J. Biochem. I l l , 186-190 (1992)
Phosphorylation of Ryanodine Receptors in Rat Myocytes during #-Adrenergic Stimulation Akira Yoshida,* 1 Masami Takahashi," Toshiaki Imagawa,**' Munekazu Shigekawa,*'* Harohiko Takisawa,' and Takao Nakamura*
Received for publication, August 26, 1991
We studied /9-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and Immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of c AMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [ /- 3 2 P] ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the /9-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The ^-agonistinduced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during /9-adrenergic stimulation of cardiac myocytes.
Intracellular Ca2+ is important in the regulation of cardiac muscle contraction. Entry of Ca2+ through dihydropyridine-sensitive Ca2+ channels triggers cardiac muscle contraction. However, most of the Ca2+ required for contraction is supplied by the sarcoplasmic reticulum (SR), possibly via a Ca2+-induced Ca2+-release mechanism (1, 2). Ca2+ release channels have been purified from skeletal and cardiac muscles as receptors of a plant alkaloid, ryanodine (3-7). The incorporation of the receptor into a lipid bilayer reconstituted ryanodine-sensitive Ca2+ conductance (810). Cloning and sequence analysis of cDNAs encoding these ryanodine receptors revealed that the skeletal and cardiac receptors are distinct gene products (11-13). In cardiac muscle, /9-adrenergic stimulation increases the rate of force development and the maximal force of contraction as well as the rate of relaxation. Much of these /9-adrenergic agonist effects can be explained by an alteration of the intracellular Cas+ transient, because the rates of the rise and fall of intracellular free Ca2+ ([Ca2+],) and the maximal level of [Ca2+]i increase markedly after /9-adrenergic stimulation. The acceleration of Ca1+ removal from the cytoplasm is attributed to enhancement of the Ca I+ pumping activity of the SR through a phosphorylation of phospholamban, a regulatory protein of SR Ca2+-ATPase (14). However, it is not clear how /9-adrenergic agonists stimulate the rate of the [Ca1+]i increase. Previously, using canine cardiac microsomes, we showed that the ryanodine receptor was phosphorylated by cAMP-
dependent protein kinase (PKA) and that the ryanodine binding activity was greater in the phosphorylated than in the unphosphorylated receptor (15). These results suggest that cAMP-dependent phosphorylation of the cardiac ryanodine receptor may be physiologically important for the regulation of the Ca2+ release channel activity. In the present study, we show that the ryanodine receptor can be phosphorylated in rat cardiac myocytes by /9-adrenergic stimulation. EXPERIMENTAL PROCEDURES Isolation of Ventricular Myocytes—Ventricular myocytes were isolated from adult rat heart as described previously (16). The myocytes were then saponin-permeabilized as described (16) with the following modifications: the saponin concentration and incubation times were decreased to 20 //g/ml and 4 min, respectively. The isolated cells were suspended in phosphorylation assay medium (120 mM KC1,2 mM MgCl 2 ,1 mM EGTA, and 20 mM HEPES at pH 7.3) containing 0.5 mM ATP and 0.1 mM GTP and used on the day of preparation. Cell Culture of Newborn Rat Myocytes—Ventricles from 3-day-old rat hearts were cut into small fragments and the cells were dissociated by 4 trypsinization cycles (0.05% w/v, trypsin) at 37"C for 7 min each. Cell suspensions from each dissociation cycle were placed in 20 ml of cold trypsin inhibitor solution (50% precolostrum newborn calf serum and 50% Hanks' salt solution), passed through two layers of lens papier and centrifuged at 200X g for 5 min. The cells were resuspended in culture medium consisting of 5% precolostrum newborn calf serum, 5% heat-inactivated horse serum, and 90% Dulbecco's modified Eagle's medium and plated for 1 h in 100 mm petri dishes. The heart cells
1
Present address: Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194. Abbreviations: [Ca t+ ]i, intracellular free CaI+ concentration; CHAPS, 3-[(3-cholamidopropyl)dimethylaminonio]-l-propanesulfonate; PC, phoephatidylcholine; PKA, cAMP-dependent protein kinase; SR, sarcoplasmic reticulum.
186
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
'Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, 560; "Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194; and "'Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565
187
cAMP-Dependent Phosphorylation of Cardiac Ryanodine Receptor
Vol. I l l , No. 2, 1992
ed protein in the precipitate was analyzed by SDS-PAGE. Phosphorylation of the Ryanodine Receptor of Cultured Newborn Rat Myocytes—Cells were incubated with a phosphate-free, low-K+ solution (140 mM NaCl, 4.7 mM KC1, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, and 15 mM HEPES-Tris, pH 7.4) containing 50//Ci of "PO 4 for 18 h at 37*C in a final volume of 1 ml. After being washed with the phosphate-free, low-K+ solution, cells were incubated with 1 //M isoproterenol for 5 min The cells were then solubilized with NEHDPF containing 1% CHAPS and 0.5% PC. The ryanodine receptor was immunoprecipitated by Ry-4 and the phosphorylated proteins were analyzed as described above. SDS-PAGE—Proteins were separated by SDS-PAGE as described previously (18) except for the use of a linear 412% acrylamide gradient gel (Tefco). Materials—A monoclonal antibody, Ry-4, was prepared as described previously (19). The catalytic subunit of type I cAMP-dependent protein kinase was purchased from Sigma. The drugs and chemicals, and their sources were as follows: [sH]ryanodine and [y- 32 P]ATP, New England Nuclear; 9,21-didehydroryanodine, Wako Pure Chemical Industries; aflinity-purified mouse IgG, Zymed Laboratories; protein A-Sepharose, Pharmacia LKB Biotechnology; all other chemicals, Wako, Sigma, and Bio-Rad Laboratories. RESULTS Isolation of Ryanodine Receptor from Rat Cardiac Myocytes by Monoclonal Antibody—Rat cardiac myocytes solubilized with 1% CHAPS were treated with either a monoclonal antibody against the canine ryanodine receptor, Ry-4, or control mouse IgG. The antibody-antigen complexes were then isolated using protein A-Sepharose. When incubated with [3H]ryanodine in the presence or absence of excess unlabeled 9,21-didehydroryanodine, specific [ J H]ryanodine binding activity was observed only in the immunoprecipitate treated with Ry-4 (Fig. 1 A). The immunoprecipitates were incubated with the catalytic subunit of PKA and [y-"P]ATP, and then separated by SDS-PAGE. As shown in Fig. IB, a phosphorylated polypeptide having the same molecular weight as that of the phosphorylated canine cardiac ryanodine receptor appeared only in the immunoprecipitate treated with Ry-4. These results indicate that the ryanodine receptor from rat cardiac myocytes is similar to the canine cardiac ryanodine receptor (19) with respect to molecular weight, cAMP-dependent phosphorylation, and immunoreactivity with Ry-4. /3-Adrenergic Receptor-Mediated Phosphorylation of Ryanodine Receptor in Rat Myocytes—The myocytes were pretreated with saponin to permeabilize the plasma membrane, then incubated with [ y-"P] ATP for various periods in the presence or absence of 1 //M isoproterenol. After solubilization of the cells with CHAPS, the ryanodine receptor was immunoprecipitated with Ry-4 and the amount of 32P incorporated into the receptor was determined as described under "EXPERIMENTAL PROCEDURES.' As shown in Fig. 2, the amount of 3r P incorporated into the ryanodine receptor after a 4-min incubation with isoproterenol was 40% greater compared with controls. This stimulation of phosphorylation by 1 //M isoproterenol was completely blocked by 2.5 ^M propranolol (Fig. 3), indicating
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
which were not attached to the glass plate were collected and plated in collagen-coated 35 mm plastic tissue culture dishes at a density of 5.3 X10*/cm2. Cells were grown in a humidified 10% CO2-90% air atmosphere at 37"C. Solubilization of the Ryanodine Receptor from Rat Myocytes—The myocytes suspended in a solution (0.9 M NaCl, 50 mM Na-phosphate, 2.5 mM EDTA, 20 mM NaF, and 50 mM HEPES-Tris at pH 7.4) (NEHDPF solution) containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 /*M pepstatin A, 1 mM 1,10-phenanthroline, 1 fig/ml antipain, 1 /*g/ml leupeptin) were sonicated then centrifuged at 540,000 x g for 15 min The precipitate was solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-l-propanesulfonate (CHAPS), 0.5%phosphatidylcholine (PC) in NEHDPF solution containing the protease inhibitors. [3H]Ryanodine Binding Assay—Solubilized myocytes were incubated for 2 h at room temperature with 6 fig of either Ry-4, a monoclonal antibody against the cardiac ryanodine receptor, or control mouse IgG in a final volume of 1 ml. Three milligrams of protein A-Sepharose equilibrated with phosphate-buffered saline containing 1% bovine serum albumin was added to the antigen-antibody complex and the mixture was incubated for 1 h at 4"C. The immunoprecipitate was isolated by centrifugation and resuspended in binding assay solution (1 M NaCl, 1.05 mM CaCl2, 1 mM EGTA, 1% CHAPS, 0.5% PC, 2 mM DTT, 50 mM HEPES-Tris, pH7.4). The immunoprecipitate was then incubated with 16 nM [SH] ryanodine in the presence or absence of 50//M didehydroryanodine for 2 h at room temperature. After washing of the immunoprecipitate three times with the binding solution, the radioactivity in the immunoprecipitate was determined by using a scintillation counter. Phosphorylation of Immuno-Isolated Ryanodine Receptoi—The ryanodine receptor was isolated from solubilized myocytes by immunoprecipitation as described above. The immunoprecipitate was washed three times by centrifugation with 1% CHAPS, 0.5% PC, 6 mM MgCl2,6 mM EGTA, protease inhibitors, and 50 mM HEPES-Tris (pH 7.4) and suspended in 100 //I of the same solution containing 2 /*M [y- 32 P]ATP (10MCi) and 16 units of the PKA catalytic subunit. After 10 min incubation at 37*C, phosphorylation was terminated by adding 1 ml of cold NEHDPF solution containing 1% CHAPS and 0.5% PC. The sample then was washed three times with the same NEHDPF solution and separated by SDS-PAGE. The phosphorylated protein in the gel was analyzed using a Fuji Bioimageanalyzer (17). Phosphorylation of Ryanodine Receptor in SaponinTreated Rat Cardiac Myocytes—Saponin-treated myocytes were incubated with phosphorylation assay medium containing 0.5 mM [y-"P]ATP (50//Ci), 0.1 mM GTP, and drugs in a final volume of 0.2 ml at 37*C. The reaction was terminated by adding 0.8 ml of cold NEHDPF solution. The cells were collected by centrifugation and solubilized in 0.6 ml of NEHDPF containing 1% CHAPS and 0.5% PC. After removal of insoluble material by centrifugation, the ryanodine receptor was immunoprecipitated using Ry-4 and protein A-Sepharose as described above. The immunoprecipitate was washed three times with NEHDPF containing 1% CHAPS and 0.5% PC and once with 1% CHAPS, 120 mM NaCl, 50 mM Na-phosphate, 2.5 mM EDTA, 20 mM NaF, and 50 mM HEPES-Tris (pH 7.4). The phosphorylat-
188
A. Yoshida et aL
B.
Isoproterenol
1 2 4 8 1 2 4 8
20000
Time (min)
10000
I a
-
+ K j 4
ioo
+
Control
""•
Fig. 1. Immunoprecipitation of the ryanodine receptor from rat cardiac myocytes. (A) [ J H] Ryanodine binding to the receptor immunoprecipitated by a specific monoclonal antibody, Ry-4. The rat cardiac myocytes were solubilized with CHAPS and incubated with either Ry-4 or control mouse IgG. The resulting antigen-antibody complex was isolated with protein A-Sepharose. The immunoprecipitate was then incubated with 16 nM ['H]ryanodine in the presence ( + ) or absence ( —) of 50 ^M 9,21-didehydroryanodine. After washing, the radioactivity recovered in the immunoprecipitate was determined by liquid scintillation counting. (B) Phosphorylation of the ryanodine receptor by PKA. Purified canine cardiac ryanodine receptor phosphorylated by catalytic subunit of PKA and [ y-"P] ATP (lane 1). The ryanodine receptors solubilized from rat cardiac myocytes were immunoprecipitated with Ry-4 (lane 3) and control mouse IgG (lane 2). The immunoprecipitates were incubated with [y-"P]ATP and the catalytic subunit of PKA for 5 min at 30'C, then the phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. The migrating position of the phosphorylated canine cardiac ryanodine receptor is indicated by an arrow.
that isoproterenol exerts its action through binding to the ^-adrenergic receptor. Cyclic-AMP-Mediated Phosphorylation of Ryanodine Receptor in Cardiac Myocytes—Activation of the yS-adrenergic receptor induces accumulation of intracellular cAMP. As shown in Fig. 4, exogenously added cAMP also stimulated phosphorylation of the ryanodine receptor in permeabilized cardiac myocytes. The extent of stimulation by cAMP was much larger than that by isoproterenol; up to a 4-fold stimulation of J!P-incorporation was observed in the presence of cAMP. The effect of cAMP appeared at concentrations above 0.1 //M and reached maximum above 2/*M (Fig. 5). We have shown that cAMP-dependent protein kinase phosphorylated purified canine cardiac ryanodine receptor at a ratio of 0.70 to 0.77 mol per mol of ryanodine-binding site {15). In order to estimate the molar ratio of 32P incorporated in the ryanodine receptor to [*H]ryanodine binding site in the isolated myocytes, the [*H]ryanodine binding in the immunoprecipitate was measured in parallel experiments, in which the myocytes were incubated with unlabeled ATP instead of [y-"P]ATP. The values of the ratio after 4 min incubation with ATP were 0.45 and 0.15 in the presence and absence of cAMP, respectively. Phosphorylation of Ryanodine Receptor in Intact Newborn Rat Myocytes—To determine whether the cardiac ryanodine receptor can be phosphorylated by /?-adrenergic
0* 0 Time (min) Fig. 2. /?-Adrenergic agonist-induced phoephorylation of the ryanodine receptor In rat cardiac myocytes. (A) Saponin-treated rat cardiac myocytes were incubated with 0.6 mM [y-"P]ATP and 0.1 mM GTP in the presence or absence of 5 y. M isoproterenol at 37*C. At the indicated times, aliquots of the cell suspension were mixed with cold NEHDPF to stop phosphorylation. The ryanodine receptor was immunoprecipitated with Ry-4 and protein A-Sepharose and separated by SDS-PAGE as described under "EXPERIMENTAL PROCEDURES." The figure shows images of autoradiograms obtained by a Fuji Bioimageanalyzer. (B) The amounts of "P incorporated into the ryanodine receptor depicted in panel A were quantified by using the Fuji Bioimageanalyzer and are expressed as arbitrary units. O, without isoproterenol; • , with 5^M isoproterenol.
B. Control
Iso.
Iso.+Pro.
-S
200
ioo .
a Up+Pro.
Fig. 3. Inhibition of isoproterenol-induced phosphorylation of the ryanodine receptor in cardiac myocytes by a /9-adrenergic antagonist. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [ y-"P] ATP and 0.1 mM GTP for 5 min at 37'C in the presence or absence of 1 ^M isoproterenol (Iso.) and/or 2.5 ^M propranolol (Pro.). The reaction was stopped at the indicated times. The phosphorylation of the ryanodine receptor in the immunoprecipitate was analyzed by SDS-PAGE and the Fuji Bioimageanalyzer as described in the legend to Fig. 2. (A) Gel autoradiograms; (B) "P levels incorporated into ryanodine receptors. These results are means ± S D of triplicate determinations. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
200
cAMP-Dependent Phosphorylation of Cardiac Ryanodine Receptor A.
cAMP
—
Time(min)
1
2
4
189
+
6
1 01
2
4
6
1 0
-» «*.«* *~ i>» * * *
•
«
•
Control ISP ISP+PRO Fig. 6. ^-Adrenergic agonist-induced phosphorylation of the ryanodine receptor in rat neonatal cardiac myocytes. Cells were incubated with "PO4 for 18 h then incubated with 1 //M isoproterenol for 5 min at 37'C in the presence or absence of 5 n M propranolol. The labeled cells were solubilized with CHAPS. The ryanodine receptors were immunoprecipitated and separated by SDS-PAGE as described under "EXPERIMENTAL PROCEDURES." The radioactivity incorporated into the protein band corresponding to the ryanodine receptor was determined by a Fuji Bioimageanalyzer and is expressed as arbitrary units. These results are means ± SD of triplicate determinations. Time (min) Fig. 4. Cyclic AMP-induced phosphorylation of the ryanodine receptor in rat cardiac myocytes. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [y-"P]ATP and 0.1 mM GTP in the presence (•) or absence (O) of 2 // M cAMP at 37'C. The reaction was stopped at the indicated time. The phosphorylation of the ryanodine receptor in the immunoprecipitate was analyzed as described in the legend to Fig. 2. (A) Gel autoradiograma; (B) "P levels incorporated into ryanodine receptors.
"P-inorganic phosphate for 18 h, then incubated them with 1 fiM isoproterenol for 5 min. As shown in Fig. 6, the incorporation of " P into the ryanodine receptor was more than two times greater in those cells treated with isoproterenol than in the controls. This effect was completely blocked by 5 //M propranolol. DISCUSSION
o
| I0 °
cAMP(jiM)
Fig. 5. Effect of increasing cAMP concentrations on phosphorylation of ryanodine receptors. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [y-"P]ATPand0.1 mM GTP in the presence of various concentrations of cAMP for 5 min at 37*C. The amount of "P incorporated into ryanodine receptors was determined as described in the legend to Fig. 2.
stimulation in intact cells, heart cells from newborn rats cultured for 6 days were used. The beating rate of the cultured heart cells increased upon addition of 1 jiM isoproterenol to the culture medium, indicating that the /3-adrenergic receptor was expressed in these cells (data not shown). We preincubated cultured heart cells with Vol. I l l , No. 2, 1992
In the present study, we showed that isoproterenol-induced phosphorylation of the ryanodine receptor occurred in intact as well as saponin-permeabilized rat cardiac myocytes (Figs. 2, 3, and 6). Since this effect was abolished by propranolol (Figs. 3 and 6), it was concluded that isoproterenol exerts its action through the activation of the padrenergic receptor. It is well known that stimulation of the 0- adrenergic receptor in cardiac cells induces the activation of adenylate cyclase, which causes the elevation of intracellular cAMP, which in turn activates PKA activity (20). The following observations suggest that the ryanodine receptor of the cardiac myocytes was phosphorylated by endogenous PKA during /?-adrenergic stimulation. (A) The ryanodine receptor in cardiac microsomes served as a good substrate for the catalytic subunit of PKA (Ref. 15 and Fig. IB of the present study). (B) Isoproterenol enhanced the "P-incorporation into the ryanodine receptor in the permeabilized myocytes (Figs. 2 and 3). (C) Exogenous cAMP mimicked the effect of isoproterenol in permeabilized myocytes (Figs. 4 and 5). The stimulatory effect of isoproterenol was smaller than that of cAMP, which could be due to partial damage of the /9-adrenergic receptor caused by collagenase during the isolation of myocytes and/or to the loss of cAMP by diffusion through the permeabilized plasma membrane. The amount of 32P-incorporation into the ryanodine receptor in myocytes (0.45 mol/mol) was smaller than that into the receptor in vitro (0.7 mol/mol). It is quite likely that a partial dephosphorylation by a phosphatase may occur in myocytes.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
B.
190
REFERENCES 1. Endo, M. (1977) PhynioL Rev. 57, 71-108 2. Fabiato, A. & Fabiato, F. (1977) Ore. Ret. 40, 119-129 3. Inui, M., Saito, A., & Fleischer, S. (1987) J. BioL Chem. 282, 1740-1747 4. Inui, M., Saito, A., ft Fleischer, S. (1987) J. BioL Chem. 282, 15637-15642 5. Campbell, K.P., Knudson, CM., Imagawa, T., Leung, A.T., Sutko, J.L., Kahl, S.D., Raab, C.R., & Madron, L. (1987) J. Biol. Chem. 262, 6460-6463 6. Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.-Y., & Meissner, G. (1988) Nature 331, 315-319 7. Lai, F.A., Anderson, K., Rousseau, E., Liu, Q.-Y., & Meissner, G. (1988) Biochem. Biophys. Res. Commun. 243, 704-709 8. Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P., & Coronado, R. (1988) J. Gen. PhysioL 92, 1-26 9. Hymel, L., Inui, M., Fleischer, S., & Schindler, H. (1988) Proc NatL Acad. Sci. U.S.A. 85, 441-445 10. Anderson, K., Lai, F.A., Liu, Q.-Y., Rousseau, E., Erickson, H.P., & Meissner, G. (1989) J. BioL Chem. 264, 1329-1335 11. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kanagawa, K., Minamino, N., Matsuo, H., Ueda, M., Masao, H., Hirose, T., & Numa, S. (1989) Nature 339, 439-445 12. Otsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M., & MacLennan, D.H. (1990) J. BioL Chem. 266, 13472-13483 13. Nakai, J., Imagawa, T., Hakamata, Y., Shigekawa, M., Takeahima, H., & Numa, S. (1990) FEBS Lett. 271, 169-177 14. Tada, M. ft Kate, A.M. (1982) Annu. Rev. PhysioL 44, 401-423 15. Takasago, T., Imagawa, T., & Shigekawa, M. (1989) J. Biochem. 106, 872-877 16. Miyakoda, G., Yoshida, A., Takisawa, H., & Nakamura, T. (1987) J. Biochem. 102, 211-224 17. Amemiya, Y. & Miyahara, J. (1988) Nature 338, 89-90 18. Yoshida, A., Takahashi, M., Fujimoto, Y., Takisawa, H., & Nakamura, T. (1990) J. Biochem. 107, 608-612 19. Imagawa, T., Takasago, T., & Shigekawa, M. (1989) J. Biochem. 106, 342-348 20. Sutherland, E.W. (1972) Science 177, 401-408 21. Allen, D.G. & Blink.. J.R. (1978) Nature 273, 509-513 22. Rossie, S. & Catterall, W.A. (1987) Enzymes 18, 335-358 23. Reuter, H. (1974) J. PhysioL (London) 242, 429-451 24. Rousseau, E. & Meissner, G. (1989) Am. J. PhysioL 266, H328H333
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
/?-Adrenergic stimulation dramatically changes the [Ca2+]i transient in cardiac cells (21). The rates of the rise and fall of [Ca 2+ ], and the maximal level of transient [Ca2+]i are increased by y9-adrenergic stimulation. Since the major source of CaJ+ is the SR, the activities of some functional proteins localized there may be modified after the activation of the /9-adrenergic receptor. Previously, we showed that phospholamban, a regulatory protein for the SR Ca I+ -pump ATPase of cardiac muscle, was phosphorylated by ^-adrenergic stimulation in myocytes (16). This observation provided an important insight into the mechanism of the ft-agonist-induced acceleration of the falling phase of transient [Ca !+ ]i, since the phosphorylation of phospholamban activates the Ca2+-pump of cardiac SR. In contrast, the molecular mechanism for the acceleration of the rising phase of the [Ca 2+ ] ( transient remains unclear. The activities of many kinds of ion channels and receptors are regulated by protein phosphorylation (22). In cardiac cells, the function of dihydropyridine-sensitive Ca2+ channels in sarcolemma is stimulated by cAMP-dependent phosphorylation (23). On the other hand, little is known about the regulation of the SR Ca2+ release channel. Previously, we observed that the flUj value for [ 3 H]ryanodine binding to canine cardiac microsomal membranes was increased by cAMP-dependent phosphorylation (15). Because ryanodine is believed to bind preferentially to open Ca2+ release channels (15, 24), this observation suggests that cAMP-dependent phosphorylation of the ryanodine receptor up-regulates Ca2+ channel activity. The present finding that the ryanodine receptor in the intact cardiac myocyte was phosphorylated by the /?-adrenergic stimulation suggests that cAMP-dependent phosphorylation may be a physiologically important mechanism for regulation of SR Ca2+-release channel activity. Thus, the cAMP-dependent phosphorylation of the SR Ca2+-release channel may account at least partly for the acceleration of the rising phase of transient [Ca 2+ ], in /3-agonist-treated cardiac myocytes.
A. Yoshida et al.
Phosphorylation of Ryanodine Receptors in Rat Myocytes during #-Adrenergic Stimulation Akira Yoshida,* 1 Masami Takahashi," Toshiaki Imagawa,**' Munekazu Shigekawa,*'* Harohiko Takisawa,' and Takao Nakamura*
Received for publication, August 26, 1991
We studied /9-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and Immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of c AMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [ /- 3 2 P] ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the /9-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The ^-agonistinduced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during /9-adrenergic stimulation of cardiac myocytes.
Intracellular Ca2+ is important in the regulation of cardiac muscle contraction. Entry of Ca2+ through dihydropyridine-sensitive Ca2+ channels triggers cardiac muscle contraction. However, most of the Ca2+ required for contraction is supplied by the sarcoplasmic reticulum (SR), possibly via a Ca2+-induced Ca2+-release mechanism (1, 2). Ca2+ release channels have been purified from skeletal and cardiac muscles as receptors of a plant alkaloid, ryanodine (3-7). The incorporation of the receptor into a lipid bilayer reconstituted ryanodine-sensitive Ca2+ conductance (810). Cloning and sequence analysis of cDNAs encoding these ryanodine receptors revealed that the skeletal and cardiac receptors are distinct gene products (11-13). In cardiac muscle, /9-adrenergic stimulation increases the rate of force development and the maximal force of contraction as well as the rate of relaxation. Much of these /9-adrenergic agonist effects can be explained by an alteration of the intracellular Cas+ transient, because the rates of the rise and fall of intracellular free Ca2+ ([Ca2+],) and the maximal level of [Ca2+]i increase markedly after /9-adrenergic stimulation. The acceleration of Ca1+ removal from the cytoplasm is attributed to enhancement of the Ca I+ pumping activity of the SR through a phosphorylation of phospholamban, a regulatory protein of SR Ca2+-ATPase (14). However, it is not clear how /9-adrenergic agonists stimulate the rate of the [Ca1+]i increase. Previously, using canine cardiac microsomes, we showed that the ryanodine receptor was phosphorylated by cAMP-
dependent protein kinase (PKA) and that the ryanodine binding activity was greater in the phosphorylated than in the unphosphorylated receptor (15). These results suggest that cAMP-dependent phosphorylation of the cardiac ryanodine receptor may be physiologically important for the regulation of the Ca2+ release channel activity. In the present study, we show that the ryanodine receptor can be phosphorylated in rat cardiac myocytes by /9-adrenergic stimulation. EXPERIMENTAL PROCEDURES Isolation of Ventricular Myocytes—Ventricular myocytes were isolated from adult rat heart as described previously (16). The myocytes were then saponin-permeabilized as described (16) with the following modifications: the saponin concentration and incubation times were decreased to 20 //g/ml and 4 min, respectively. The isolated cells were suspended in phosphorylation assay medium (120 mM KC1,2 mM MgCl 2 ,1 mM EGTA, and 20 mM HEPES at pH 7.3) containing 0.5 mM ATP and 0.1 mM GTP and used on the day of preparation. Cell Culture of Newborn Rat Myocytes—Ventricles from 3-day-old rat hearts were cut into small fragments and the cells were dissociated by 4 trypsinization cycles (0.05% w/v, trypsin) at 37"C for 7 min each. Cell suspensions from each dissociation cycle were placed in 20 ml of cold trypsin inhibitor solution (50% precolostrum newborn calf serum and 50% Hanks' salt solution), passed through two layers of lens papier and centrifuged at 200X g for 5 min. The cells were resuspended in culture medium consisting of 5% precolostrum newborn calf serum, 5% heat-inactivated horse serum, and 90% Dulbecco's modified Eagle's medium and plated for 1 h in 100 mm petri dishes. The heart cells
1
Present address: Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194. Abbreviations: [Ca t+ ]i, intracellular free CaI+ concentration; CHAPS, 3-[(3-cholamidopropyl)dimethylaminonio]-l-propanesulfonate; PC, phoephatidylcholine; PKA, cAMP-dependent protein kinase; SR, sarcoplasmic reticulum.
186
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
'Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, 560; "Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194; and "'Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565
187
cAMP-Dependent Phosphorylation of Cardiac Ryanodine Receptor
Vol. I l l , No. 2, 1992
ed protein in the precipitate was analyzed by SDS-PAGE. Phosphorylation of the Ryanodine Receptor of Cultured Newborn Rat Myocytes—Cells were incubated with a phosphate-free, low-K+ solution (140 mM NaCl, 4.7 mM KC1, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, and 15 mM HEPES-Tris, pH 7.4) containing 50//Ci of "PO 4 for 18 h at 37*C in a final volume of 1 ml. After being washed with the phosphate-free, low-K+ solution, cells were incubated with 1 //M isoproterenol for 5 min The cells were then solubilized with NEHDPF containing 1% CHAPS and 0.5% PC. The ryanodine receptor was immunoprecipitated by Ry-4 and the phosphorylated proteins were analyzed as described above. SDS-PAGE—Proteins were separated by SDS-PAGE as described previously (18) except for the use of a linear 412% acrylamide gradient gel (Tefco). Materials—A monoclonal antibody, Ry-4, was prepared as described previously (19). The catalytic subunit of type I cAMP-dependent protein kinase was purchased from Sigma. The drugs and chemicals, and their sources were as follows: [sH]ryanodine and [y- 32 P]ATP, New England Nuclear; 9,21-didehydroryanodine, Wako Pure Chemical Industries; aflinity-purified mouse IgG, Zymed Laboratories; protein A-Sepharose, Pharmacia LKB Biotechnology; all other chemicals, Wako, Sigma, and Bio-Rad Laboratories. RESULTS Isolation of Ryanodine Receptor from Rat Cardiac Myocytes by Monoclonal Antibody—Rat cardiac myocytes solubilized with 1% CHAPS were treated with either a monoclonal antibody against the canine ryanodine receptor, Ry-4, or control mouse IgG. The antibody-antigen complexes were then isolated using protein A-Sepharose. When incubated with [3H]ryanodine in the presence or absence of excess unlabeled 9,21-didehydroryanodine, specific [ J H]ryanodine binding activity was observed only in the immunoprecipitate treated with Ry-4 (Fig. 1 A). The immunoprecipitates were incubated with the catalytic subunit of PKA and [y-"P]ATP, and then separated by SDS-PAGE. As shown in Fig. IB, a phosphorylated polypeptide having the same molecular weight as that of the phosphorylated canine cardiac ryanodine receptor appeared only in the immunoprecipitate treated with Ry-4. These results indicate that the ryanodine receptor from rat cardiac myocytes is similar to the canine cardiac ryanodine receptor (19) with respect to molecular weight, cAMP-dependent phosphorylation, and immunoreactivity with Ry-4. /3-Adrenergic Receptor-Mediated Phosphorylation of Ryanodine Receptor in Rat Myocytes—The myocytes were pretreated with saponin to permeabilize the plasma membrane, then incubated with [ y-"P] ATP for various periods in the presence or absence of 1 //M isoproterenol. After solubilization of the cells with CHAPS, the ryanodine receptor was immunoprecipitated with Ry-4 and the amount of 32P incorporated into the receptor was determined as described under "EXPERIMENTAL PROCEDURES.' As shown in Fig. 2, the amount of 3r P incorporated into the ryanodine receptor after a 4-min incubation with isoproterenol was 40% greater compared with controls. This stimulation of phosphorylation by 1 //M isoproterenol was completely blocked by 2.5 ^M propranolol (Fig. 3), indicating
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
which were not attached to the glass plate were collected and plated in collagen-coated 35 mm plastic tissue culture dishes at a density of 5.3 X10*/cm2. Cells were grown in a humidified 10% CO2-90% air atmosphere at 37"C. Solubilization of the Ryanodine Receptor from Rat Myocytes—The myocytes suspended in a solution (0.9 M NaCl, 50 mM Na-phosphate, 2.5 mM EDTA, 20 mM NaF, and 50 mM HEPES-Tris at pH 7.4) (NEHDPF solution) containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 /*M pepstatin A, 1 mM 1,10-phenanthroline, 1 fig/ml antipain, 1 /*g/ml leupeptin) were sonicated then centrifuged at 540,000 x g for 15 min The precipitate was solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-l-propanesulfonate (CHAPS), 0.5%phosphatidylcholine (PC) in NEHDPF solution containing the protease inhibitors. [3H]Ryanodine Binding Assay—Solubilized myocytes were incubated for 2 h at room temperature with 6 fig of either Ry-4, a monoclonal antibody against the cardiac ryanodine receptor, or control mouse IgG in a final volume of 1 ml. Three milligrams of protein A-Sepharose equilibrated with phosphate-buffered saline containing 1% bovine serum albumin was added to the antigen-antibody complex and the mixture was incubated for 1 h at 4"C. The immunoprecipitate was isolated by centrifugation and resuspended in binding assay solution (1 M NaCl, 1.05 mM CaCl2, 1 mM EGTA, 1% CHAPS, 0.5% PC, 2 mM DTT, 50 mM HEPES-Tris, pH7.4). The immunoprecipitate was then incubated with 16 nM [SH] ryanodine in the presence or absence of 50//M didehydroryanodine for 2 h at room temperature. After washing of the immunoprecipitate three times with the binding solution, the radioactivity in the immunoprecipitate was determined by using a scintillation counter. Phosphorylation of Immuno-Isolated Ryanodine Receptoi—The ryanodine receptor was isolated from solubilized myocytes by immunoprecipitation as described above. The immunoprecipitate was washed three times by centrifugation with 1% CHAPS, 0.5% PC, 6 mM MgCl2,6 mM EGTA, protease inhibitors, and 50 mM HEPES-Tris (pH 7.4) and suspended in 100 //I of the same solution containing 2 /*M [y- 32 P]ATP (10MCi) and 16 units of the PKA catalytic subunit. After 10 min incubation at 37*C, phosphorylation was terminated by adding 1 ml of cold NEHDPF solution containing 1% CHAPS and 0.5% PC. The sample then was washed three times with the same NEHDPF solution and separated by SDS-PAGE. The phosphorylated protein in the gel was analyzed using a Fuji Bioimageanalyzer (17). Phosphorylation of Ryanodine Receptor in SaponinTreated Rat Cardiac Myocytes—Saponin-treated myocytes were incubated with phosphorylation assay medium containing 0.5 mM [y-"P]ATP (50//Ci), 0.1 mM GTP, and drugs in a final volume of 0.2 ml at 37*C. The reaction was terminated by adding 0.8 ml of cold NEHDPF solution. The cells were collected by centrifugation and solubilized in 0.6 ml of NEHDPF containing 1% CHAPS and 0.5% PC. After removal of insoluble material by centrifugation, the ryanodine receptor was immunoprecipitated using Ry-4 and protein A-Sepharose as described above. The immunoprecipitate was washed three times with NEHDPF containing 1% CHAPS and 0.5% PC and once with 1% CHAPS, 120 mM NaCl, 50 mM Na-phosphate, 2.5 mM EDTA, 20 mM NaF, and 50 mM HEPES-Tris (pH 7.4). The phosphorylat-
188
A. Yoshida et aL
B.
Isoproterenol
1 2 4 8 1 2 4 8
20000
Time (min)
10000
I a
-
+ K j 4
ioo
+
Control
""•
Fig. 1. Immunoprecipitation of the ryanodine receptor from rat cardiac myocytes. (A) [ J H] Ryanodine binding to the receptor immunoprecipitated by a specific monoclonal antibody, Ry-4. The rat cardiac myocytes were solubilized with CHAPS and incubated with either Ry-4 or control mouse IgG. The resulting antigen-antibody complex was isolated with protein A-Sepharose. The immunoprecipitate was then incubated with 16 nM ['H]ryanodine in the presence ( + ) or absence ( —) of 50 ^M 9,21-didehydroryanodine. After washing, the radioactivity recovered in the immunoprecipitate was determined by liquid scintillation counting. (B) Phosphorylation of the ryanodine receptor by PKA. Purified canine cardiac ryanodine receptor phosphorylated by catalytic subunit of PKA and [ y-"P] ATP (lane 1). The ryanodine receptors solubilized from rat cardiac myocytes were immunoprecipitated with Ry-4 (lane 3) and control mouse IgG (lane 2). The immunoprecipitates were incubated with [y-"P]ATP and the catalytic subunit of PKA for 5 min at 30'C, then the phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. The migrating position of the phosphorylated canine cardiac ryanodine receptor is indicated by an arrow.
that isoproterenol exerts its action through binding to the ^-adrenergic receptor. Cyclic-AMP-Mediated Phosphorylation of Ryanodine Receptor in Cardiac Myocytes—Activation of the yS-adrenergic receptor induces accumulation of intracellular cAMP. As shown in Fig. 4, exogenously added cAMP also stimulated phosphorylation of the ryanodine receptor in permeabilized cardiac myocytes. The extent of stimulation by cAMP was much larger than that by isoproterenol; up to a 4-fold stimulation of J!P-incorporation was observed in the presence of cAMP. The effect of cAMP appeared at concentrations above 0.1 //M and reached maximum above 2/*M (Fig. 5). We have shown that cAMP-dependent protein kinase phosphorylated purified canine cardiac ryanodine receptor at a ratio of 0.70 to 0.77 mol per mol of ryanodine-binding site {15). In order to estimate the molar ratio of 32P incorporated in the ryanodine receptor to [*H]ryanodine binding site in the isolated myocytes, the [*H]ryanodine binding in the immunoprecipitate was measured in parallel experiments, in which the myocytes were incubated with unlabeled ATP instead of [y-"P]ATP. The values of the ratio after 4 min incubation with ATP were 0.45 and 0.15 in the presence and absence of cAMP, respectively. Phosphorylation of Ryanodine Receptor in Intact Newborn Rat Myocytes—To determine whether the cardiac ryanodine receptor can be phosphorylated by /?-adrenergic
0* 0 Time (min) Fig. 2. /?-Adrenergic agonist-induced phoephorylation of the ryanodine receptor In rat cardiac myocytes. (A) Saponin-treated rat cardiac myocytes were incubated with 0.6 mM [y-"P]ATP and 0.1 mM GTP in the presence or absence of 5 y. M isoproterenol at 37*C. At the indicated times, aliquots of the cell suspension were mixed with cold NEHDPF to stop phosphorylation. The ryanodine receptor was immunoprecipitated with Ry-4 and protein A-Sepharose and separated by SDS-PAGE as described under "EXPERIMENTAL PROCEDURES." The figure shows images of autoradiograms obtained by a Fuji Bioimageanalyzer. (B) The amounts of "P incorporated into the ryanodine receptor depicted in panel A were quantified by using the Fuji Bioimageanalyzer and are expressed as arbitrary units. O, without isoproterenol; • , with 5^M isoproterenol.
B. Control
Iso.
Iso.+Pro.
-S
200
ioo .
a Up+Pro.
Fig. 3. Inhibition of isoproterenol-induced phosphorylation of the ryanodine receptor in cardiac myocytes by a /9-adrenergic antagonist. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [ y-"P] ATP and 0.1 mM GTP for 5 min at 37'C in the presence or absence of 1 ^M isoproterenol (Iso.) and/or 2.5 ^M propranolol (Pro.). The reaction was stopped at the indicated times. The phosphorylation of the ryanodine receptor in the immunoprecipitate was analyzed by SDS-PAGE and the Fuji Bioimageanalyzer as described in the legend to Fig. 2. (A) Gel autoradiograms; (B) "P levels incorporated into ryanodine receptors. These results are means ± S D of triplicate determinations. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
200
cAMP-Dependent Phosphorylation of Cardiac Ryanodine Receptor A.
cAMP
—
Time(min)
1
2
4
189
+
6
1 01
2
4
6
1 0
-» «*.«* *~ i>» * * *
•
«
•
Control ISP ISP+PRO Fig. 6. ^-Adrenergic agonist-induced phosphorylation of the ryanodine receptor in rat neonatal cardiac myocytes. Cells were incubated with "PO4 for 18 h then incubated with 1 //M isoproterenol for 5 min at 37'C in the presence or absence of 5 n M propranolol. The labeled cells were solubilized with CHAPS. The ryanodine receptors were immunoprecipitated and separated by SDS-PAGE as described under "EXPERIMENTAL PROCEDURES." The radioactivity incorporated into the protein band corresponding to the ryanodine receptor was determined by a Fuji Bioimageanalyzer and is expressed as arbitrary units. These results are means ± SD of triplicate determinations. Time (min) Fig. 4. Cyclic AMP-induced phosphorylation of the ryanodine receptor in rat cardiac myocytes. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [y-"P]ATP and 0.1 mM GTP in the presence (•) or absence (O) of 2 // M cAMP at 37'C. The reaction was stopped at the indicated time. The phosphorylation of the ryanodine receptor in the immunoprecipitate was analyzed as described in the legend to Fig. 2. (A) Gel autoradiograma; (B) "P levels incorporated into ryanodine receptors.
"P-inorganic phosphate for 18 h, then incubated them with 1 fiM isoproterenol for 5 min. As shown in Fig. 6, the incorporation of " P into the ryanodine receptor was more than two times greater in those cells treated with isoproterenol than in the controls. This effect was completely blocked by 5 //M propranolol. DISCUSSION
o
| I0 °
cAMP(jiM)
Fig. 5. Effect of increasing cAMP concentrations on phosphorylation of ryanodine receptors. Saponin-treated rat cardiac myocytes were incubated with 0.5 mM [y-"P]ATPand0.1 mM GTP in the presence of various concentrations of cAMP for 5 min at 37*C. The amount of "P incorporated into ryanodine receptors was determined as described in the legend to Fig. 2.
stimulation in intact cells, heart cells from newborn rats cultured for 6 days were used. The beating rate of the cultured heart cells increased upon addition of 1 jiM isoproterenol to the culture medium, indicating that the /3-adrenergic receptor was expressed in these cells (data not shown). We preincubated cultured heart cells with Vol. I l l , No. 2, 1992
In the present study, we showed that isoproterenol-induced phosphorylation of the ryanodine receptor occurred in intact as well as saponin-permeabilized rat cardiac myocytes (Figs. 2, 3, and 6). Since this effect was abolished by propranolol (Figs. 3 and 6), it was concluded that isoproterenol exerts its action through the activation of the padrenergic receptor. It is well known that stimulation of the 0- adrenergic receptor in cardiac cells induces the activation of adenylate cyclase, which causes the elevation of intracellular cAMP, which in turn activates PKA activity (20). The following observations suggest that the ryanodine receptor of the cardiac myocytes was phosphorylated by endogenous PKA during /?-adrenergic stimulation. (A) The ryanodine receptor in cardiac microsomes served as a good substrate for the catalytic subunit of PKA (Ref. 15 and Fig. IB of the present study). (B) Isoproterenol enhanced the "P-incorporation into the ryanodine receptor in the permeabilized myocytes (Figs. 2 and 3). (C) Exogenous cAMP mimicked the effect of isoproterenol in permeabilized myocytes (Figs. 4 and 5). The stimulatory effect of isoproterenol was smaller than that of cAMP, which could be due to partial damage of the /9-adrenergic receptor caused by collagenase during the isolation of myocytes and/or to the loss of cAMP by diffusion through the permeabilized plasma membrane. The amount of 32P-incorporation into the ryanodine receptor in myocytes (0.45 mol/mol) was smaller than that into the receptor in vitro (0.7 mol/mol). It is quite likely that a partial dephosphorylation by a phosphatase may occur in myocytes.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
B.
190
REFERENCES 1. Endo, M. (1977) PhynioL Rev. 57, 71-108 2. Fabiato, A. & Fabiato, F. (1977) Ore. Ret. 40, 119-129 3. Inui, M., Saito, A., & Fleischer, S. (1987) J. BioL Chem. 282, 1740-1747 4. Inui, M., Saito, A., ft Fleischer, S. (1987) J. BioL Chem. 282, 15637-15642 5. Campbell, K.P., Knudson, CM., Imagawa, T., Leung, A.T., Sutko, J.L., Kahl, S.D., Raab, C.R., & Madron, L. (1987) J. Biol. Chem. 262, 6460-6463 6. Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.-Y., & Meissner, G. (1988) Nature 331, 315-319 7. Lai, F.A., Anderson, K., Rousseau, E., Liu, Q.-Y., & Meissner, G. (1988) Biochem. Biophys. Res. Commun. 243, 704-709 8. Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P., & Coronado, R. (1988) J. Gen. PhysioL 92, 1-26 9. Hymel, L., Inui, M., Fleischer, S., & Schindler, H. (1988) Proc NatL Acad. Sci. U.S.A. 85, 441-445 10. Anderson, K., Lai, F.A., Liu, Q.-Y., Rousseau, E., Erickson, H.P., & Meissner, G. (1989) J. BioL Chem. 264, 1329-1335 11. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kanagawa, K., Minamino, N., Matsuo, H., Ueda, M., Masao, H., Hirose, T., & Numa, S. (1989) Nature 339, 439-445 12. Otsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M., & MacLennan, D.H. (1990) J. BioL Chem. 266, 13472-13483 13. Nakai, J., Imagawa, T., Hakamata, Y., Shigekawa, M., Takeahima, H., & Numa, S. (1990) FEBS Lett. 271, 169-177 14. Tada, M. ft Kate, A.M. (1982) Annu. Rev. PhysioL 44, 401-423 15. Takasago, T., Imagawa, T., & Shigekawa, M. (1989) J. Biochem. 106, 872-877 16. Miyakoda, G., Yoshida, A., Takisawa, H., & Nakamura, T. (1987) J. Biochem. 102, 211-224 17. Amemiya, Y. & Miyahara, J. (1988) Nature 338, 89-90 18. Yoshida, A., Takahashi, M., Fujimoto, Y., Takisawa, H., & Nakamura, T. (1990) J. Biochem. 107, 608-612 19. Imagawa, T., Takasago, T., & Shigekawa, M. (1989) J. Biochem. 106, 342-348 20. Sutherland, E.W. (1972) Science 177, 401-408 21. Allen, D.G. & Blink.. J.R. (1978) Nature 273, 509-513 22. Rossie, S. & Catterall, W.A. (1987) Enzymes 18, 335-358 23. Reuter, H. (1974) J. PhysioL (London) 242, 429-451 24. Rousseau, E. & Meissner, G. (1989) Am. J. PhysioL 266, H328H333
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/2/186/873742 by Stockholm University Library user on 21 January 2019
/?-Adrenergic stimulation dramatically changes the [Ca2+]i transient in cardiac cells (21). The rates of the rise and fall of [Ca 2+ ], and the maximal level of transient [Ca2+]i are increased by y9-adrenergic stimulation. Since the major source of CaJ+ is the SR, the activities of some functional proteins localized there may be modified after the activation of the /9-adrenergic receptor. Previously, we showed that phospholamban, a regulatory protein for the SR Ca I+ -pump ATPase of cardiac muscle, was phosphorylated by ^-adrenergic stimulation in myocytes (16). This observation provided an important insight into the mechanism of the ft-agonist-induced acceleration of the falling phase of transient [Ca !+ ]i, since the phosphorylation of phospholamban activates the Ca2+-pump of cardiac SR. In contrast, the molecular mechanism for the acceleration of the rising phase of the [Ca 2+ ] ( transient remains unclear. The activities of many kinds of ion channels and receptors are regulated by protein phosphorylation (22). In cardiac cells, the function of dihydropyridine-sensitive Ca2+ channels in sarcolemma is stimulated by cAMP-dependent phosphorylation (23). On the other hand, little is known about the regulation of the SR Ca2+ release channel. Previously, we observed that the flUj value for [ 3 H]ryanodine binding to canine cardiac microsomal membranes was increased by cAMP-dependent phosphorylation (15). Because ryanodine is believed to bind preferentially to open Ca2+ release channels (15, 24), this observation suggests that cAMP-dependent phosphorylation of the ryanodine receptor up-regulates Ca2+ channel activity. The present finding that the ryanodine receptor in the intact cardiac myocyte was phosphorylated by the /?-adrenergic stimulation suggests that cAMP-dependent phosphorylation may be a physiologically important mechanism for regulation of SR Ca2+-release channel activity. Thus, the cAMP-dependent phosphorylation of the SR Ca2+-release channel may account at least partly for the acceleration of the rising phase of transient [Ca 2+ ], in /3-agonist-treated cardiac myocytes.
A. Yoshida et al.
