scopus (14) Google Scholar, 8.Hilal-Dandan R. Merck D.T. Lujan J.P. Brunton L.L. Mol. Pharmacol. 1994; 45: 1183-1190PubMed Google Scholar) raises the question of why adenosine A1 receptor activation is associated with so few effects. One explanation for the lack of direct adenosine A1 receptor effects may be the relatively low receptor density in ventricular myocardium compared with atrial tissue (9.Musser B. Morgan M.E. Leid M. Murray T.F. Linden J. Vestal R.E. Eur. J. Pharmacol. 1993; 246: 105-111Crossref PubMed Scopus (38) Google Scholar). Another possibility is that the receptor is inefficiently coupled to Gi proteins and second messengers. Coupling efficiency may be due, in part, to the grouping of receptors and second messengers in microdomains for rapid and selective activation or deactivation of intracellular signaling mechanisms. Caveolae are cholesterol/sphingomyelin-rich plasma membrane microdomains that have been shown to serve as a scaffold for receptors and second messengers (10.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar). Caveolae are present in most cell types and are abundant in cardiomyocytes (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). There are several reports that G protein-coupled receptors redistribute to caveolae in response to receptor occupation (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 12.Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Crossref PubMed Scopus (170) Google Scholar, 13.Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar). In addition, other studies have documented that specific second messenger systems, including heterotrimeric G proteins, are concentrated in caveolae (for review, see Ref. 10.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar). Although the role of caveolae in cardiac signal transduction has not been well studied, it has been reported that muscarinic M2 cholinergic receptors in rat ventricular cardiomyocytes localize to caveolae after agonist stimulation (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Because adenosine and acetylcholine exert similar effects in ventricular myocardium (14.Linden J. Hollen C.E. Patel A. Circ. Res. 1985; 56: 728-735Crossref PubMed Scopus (91) Google Scholar), we hypothesized that stimulation of adenosine A1 receptors would promote translocation of the receptors into caveolae. Surprisingly, we found that in rat ventricular cardiomyocytes, adenosine A1 receptors were associated with caveolae in unstimulated cells, whereas activation of the adenosine A1 receptors induced the receptors to translocate out of caveolae. Mouse IgGs directed against caveolin-3, clathrin, and eNOS were obtained from Transduction Laboratories (Lexington, KY). Rabbit IgGs directed against the adenosine A1 receptor were supplied by Alpha Diagnostic International (San Antonio, TX) and Chemicon International (Temecula, CA). Mouse IgG directed against the human transferrin receptor was supplied by Zymed Laboratories Inc. (San Francisco, CA). Horseradish peroxidase-conjugated IgGs were supplied by Cappel (West Chester, PA). Super Signal® chemiluminescent substrate was purchased from Pierce. The cholesterol determination kit was from Wako (Richmond, VA). [3H]Acetate (5.21 Ci/mmol) were supplied by Amersham Pharmacia Biotech. Adenosine receptor agonists (CCPA,1 CGS 21680) and antagonist (DPCPX) were obtained from Research Biochemicals International (Natick, MA). Adenosine deaminase and dimethyl sulfoxide (Me2SO) were obtained from Sigma. All animals in this study received humane care according to the guidelines set forth by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication 86-23, revised 1996). Ventricular myocytes were dissociated from adult, male Sprague-Dawley rats (350–400 g) using collagenase and hyaluronidase as described previously (15.Narayan P. Valdivia H.H. Mentzer R.M. Lasley R.D. J. Mol. Cell Cardiol. 1998; 30: 913-921Abstract Full Text PDF PubMed Scopus (11) Google Scholar). The resulting cell suspension was washed twice with enzyme-free buffer, and suspended in normal HEPES buffer (1.0 mm CaCl2, pH = 7.4) to a final concentration of ∼1 mg/ml. Typically, over 70% of the cells were rod-shaped, trypan blue-excluding ventricular myocytes. Caveolae were isolated as described previously (16.Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Ten to 15 μg of purified caveolae were isolated from three rat heart preparations with this method. Alkaline phosphatase was measured by the method of Engstrom (17.Engstrom L. Biochim. Biophys. Acta. 1961; 52: 36-48Crossref PubMed Scopus (98) Google Scholar). Galactosyltransferase and NADPH-cytochromec reductase were assayed using methods adapted from Graham (18.Graham J. Higgins J. Methods in Molecular Biology. 1993; 19Google Scholar). Lactate dehydrogenase was measured with a standard kit from Sigma. The mass of cholesterol was determined as described previously (19.Carr T.P. Andresen C.J. Rudel L.L. Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (485) Google Scholar). Cellular fractions were processed as described previously (16.Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Three groups (n = 2–3 experiments/group) of cells were studied. To provide enough protein sample for the fractionation of cell lysates, the cardiomyocyte yield from three hearts was combined in a single experiment. All myocyte suspensions were performed in the presence of adenosine deaminase (2 units/ml) to degrade endogenous adenosine. Control myocytes were suspended at room temperature in 7 ml of HEPES buffer (∼3–4 mg of protein/ml) with periodic mixing. A second group of myocytes was incubated with the adenosine A1 receptor agonist 2-chloro-N 6-cyclopentyladenosine (CCPA, 200 nm) for 15 min. The suspension was then centrifuged (1000 × g), and the pellet was washed three times with normal HEPES buffer (10 ml). After the final wash, the myocyte pellet was processed to isolate caveolae as described above. Group 3 myocytes were initially pretreated with the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 200 nm) for 5 min prior to the addition of CCPA (200 nm). One additional experiment was performed in which myocytes were incubated with the adenosine A2a receptor agonist 2-p-(2-carboxyethyl)- phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS 21680, 100 nm) as described for CCPA. Immunogold localization of caveolin-3 (20 nm gold) and A1 receptors (5 nm gold) was performed using whole mount plasma membrane preparations as described previously (20.Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y.-S. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). We defined the presence of caveola in ventricular myocytes by an established isolation procedure (16.Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In brief, ventricular myocytes were lysed and a postnuclear supernatant generated. The postnuclear supernatants were separated into cytosol, plasma membrane, and intracellular membrane (endoplasmic reticulum, Golgi, etc.) fractions by centrifugation in Percoll. The plasma membranes were then sonicated and fractionated by density gradient centrifugation to isolate caveolae. Similar to other cell type studies, the caveola fraction from ventricular myocytes contained less than 0.4% of the protein found in the initial postnuclear supernatant fraction (TableI).Table ICharacterization of rat ventricular cardiomyocyte subcellular fractionsSubcellular fractionProteinGalactosyltransferaseNADPH- cytochromec reductaseμg%%Postnuclear supernatant3210 ± 123100 ± 7.0100 ± 9.3Cytosol1502 ± 638 ± 0.516 ± 7.4Intracellular membranes1463 ± 8691 ± 8.282 ± 9.6Plasma membranes192 ± 261.1 ± 0.21.8 ± 0.4Caveola membranes9 ± 1.8NDNDVentricular cardiomyocytes were subfractionated as described under “Experimental Procedures.” The amount of protein in each subcellular fraction was determined by Lowry assay. Galactosyltransferase and NADPH-cytochrome c reductase activities were measured in each fraction and normalized with respect to the values obtained for the postnuclear supernatant fractions. ND, level of enzymatic activity was below the sensitivity of the assay. Presented data are the mean ± S.E. from three independent experiments, n = 3. Open table in a new tab Ventricular cardiomyocytes were subfractionated as described under “Experimental Procedures.” The amount of protein in each subcellular fraction was determined by Lowry assay. Galactosyltransferase and NADPH-cytochrome c reductase activities were measured in each fraction and normalized with respect to the values obtained for the postnuclear supernatant fractions. ND, level of enzymatic activity was below the sensitivity of the assay. Presented data are the mean ± S.E. from three independent experiments, n = 3. The most probable contaminates of the plasma membrane and caveola fractions are the endoplasmic reticulum and Golgi. Therefore, we measured the amount of NADPH-cytochrome reductase (endoplasmic reticulum) and galactosyltransferase (Golgi) activity in each of the subcellular fractions (Table I). The majority of the activities were associated with the intracellular membranes. The plasma membrane contained less than 2% of the total postnuclear supernatant activities, and the caveola fraction did not contain any detectable NADPH cytochrome reductase or galactosyltransferase activity. To ensure that the caveola fraction was not contaminated with other plasma membrane domains, 5 μg (Fig.1 A) or 3% (Fig.1 B) of each subcellular fraction was resolved by SDS-PAGE and immunoblotted for various caveola and non-caveola proteins. The equal protein loads in Fig. 1 A illustrate the relative enrichment of each protein in the caveola fraction, whereas the proportional protein loads in Fig. 1 B illustrate total protein distribution. Caveolin-3, a marker for muscle caveolae, was highly enriched in the caveola fraction. In addition, eNOS, which has been shown to directly interact with caveolin (21.Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), was also enriched in the caveola fraction. The non-caveola proteins, clathrin and transferrin, were excluded from the caveola fraction. The yield of caveolin-3 and eNOS in the caveola fraction was determined by immunoblot analysis in the linear range of detection (data not shown). The estimated yield, with respect to the plasma membrane fraction, of caveolin-3 was 61 ± 6% and eNOS was 69 ± 8%. Caveolae are highly enriched in cholesterol (16.Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). To further characterize the myocyte caveola fraction, we used an enzymatic kit to determine the mass of cholesterol associated with each fraction. Fig.2 demonstrates that the caveola fraction, which contained 24 μg of cholesterol, was 7.4-fold more enriched in cholesterol than the plasma membrane fraction, which contained 79 μg of cholesterol. The subcellular distribution of A1receptors under basal conditions was determined in ventricular myocytes incubated in HEPES buffer supplemented with adenosine deaminase. Three percent of each subcellular fraction was analyzed for the presence of A1 receptors by SDS-PAGE and immunoblotting. Under these conditions, A1 receptors were highly concentrated (67 ± 5% with respect to the plasma membrane) in the caveola fraction (Fig. 3 A, Buffer). Similar results were obtained in two other groups of control myocytes. We next determined the subcellular localization of A1receptors after incubating myocytes with the A1 receptor agonist CCPA. Treatment with CCPA caused the quantitative removal of A1 receptors out of the caveola fraction (Fig.3 A, CCPA). Similar results were obtained when this treatment was repeated in additional myocytes. Quantification of the immunoreactive bands demonstrated that greater than 90% of the signal originally detected in the caveola fraction was now detected in the plasma membrane fraction (data not shown). To verify that this caveola to plasma membrane translocation was selective for A1 receptors, two additional protocols were performed. In the first, pretreatment of myocytes with the A1 receptor antagonist DPCPX prior to addition of CCPA prevented the redistribution of the receptor (Fig. 3 A, CCPA +DPCPX). Finally, treatment of cells with the A2aagonist CGS 21680 did not affect the caveola localization of the A1 receptor (Fig. 3 A, CGS). Immunoblots for caveola and non-caveola marker proteins demonstrated that CCPA treatment did not alter the yield or purity of caveolae (Fig.3 B). Recently, Stan et al. (22.Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (177) Google Scholar) suggested that subcellular fractionation methods to isolate caveolae are contaminated with vesicles with similar biophysical properties as caveolae. Therefore, to verify that A1 receptors are associated with caveolae, we used caveolin-3 IgG to immunoprecipitate caveola membranes from isolated caveolae. Isolated ventricular myocytes were incubated in control or CCPA-supplemented HEPES buffer for 15 min, then processed to immunoprecipitate caveolae with caveolin-3 IgG. The precipitated material and the remaining supernatants were resolved by SDS-PAGE and immunoblotted with IgGs for eNOS, A1 receptor, and caveolin-3. Caveolin-3 co-immunoprecipitated eNOS and the A1 receptor from caveolae isolated from cells treated with buffer (Fig. 4, Buffer). However, caveolin-3 only co-immunoprecipitated eNOS from caveolae isolated from cells treated with CCPA (Fig. 4, CCPA). Approximately 10% of caveolin-3, eNOS, and A1 receptor was not precipitated with caveolin-3 IgG. We have shown by subfractionation and immunoprecipitation that A1 receptors are localized to caveolae. To confirm these findings in an independent manner, A1 receptors were localized by immunoelectron microscopy. Cells were treated with buffer (Fig. 5 A) or CCPA (Fig.5 B) as described above. The membranes were then processed to localize caveolin-3 (arrowheads) and A1receptors (arrows). Caveolin-3 and A1 receptors were found over small invaginations on the membrane surface that had the characteristic appearance of caveolae in buffer-treated cells. However, after CCPA treatment, only caveolin-3 was associated with these small invaginations. This is the first report of adenosine receptor association with caveolae. Under basal conditions, ventricular myocyte A1receptors were localized primarily in caveolae. However, after agonist (CCPA) binding, A1 receptors translocated from the caveolae to the plasma membrane fraction. This effect was blocked by prior treatment with the selective A1 receptor antagonist DPCPX, indicating that this phenomenon is agonist-specific. This unique pattern of receptor localization may explain, in part, the lack of direct effects of A1 receptor in ventricular myocytes. The A1 receptor is a member of the family of G protein-coupled receptors containing a seven-transmembrane-spanning domain. There are several reports that similar G protein-coupled receptors, e.g. angiotensin II (12.Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Crossref PubMed Scopus (170) Google Scholar), bradykinin B2 (13.Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar), and endothelin A (23.Chun M. Liyanage U.D. Lisanti M.P. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11728-11732Crossref PubMed Scopus (326) Google Scholar), and muscarinic receptors (13.Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar) are present in caveolae. However, the present results indicate that, in contrast to these receptors, the A1 receptor translocates out of, rather than into, caveolae with receptor activation. There are at least two differences between ventricular myocyte A1 receptors and these other G protein-coupled receptors, which may explain this unique relationship of A1receptors with caveolae. First, angiotensin II, bradykinin, and endothelin have been reported to increase intracellular calcium and exert direct inotropic effects in ventricular myocytes (5.Brown J.H. Buxton I.L. Brunton L.L. Circ. Res. 1985; 57: 532-537Crossref PubMed Google Scholar, 6.Minshall R.D. Nakamura F. Becker R.P. Rabito S.F. Circ. Res. 1995; 76: 773-780Crossref PubMed Scopus (82) Google Scholar, 7.Barry W.H. Matsui H. Bridge J.H. Spitzer K.W. Adv. Exp. Med. Biol. 1995; 382: 31-39Crossref PubMed Scopus (14) Google Scholar). In contrast, A1 receptor occupation is not associated with these effects (4.Song Y. Belardinelli L. Am. J. Physiol. 1996; 271: C1233-C1243Crossref PubMed Google Scholar). Another difference is the phenomenon of receptor desensitization. Angiotensin II, bradykinin B2, and endothelin A receptors all desensitize within minutes of receptor activation (24.Abdellatif M.M. Neubauer C.F. Lederer W.J. Rogers T.B. Circ. Res. 1991; 69: 800-809Crossref PubMed Scopus (68) Google Scholar, 25.Blaukat A. Abd Alla S.A. Lohse M.J. Muller-Esterl W. J. Biol. Chem. 1996; 271: 32366-32374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26.Cyr C. Kris R.M. J. Cardiovasc. Pharmacol. 1993; 22 Suppl. 8: S11-S14Crossref PubMed Scopus (7) Google Scholar). In fact, it has been hypothesized that receptor movement into caveolae may play a role in this phenomenon (27.de Weerd W.F. Leeb-Lundberg L.M.F. J. Biol. Chem. 1997; 272: 17858-17866Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Cardiac A1 receptors desensitize only after several hours to days of continuous agonist treatment (28.Lee H.T. Thompson C.I. Hernandez A. Lewy J.L. Belloni F.L. Am. J. Physiol. 1993; 265: H1916-H1927Crossref PubMed Google Scholar). It should be noted that movement out of caveolae is also associated with desensitization because agonist-induced translocation of epidermal growth factor receptor out of caveolae is associated with rapid desensitization (29.Mineo 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). Our observation of A1 receptor translocation from caveolae to sarcolemma after agonist treatment is the opposite of that reported for the ventricular myocyte muscarinic M2 receptor (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). This is surprising, given the fact that A1 and muscarinic M2 receptors exert a nearly identical pertussis toxin-sensitive anti-adrenergic effect in ventricular myocytes. However, muscarinic receptors have several properties unique from A1 receptors, which may explain these different associations with caveolae. The muscarinic agonist carbachol, at doses similar to that used by Feron et al. (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), increases inositol phosphate turnover, intracellular Ca2+, and contractility in ventricular myocytes (5.Brown J.H. Buxton I.L. Brunton L.L. Circ. Res. 1985; 57: 532-537Crossref PubMed Google Scholar). In addition, it appears that muscarinic M2 receptors may exert direct effects in ventricular myocytes via coupling to eNOS (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Similar to the observations of Feron et al. (11.Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), we observed in the present study that eNOS is localized in caveolae. Finally, it has been reported that muscarinic M2 receptors can be desensitized with brief agonist exposure (30.Pals-Rylaarsdam R. Hosey M.M. J. Biol. Chem. 1997; 272: 14152-14158Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The A1 receptor antibody used in this study (Chemicon International) was raised against the third extracellular domain of the rat adenosine A1 receptor gene (amino acids 163–176). In addition to the 36-kDa band (the A1 receptor is a 36-kDa protein), the antibody also cross-reacted with a 74-kDa band. Ciruelaet al. (31.Ciruela F. Casado V. Mallol J. Canela E.I. Lluis C. Franco R. J. Neurosci. Res. 1995; 42: 818-828Crossref PubMed Scopus (123) Google Scholar) also observed a 74-kDa band in several tissues (brain, kidney, lung) in several species (rat, pig, lamb) using a rabbit polyclonal antibody raised against the same peptide sequence of the rat A1 receptor. They reported that this band was converted to a 39-kDa form after agonist binding. We observed similar results in the present study, although agonist binding did not convert all of the higher molecular weight band to the lower molecular weight form. The mechanistic explanation for this observation is not known. Identical results were obtained with an Alpha Diagnostic International antibody generated against the same epitope (data not shown). Several lines of evidence support the hypothesis that A1receptor movement between caveolae and sarcolemma is dependent on occupation of the receptor by an A1 agonist. First, all studies were performed in the presence of adenosine deaminase to degrade endogenously released adenosine. The subsequent addition of the A1 receptor agonist CCPA, which is relatively selective for A1 receptors, resulted in the movement of the receptor from caveolae to sarcolemmal membranes. At the concentration used in the present study, there is no evidence that CCPA activates cardiac myocyte A2a or A3 receptors. In addition, the A2a agonist CGS 21680 did not induce A1receptor translocation from caveolae. Finally, DPCPX, which is a highly selective adenosine A1 receptor antagonist (32.Martens D. Lohse M.J. Schwabe U. Circ. Res. 1988; 63: 613-620Crossref PubMed Scopus (34) Google Scholar), completely blocked the effects of CCPA. Since this is the first report of the localization of the A1 receptor in caveolae, it is not known whether A1 receptor translocation out of caveolae activates or terminates signaling. There are reports that inhibitory G protein subunits are localized in caveolae (33.Song K.S. Lisanti M.P. Parenti M. Galbiati F. Sargiacomo M. Cell Mol. Biol. 1997; 43: 293-303PubMed Google Scholar), and cardiac A1receptors couple primarily to Gαi subunits (34.Figler R.A. Graber S.G. Lindorfer M.A. Yasuda H. Linden J. Garrison J.C. Mol. Pharmacol. 1996; 50: 1587-1595PubMed Google Scholar). Two components of the only known pathway that A1 receptor activation modulates in ventricular myocardium, β-adrenergic receptors and adenylyl cyclase, have been reported to be localized in light vesicular fractions which may be caveolae (35.Maisel A.S. Motulsky H.J. Insel P.A. Science. 1985; 230: 183-186Crossref PubMed Scopus (134) Google Scholar, 36.Huang C. Hepler J.R. Chen L.T. Gilman A.G. Anderson R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (189) Google Scholar). Furthermore, cardiac adenylyl cyclase can be inhibited in vitro by the caveolin-3 scaffolding domain peptide (37.Toya Y. Schwencke C. Couet J. Lisanti M.P. Ishikawa Y. Endocrinology. 1998; 139: 2025-2031Crossref PubMed Google Scholar). Therefore, the location of A1 receptors is likely to significantly influence the ability of the receptors to couple to downstream signaling events. It has recently been reported that stimulation of rat cardiac myocytes with endothelin is associated with the recruitment of PKC-α and -ε isoforms to the caveolae fraction (38.Rybin V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: 980-988Crossref PubMed Scopus (143) Google Scholar). Henry et al. (39.Henry P. Demolombe S. Puceat M. Escande D. Circ. Res. 1996; 78: 161-165Crossref PubMed Scopus (70) Google Scholar) previously reported that A1 receptor stimulation of ventricular myocytes induced a transient cytosol to membrane translocation of PKC-δ. Since endothelin, but not A1, receptor stimulation is associated with direct effects in myocytes, it is possible that A1 receptor stimulation may recruit PKC- δ out of caveolae, where PKC signaling may be physiologically important. In summary, we have demonstrated that, in contrast to other G protein-coupled receptors, A1 receptors translocate out of caveolae with agonist binding. The molecular consequences of this translocation are not known. Future studies will elucidate the relationship between A1 receptors, caveolae, and signal transduction." @default.
• W2022992564 created "2016-06-24" @default.
• W2022992564 creator A5024547721 @default.
• W2022992564 creator A5044742086 @default.
• W2022992564 creator A5061836988 @default.
• W2022992564 creator A5067014042 @default.
• W2022992564 date "2000-02-01" @default.
• W2022992564 modified "2023-10-10" @default.
• W2022992564 title "Activated Cardiac Adenosine A1 Receptors Translocate Out of Caveolae" @default.
• W2022992564 cites W133118496 @default.
• W2022992564 cites W1535611266 @default.
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