Reversible Inhibition of the Calcium-pumping ATPase in Native Cardiac Sarcoplasmic Reticulum by a Calmodulin-binding Peptide
2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês
10.1074/jbc.275.6.4407
ISSN1083-351X
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoCalmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca2+cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca2+ pump (Ca2+-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca2+uptake, Ca2+-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca2+ uptake and Ca2+-ATPase activities of SR (50% inhibition at ∼2–3 μm CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease inV max of Ca2+ uptake, unalteredK 0.5 for Ca2+ activation of Ca2+ transport, and a 10-fold decrease in the apparent affinity of the Ca2+-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca2+-binding sites of the Ca2+-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca2+-ATPase, phospholamban, and ryanodine receptor-Ca2+ release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca2+ pump function and protein phosphorylation was fully reversed by exogenous CaM (1–3 μm). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca2+ transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca2+pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca2+ pump function, CaM BP (1–2.5 μm) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM. Calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca2+cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca2+ pump (Ca2+-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca2+uptake, Ca2+-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca2+ uptake and Ca2+-ATPase activities of SR (50% inhibition at ∼2–3 μm CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease inV max of Ca2+ uptake, unalteredK 0.5 for Ca2+ activation of Ca2+ transport, and a 10-fold decrease in the apparent affinity of the Ca2+-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca2+-binding sites of the Ca2+-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca2+-ATPase, phospholamban, and ryanodine receptor-Ca2+ release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca2+ pump function and protein phosphorylation was fully reversed by exogenous CaM (1–3 μm). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca2+ transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca2+pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca2+ pump function, CaM BP (1–2.5 μm) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM. sarcoplasmic reticulum ryanodine receptor-Ca2+ release channel Ca2+/calmodulin-dependent protein kinase II calmodulin-binding peptide phosphate-buffered saline bovine serum albumin By regulating cytosolic Ca2+ concentration, the sarcoplasmic reticulum (SR)1plays a central role in the contraction-relaxation cycle of heart muscle. Upon excitation of the cardiomyocyte, Ca2+ is released from the SR through Ca2+-release channels (known as RYR-CRC) to initiate muscle contraction (1.Feher J.J. Fabiato A. Langer G.A. Calcium and the Heart. Raven Press, Ltd., New York1990: 199-268Google Scholar, 2.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (445) Google Scholar, 3.Cannell M.B. Cheng H. Lederer W.J. Science. 1995; 268: 1045-1049Crossref PubMed Scopus (514) Google Scholar, 4.Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Publishers Group, Dordrecht, Netherlands1991Google Scholar, 5.Solaro R.J. Rarick H.M. Circ. Res. 1998; 83: 471-480Crossref PubMed Scopus (292) Google Scholar). Subsequent muscle relaxation occurs upon sequestration of Ca2+ back into the SR lumen by a Ca2+-pumping ATPase (Ca2+-ATPase) present in the SR (1.Feher J.J. Fabiato A. Langer G.A. Calcium and the Heart. Raven Press, Ltd., New York1990: 199-268Google Scholar, 4.Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Publishers Group, Dordrecht, Netherlands1991Google Scholar, 6.Inesi G. Submilla C. Kirtley M.E. Physiol. Rev. 1990; 70: 749-760Crossref PubMed Scopus (152) Google Scholar, 7.MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). A well known mechanism for the regulation of the cardiac SR Ca2+-ATPase involves phosphorylation of another intrinsic SR protein, phospholamban (8.Tada M. Katz A.M. Annu. Rev. Physiol. 1982; 44: 401-423Crossref PubMed Scopus (323) Google Scholar, 9.Colyer J. Cardiovasc. Res. 1993; 27 (1711): 1766Crossref PubMed Scopus (52) Google Scholar, 10.Kadambi V.J. Kranias E.G. Biochem. Biophys. Res. Commun. 1997; 239: 1-5Crossref PubMed Scopus (53) Google Scholar, 11.Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar). In its unphosphorylated state, phospholamban is thought to interact with the Ca2+-ATPase exerting an inhibitory effect; phosphorylation of phospholamban by cAMP-dependent protein kinase or CaM kinase is thought to disrupt this interaction resulting in stimulation of Ca2+ pump activity (8.Tada M. Katz A.M. Annu. Rev. Physiol. 1982; 44: 401-423Crossref PubMed Scopus (323) Google Scholar, 9.Colyer J. Cardiovasc. Res. 1993; 27 (1711): 1766Crossref PubMed Scopus (52) Google Scholar, 10.Kadambi V.J. Kranias E.G. Biochem. Biophys. Res. Commun. 1997; 239: 1-5Crossref PubMed Scopus (53) Google Scholar, 11.Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar). In cardiac SR, the RYR-CRC also undergoes phosphorylation by CaM kinase (12.Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar, 13.Takasago T. Imagawa T. Furukawa K. Ogurusu T. Shigekawa M. J. Biochem. (Tokyo). 1991; 109: 163-170Crossref PubMed Scopus (172) Google Scholar, 14.Xu A. Hawkins C. Narayanan N. J. Biol. Chem. 1993; 268: 8394-8397Abstract Full Text PDF PubMed Google Scholar), and this may result in stimulation of Ca2+ release from the SR (12.Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar, 15.Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 16.Dubell W.H. Lederer W.J. Rogers T.B. J. Physiol. (Lond.). 1996; 493: 793-800Crossref Scopus (80) Google Scholar, 17.Li L. Satoh H. Ginsburg K.S. Bers D.M. J. Physiol. (Lond.). 1997; 501: 17-32Crossref Scopus (151) Google Scholar). Recent studies from this laboratory (14.Xu A. Hawkins C. Narayanan N. J. Biol. Chem. 1993; 268: 8394-8397Abstract Full Text PDF PubMed Google Scholar, 18.Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Abstract Full Text PDF PubMed Google Scholar, 19.Xu A. Hawkins C. Narayanan N. J. Mol. Cell. Cardiol. 1997; 29: 405-418Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 20.Xu A. Narayanan N. Am. J. Physiol. 1998; 275: H2087-H2094PubMed Google Scholar, 21.Netticadan T. Xu A. Narayanan N. Arch. Biochem. Biophys. 1996; 333: 368-376Crossref PubMed Scopus (20) Google Scholar, 22.Xu A. Narayanan N. Biochem. Biophys. Res. Commun. 1999; 258: 66-72Crossref PubMed Scopus (46) Google Scholar) and other laboratories (23.Toyofuku T. Kurzydlowski K. Narayanan N. MacLennan D.H. J. Biol. Chem. 1994; 269: 26492-26496Abstract Full Text PDF PubMed Google Scholar, 24.Odermatt A. Kurzydlowski K. MacLennan D.H. J. Biol. Chem. 1996; 271: 14206-14213Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 25.Allen B.G. Katz S. Mol. Cell. Biochem. 1996; 155: 91-103Crossref PubMed Google Scholar, 26.Osada M. Netticadan T. Tamura K. Dhalla N.S. Am. J. Physiol. 1998; 247: H2025-H2034Google Scholar) have demonstrated that in cardiac SR, a membrane-associated CaM kinase phosphorylates the Ca2+-ATPase in addition to RYR-CRC and phospholamban. The phosphorylation occurred at a serine residue and was specific for the cardiac/slow-twitch muscle isoform (SERCA2a) of the Ca2+-ATPase (18.Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis studies by Toyofuku et al. (23.Toyofuku T. Kurzydlowski K. Narayanan N. MacLennan D.H. J. Biol. Chem. 1994; 269: 26492-26496Abstract Full Text PDF PubMed Google Scholar) resulted in the identification of Ser38 as the site in SERCA2a that is phosphorylated by CaM kinase. Studies using native cardiac SR vesicles (14.Xu A. Hawkins C. Narayanan N. J. Biol. Chem. 1993; 268: 8394-8397Abstract Full Text PDF PubMed Google Scholar), purified SR Ca2+-ATPase preparations (14.Xu A. Hawkins C. Narayanan N. J. Biol. Chem. 1993; 268: 8394-8397Abstract Full Text PDF PubMed Google Scholar, 18.Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Abstract Full Text PDF PubMed Google Scholar), and SERCA2a expressed in HEK-293 cells (23.Toyofuku T. Kurzydlowski K. Narayanan N. MacLennan D.H. J. Biol. Chem. 1994; 269: 26492-26496Abstract Full Text PDF PubMed Google Scholar) suggested that Ser38 phosphorylation of the Ca2+-ATPase results in activation of the V max of Ca2+ transport. Some studies have, however, questioned the physiological role of Ca2+-ATPase phosphorylation. Thus, a study by Odermatt et al. (24.Odermatt A. Kurzydlowski K. MacLennan D.H. J. Biol. Chem. 1996; 271: 14206-14213Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) showed CaM kinase-mediated phosphorylation of the Ca2+-ATPase in native rabbit cardiac SR as well as SERCA2a expressed in HEK-293 cells but failed to observe a significant stimulatory effect of phosphorylation on Ca2+-ATPase function. Another study by Reddy et al. (27.Reddy L.G. Jones L.R. Pace R.C. Stokes D.L. J. Biol. Chem. 1996; 271: 14964-14970Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) reported failure to observe phosphorylation of the Ca2+-ATPase in canine cardiac SR or purified Ca2+-ATPase reconstituted in lipid vesicles. These studies have attributed the stimulatory effect of CaM kinase to the phosphorylation of phospholamban and a consequent increase in Ca2+ affinity of the Ca2+-ATPase. In native cardiac SR, analysis of the selective effect of Ca2+-ATPase phosphorylation on Ca2+-pumping activity of this enzyme is hampered by the concomitant phosphorylation of phospholamban and RYR-CRC by the membrane-bound CaM kinase. Recently, we achieved selective phosphorylation of the Ca2+-ATPase by the SR-associated CaM kinase by utilizing a phospholamban monoclonal antibody, which inhibits phospholamban phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which was found to inhibit RYR-CRC phosphorylation (22.Xu A. Narayanan N. Biochem. Biophys. Res. Commun. 1999; 258: 66-72Crossref PubMed Scopus (46) Google Scholar). Under these conditions, Ca2+-ATPase phosphorylation by endogenous CaM kinase resulted in enhanced V max of Ca2+transport (22.Xu A. Narayanan N. Biochem. Biophys. Res. Commun. 1999; 258: 66-72Crossref PubMed Scopus (46) Google Scholar). During the course of these studies we have found that, in addition to the endogenous CaM kinase, SR vesicles isolated from cardiac muscle contains significant amount of calmodulin that is resistant to extraction with high salt (0.6 m KCl). The presence of calmodulin in isolated SR vesicles may mask the true potential of calmodulin-dependent regulation of SR function in in vitro experiments. For example, since both calmodulin and CaM kinase are structured in the SR, introduction of Ca2+ to the assay medium to measure Ca2+transport would also result in concurrent activation of CaM kinase and other Ca2+/calmodulin-dependent membrane events. This issue assumes a higher level of complexity given that CaM kinase, once activated, undergoes autophosphorylation and retains activity independently of Ca2+/calmodulin (28.Bruan A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar). In the present study, we utilized a previously characterized amphiphilic, high affinity calmodulin-binding peptide (29.DeGrado W.F. Prendergast F.G. Wolfe Jr, H.R. Cox J.A. J. Cell. Biochem. 1985; 29: 83-93Crossref PubMed Scopus (48) Google Scholar) to unmask the potential influence of SR-associated calmodulin on cardiac SR Ca2+-ATPase function. The results presented here demonstrate a strong inhibitory action of CaM BP on the Ca2+ ion-transporting as well as energy-transducing functions of the Ca2+-ATPase. This inhibition stems from the association of CaM BP with SR membrane target(s) and is readily reversed by calmodulin. These findings imply that a calmodulin-dependent process controls the velocity of Ca2+ pumping in native cardiac SR. 45CaCl2 was purchased from NEN Life Science Products, and [γ-32P]ATP was from Amersham Pharmacia Biotech. Reagents for electrophoresis were obtained from Bio-Rad. Monoclonal antibody against calmodulin was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). All other chemicals were from Sigma. A 17-amino acid high affinity calmodulin-binding peptide (designated CaM BP in this report), designed and characterized previously by DeGrado et al.(29.DeGrado W.F. Prendergast F.G. Wolfe Jr, H.R. Cox J.A. J. Cell. Biochem. 1985; 29: 83-93Crossref PubMed Scopus (48) Google Scholar), and three fragments of this peptide with overlapping residues were synthesized by the University of Victoria Protein Micro-chemistry Center using a model 430A Applied Biosystems peptide synthesizer. The C termini of the peptides were amidated. All peptides were purified by high performance liquid chromatography, analyzed by mass spectrometry, and sequenced on Applied Biosystems model 473A protein sequencer. The sequences included the following: CaM BP, LKWKKLLKLLKKLLKLG; fragment A, LKWKKLL; fragment B, LLKLLKK; and fragment C, KKLLKLG. SR membrane vesicles were prepared from heart ventricles and fast-twitch (adductor magnus) skeletal muscle of New Zealand White rabbits (body weight 2.5–3 kg) as described previously (30.Jones D.L. Narayanan N. Am. J. Physiol. 1998; 274: H98-H105Crossref PubMed Google Scholar). Following isolation, the SR vesicles were suspended in 10 mm Tris maleate (pH 6.8) containing 100 mm KCl and stored at −80 °C after quick-freezing in liquid N2. Protein concentration was determined by the method of Lowry et al. (31.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using BSA as standard. ATP-dependent, oxalate-facilitated Ca2+ uptake by SR was determined using a Millipore filtration technique as described previously (32.Narayanan N. Biochim. Biophys. Acta. 1981; 678: 442-459Crossref PubMed Scopus (76) Google Scholar). The standard incubation medium for Ca2+ uptake (total volume 250 μl) contained 50 mm HEPES (pH 7.2), 5 mmMgCl2, 5 mm NaN3, l20 mm KCl, 0.1 mm EGTA, 5 mm potassium oxalate, 5 mm ATP, 0.l mm45CaCl2 (∼8000 cpm/nmol; free Ca2+, 7.5 μm), 25 μm ruthenium red, and SR (6 μg of protein). In experiments where Ca2+concentration dependence was studied, the EGTA concentration in the assay medium was held at 0.1 mm, and the amount of total45CaCl2 added was varied in the range 1 to 200 μm to yield the desired free Ca2+. Modifications to the standard incubation medium are specified in the figure legends. Unless indicated otherwise, all assays were carried out at 37 °C; the Ca2+ transport reaction was initiated by the addition of SR vesicles after preincubation of the rest of the assay components for 3 min. The initial free Ca2+concentrations in the assay medium were determined using the computer program of Fabiato (33.Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (977) Google Scholar). The data on Ca2+concentration dependence on Ca2+ uptake were analyzed by nonlinear regression curve fitting using SigmaPlot scientific graph program (Jandel Scientific) run on an IBM-PC computer. The data were fit to Equation 1, ν=Vmax[Ca2+] n/(K0.5n+[Ca2+] n)Equation 1 where v is the measured Ca2+ uptake activity at a given Ca2+ concentration;V max is the maximum activity reached;K 0.5 is the Ca2+ concentration giving half of V max, and n is the equivalent to the Hill coefficient. Ca2+-ATPase activity of SR membranes was quantified as described previously (34.Narayanan N. Su N. Bedard P. Biochim. Biophys. Acta. 1991; 1070: 83-91Crossref PubMed Scopus (31) Google Scholar) using an incubation medium identical to that used for Ca2+ uptake except that [γ-32P]ATP was used instead of nonradioactive ATP and nonradioactive CaCl2 was used instead of 45CaCl2. Parallel assays were also performed in the absence of Ca2+(i.e. in the presence of 0.2 mm EGTA with no CaCl2 added to the assay medium), and the Ca2+-ATPase activity was defined as the difference in ATP hydrolysis (liberation of 32Pi) measured in the absence and presence of Ca2+. The ATPase reaction was initiated by the addition of SR vesicles after preincubation of the rest of the assay components for 3 min at 37 °C and was allowed to proceed for 2 min. Western blotting analysis of endogenous calmodulin in SR vesicles was performed using a monoclonal antibody specific for calmodulin (35.Sacks D.B. Porter S.E. Ladenson J.H. McDonald J.M. Anal. Biochem. 1991; 194: 369-377Crossref PubMed Scopus (79) Google Scholar). SR proteins were fractionated on SDS-polyacrylamide (15%, homogenous) mini-gels and then electroblotted to nitrocellulose sheets. The sheets were incubated in 0.2% glutaraldehyde in PBS for 45 min at 24 °C and then rinsed in PBS. Nonspecific binding sites were blocked with 2% BSA, 0.1% gelatin in PBS for 60 min at 37 °C. Following three 15-min washes with 0.05% Tween 20 in PBS, the sheets were incubated with anti-calmodulin monoclonal antibody (0.5 μg/ml in PBS) for 60 min at 37 °C and then with alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody (dilution 1:1000). After five 10-min washes in PBS/Tween, the sheets were rinsed with deionized water, and the immunoreactive peptide band representing calmodulin was visualized following color development using a Bio-Rad assay kit. Endogenous CaM kinase-catalyzed SR protein phosphorylation was measured as described previously (18.Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Abstract Full Text PDF PubMed Google Scholar). The standard incubation medium (total volume 50 μl) for phosphorylation by endogenous CaM kinase contained 50 mm HEPES (pH 7.4), 10 mm MgCl2, 0.1 mm CaCl2, 0.1 mm EGTA, 1 μm calmodulin, 0.8 mm [γ-32P]ATP (specific activity, 300–400 cpm/pmol), and SR (30 μg of protein). The phosphorylation reaction was initiated by the addition of SR after preincubation of the rest of the assay components for 3 min at 37 °C. The reaction was terminated after 2 min by the addition of 15 μl of SDS sample buffer, and the samples were analyzed in 4–18% SDS-polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue, dried, and autoradiographed. Quantification of phosphorylation was carried out by liquid scintillation counting after excision of the radioactive bands from the gels (18.Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Abstract Full Text PDF PubMed Google Scholar). Rabbits were anesthetized with sodium pentobarbital (35 mg/kg, intravenously), and the hearts were excised and immediately cannulated for retrograde aortic perfusion of the coronary arteries with mammalian Ringer solution consisting of 154 mm NaCl, 5 mm KCl, 2.2 mm CaCl2, 6 mm NaHCO3, and 5.5 mm dextrose. The perfusion buffer was equilibrated with 95% O2, 5% CO2, which maintained a pH of 7.4; the perfusion temperature was set at 37 ± 0.2 °C. The hearts were perfused at a constant flow rate of 25 ml/min using a peristaltic pump. After an initial 15–20 min of perfusion, when the spontaneous beating had stabilized, a latex balloon-tipped cannula filled with degassed H2O was inserted into the lumen of the left ventricle for obtaining systolic left ventricular pressure development. The cannula was connected via a pressure transducer (COBE, Bramalea, Canada) to a BioPac System Digital Monitor (model MP100) and a personal computer that allowed on-line monitoring of left ventricular pressure and off-line calculation of developed pressure, rate of pressure development (+dP/dt), and rate of relaxation (−dP/dt). Unless specified otherwise, the experimental values represent the average of at least three independent experiments using separate SR preparations performed in duplicate. The data are presented as mean ± S.E. The ATP-dependent, oxalate-facilitated Ca2+ uptake by SR vesicles is a useful, commonly used parameter to measure the Ca2+ pump (Ca2+-ATPase) function of SR in vitro (6.Inesi G. Submilla C. Kirtley M.E. Physiol. Rev. 1990; 70: 749-760Crossref PubMed Scopus (152) Google Scholar). The results presented in Fig. 1 demonstrate the effects of varying concentrations of CaM BP and its fragments on ATP-dependent Ca2+ uptake by cardiac SR vesicles measured in the absence and presence of calmodulin in the assay medium. When assays were performed in the absence of calmodulin, CaM BP caused strong, concentration-dependent inhibition of Ca2+ uptake by SR with virtually complete inhibition occurring at <5 μm CaM BP (Fig. 1 A). Fragmented molecules of CaM BP (CaM BP fragments A–C) failed to inhibit Ca2+ uptake by SR (Fig. 1 A, inset). Addition of low micromolar concentrations of calmodulin to the assay medium prevented the inhibitory action of CaM BP on Ca2+uptake by SR, in a concentration-dependent manner, and caused appreciable stimulation (∼40–55%) of Ca2+ uptake (Fig. 1, A and B, inset). The results presented in Fig. 1 A and the inset in Fig.1 B were obtained under the standard Ca2+ uptake assay conditions with 6 μg of SR protein in the assay medium (see under "Experimental Procedures"). In additional experiments, the effect of CaM BP on Ca2+ uptake by SR was determined with varying amounts of SR in the assay medium. The results presented in Fig. 1 B show that the concentration dependence curve for CaM BP inhibition of Ca2+ uptake is progressively shifted to the right with increasing concentration of SR in the assay. These findings suggest that the inhibitory action of CaM BP stems from its apparently stoichiometric interaction with one or more targets in the SR, and such interaction is prevented by calmodulin. Since CaM BP inhibited ATP-dependent Ca2+ uptake by cardiac SR, the effect of CaM BP on Ca2+-ATPase activity (ATP hydrolysis) was investigated. The results presented in Fig. 2 show that, under the assay conditions identical to that used for Ca2+uptake, CaM BP caused concentration-dependent inhibition of Ca2+-stimulated ATPase activity. The inhibition of Ca2+-ATPase activity and Ca2+ uptake by CaM BP occurred at similar concentration range with only a minor difference inK i values for Ca2+ uptake (50% inhibition at ∼2 μm CaM BP) and Ca2+-ATPase activity (50% inhibition at ∼2.8 μm CaM BP) (Fig. 2,inset). Thus the observed reduction in Ca2+uptake is mainly a consequence of a primary inhibition of ATPase activity by CaM BP. Addition of calmodulin (3 μm) to the assay medium reversed the inhibitory effect of CaM BP on Ca2+-ATPase activity (Fig. 2). Fig. 3 A shows the time course of ATP-dependent Ca2+ uptake by cardiac SR measured in the absence of CaM BP and in the presence of two selected concentrations of CaM BP (2 and 4 μm) with or without calmodulin. The rates of Ca2+ uptake by SR is strongly inhibited by CaM BP; the inhibition was of rapid onset (manifested within 30 s) and the degree of inhibition increased with increasing concentration of CaM BP. Addition of calmodulin (3 μm) to the assay medium prevented the inhibitory action of CaM BP. In the experiments described thus far, the effect of CaM BP was assessed by adding this peptide directly to the Ca2+ uptake assay medium. In order to determine whether the inhibitory action of CaM BP results from its association with SR membrane target(s), in subsequent experiments, the time course of Ca2+ uptake was measured using CaM BP-pretreated and control SR vesicles obtained as follows. Cardiac SR vesicles were incubated with 5 μm CaM BP in the absence of calmodulin and in the presence of 3 μm calmodulin for 10 min at 24 °C. Subsequently, the SR vesicles were recovered by centrifugation, washed to remove free CaM BP and calmodulin, and then the time course of Ca2+ uptake was determined under standard assay conditions in the absence of calmodulin or in the presence of 3 μm calmodulin. SR vesicles subjected to the same experimental protocol but without CaM BP in the incubation medium served as control for these experiments. The results from these experiments showed that pretreatment of SR with CaM BP leads to markedly reduced rates of Ca2+ uptake (Fig. 3,B and C); this decline in Ca2+ uptake rates is not observed when CaM BP-pretreatment of SR is performed in the presence of calmodulin (Fig. 3 C) or when Ca2+ uptake assays with CaM BP-pretreated SR is performed in the presence of calmodulin (Fig. 3 B). These findings suggest that the inhibitory action of CaM BP is dependent on its association with the SR membrane and that calmodulin is able to prevent the onset of CaM BP-mediated inhibition as well as reverse pre-existing inhibition induced by CaM BP. To investigate the effect of CaM BP on cardiac SR Ca2+-ATPase during its turnover, CaM BP was added to the Ca2+ uptake assay medium 3 min 15 s after initiating Ca2+-ATPase turnover. The time course of Ca2+ uptake was monitored prior to and following the addition of CaM BP for several minutes. It was found that addition of CaM BP (3 μm) during the turnover cycle of Ca2+-ATPase resulted in an apparently instantaneous and short-lived release of a small fraction (∼25%) of the pre-existing SR Ca2+ load as well as complete cessation of further Ca2+ uptake by SR vesicles (Fig.4). Addition of calmodulin (3 μm) together with CaM BP (3 μm) prevented the above effects of CaM BP (Fig. 4). In additional experiments, it was found that the fractional Ca2+ release induced by CaM BP did not exceed 25% of the pre-existing SR Ca2+ load at higher concentrations (up to 5 μm) of CaM BP (data not shown). These findings suggest that about 75% of the inhibitory effect of CaM BP on the measured Ca2+ uptake activity of SR stems from inhibition of the SR Ca2+ pump (Ca2+-ATPase); the remaining 25% of the inhibitory effect may be attributed to CaM BP-induced Ca2+ release. Since calmodulin prevented the effects of CaM BP, it is likely that CaM BP exerts its effects by interfering with calmodulin-dependent processes that are normally involved in the control of Ca2+sequestering and Ca2+ release functions of the SR. The results presented in Fig. 5 show the effect of two selected concentrations of CaM BP (1.5 and 3 μm) on Ca2+ uptake by cardiac SR at a wide range of Ca2+ concentrations (9 nm to 67 μm). CaM BP inhibited Ca2+ uptake at all Ca2+ concentrations tested. At the submaximally effective concentrations of CaM BP used, the inhibitory effect could not be overcome with increasing Ca2+ concentration. On the other hand, addition of calmodulin (3 μm) to the assay medium fully reversed the inhibitory effect of CaM BP. The kinetic parameters derived from the data shown in Fig. 5 are summarized in TableI. It can be seen that the inhibitory action of CaM BP is associated with a decrement inV max without appreciable changes in the apparent affinity of the Ca2+-ATPase for Ca2+ or the Hill coefficient (n H) for Ca2+.Table IEffect of CaM BP on the kinetic parameters of Ca 2+ uptake by SRParameterControl1.5 μmCaM BP3 μm CaM BP3 μm CaM BP + 3 μm calmodulinV max, nmol Ca2+ · mg of protein−1 · min−1489 ± 85362 ± 68255 ± 46616 ± 76K 0.5 for Ca2+ (μm)0.62 ± 0.070.55 ± 0.080.54 ± 0.040.91 ± 0.10Hill coefficient (n H)1.40 ± 0.041.47 ± 0.131.35 ± 0.051.20 ± 0.02The ki
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