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Protein kinase regulation of sarcoplasmic reticulum function in isolated adult rat ventricular myocytes Wientzek, Monika 1992

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PROTEIN KINASE REGULATION OFSARCOPLASMIC RETICULUM FUNCTION INISOLATED ADULT RAT VENTRICULAR MYOCYTESByMONIKA WIENTZEKB.Sc.(Hons.), The University of Manitoba, 1984.M.Sc., The University of Manitoba, 1986.A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Faculty of Pharmaceutical Sciences)(Division of Pharmacology and Toxicology)We accept this thesis as conformingto the standard required.THE UNiVERSITY OF BRITISH COLUMBIAApril, 1992© MONIKA WIENTZEK, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Pharmaceutical SciencesThe University of British ColumbiaVancouver, CanadaDate April 27, 1992DE-6 (2/88)11ABSTRACTThe sarcoplasmic reticulum (SR) is one of the major regulators of thecytosolic Ca2+ concentration in cardiac ventricular muscle cells. In themyocardium, relaxation results from a decrease in cytoplasmic free Ca2+ levelsmediated through an efflux of Ca2 from the cell via the sarcolemmal Na/Ca2-exchanger and by the sequestration of0a2+by the network SR membranes throughthe actions of a calcium pump (a Mg+dependent, Ca2+/K+activated adenosinetriphosphatase;Ca2’iK-ATPase). During an action potential, the Ca2 stored inthe SR is released to the cytoplasm via a Ca2+rele se channel present in thejunctional SR and Ca2 also enters the cell through voltage-controlled Ca2channels in the sarcolemmal membrane. These two processes result in an increasein cytoplasmic Ca2+ concentration which leads to contraction of the myocardium.SR membrane function is regulated in part by the phosphorylation ofproteins present in this membrane. Subsequent to 3-adrenergic stimulation of theheart by catecholamines, levels of cAMP are increased leading to the activation ofcAMP-dependent protein kinase (PK A). In isolated cardiac SR membrane vesiclesand perfused hearts, phosphorylation of an indigenous SR protein, phospholamban,is mediated by PK A. Phosphorylated phospholamban acts as a modulator of theCa2/K-ATPase to stimulate active Ca2 uptake by increasing the affinity of thisenzyme for Ca2. This stimulation of Ca2-uptake is the main mechanism bywhich catecholamines accelerate relaxation in the heart. When phospholamban isin the dephosphorylated state, a cytoplasmic portion of the molecule interacts nearthe phosphorylation site of the Ca2-pump to inhibitCa2-transport.Although a number of studies have examined the phosphorylation ofphospholamban in different experimental models, there appears to be anincongruity between the results obtained from isolated SR vesicles and perfusedwhole hearts. In isolated SR membranes, phospholamban can be phosphorylated by111PK A,Ca2/calmodulin-dependent protein kinase (CAM PK), cGMP-dependentprotein kinase (PK G) andCa2/phospholipid-dependent protein kinase (PK C).However, in isolated perfused hearts, only PK A and CAM PK (only after activationof PK A) are found to phosphorylate phospholamban. Thus, in a cardiac cell, theremay be a functional basis for the phosphorylation of phospholamban by severaldifferent types of protein kinases. Further, the protein kinase pathways may notact independently of one another but may act synergistically or antagonistically tomodulate SR function.In this study, isolated adult rat ventricular myocytes were used to examinethe phosphorylation of proteins in response to activators of PK A and PK C. Aswell, the activation of PK C was investigated in the isolated myocytes. Experimentswere also carried out to analyze the oligomeric species of phosphorylatedphospholamban obtained in the presence of activators of PK A, PK C and PK A andC, together.In the present study, a method for the isolation of a high number of viableadult rat ventricular myocytes was established. The availability of these myocytesenabled the development of methods for myocyte homogenization and isolation ofSR membranes from these homogenates. This study describes, for the first time,the isolation of sarcoplasrnic reticulum (SR) membranes from adult rat ventricularmyocytes obtained from a single rat heart. The myocyte SR preparation exhibitssimilarCa2-transport andCa2/K-ATPase activity as well as a similar proteinprofile to SR membranes isolated from intact rat heart tissue. This SR preparationexhibited a Ca2iK-ATPase activity of 414 ± 62 nmollminlmg protein (mean ±S.E.M.; n=6) and an oxalate-stimulated Ca2-uptake activity of 107 ± 4nmollmin/mg protein (mean ± S.E.M.; n=6). Pretreatment of the SR vesicles with 5jiM ruthenium red increased the oxalate-stimulated Ca2-uptake to 208 ± 10nmollminlmg protein demonstrating the presence of junctional SR membranes.Sodium dodecylsulphate polyacrylamide gel electrophoresis shows that the isolatedivSR membranes contained protein bands at 100 (Ca2fK-ATPase), 55(calsequestrin andJor caireticulin) and 53 kDa (glycoprotein). Western blots ofmyocyte SR membranes stained with ruthenium red detected 2 majorCa2-binclingprotein bands in this preparation at 53-55 kDa (calsequestrin and/or caireticulin)and 97-100 kfla(Ca2fK-ATPase). The presence of phospholamban was confirmedin the myocyte SR membranes on immunoblots probed with a monoclonal antibodyto phospholamban.The availability of purified SR membranes from adult rat ventricularmyocytes provided a useful model for the study of the regulation of SR function byprotein phosphorylation. For these studies, myocyte SR membranes were isolatedand characterized in buffers developed to prevent the dephosphorylation of proteins.These SR membranes exhibited a protein profile similar to those isolated in controlbuffers, less contamination by enzymatic activities from other cellular membranesand lower recovery ofCa2-uptake andCa2/K-ATPase activities. Three distinctproteins (phospholamban, a 31 and a 152 kDa protein) were phosphorylated by PKA in homogenates and SR membranes from adult rat myocytes stimulated withisoproterenol or forskolin. The stimulation of protein phosphorylation in myocytehomogenates and SR membranes by isoproterenol was not affected by two differentinhibitors of PK A. Also, an inhibitor of CAM PK did not affect the stimulation ofprotein phosphorylation in myocyte homogenates and SR membranes byisoproterenol. Treatment of isolated adult rat myocytes with DMSO(climethylsulfoxide) or phorbol esters dissolved in DMSO resulted in thephosphorylation of phospholamban in myocyte homogenates and SR membranes.When OAG (1-oleoyl-2-acetylglycerol) and isoproterenol were used together tostimulate protein phosphorylation in isolated adult rat myocytes, the same proteinswere phosphorylated as were found in homogenates and SR membranes treatedwith isoproterenol alone. In cytosolic fractions isolated from isoproterenol and OAGplus isoproterenol-treated myocytes, the phosphorylation of a 21, 31 and 152 kDaVprotein was stimulated. The phosphorylation of a 24 kiJa protein appeared to bedecreased in myocytes treated with isoproterenol, OAG and isoproterenol plus OAG.The separation of phosphorylated pentameric species of phospholamban fromrat myocyte SR was found to be more difficult to achieve than from SR membranesprepared from canine heart. In control and OAG-treated myocytes, two species ofphosphorylated pentameric phospholamban were obtained. In myocytes treatedwith isoproterenol or OAG plus isoproterenol, five species of phosphorylatedpentameric phospholamban were obtained.To assay the activity of PK C from myocyte cytosol and membrane fractions,FPLC fractionation to remove an inhibitory or interfering activity and the inclusionof the peptide inhibitor of PK A were required. The specific activity of PK C inmyocyte cytosol was found to be much higher than that previously found fromcytosolic fractions of canine, rat or guinea pig heart. Three peaks of Ca2 and lipid-dependent PK C activity were found in cytosolic fractions isolated from control,isoproterenol, OAG and isoproterenol plus OAG-treated myocytes. The main peakof activity contained type II and type III isozymes of PK C, in perhaps in bothautophosphorylated and nonphosphorylated states. The increase in molecularweight upon autophosphorylation of type III PK C has not been documentedpreviously. The second major peak may contain only nonphosphorylated forms oftype III PK C in control myocytes and type II and III PK C from OAG-treatedmyocytes. The third peak of PK C activity in the cytosol did not contain type III PKC protein. Two peaks of Ca2 and lipid-dependent PK C activity were found inmembranes isolated from control, isoproterenol, OAG and isoproterenol plus OAGtreated myocytes. The main peak of activity contained type III PK C, in perhapsboth autophosphorylated and nonphosphorylated states. The second peak whichcontained a large Ca2 and lipid-independent kinase activity contained type III PKC only, in perhaps the nonphosphorylated and proteolyzed PK M form. There wereno differences in the number or types of PK C formed with respect to incubationvitime. There was significantly less PK C activity in membrane fractions frommyocytes that had been treated with ethanol. As well, this study demonstrates thatin isolated adult rat ventricular myocytes, OAG was not able to activate PK C asdetermined by translocation, autophosphorylation and protein phosphorylationstudies.viiTABLE OF CONTENTSABSTRACTTABLE OF CONTENTS viiLIST OF TABLES xiLIST OF FIGURES xiiLIST OF ABBREVIATIONS xvACKNOWLEDGEMENTS xixDEDICATION xx1. INTRODUCTION 11.1. Regulation of Myocardial Contractility and Relaxation 11.1.1. Primary Control 11.1.2. Excitation-Contraction Coupling 21.1.3. Relaxation 41.2. The Sarcoplasmic Reticulum 51.2.1. Structure of the Sarcoplasmic Reticulum 51.2.2. Function of Sarcoplasmic Reticulum Proteins 71.2.2.1. Storage ofCa2 71.2.2.2. Release ofCa2 71.2.2.3. Accumulation of Ca2 81.2.3. Regulation of Sarcoplasmic Reticulum Function 81.2.3.1. Regulation of Ca2 Storage and Release 81.2.3.2. Regulation of Ca2Accumulation 91.3. Experimental Models of the Myocardium 121.4. Hypothesis, Rationale and Aims of This Study 171.4.1. Hypothesis and Rationale 171.4.2. Specific Aims 192. MATERIALS AND METHODS 202.1. Materials 202.1.1. Animals 202.1.2. Chemicals 202.1.2.1. Materials for Isolation of Adult Rat Ventricular Myocytes 202.1.2.2. Chemicals for the Treatment of Isolated Adult Rat VentricularMyocytes 21v1u2.1.2.3. Radioactivity and Autoradiography 212.1.2.4. Materials for the Isolation and Characterization of MyocyteHomogenates and SR Membranes 222.1.2.5. Materials for the Isolation and Quantitation of PK C 232.2. Methods 232.2.1. Isolation of Adult Rat Ventricular Myocytes 232.2.1.1. Modified Method of Piper et al. 232.2.1.2. Modified Method of Li et al. and Wimsatt et al. 242.2.2. Characterization of Isolated Adult Rat Ventricular Myocytes 262.2.2.1. Cell Counting, Morphology and Maintenance of Contractile Function 262.2.2.2. Quantitation of cAMP 272.2.3. Homogenization of Isolated Adult Rat Ventricular Myocytes 272.2.3.1. Preliminary Homogenization Methods 272.2.3.2. Successful Homogenization Methods 282.2.4. Isolation of Sarcoplasmic Reticulum Membranes from Isolated AdultRat Ventricular Myocytes 292.2.4.1. Preliminary Methods for the Isolation of SR Membranes fromIsolated Myocytes 292.2.4.2. Final Method for the Isolation of SR Membranes from IsolatedMyocytes 312.2.5. Characterization of SR Membranes from Isolated Myocytes 322.2.5.1. Biochemical Assays 322.2.5.1.1. Determination ofCa2-Transport Activity 322.2.5.1.2. Determination ofCa/K and Na/K-ATPase Activities 332.2.5.1.3. Determination of Cytochrome C Oxidase Activity 342.2.5.1.4. Determination of Protein Concentration 352.2.5.1.5. Determination of Free Calcium Concentrations 362.2.5.2. Electrophoretic Methods 362.2.5.2.1. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis 362.2.5.2.1.1. Polyacrylamide Gradient (5 -20% and 10 - 20%) Gels 362.2.5.2.1.2. Polyacrylamide and Bis-acrylamide Gradient Gels 382.2.5.2.1.3. Polyacrylamide Non-Gradient Gels 382.2.5.2.2. Staining of Sodium Dodecylsulfate Polyacrylamide Gels 392.2.5.2.2.1. Coomassie Brilliant Blue Stain 392.2.5.2.2.2. Stainsall Stain 392.2.5.2.3. Drying of SDS-PAGE Gels 402.2.5.2.4. Western Blotting 402.2.5.2.4.1. Antibody Localization 402.2.5.2.4.2. Ruthenium Red Staining 422.2.5.2.5. Analysis of SDS-PAGE Gels, Autoradiographs and Western Blots 42ix2.2.5.2.6. Autoradiography 422.2.6. Isolation and Quantitation of Protein Kinase C 432.2.6.1. Preparation of Myocyte Cytosol and Membrane Fractions 432.2.6.2. Preparation of Cytosol from Rat Brain and Cytosol and MembraneFractions from Bovine Trachea 442.2.6.3. DEAE-Cellulose Chromatography 452.2.6.4. Fast Protein Liquid Chromatography 462.2.6.5. Determination of Protein Kinase C Activity 462.2.6.6. Protein Concentration of PK C Activity Peaks 482.2.7. Phosphorylation of Proteins 492.2.7.1. Phosphorylation of Proteins in Isolated Myocytes 492.2.7.2. Phosphorylation and Dephosphorylation of Proteins in Isolated SRVesicles 503. RESULTS 513.1. Characterization of Isolated Adult Rat Ventricular Myocytes 513.1.1. General Viability Studies 513.1.2. Maintenance of Hormone Responsiveness 543.2. Homogenization of Isolated Adult Rat Ventricular Myocytes 543.3. Characteristics of SR Membranes Isolated From Adult RatVentricular Myocytes 573.3.1. Preparation of SR Membranes from Isolated Myocytes 573.3.2. Marker Enzyme Activities 573.3.2.1. SR Membranes Prepared in Control Buffers 583.3.2.2. SR Membranes Prepared in Buffers That Prevent Dephosphorylation 643.3.3. Protein Profile of SR Membranes 673.3.3.1. SR Membranes Prepared in Control Buffers 673.3.3.2. SR Membranes Prepared in Buffers That Prevent Dephosphorylation 713.4. Phosphorylation of Proteins in Isolated Intact Adult Rat VentricularMyocytes 753.4.1. Proteins Phosphorylated in Response to PK A Activation 753.4.1.1. Effect of PK A Inhibitors on PK A Stimulated ProteinPhosphorylation 813.4.2. Proteins Phosphorylated in Response to PK C Activation 873.4.3. Concerted Kinase Regulation of Protein Phosphorylation 903.4.3.1. Possible Involvement of Calcium/Calmodulin-Dependent ProteinKinase 903.4.3.2. Possible Involvement of Protein Kinase C 943.5. Phosphorylated Species of Phospholamban 101x3.6. Stimulation of Protein Kinase C in Adult Rat Ventricular Myocytes 1033.6.1. Activation of Protein Kinase C in Adult Rat Ventricular Myocytes 1033.6.1.1. Isolation and Quantitation of PK C After DEAE-CelluloseChromatography 1063.6.1.2. Isolation and Quantitation of PK C After Fast Protein LiquidChromatography 1064. DISCUSSION 1234.1. Isolated Adult Rat Ventricular Myocytes 1234.2. Sarcoplasmic Reticulum Membranes Isolated from Adult RatVentricular Myocytes 1254.2.1. Homogenization of Isolated Adult Rat Ventricular Myocytes 1254.2.2. Preparation of Sarcoplasniic Reticulum Membranes from IsolatedMyocytes 1254.2.3. Enzymatic Activities and Protein Profile of Myocyte SarcoplasmicReticulum 1274.2.4. Comparison of Myocyte Sarcoplasmic Reticulum MembranesPrepared in Control Buffers Versus Buffers to PreventDephosphorylation 1314.3. Protein Phosphorylation in Isolated Adult Rat VentricularMyocytes Stimulated with PK A and PK C 1334.3.1. Proteins Phosphorylated in Response to PK A Activation 1344.3.2. Proteins Phosphorylated in Response to PK C Activation 1364.3.3. CalciumJCalmodulin-dependent Protein Kinase Involvement 1404.3.4. Protein Phosphorylation in Response to PK A and PK C Activation 1414.4. Phosphorylated Species of Oligomeric Phospholamban 1434.5. Activation of PK C in Isolated Adult Ventricular Myocytes 1464.5.1. Translocation of PK C in Isolated Adult Rat Ventricular Myocytes 1474.5.2. Autophosphorylation of PK C in Isolated Adult Rat VentricularMyocytes 1525. SUMMARY AND CONCLUSIONS 1556. REFERENCES 159xiLIST OF TABLESTABLE 1. cA1VIP accumulation in isolated adult rat ventricular myocytes. 55TABLE 2.CaiK-ATPase activities of several preparations obtained bydifferent myocyte homogenization and SR centrifugation protocols. 56TABLE 3. Ca2-uptake activities of fractions obtained during the isolation ofSR membranes from adult rat ventricular myocytes. 59TABLE 4.Ca2/K-ATPase activities of fractions obtained during the isolationof SR membranes from adult rat ventricular myocytes. 61TABLE 5.Ca2-ATP se andCa2-uptake activities of SR membranes isolatedfrom whole adult rat hearts compared to those from isolated adultrat ventricular myocytes. 62TABLE 6. Marker enzyme activities of homogenate and SR membranes isolatedfrom adult rat ventricular myocytes. 63TABLE 7. Ca2-uptake activities of fractions obtained during the isolation ofSR membranes from adult rat ventricular myocytes using buffers toprevent dephosphorylation. 65TABLE 8.Ca2iK-ATPase activities of fractions obtained during the isolationof SR membranes from adult rat ventricular myocytes using buffersto prevent dephosphorylation. 66TABLE 9. Marker enzyme activities of homogenate and SR membranes isolatedfrom adult rat ventricular myocytes using buffers to preventdephosphorylation. 68TABLE 10. Protein Kinase C Activities. 107xiiLIST OF FIGURESFigure 1. Photograph of a crude preparation of adult rat ventricular myocytes. 52Figure 2. Trypan blue staining of a crude preparation of adult rat ventricularmyocytes. 53Figure 3. Coomassie blue-stained SDS-PAGE gel of homogenates and SRmembranes from whole rat heart and isolated rat ventricularmyocytes. 69Figure 4. Stainsall stained SDS-PAGE gel of homogenates and SR membranepreparations from whole rat heart and isolated rat ventricularmyocytes. 70Figure 5. Ca2-binding proteins of SR membranes purified from isolated ratventricular myocytes and whole rat heart. 72Figure 6. Identification of phospholamban in SR membranes purified fromisolated rat ventricular myocytes and whole rat heart. 73Figure 7. Coomassie blue-stained SDS-PAGE gel and western blot of myocytehomogenates and SR membranes prepared in buffers to preventdephosphorylation. 74Figure 8.A. Autoradiograph of homogenates and SR membranes from controland isoproterenol-stimulated rat ventricular myocytes. 76Figure 8.B. SDS-PAGE gel of homogenates and SR membranes from controland isoproterenol-stimulated rat ventricular myocytes. 78Figure 9. Western blot of homogenates and purified SR membranes fromcontrol and isoproterenol-stimulated rat ventricular myocytes. 79Figure 10.A. Autoradiograph of crude membranes from control, isoproterenoland forskolin-stimulated myocytes. 80Figure 10.B. SDS-PAGE gel of crude membranes from control, isoproterenoland forskolin-stimulated myocytes. 82Figure 11.A. Autoradiograph of homogenates and SR membranes isolated fromisoproterenol-stimulated myocytes in the absence and presence ofHA1004. 83Figure 11.B. SDS-PAGE gel of homogenates and SR membranes isolated fromisoproterenol-stimulated myocytes in the absence and presence ofHA1004. 84Figure 12.A. Autoradiograph of homogenates and SR membranes isolated fromisoproterenol-stimulated myocytes in the absence and presence ofHAlOO4andH-8. 85Figure 12.B. SDS-PAGE gel of homogenates and SR membranes isolated fromisoproterenol-stimulated myocytes in the absence and presence ofHA1004 and H-8. 86xiiiFigure 13. Autoradiograph of homogenates and SR membranes isolated frommyocytes stimulated with isoproterenol, x-phorbol didecanoate andphorbol myristate acetate. 88Figure 14. Autoradiograph of homogenates and SR membranes isolated frommyocytes stimulated with isoproterenol, DMSO and OAG (in DMSO). 89Figure 15.A. Autoradiograph of homogenates and SR membranes isolated frommyocytes stimulated with isoproterenol, ethanol and OAG (inethanol). 91Figure 15.B. SDS-PAGE gel of homogenates and SR membranes isolated frommyocytes stimulated with isoproterenol, ethanol and OAG (inethanol) 92Figure 16. Autoradiograph of homogenates and SR membranes isolated fromcontrol myocytes and myocytes stimulated with OAG. 93Figure 17. Autoradiograph of homogenates and SR membranes isolated fromDMSO and isoproterenol-stimulated myocytes in the absence andpresence of CGS 9343 B. 95Figure 18. Autoradiograph of homogenates and SR membranes isolated frommyocytes stimulated with isoproterenol and with OAG andisoproterenol. 96Figure 19. Incorporation of radioactivity into 27 and 8.5 kiJa protein bandsfrom SDS-PAGE gels of homogenates and SR membranes fromcontrol myocytes and myocytes stimulated with isoproterenol, OAGand OAG & isoproterenol. 97Figure 20. Incorporation of radioactivity into 31 and 152 kDa protein bandsfrom SDS-PAGE gels of homogenates and SR membranes fromcontrol myocytes and myocytes stimulated with isoproterenol, OAGand OAG & isoproterenol. 99Figure 21. Autoradiograph of myocyte cytosolic fraction isolated from control,isoproterenol, isoproterenol & OAG and OAG-treated myocytes. 100Figure 22. Western blot and SDS-PAGE gel of canine cardiac ventricular SRmembranes probed with a monoclonal antibody to phospholamban. 102Figure 23. Western blot and SDS-PAGE gel of rat myocyte and canine cardiacventricle SR membranes probed with a monoclonal antibody tophospholamban. 104Figure 24. Western blot of homogenates and SR membranes from control,isoproterenol, OAG and isoproterenol & OAG-stimulated myocytes. 105Figure 25. FPLC profile of purified rat brain PK C. 109Figure 26. Protein elution FPLC profile of control myocyte cytosol andmembrane fractions. 110xivFigure 27. PK C activity of FPLC profiles of control myocyte cytosolic fractionsand membranes assayed in the absence and presence of cAMP-dependent protein kinase inhibitor. 111Figure 28. PK C activities of FPLC profiles of cytosolic and membrane fractionsfrom control myocytes incubated for 1, 5 and 10 mm. 112Figure 29. Area under the curve (AUC) of PK C activity from FPLC fractions27 - 31 of myocyte cytosol and membrane fractions. 113Figure 30. Western blots of PK C activity peaks from control (1 mm) myocytecytosol and membrane fractions, probed with antibodies to Type IIand Type III PK C. 115Figure 31. PK C activities of FPLC profiles of cytosol and membrane fractionsfrom ethanol-treated myocytes incubated for 1, 5 and 10 mm. 116Figure 32. PK C activities of FPLC profiles of cytosol and membrane fractionsfrom OAG-treated myocytes incubated for 1, 5 and 10 mm. 118Figure 33. Western blots of PK C activity peaks from OAG-treated (10 mm)myocyte cytosol and membrane fractions, probed with antibodies toType II and Type III PK C. 120Figure 34. PK C activities of FPLC profiles of cytosol and membrane fractionsfrom R59022 and OAG-treated myocytes incubated for 1, 5 and 10mm. 121xvLIST OF ABBREVIATIONS& and% percentphosphorus-32tritium45Ca calcium-45x-PDD 4c-phorbol didecanoateANOVA analysis of varianceATP adenosine 5’-triphosphateATPase adenosine triphosphataseAUC area under the curveAV atrioventricularBIS N,N’-methylene-bis-acrylamideBCIP 5-bromo-4-chloro-3-indoyl phosphate p-toluidine saltBSA bovine serum albuminCa2+ free calcium ionsCafK-ATPase calcium-dependentJpotassium-stimulated-ATPaseCAM calmodulinCAM PK Ca2/calmodu1in-dependent protein kinasecAMP adenosine 3 ‘:5 ‘-cyclic monophosphateCGS 9343 B 1,3-dihydro-1-(1-((4-methyl-4H, 6H-pyrrolo(1,2-a)-(4, 1)benzoxazepin-4-yl)methyl)-4-piperindinyl)-2H-benzimidazol-2-one maleateCi Curiecpm counts per minuteDAG diacyiglycerolddfT2O distilled, deionized waterDEAE diethylaminoethylxviDM50 dimethylsulfoxidedpm disintegrations per minuteDTT dithiothreitolE-P phosphoenzyme intermediate of the SRCa2fK-ATPaseE1,E2 conformational states of the SRCa2/K-ATPaseEDTA ethylenediaminetetraacetic acidEGTA ethyleneglycol bis(f3-aminoethylether)-N,N,N’N’-tetraacetic acidETOH ethanolForsk. forskolinFPLC fast protein liquid chromatographyg gravitational forceg gramgmax gravitational force at maximum radiusH-8 N-(2-(methylamino) ethyl)-5-isoquinolinesulfonamidedihydrochiorideHA1004 N-(2-guanidinoethyl)-5-isoquinolinesulfonaniidehydrochlorideHEPES N-(2-hydroxyethyl)piperazine-N’(2-ethanesulfonic acid)HPL high molecular weight form of phospholambanIBMX isobutylmethyixanthine1P3 inositol- 1 ,4,5-trisphosphateISO. isoproterenolK-H Krebs-Henseleit bufferkDa kilodaltonsKj dissociation constant for inhibitorKm Michealis constant1 litrexviiLDL low density lipoproteinLPL low molecular weight form of phospholambanM molarmA milliampsMEM Joklik-Modified minimum essential mediumMES 2-(N-morpholino)ethanesulfonic acidmg milligramMgATP magnesium adenosine triphosphatemm minuteml millilitremol moleMOPS 3-(N-morpholino)propanesulfonic acidn number of separate experimentsNafK-ATPase sodiumlpotassium-dependent-ATPaseOAG 1-oleoyl-2-acetylglycerolPAGE polyacrylamide gel electrophoresispH negative logarithm of the hydrogen ion activityP inorganic phosphate (PO43PK A cAMP-dependent protein kinasePK C Ca2/phospholipid-dependent protein kinasePK M catalytic fragment of PK CPMA phorbol 12-myristate 13-acetatePMSF phenylmethylsulfonyl fluoridePS phosphatidylserinePTFE TeflonR59022 6-(2-(4-((p-fluorophenyl)-phenylmethylene)-1-piperidinyl)-ethyl)-7-methyl-5-H-thiazolo-3 ,2-a-pyrimidine-5-oneray average radiusxviiirpm revolutions per minutes secondS.D. standard deviationS.E.M. standard error of the meanSA sinoatrialSAG 1-stearoyl-2-aracbidonylglycerolSDS sodium dodecylsulfateSL sarcolemmaSR sarcoplasmic reticulumSTI soybean trypsin inhibitort-tubule transverse tubuleTBS TRIS-buffered salineTCA trichioroacetic acidTEMED N,N,N’,N’-tetramethylethylenediamineTPCK L- 1-tosylamide-2-phenylethyl chioromethyl ketoneTRIS tris(hydroxymethyl)aminomethaneTTBS TRIS-buffered saline containing 0.2% Tween 20microV voltxixACKNOWLEDGEMENTSI would like to sincerely thank my supervisor Dr. Sidney Katz for hisguidance and interest in these studies. I am also very grateful for hisencouragement during the difficult periods of this work. Also, I would like to thankthe members of my supervisory committee, Dr. Stelvio Bandiera, Dr. KeithMcErlane, Dr. Kath MacLeod, Dr. Glen Tibbits and Dr. Michael Walker for theirhelpful suggestions over the course of these studies.Very special thanks to my husband, Matthew, for his encouragement andmany helpful discussions during the period of this work and for his assistance withthe statistical analysis, typing, library work and editing during the production ofthis thesis.Many thanks to my friends and collegues in the laboratory, Dr. Bruce Allen,Dr. Shawn Black, Dr. James Gilchrist, Dr. Raj Mahey, Ms. Ling Wu and Dr. KathQuayle. A special thankyou to Ms. Gaye McDonald-Jones for many helpfuldiscussions and expert technical assistance.I would like to acknowledge the generous gifts of Dr. Jerry Wang (Dept. ofBiochemistry, University of Calgary) who provided the Al monoclonal antibody tophospholamban, of Dr. Christopher Wilson (Dept. of Pediatrics, University ofWashington) who provided the antisera against PK C Type II and Type III and Dr.Bruce Allen (Dept. of Biochemistry, University of Calgary) who provided caninecardiac SR membrane vesicles and purified rat brain PK C.I also gratefully acknowledge the receipt of my studentship from the Heartand Stroke Foundation of Canada.DEDICATIONThis thesis is dedicated to my husband, Dr. Matthew Rowland Wright, for hispatience, love, support and encouragement.xx11. INTRODUCTION1.1. Regulation ofMyocardial Contractility and Relaxation1.1.1. Primary ControlAt rest, the heart can generate and conduct electrical impulses which allowthe myocardial cells of the heart to contract and relax as a functional syncytium.There are three pacemaker regions in cardiac muscle, the SA (sinoatrial) node, theAV (atrioventricular) node and the His-Purkinje system. These specialized cells arecapable of spontaneous depolarization to the threshold of initiation of an actionpotential. The SA node generates impulses at the fastest rate (70/mm at rest inman) and dominates the other pacemakers to determine the heart rate. Normally,the electrical impulse from the SA node is transmitted throughout the cells of theatria, which are stimulated to contract, to the AV node. The AV node conducts theimpulse from the atria to the ventricles, slowing the conduction from the SA nodeand by genesis of its own impulses at 50 - 60/mm. From the AV node, the impulsetravels down the bundle of His to the ventricles, causing them to contract. Whenthe rate of impulse generation by the SA node is slowed, due to disease or intensevagal stimulation, the AV node becomes the dominant pacemaker of the heart. Ifthere is failure of both the SA and AV nodes, the His-Purkinje system will generateimpulses at 30 - 40/mm which may be too low a rate to sustain life.The heart is regulated by the autonomic nervous system and it has anintrinsic ability to regulate its own function by responding to changes in venousreturn. This matching of venous return to cardiac output is known as the Frank-Starling law of the heart [Frank (1895); Patterson et al. (19 14)1 and is the primarymechanism for the control of cardiac output in the resting state. Briefly, anincreased amount of blood entering the heart causes an increased stretch of cardiac2muscle. The stretched muscle contracts with a greater force hence, pumping theextra blood out of the heart.In addition, the contractile force of the heart and heart rate may also becontrolled by the autonomic nervous system. Sympathetic nerves, releasingnoradrenaline, increase both contractility and heart rate. There are a large numberof sympathetic inputs into both the atria and ventricles. Cholinergic nerves,releasing acetylcholine, decrease both contractility and heart rate. The two atriaare well supplied by parasympathetic nerves but the ventricles have fewparasympathetic fibers. The SA and AV nodes are also innervated cholinergically,by branches of the vagus nerve.1.1.2. Excitation-Contraction CouplingWhen an electrical impulse reaches a contractile cell of the heart, the cellbecomes excited and an action potential is generated. The action potential consistsof depolarization by a fast inward Na+ current followed by an inward Ca2+ current.These two depolarizing currents are balanced by a repolarizing current, theinwardly rectifying K+ current. Repolarization then occurs due to a decay of theinward Ca2+ current and activation of an outward delayed rectifying K+ current.The action potential travels along the sarcolemma (SL) and down the transversetubules (t-tubules), causing Ca2 to enter the cell through voltage-controlled Ca2-channels in the SL membrane. Ca-induced Ca2 release [Fabiato (1983); Fabiatoand Fabiato (1975)] results from the increase of cytosolic Ca2 which triggers alarge release of Ca2+ from the sarcoplasmic reticulum (SR). Upon influx of Ca2+from outside the cell and release of Ca2 from the SR, the intracellular Ca2concentration rises from a resting level of 0.1 to a maximum of 5 .tM [Blinks (1986)].The Ca2 in the cytosol binds to the Ca2 binding site on troponin C causing thisprotein to interact more strongly with troponin I, thereby terminating the inhibition3of the interaction between actin with myosin. This conformational change displacesthe troponin-tropomyosin complex from actin and allows actin to interact withmyosin, consequently allowing force generation [Bers (1991)1. Contraction of themyofibril then occurs according to the sliding filament crossbridge attachmenttheory of force generation elaborated by Huxley and Simmons (1971).Myocardial contractility can be regulated by both branches of the autonomicnervous system, the sympathetic and parasympathetic nervous system.Sympathetic stimulation via the interaction of noradrenaline and adrenaline withc- and 3-adrenergic receptors on the surface of the SL membrane causes an increasein both the Ca2+ and the K+ currents. 3-receptors are linked to adenylate cyclasethrough a stimulatory G-protein and when activated, adenylate cyclase producescAMP (cyclic adenosine-monophosphate) which is then available to activate thecAMP-dependent protein kinase (PK A). When active, PK A phosphorylates thevoltage-dependent Ca2+ channel in the SL which results in an increase in theamount of Ca2 entering the cell [Hartzell et at. (1991)1. Increased Ca2 in thecytosol of the cell leads to a stimulation of the rate and force of contraction due to agreater activation of troponin C. The stimulation of x-receptors results in positiveinotropy by increasing the sensitivity of the contractile proteins to Ca2 [Endoh andBlinks (1988)1 and by an increase in cytoplasmic Ca2 caused, perhaps, by releaseof Ca2 from the SR via an inositol(1,4,5)-trisphosphate-sensitive receptor [Scholzet at. (1988)1. Activation of x-receptors also prolongs the action potential durationby decreasing an outward K+ current, which may also lead to an increase incontractility [Fedida et at. (1989)]. There are still many unresolved questionsregarding the mechanism of positive inotropy caused by ce-receptor activation [Bers(1991)]. Activation of muscarinic receptors by acetylcholine causes an inhibition ofthe stimulation by (3-receptor agonists of adenylate cyclase via an inhibitory Gprotein and thus, attenuation of the phosphorylation of proteins by decreasing4cAMP production [Fleming et at. (1987)]. Therefore, parasympathetic stimulationresults in a decrease in contractility.1.1.3. RelaxationRelaxation of the myocardium occurs as the cell membrane repolarizes andby the removal of Ca2+ from the cytosol of the cell. The decrease in cytosolic Ca2+concentration causes Ca2+ to dissociate from troponin C, which allows troponin I toinhibit actin and thus, disrupt its interaction with myosin. Ca2 is removed mainlyfrom the cytosol by sequestration into the SR by a Ca2-pump, the Ca2fK-ATPase (a Mg2-dependent, Ca2/K-activated adenosine triphosphatase) andthrough efflux from the cell via the Na/Ca-exchanger [Bers (1991)].Cardiac relaxation may also be regulated by sympathetic andparasympathetic influences. Noradrenaline, via the activation of 3-receptors andcAMP production, stimulates the PK A phosphorylation of several proteins involvedin relaxation. The major cytosolic proteins phosphorylated include troponin I andC-protein (a protein associated with myofibrils) and from the SR, a protein namedphospholamban. Phosphorylation of troponin I results in an increased rate of Ca2+dissociation from troponin C thus, separating troponin I from C and allowingtroponin I to once again inhibit the interaction of actin with myosin. When C-protein is phosphorylated it is not as effective in stimulating the myosin ATPaseand thus, promotes relaxation [Hartzell (1985)]. The phosphorylation ofphospholamban has been shown to activate theCa2/K-ATPase by increasing itsaffinity for Ca2 and thus, stimulating Ca2 uptake into the SR leading to anacceleration of relaxation [Tada et at. (1982)]. This stimulation ofCa2-uptake isthe main mechanism by which sympathetic neurotransmitters accelerate relaxationin the heart. Parasympathetic stimulation results in a negative chronotropic effectdue to hyperpolarization of SA node cells and a decrease in conduction velocity in5the AV node and Purkinje fibers [Hutter and Trautwein (1956); Taylor (1985)1.Further, acetyicholine interacts with muscarinic receptors on the surface of the SLmembrane leading to modulation of a K+ channel directly through a G protein[Pfaffinger et al. (1985)]. This results in an increase in the K conductance throughthe channel and subsequently, faster hyperpolarization of the membrane.1.2. The Sarcoplasmic Reticulum1.2.1. Structure of the Sarcoplasmic ReticulumThe sarcoplasmic reticulum (SR) is an intracellular tubular membranousnetwork analogous to the endoplasmic reticulum of noncontracting cells. The SRcan be separated into two main types: a) network (longitudinal or free SR), ameshwork surrounding the myofilaments and b) junctional SR [Katz et al. (1986)].Continuous with the network SR, the junctional cisternae are closely associatedwith sarcolemmal and t-tubule membranes. Between the junctional SR and the ttubule membrane are regularly spaced, densely staining processes, called “feet”[Franzini-Armstrong (1970)1. These processes suggest a continuity between thejunctional SR and the t-tubule membrane. Ultrastructurally, another specializedtype of junctional SR can be recognized known as corbular SR [Dolber and Sommer(1981)]. Corbular SR does not associate with the SL or t-tubule membrane butappears to bud from the network SR and is associated with Z and I bands[Jorgensen et al. (1988)]. The lumen of junctional and corbular SR contain electron-dense granules, which are not present in network SR [Sommer and Jennings(1986)].In the myocardium, proteins specific to the SR may be uniformly distributedor localized to certain types of SR. TheCa2fK-ATPase is uniformly distributed inthe membrane of network SR but in junctional SR is not present in areas associated6with SL or t-tubule membranes [Jorgensen et at. (1982)]. CaIK-ATPase is alsonot found in corbular SR membranes. A 53 kDa glycoprotein has beendemonstrated in both network and junctional SR but in network SR it appears toco-localize with theCa21K-ATPase [Leberer et at. (1990)]. The SR also contains a130 kDa glycoprotein, which is immunochemically similar to the 53 kflaglycoprotein [Campbell et at. (1983)]. Phospholamban, the Ca2-pump regulatorprotein, has been found to be uniformly distributed throughout the membranes ofboth network and corbular SR but in junctional SR is not present in areasassociated with SL or t-tubule membranes [Jorgensen and Jones (1987)1. Theelectron-dense granules present in the lumen of both junctional and corbular SRhave been identified as the Ca2-binding protein, calsequestrin [Jorgensen et at.(1988)]. Calreticulin, the 55 kDa high affinity Ca2-binding protein of SR and a165 kDa Ca2-binding protein that also binds LDL were found to be distributedthroughout the lumen of network and junctional SR [Fliegel et at. (1989); Hofmannet at. (1989)1. The “feet” structures between the junctional and t-tubule membraneshave recently been identified as oligomers of the SR Ca2+rele se channel (alsoknown as the cardiac ryanodine receptor) [Inui et at. (1987)1. There are severalother ion channels in the membranes of the SR including a chloride channel thathas been found to be randomly distributed throughout the SR [Rousseau et at.(1988)] and a potassium channel that has been isolated from network SR [Hill et at.(1989)]. ACa2/calmodulin-dependent protein kinase [Molla and Demaille (1986)]and a phosphatase [Kranias and Di Salvo (1986)] are also associated with SRmembranes but the localization of these proteins in the SR is unknown.Recently, in human SR membranes, the following proteins have beenidentified: the Ca2hiK+ATPase, calsequestrin, Ca2+rele se channel,phospholamban and glycoproteins of 53, 155 and 165 kDa [Movsesian et at. (1990)].71.2.2. Function of Sarcoplasmic Reticulum ProteinsAlthough cardiac SR performs some functions similar to the endoplasmicreticulum, such as lipid metabolism [Cornell and MacLennan (1985); Kasinathanand Kirchberger (1985)] and glycolysis [Entman et al. (1976)1, the main functionsare to store, release and accumulate Ca2+.1.2.2.1. Storage ofCa2Ca2 is stored in the lumen of the SR by binding mainly to two proteins: 1) alow affinity, high capacity Ca2-binding protein, calsequestrin and 2) caireticulin,which exhibits both high affinity, low capacity and low affinity, high capacity Ca2-binding sites. Calsequestrin is a 55 kDa protein that can bind 12 - 40 moles ofCa2/mole with an affinity (Kd) of 0.1 - 1.0 mM [Mitchell et al. (1988)]. Ascalsequestrin is localized to junctional and corbular SR, it is available to releaseCa2 near theCa2-rele se channels of the junctional SR. Caireticulin can bind 25moles of Ca2/mole at the high capacity site and 1 mole of Ca2fmo1e at the lowcapacity site [Ostwald and MacLennan (1974)]. The role of the 165 kDa Ca2-binding protein of the SR remains unknown.1.2.2.2. Release of Ca2Ca2+ is mainly released from cardiac SR via the Ca2+ release channelpresent in the junctional SR. This channel exhibits a molecular weight of 350 - 450kDa and the homotetramer protein forms a quatrefoil structure. In electronmicrographs, the quatrefoil structure was found to be identical to the “feet”structures observed between the junctional SR and the t-tubule membrane [Saito etal. (1988)]. A minor pathway of Ca2 release from cardiac SR involves the8activation of an inositol(1,4,5)-trisphosphate-sensitive receptor [Hirata et al.(1984)]. This release of Ca2 by 1P3 has been found to be too slow and too small tobe a primary mechanism in cardiac excitation-contraction coupling [Feher andFabiato (1990)1.1.2.2.3. Accumulation of Ca2The accumulation of Ca2+ into the cardiac SR occurs via the action of acalcium pump, theCa2/K-ATPase. The ATPase has a molecular weight of 110kDa and transports 2 moles of Ca2 for every mole of MgATP hydrolyzed [Yamadaet al. (1970)]. In the basic reaction sequence, the enzyme undergoes an E1 to E2conversion with the formation of an E-P acyiphosphate intermediate [Tada et al.(1988)]. For charge balance, duringCa2-uptake, the counter-transport of positivecharges or co-transport of negative charges may be mediated by the anion andcation channels present in the SR membrane [Feher and Fabiato (1990)]. Recently,theCa2+/K+ATPase has been shown to be able to operate as a Ca2+ channel forrapid Ca2+ efflux, under conditions where certain drugs have uncoupled theenzyme [de Meis (1991)].1.2.3. Regulation of Sarcoplasmic Reticulum Function1.2.3.1. Regulation of Ca2 Storage and ReleaseThe regulation of the storage of Ca2 in the SR by its binding and release toCa2-binding proteins is not well understood. It has been suggested that thepotassium concentration within the SR modulates the Ca2-binding properties ofcalsequestrin, since the affinity of calsequestrin for Ca2+ is reduced by potassium[Slupsky et al. (1987)]. In junctional SR, there is a 26 kDa protein which binds to9calsequestrin. It is not known if this protein serves to anchor calsequestrin to theinside of the SR membrane or whether it is involved in the interaction betweenCa2 storage and release mechanisms [Mitchell et at. (1988)]. Calsequestrin can bephosphorylated in vitro by casein kinase II but this phosphorylation was found notto affect its function [Cala and Jones (199 1)1.The regulation ofCa2+rele se from the SR is mainly a result of effects onthe Ca2+rele se channel. The channel is activated to release Ca2+ atsubmicromolar concentrations of cytosolic Ca2+ [Meissner et at. (1988)], by mMconcentrations of adenine nucleotides (only when Ca2+ had already partiallyactivated the channel; Rousseau et at. (1986)) and caffeine [Rousseau et at. (1988)].Ruthenium red and magnesium (both iM; Rousseau and Meissner (1989)),calmodulin (1 - 4 pM; Meissner and Henderson (1987)) and decreasing pH [Ma et al.(1988)] inhibit the activity of the channel. Recently, the Ca2-rele se channel hasbeen shown to be a preferred substrate forCa2+/calmodulindependent proteinkinase (CAM PK) and when phosphorylated, the channel became active [Witcher etat. (1991)].1.2.3.2. Regulation of Ca2AccumulationThe regulation of Ca2-uptake into the SR is primarily effected throughactions on theCa2/K-ATPase. Monovalent cations (10 - 20 mM) can stimulatethe activity of the pump with the most effective being K [Jones et at. (1977)].There is some controversy as to the function of the 53 kDa glycoprotein that colocalizes with theCa2/K-ATPase and whether it has any effect upon the functionof the Ca2-pump [Leberer et at. (1989)]. As described previously, the activity ofthe CafK-ATPase is modulated by phospholamban. Phospholamban is ahomopentamer with an apparent molecular mass of 25 - 27 kDa as determined byalkaline SDS-PAGE [Jones et at. (1985)]. Each monomer (obtained upon boiling in10SDS) has an approximate molecular mass of 8.5 kDa as determined by alkalineSDS-PAGE and 6 kfla as inferred from the amino acid sequence. Recently,Watanabe et at. (1991) have used low-angle laser light scattering photometry tomore accurately determine the molecular mass of phospholamban. Theseinvestigators found that the phospholamban oligomer had a molecular mass of 30.4kDa and confirmed that it was composed of 5 subunits.Phospholamban stimulates the Ca27X-ATPase by lowering its Km forCa2, only when it is phosphorylated [Le Peuch et at. (1980)]. Recently, in humancardiac SR membranes, phospholamban was found to stimulate Ca2+upt ke byincreasing the apparent affinity of theCa2[K-ATPase for Ca2 {Movsesian et at.(1990)]. When phospholamban is in the dephosphorylated state, a cytosolic portionof the monomer interacts near the phosphorylation site of the Ca2 pump to inhibitCa2 transport [Kirchberger (1991)1. Upon phosphorylation, phospholambanundergoes a conformational change and no longer interacts with the Ca2+ pump.Removal of the cytoplasmic portion of phospholamban by proteolytic cleavage alsoleads to activation ofCa2-uptake [Kirchberger et at. (1986)]. Direct evidence foran interaction of phospholamban with the Ca2/K-ATPase has come from SRreconstitution studies and other studies employing a monoclonal antibody tophospholamban or crosslinking reagents. In the reconstitution studies [Kim et at.(1990)1, purified phospholamban and Ca2/K-ATPase were reconstituted intophospholipid vesicles and the Ca2 transport of these vesicles modulated by thestate of phospholamban phosphorylation. Crosslinking of purified phospholambanandCa27X-ATPase identified a region close to the phosphorylation site of thisenzyme which bound to dephosphorylated, but not phosphorylated, phospholamban[James et at. (1989)1. By incubating SR vesicles with a monoclonal antibody tophospholamban, Suzuki and Wang (1986) found Ca2-uptake to be increased to aslightly higher level than that produced by PK A phosphorylation. More recently,these investigators have demonstrated that phospholamban amino acid residues 711to 16 are involved in the regulation of theCa2fK-ATPase [Morris et al. (1991)].Dialysis of isolated ventricular myocytes with a monoclonal antibody tophospholamban was shown to result in an increase in Ca2+upt ke and release inresponse to depolarizing pulses and inhibited the ability of isoproterenol (anonspecific p-receptor agonist) to stimulate Ca2+upt ke and release [Sham et al.(1991)].Phospholamban was found to be phosphorylated at serine 16 [Wegener et al.(1989)] by cAMP-dependent protein kinase in response to f3-receptor agonists inintact perfused hearts [Kranias and Solaro (1982); Lindemann et al. (1983)]. Also,PK A was found to phosphorylate phospholamban in isolated adult rat ventricularmyocytes [Blackshear et al. (1984)] and isolated SR vesicles [Kirchberger and Tada(1976)). Ca2/calmodulin-dependent protein kinase (CAM PK) also phosphorylatesphospholamban in the perfused heart and in isolated SR vesicles, on the threonineat position 17 [Simmerman et al. (1986)]. The physiological significance of CAM PKphosphorylation is not understood as, in the perfused heart, it only occurs after 13-receptor activation [Lindemann and Watanabe (1985)]. In experiments in isolatedSR vesicles,Ca2/phospholipid-dependent protein kinase (PK C) was also shown tophosphorylate phospholamban with a concurrent increase in Ca2+upt ke[Movesian et al. (1984)1. However, in perfused adult rat hearts, activators of PK Chave been found to decrease the rate and force of contraction and phospholambanwas found not to be phosphorylated [Edes and Kranias (1990)]. A recent study castdoubt on the observation that activation of PK C causes a decrease in contractility[MacLeod and Harding (1991)]. These authors found that in isolated adult rat andguinea pig ventricular myocytes, activation of PK C caused a positive inotropiceffect by increasing systolic Ca2. In isolated SR vesicles, cGMP-dependent proteinkinase (PK G) was also found to phosphorylate phospholamban [Raeymaekers et al.(1988)], however, in intact perfused hearts, phospholamban was not phosphorylatedin response to agents that increased intracellular cGMP [Huggins et al. (1989)1.12The majority of phospholamban was shown to be dephosphorylated byprotein phosphatase 1 activity in isolated SR vesicles [MacDougall et al. (1991)1. Aswell, both an inhibitor of protein phosphatase 1 and protein phosphatase 1 itself,have been shown to be phosphorylated by PK A in perfused guinea pig heart andisolated SR membranes [MacDougall et al. (1991); Neumann et al. (1991)]. Thesephosphorylations prevent protein phosphatase 1 from dephosphorylatingphospholamban, thus, contributing to the positive inotropic effect of f-adrenergicstimulation.In isolated SR vesicles, the rate of Ca2+upt ke can be correlated to thephosphorylated and dephosphorylated levels of phospholamban [Kasinathan et al.(1988)]. Since each monomer of phospholamban contains two phosphorylation sites,a pentamer of phospholamban contains 10 sites in total. Phosphorylation ofphospholamban reduces its electrophoretic mobility on SDS-PAGE gels, undercertain conditions [Wegener and Jones (1984)]. Western immunoblots of these gelsdemonstrate multiple bands of the phospholamban pentamer as a result of fillingone or both sites of phosphorylation in each of the monomers. Therefore, 6 bandsare observed following phosphorylation by PK A, corresponding to thephosphorylation of monomers 0 to 5 at a single site [Li et al. (1990)]. Eleven bandsare observed following phosphorylation by both PK A and CAM PK, indicating thephosphorylation of monomers 0 to 5 at both sites [Wegener et al. (1989)].1.3. Experimental Models of the MyocardiumCommonly used experimental models of the myocardium include: the wholeheart in vivo, isolated perfused hearts, tissue slices, organ culture, isolatedventricular myocytes, heart homogenates and intracellular organelles isolated fromhearts. Each model has distinct properties that are advantageous for the13performance of certain types of experiments and disadvantageous for others[Jennings and Morgan (1986)].From the whole heart in vivo, in closed-chest experiments, one can measureheart sounds and electrical activity by monitoring echo- and electrocardiograms[Shepherd and Vanhoutte (1979)]. It is also possible to measure arteriovenousdifferences across the myocardium of endogenous substrates, hormones, drugs ortoxins [Jennings and Morgan (1986)]. In open-chested experiments, it is possible toobtain samples of tissue from specific areas of the heart through biopsy before,during and after the experiment, as well as sampling from coronary arteries andveins. Because of these features, the open-chested dog is often utilized as a model ofregional ischemia. Disadvantages include stability of the test subject during thestudy (for example: heart failure, arrhythmia or ischemia that may develop uponapplication of hormones, drugs toxins, etc.), tissue heterogeneity which makesstudies on metabolism difficult to interpret and finally, reactions can only bestudied through flux of the entire pathway rather than the individual steps of thepathway or process.Isolated perfused hearts can be prepared for perfusion in a Langendorff[Langendorif (1895)] or working heart mode [Neeley et al. (1967)]. The Langendorifmode maintains the intrinsic beating rate of the heart and the buffers are perfusedthrough the coronary arteries via the aorta in a retrograde fashion. Langendorffperfusion is easy to perform and useful for studies involving the incorporation ofradioactivity into phospholipids, proteins, etc. The working heart is a left heartpreparation where the left atrium and subsequently, the left ventricle is perfusedvia the pulmonary vein and the myocardial tissue is perfused via the coronaryarteries from outflow through the aorta. Contraction is stimulated electrically andboth the preload and afterload pressures can be varied. Thus, the working heart isuseful for studies where mechanical performance and metabolism are correlatedfollowing the addition of drugs, hormones or substrates. Advantages of the perfused14heart model include: i) compound delivery via the normal capillary bed, ii) intactcells, iii) stability of the preparation is easily measured, iv) mechanical function isstable for hours and v) the heart can be rapidly frozen after the study for themeasurement of metabolites. Perfused hearts have several disadvantagesincluding: i) the cellular heterogeneity of the heart, ii) coronary flow is increasedgreatly over the in vivo situation, iii) events that require 3 to 6 hrs or longer todevelop in vivo can not be measured, iv) the identity of a single hormone or agentmust be known in order to add it to the perfusate and v) in ischemic studies, theheart develops contracture-rigor and the no-flow phenomenon develops [Jenningsand Morgan (1986)].Handcut tissue slices of atrial, ventricular or papillary muscle are especiallyuseful for studies on the regulation of cell volume of the adult heart [Grochowski etal. (1976)]. These slices are also useful in suspension for studying the effect ofagents on integrated metabolism. Tissue slices have the following advantages: i)they maintain cell volume, ultrastructure and ion gradients, ii) there is nodependence on vascular integrity for substrate supply or loss of metabolic productsand iii) the slice functions in an integrated way, therefore, studies on anoxia andosmolarity are possible. The major disadvantages of tissue slices include: i) theheterogeneity of the tissue, ii) the impossibility of assessing contractile functionwithin the slice and iii) the slice is diffusion limited.Fetal mouse hearts can be cultured intact for 24 to 48 hrs [Wildenthal(1971)]. An advantage over tissue slices is that all cells are intact and experimentscan be performed over a much longer period of time. Disadvantages of fetal organculture include: i) limitations by diffusion leading to necrosis at the centre if smallhearts are not used, ii) cellular heterogeneity and iii) the fact that myocytes arefetal and not adult.Fetal, neonatal and adult hearts can be used to isolate cardiac muscle cells(myocytes) by digestion of the hearts with proteolytic enzymes [Harary and Farley15(1960); Jacobson (1989)]. Fetal or neonatal myocytes can be grown in culture forextended periods of time making them useful for studies involving hypertrophy anddifferentiation. However, because the cells are growing and dividing they have thedisadvantage of de-differentiation during their time in culture and overgrowth byfibroblasts interferes with the interpretation of data from metabolic studies.Myocytes from adult hearts do not divide and are usually utilized as freshpreparations. Isolated myocytes offer the advantage of being a homogeneous cellpopulation as well as being free of neural and humoral influences. The ability tolocalize a metabolic or other activity to the cardiac muscle cell is the mainadvantage in the use of this model. They are an intact, viable cellular preparationthat can be easily manipulated. The medium surrounding isolated myocytes can becontrolled and quickly changed to facilitate treatment with different agents. As aresult, myocytes (especially adult ventricular cells; Watanabe et al. (1986)) arewidely used as a model for: i) receptor binding studies [ex. Martens et al. (1987)1, ii)studies on hormone-receptor interaction [ex. Bode and Brunton (1989)], iii)metabolic studies [ex. Hee-Cheong and Severson (1989)1, iv) studies on proteinphosphorylation [ex. Robinson-Steiner and Corbin (1986)], v) ion flux studies [ex.Brierly et al. (1983)1, vi) electrophysiological studies on ion channels and currentsincluding patch-clamping and techniques for internal dialysis of the cell [ex. Harveyand Hume (1989); Sham et al. (1991)1, vii) contractility studies utilizing single celledge detection [ex. MacLeod and Harding (1991)1, viii) drug uptake studies [ex.Cramb and Dow (1983)1 and ix) in studies utilizing calcium imaging techuiques tomonitor spontaneousCa2+oscillations andCa2+inducedCa2+rele se from the SR[ex. Berridge and Galione (1988); Takamatsu et al. (1990)]. Disadvantages ofisolated myocytes include: i) the viability of the preparation initially and duringexperimentation, ii) enzymes used for isolation may damage the glycocalyx and iii)intact quiescent adult myocytes do not spontaneously contract (i.e. they do notperform the work of contraction and relaxation). However, adult myocytes can be16electrically stimulated to contract at rates of perfused hearts and studies haveshown that they will consume the same amount of oxygen as the perfused heart[Haworth et at. (1983)]. Further, it has been observed that isolated adult myocyteswhich spontaneously contract after isolation are electrochemically shunted andfreely permeable to Ca2 [Dani et at. (1979)1. Neonatal and fetal myocytes willspontaneously contract in culture and observations and measurements ofcontractile function can be made.Heart homogenates can be prepared by grinding the tissue in various ways.Homogenates can be used to study metabolism but the results obtained from thesestudies are difficult to interpret since all cells are broken and cellular organelles arearranged randonily in solution. As well, it is not possible to localize an enzymaticactivity or organelle to a specific cell type. Homogenates are most often used toprepare intracellular organelles such as nuclei, mitochondria, myofibrils, lysosomes,sarcolemma and sarcoplasmic reticulum membranes.Isolated cellular organelles can be separated from homogenized heart tissueusually by differential centrifugation. Little work has been done on organellesprepared from isolated myocytes. In an isolated organelle, one can determine theorigin of a function or enzymatic activity and can study the regulation of specificfunctions without interference from other cell components. Disadvantages in usingisolated organelles include the yield and purity of the fraction obtained. As well,only the function of the isolated organelle itself can be investigated.171.4. Hypothesis, Rationale and Aims of This Study1.4.1. Hypothesis and RationaleAlthough a number of studies have examined the phosphorylation ofphospholamban in different experimental models (see section 1.2.3.2.), thereappears to be some incongruity between the results obtained from isolated SRmembrane vesicles and perfused whole hearts. In isolated SR vesicles, PK A, PK C,CAM PK and PK G all phosphorylate phospholamban. However, in isolatedperfused hearts, only PK A and CAM PK (only after activation of PK A) are found tophosphorylate phospholamban. Thus, in the present study, it was hypothesizedthat in a cardiac cell, there may be a functional basis for the phosphorylation ofphospholamban by several different types of protein kinases and further, that theprotein kinase pathways may not act independently of one another but maysynergize or antagonize each other to modulate SR function.For the present studies, the isolated adult rat ventricular myocyte waschosen as the model system. Ventricular myocytes were chosen since these are thecontractile cells of the heart and as such, regulation of their SR function is of primeinterest. Adult myocytes were chosen since the SR of neonatal cells is poorlydeveloped and t-tubules are almost nonexistent [Hirakow and Gotoh (1975)]. Arecent study shows that in newborn myocardium the SR plays a negligible role inexcitation-contraction coupling [Klitzner and Friedman (1989)]. Isolated myocytesare intact cells that possess the internal regulatory pathways used in the functionalmyocardium. In the intact heart, myocytes represent only 20% of the cellularpopulation but 80% of the heart mass. The remaining cells (80%) are neuronal,fibroblastic, smooth muscle, endothelial and epithelial. Due to this cellularheterogeneity it is difficult to identify a population of cells responsible for a specificproperty of the myocardium. Isolated adult ventricular myocyte preparations offer18the advantage of being a more homogeneous cell population than is present in theisolated perfused heart thereby allowing a particular function to be localized to themuscle cell. As well, complex regulatory mechanisms may be studied in themyocyte without the additional complications brought about by the neuronal andhormonal effects that exist in intact myocardium. For our experiments, an addedadvantage was that the medium surrounding the isolated myocytes can becontrolled and quickly changed to facilitate treatment with various agonists andantagonists.Several studies on protein phosphorylation in adult cardiac ventricularmyocytes have been published. In an early study utilizing adult rat ventricularcells, isoproterenol stimulated the phosphorylation of 5 proteins (150, 94, 33, 28,and 12 kDa), none of which were thought to be phospholamban [Onorato andRudolph (1981)1. Another study utilized several methods of compromising thecellular integrity (coichicine and saponin treatment) of the adult myocytes andfound that isoproterenol-stimulation increased the phosphorylation of 3 proteins(150, 28 and 26 kDa) and that phospholamban phosphorylation was a minorcomponent of the overall phosphorylation pattern observed [MacKay and Sulakhe(1988)1. In saponin permeabilized, spontaneously beating adult rat myocytes,elevated cAMP levels were also found to increase the phosphorylation of 5 proteins(150, 28, 24, 15 and 12 kDa). The 24 kiJa protein was identified as phospholamban[Miyakoda et al. (1987)]. Blackshear et al. (1984) demonstrated the phosphorylationof 13 proteins in response to isoproterenol treatment of isolated myocytes, one ofwhich was identified as phospholamban. Recently, George et al. (1991) found 3proteins (155, 31 and 6 kiJa) phosphorylated in response to isoproterenol treatmentof isolated myocytes.In this study, isolated adult rat ventricular myocytes were used to study thephosphorylation of proteins in response to activators of PK A and PK C. SR19membranes were isolated from the myocytes to enable the localization ofphosphorylated proteins to this organelle.1.4.2. Specific Aims1) To establish a method for the isolation of adult rat ventricular myocytes from asingle rat heart that would result in a preparation with a high number ofviable, rod-shaped cells.2) To develop a method for the isolation of purified SR membranes from adult ratventricular myocytes.3) To characterize the SR membranes obtained in regards to Ca2+transportactivity,Ca2/K-ATPase activity, purity and protein profile.4) To determine the proteins phosphorylated in the intact myocyte subsequent tothe stimulation of the cells with isoproterenol (a non-specific f3-agonist).5) To determine the proteins phosphorylated in the intact myocyte subsequent tothe stimulation of the cells with PK C activators and to study the activation ofthis kinase in the myocytes.6) To analyze the oligomeric species of phosphorylated phospholamban obtained inthe presence of activators of PK A, PK C and PK A and C, together.202. MATERIALS AND METHODS2.1. Materials2.1.1. AnimalsMale Sprague-Dawley rats (300 - 600 g) were used throughout this study.The animals were obtained from Animal Care, U.B.C. or from Charles River,Montreal, Que. The animals were maintained on Purina Rat Chow and tap water,ad libitum, in a light and temperature controlled room.2.1.2. Chemicals2.1.2.1. Materials for Isolation of Adult Rat Ventricular MyocytesCLS II collagenase (130 - 160 units/mg) was obtained from WorthingtonBiochemical Corp., Freehold, NJ. Bovine serum albumin (Fraction V, fatty acidfree) was obtained from Boehringer Mannheim, Dorval, Que. Joklik-modifiedminimum essential medium, Medium-199 with Earle’s salts, minimum essentialmedium non-essential amino acid solution (10 mM) (lOOx), minimum essentialmedium amino acid solution without glutamine (50x), HEPES, pyruvic acid (Nasalt), heparin (Na+ salt) (porcine intestinal mucosa, average molecular weight 4,000- 6,000), EGTA, carnitine, creatine, citric acid, tissue culture-tested insulin (bovinepancreas), CaC12 glutamine, NaHCO3 and taurine were obtained from SigmaChemical Co., St. Louis, MO. Pentobarbital sodium (65 mg/mi) was obtained fromCanada Packers Inc., Cambridge, Ont. Basal medium Eagle vitamin solution (lOOx)and trypan blue (0.4%) were obtained from Gibco Labs, Grand Island, NY. All otherchemicals were at least of AnalaRR grade from BDH Chemicals, Canada Ltd.212.1.2.2. Chemicals for the Treatment of Isolated Adult Rat Ventricular MyocytesCatalytic subunit of PK A (bovine heart), calmodulin, (±) isoproterenol-HC1,4cc-phorbol 12, 13-dlidecanoate and phorbol 12, 13-myristate acetate were obtainedfrom Sigma Chemical Co., St. Louis, MO. HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride) and H-8 (N-(2-(methylamino) ethyl)-5-isoquinolinesulfonamide dihydrochloride) were obtained from Seikagaku America,Inc., St. Petersburg, FL. CGS 9343 B (1,3.-dihydro-1-(1-((4-methyl-4H, 6H-pyrrolo(1,2-a)-(4,1)benzoxazepin-4-yl)methyl)-4-piperindinyl)-2H-benzimidazol-2-one maleate) was a kind gift from Ciba-Geigy Ltd., Basle, Switzerland. 1-Oleoyl-2-acetyiglycerol was obtained from Serdary Research Laboratories Inc., London, Ont.Ethanol and dimethylsulfoxide were AnalaRR grade and obtained from BDHChemicals Canada Inc. Forskolin (Coleus forskohlii) and diacylglycerol kinaseinhibitor (6-(2-(4-((p-fluorophenyl)-phenylmethylene)-1-piperidinyl)-ethyl)-7-methyl-5-H-thiazolo-3,2-a-pyrimidine-5-one; R59022) were obtained from Calbiochem Corp.,La Jolla, CA.2.1.2.3. Radioactivity and Autoradiography[y-32P)adenosine triphosphate in ethanol (30 Curies/mmol; 1 mCiJml), cAMPprotein binding {3H] kit and32P-orthophosphate in aqueous solution (HC1 free) (10mCiJml) were obtained from Amersham, Oakville, Ont. 45CaC12 (2 mCiJml) wasobtained from ICN Radiochemicals, Irvine, CA. Kodak X-OMAT film, Cronexintensifying screens and Kodak GBX developer and fixer (with replenisher) wereobtained from Sigma Chemical Co., St. Louis, MO. Unisolve 1 (Terochem) andReady Safe (Beckman Instruments Inc.) were used for liquid scintillation counting.222.1.2.4. Materials for the Isolation and Characterization of Myocyte Homogenatesand SR MembranesDithiothreitol, bovine serum albumin (Fraction V),phenyhnethylsulfonylfluoride (PMSF), tosyl-L-phenylalanine chioromethyl ketone(TPCK) and alkaline phosphatase were obtained from Boehringer MannheimCanada Ltd. NaHCO3,KC1, Na2HPO4methanol, acetic acid, MgCl2 sucrose,sodium molybdate, stainsall (1-ethyl-2-(3-( 1-ethylnapthol(1,2d)thiazolin-2-ylidene)-2-methylpropenyl)..napthol-(1,2d)-thiazolium bromide, Kodak Organic Chemical,No. 2718), NaP and NaCl of AnalaRR grade were obtained from BDH ChemicalsCanada Ltd. L-Histidine, MOPS (3-(N-morpholino)propanesulfonic acid), Tris base(tris(hydroxy-methyl)aminomethane), Na2EDTA, EGTA, adenosine 5’-triphosphate(Tris salt), ouabain, oxalate (Tris salt), alamethicin (antibiotic U-22324, fromTrichoderma viridae), imidazole, leupeptin, soybean trypsin inhibitor, cytochrome c(horse heart), aprotonin, pepstatin A and NaN3 were obtained from SigmaChemical Co., St. Louis, MO. Ruthenium red was obtained from Fluka ChemicalCorp., Ronkonkoma, NY. Acrylamide, bis-acrylamide, ammonium persulfate,sodium dodecylsulfate, Bradford protein reagent concentrate, bovine y-globulinprotein standard, Coomassie brilliant blue R250, glycine, TEMED, Zeta-probemembranes, SDS-PAGE high and low molecular weight standards, nitro bluetetrazolium, BCIP, alkaline phosphatase and horse radish peroxidase avidinconjugates, horse radish peroxidase color development reagent, prestained andbiotinylated SDS-PAGE high and low molecular weight standards were obtainedfrom Bio-Rad Labs., Mississauga, Ont. Goat F(ab’)2 anti-mouse (alkalinephosphatase and horse radish peroxidase-conjugated) and goat F(ab’)2 anti-rabbitIgG (G & L) (alkaline phosphatase and horse radish peroxidase-conjugated) wereobtained from Tago Immunologicals, TAGO Inc., Burlingame, CA. BioGel wrap wasobtained from BioDesign Inc., NY.232.1.2.5. Materials for the Isolation and Quantitation of PK CHistone Type 111-S from calf thymus, protein kinase inhibitor (PICI, syntheticrabbit sequence), HEPES, leupeptin, EGTA, Na2EDTA, MOPS, Na3VO4DEAEcellulose and 3-glycerophosphate were obtained from Sigma Chemical Co., St.Louis, MO. Dithiothreitol was obtained from Boehringer Mannheim Canada Ltd.P-Si (cellulose-phosphate) ion exchange chromatography paper was obtained fromWhatman International Ltd., Maidstone, England. NaC1, MgC12 KOH, CaC12 andorthophosphoric acid were obtained from BDH Chemicals Canada Ltd.Phosphatidylserine and 1-stearoyl-2-arachidonylglycerol were obtained fromSerdary Research Laboratories Inc., London, Ont.2.2. Methods2.2.1. Isolation of Adult Rat Ventricular Myocytes2.2.1.1. Modified Method of Piper et al.The following method was modified from that of Piper et al. (1982). Heartswere rapidly removed from the anesthetized rats (70 mg/kg pentobarbital sodiumand 1000 units/0.3 kg heparin), following attainment of surgical anaesthesia andrinsed with Joklik-Modified Minimum Essential Medium (MEM) containing 1 mMCaCl2, at 4°C. Hearts were cannulated via the aorta and Langendorif perfusion at37°C was started immediately with MEM (pH 7.4 at 20°C) containing the followingadditions (mM): HEPES, 21.1; NaHCO3,4.4; taurine, 60; MgCl2, 2.42; glucose, 4;creatine, 20; glutamate, 8. and carnitine, 2. This buffer (HEPES-MEM) alsocontained 1% fatty acid free BSA and essential amino acids, however, no calciumwas added. Subsequent to a 4.5 mm non-recirculating perfusion at 37°C, hearts24were perfused in a recirculating manner for 35 mm with HEPES-MEM containing25 tM CaC12 and 0.8 mg/nil collagenase. The buffers were aerated with 100% 02before use and throughout the perfusion. After perfusion, the ventricles wereminced with a scalpel and the tissue pieces incubated in a 37°C water bath for 10mm in 10 ml of the recirculation medium. Following this incubation, the tissuepieces were drawn up and down very gently (no air bubbles) with plastic transferpipettes of 3 diameters (2 to 4 mm), starting with the widest pipette, until the tissuepieces became small enough to easily pass through the narrowest diameter pipette.Subsequent to filtration through 200 jim nitex mesh, the cells were washed threetimes with HEPES-MEM containing increasing concentrations of calcium (0.2, 0.5and 1.0 mM CaC12). Rod-shaped myocytes were purified by settling through 4%BSA in Medium-199 with Earle’s salts and then were resuspended in Medium-199.From each rat heart, 3 - 5 x i6 rod shaped myocytes were isolated, of which 75 to90% were viable.2.2.1.2. Modified Method of Li et at. and Wimsatt et at.The following method was modified from that of Li et at. (1988) and Wimsattet at. (1990). Hearts were rapidly removed from anesthetized rats (70 mg/kgpentobarbital sodium and 1000 units/0.3 kg heparin), following attainment ofsurgical anaesthesia and rinsed with 1 ml of Krebs-Henseleit (K-H) buffer at 4°C.This buffer contained (mM): NaC1, 118.0; KC1, 4.8; CaC12, 1.0; MgSO4, 1.2;I(112P04,1.2; glutamine, 0.7; glucose, 11.0; Na-pyruvate, 5.0; HEPES, 25.0; pH 7.3- 7.35 at 20°C. Hearts were cannulated via the aorta and Langendorff perfusionwas started immediately in a non-recirculating mode at a flow rate of 10 mi/mm for5 mm at 37°C with K-H containing vitamins and amino acids. Subsequently, heartswere perfused (in non-recirculating mode) with K-H containing no added CaC12 and0.02 mM EGTA. Hearts were then perfused in a recirculating mode with K-H25containing 1 mg/mi BSA (fatty acid free) and 1 mg/mi collagenase. After 20 mm, 125p.1 0.1 M CaC12 was added to the perfusate to bring the Ca2 concentration to 250p.M. After 5 mm, this was repeated to bring the total Ca2 concentration to 500 p.M,then after another 5 mm, 250 p.1 of 0.1 M CaC12 was added to bring the total Ca2concentration of the perfusate to 1 mM. After 40 mm, the flow rate of the perfusionwas increased over 10 mm to 16 mllmin. Perfusion was continued until heartsbecame soft or the aorta was digested through. Usually the hearts were perfused fora total time of approximately 50 mm. The buffers were aerated with 100% 02 beforeuse and throughout the perfusion. After perfusion, the ventricles were minced witha scalpel and the tissue pieces incubated in a 37°C shaking (100 cycles/mm) waterbath under 02 for 10 mm in 10 ml of K-H containing 1 mM CaC12,vitamins, aminoacids, 0.6 p.g/ml insulin, 2% BSA and 2 mg/mi collagenase. After this incubation, thetissue pieces were drawn up and down very gently (no air bubbles) with plastictransfer pipettes of 3 diameters (2 to 4 mm), starting with the widest pipette, untilthe tissue pieces became small enough to pass through the narrowest diameterpipette. Subsequent to filtration through 200 p.m nitex mesh, the cells werecentrifuged at 400 x g for 1 mm. The pellet was gently resuspended in K-Hcontaining 1 mM CaC12, vitamins, amino acids, 0.6 p.g/ml insulin and 0.5% BSA,centrifuged as before and resuspended in the same K-H buffer with 0.5% BSA.Myocytes were purified by layering 3 ml aliquots of the above suspensiononto 10 ml of K-H containing 1 mM CaC12,vitamins, amino acids, 0.6 p.g/ml insulinand 4.0% BSA. Myocytes were allowed to settle by gravity for 5 mm, thesupernatants removed and the cell pellets resuspended in K-H containing 1 mMCaCl2, vitamins, amino acids, 0.6 p.g/ml insulin and 2.0% BSA. Finally, aftercentrifugation (400 x g for 1 mm), the myocytes were resuspended in K-Hcontaining 1 mM CaCl2, vitamins, amino acids and 0.6 p.g/ml insulin. Using thismethod, from each rat heart, 3 - 8 x i06 rod shaped myocytes were isolated, ofwhich from 75 to 90% were viable.262.2.2. Characterization of Isolated Adult Rat Ventricular Myocytes2.2.2.1. Cell Counting, Morphology and Maintenance of Contractile FunctionIsolated myocytes were quantitated using a Fuchs-Rosenthal Ultra Planecounting chamber of 2 mm depth. An aliquot (20 j.ii) of the myocyte suspension wasdrawn under the coverslip of the counting chamber by capillary action. Thecounting chamber was placed under a microscope and myocytes were counted ineach of the 1 mm2 squares in the four corners of the grid. The number of myocytesin the preparation was calculated as follows:average cell number ner 1 mm square x volume of the cell suspension (ml)volume counted (nil)The volume counted for this counting chamber is 2 x i04 ml (1 mm2x 0.2 mm).The number of viable myocytes in a preparation was determined by using thecell viability stain, trypan blue. An aliquot of the myocyte cell suspension (250 jtl)was mixed with 50 p1 of 0.1% trypan blue. After 4 mm, the viable myocytes (cellsthat did not take up the dye) were counted as described above.Electrical stimulation was used to determine whether the isolated myocyteshad retained the functional ability to contract, using the method of Haworth et al.(1980). An aliquot of the myocyte suspension (2 drops) was placed on a glass slide,covered with a coverslip and placed on the microscope stage. The two leads from aGrass stimulator (model SD9) were placed in the fluid on either side of the coverslipand attached with electrical tape. Square-wave stimulating pulses of 10 ms induration and up to 100 V were used to elicit contractions from the myocytes.272.2.2.2. Quantitation of cAMPIsolated myocytes (1.2- 1.5 x i0 rod shaped cells per condition) wereincubated at 37°C for 2 mm in Mediuni-199 containing 0.5 mM IB1VIX and either 1.tM isoproterenol or 100 tM forskolin. The reaction was terminated by the additionof 35% TCA to the reaction mixture to bring the final concentration of TCA to 7%.The tubes were left on ice for 10 mm and then centrifuged at 2500 x g for 15 mm at4°C. The supernatants were then extracted 4 times with a 5x volume of water-saturated diethyl ether. The ether phases were discarded and the residue was driedat 50°C for 30 mm in an oven and, if necessary, was lyophilized to remove anyremaining water. The amount of cAMP present was determined in a 50 j.tl aliquot ofeach sample using the cAMP protein binding assay kit from Amersham.2.2.3. Homogenization of Isolated Adult Rat Ventricular Myocytes2.2.3.1. Preliminary Homogenization MethodsSeveral different methods of myocyte homogenization were attempted beforesuitable methods were found. Methods utilizing the French Press, sonication,grinding under liquid N2, wiggle bug (dentistry amalgam mixer) and Polytronhomogenizer were all tried and found to be unsuitable.For homogenization by French Press, myocytes were resuspended in buffercontaining 0.29 M sucrose, 3 mM NaN3, 10 mM imidazole (pH 6.9 at 4°C), 0.1 MKC1, 10 mM DTT, 1.3 mM PMSF, 1 jig/nil leupeptin, 25 jig/nil STI, 1 jig/miaprotonin and 1 mM EGTA. Myocytes were passed once through the press, at apressure of 1500 psi.For sonication, the myocytes were resuspended in the same homogenizationbuffer as above but without 0.1 M KC1. Sonication was carried out with a Branson28Probe Sonicator at 4°C with four bursts of 30 s each at an output of 2, % duty of 80and output control setting at 2.5.Myocytes (frozen) were ground in liquid N2 by hand with a porcelain mortarand pestle. Subsequently, the frozen cell slurry was resuspended in the same buffer(without KC1) as was used for sonication.Homogenization with the wiggle bug (dentistry amalgam mixer) was carriedout in the following buffer: 0.29 M sucrose, 3 mM NaN3, 2.0 mlvi EGTA, 25 mMNaF, 50 mM Na2HPO4 (pH 6.9 at 4°C), 10 mM DTT, 0.1 mM PMSF, 1 .tg/mlleupeptin, 25 pg/m1 STI, 1 j.ig/ml aprotonin, 0.7 jig/m1 pepstatin and 100 .tg/mlTPCK. Myocyte pellets, frozen in liquid N2, were placed into a precooled (withliquid N2) wiggle bug. The wiggle bug was agitated for 15 s, 500 .tl homogenizationbuffer added and the bug agitated for another 15 s. The resulting cell slurry wasdiluted with homogenization buffer under slow stirring.Myocytes were homogenized by polytron at a setting of 5.5 for 9 x 10 s with aBrinkman PT1O/35 Polytron Homogenizer, in the same buffer as was used for thewiggle bug.2.2.3.2. Successful Homogenization MethodsComplete homogenization of all of the myocytes isolated was successfullycarried out at 4°C in a hypotonic buffer using 1 pass (up and down) with a ‘Zero’clearance homogenizer (Kontes) on a motorized Potter-Elvehjem, set at 3.8 onreverse. The hypotonic buffer contained (mM): Tris-maleate, 10 (pH 7.4 at 4°C);HEPES, 20; NaF, 25; EGTA, 10; PMSF, 1; and DTT, 1. Homogenization was carriedout immediately after the myocytes were isolated and after preparation, aliquots ofthe homogenate were immediately frozen in liquid N2 and stored at -70°C.When SR membranes were to be isolated (see section 2.2.4.2.) from themyocyte homogenates, the following homogenization procedure was used. Myocytes29(3 - 5 x i06 cells) in a soft pellet (approximately 0.7 ml) at 4°C were gentlyresuspended in 15 ml of Buffer 1 (10 mM NaHCO3and 4 mM DTT, pH 7.4 at 4°C).The myocytes were then homogenized 3 times (total = 90 strokes) alternating 15strokes of a 7 ml glass-glass douncer (Pestle B = small clearance) with 15 strokes ofa motorized PTFE-glass Potter-Elvehjem (50 ml) set at approximately 800 rpm. Thehomogenate was then diluted to 25 ml with Buffer 1 and gentle stirring. It wasnecessary to dilute the homogenates (final 1:35 v/v; cells/buffer 1) to preventaggregation of membranes and proteins during the SR isolation procedure. Thisprocedure homogenized approximately 20-30% of the rod-shaped cells.2.2.4. Isolation of Sarcoplasmic Reticulum Membranes from Isolated Adult RatVentricular Myocytes2.2.4.1. Preliminary Methods for the Isolation of SR Membranes from IsolatedMyocytesSeveral different methods [Chamberlain and Fleischer (1988); Jones et al.(1979)] for the isolation of SR membranes from the isolated adult rat ventricularmyocy-tes were attempted prior to the development of the final method utilized.SR membrane vesicles were isolated by a modification of the method ofChamberlain and Fleischer (1988) as follows: Myocytes were homogenized in 0.29 Msucrose, 3 mM NaN3,20 mM imidazole (pH 6.9 at 4°C), 0.1 M KC1, 10 mM DTT, 0.1mM PMSF, 1 j.tg/ml leupeptin, 25 pg/ml STI, 1 pg/ml aprotonin, 0.7 ig/ml pepstatin,100 pg/ml TPCK and 1 mM EGTA. Homogenates were centrifuged for 15 mm at5,000 rpm (3,800 x in a Beckman high speed centrifuge at 4°C. Supernatantswere then centrifuged for 15 mm at 13,000 rpm (20,000 x in the samecentrifuge. The resulting supernatant was centrifuged for 2 hrs at 55,000 rpm(120,000 x g) in a Beckman ultracentrifuge (Ty65 rotor) at 4°C. The supernatant30was discarded and the pellet was resuspended in 0.29 M sucrose, 0.65 M KC1, 3 mMNaN3,0.5 mM EGTA, 10 mM imidazole, pH 6.7 at 4°C, 5 mM DTT, 0.1 mM PMSFand the protease inhibitors (.tg/ml): aprotonin, 1; leupeptin, 1; STI, 25; pepstatin,0.7 and TPCK, 50. Resuspension of the pellet was carried out with 8 strokes of aPTFE-glass homogenizer. At this point, the resuspended pellet may have beenfrozen in liquid N2 and stored at -70°C. Subsequently, the resuspended pellet waslayered onto the top of a 20 - 45% continuous sucrose density gradient containing 10mM imidazole (pH 6.7 at 4°C), 3 mM NaN3,0.65 M KC1, 5 mM DTT and the abovelisted protease inhibitors. The gradient was centrifuged for 6.5 hrs at 28,000 rpm ina Beckman ultracentriflige (SW41 rotor). Following centrifugation, 1 ml fractionswere collected from the top of the gradient with a Hamilton syringe.When the original Chamberlain and Fleischer (1988) method was used thefollowing procedure was followed: Myocytes were homogenized in the same buffer asabove, except that the DTT concentration was 0.5 mM and no protease inhibitorswere added. The first centrifugation was the same as above, the secondcentrifugation was omitted and the third centrifugation was also the same as above.The pellets were resuspended in the same buffer except that the EGTAconcentration was 0.5 mM, the DTT concentration was 0.5 mM and no proteaseinhibitors were added. The resuspended pellet was kept on ice for 30 mm and thencentrifuged at 6,000 rpm (JA2O rotor) for 10 mm at 4°C. The supernatant was thencentrifuged for 100 mm at 55,000 rpm (Ty65 rotor) at 4°C. Final pellets wereresuspended in the same buffer, as the first resuspension, with 10 strokes of aPTFE-glass homogenizer.SR membranes were isolated by the method of Jones et al. (1979) as follows:Myocytes were homogenized in 10 mM NaHCO3,10 mM DTT, 1.3 mM PMSF, 1jig/mi leupeptin, 25 jig/mi STI, 1 jig/mi aprotonin, 0.7 jig/mi pepstatin and 1 mMEGTA. The homogenate was centrifuged at 14,000 x grn (10,500 rpm in aBeckman JA2O rotor) for 20 mm at 4°C. The pellet was dliscarded and the31supernatant was centrifuged in the same manner as the homogenate. Subsequent todiscarding the pellet, the supernatant was centrifuged at 45,000 x grn (18,500rpm JA2O rotor) for 30 mm at 4°C. The supernatant was discarded and the pelletwas resuspended in 30 mM histidine (pH 7.0 at 4°C) and 0.6 M KC1 with 10 strokesof a small handheld Teflon-glass homogenizer. The resuspended pellet wascentrifuged at 45,000 x grnax (18,500 rpm JA2O rotor) for 30 mm at 4°C. The finalpellet was resuspended in 0.25 M sucrose, 0.3 M KC1 and 0.1 M Tris (pH 7.2 at 4°C)with 10 strokes of a small hand-held PTFE-glass homogenizer.2.2.4.2. Final Method for the Isolation of SR Membranes from Isolated MyocytesThe basis for the development of a method to isolate purified SR membranesfrom myocytes was the SR preparation of Harigaya and Schwartz (1969) asmodified by Jones et at. (1979). Intact adult rat ventricular myocytes werehomogenized in Buffer 1 (10 mM NaHCO3 and 4 mM DTT, pH 7.4 at 4°C) asdescribed in section 2.2.3.2. All subsequent steps were carried out at 4°C and allcentrifugations were done in Beckman centrifuges. Myocyte homogenates werecentrifuged at 330 x g (ray = 7.0 cm; 2,000 rpm JA2O rotor) for 15 mm to pelletunbroken cells. Supernatants were centrifuged at 5,000 x g (ray = 7.0 cm; 8,000 rpmJA2O rotor) for 15 mm to pellet intact nuclei and mitochondria. The resultantsupernatant was then centrifuged at 23,000 x g (ray = 7.0 cm; 17,000 rpm JA2Orotor) for 30 mm to pellet SR membranes. The pellets were resuspended in Buffer 2(30 mM histidine-Cl, 0.6 M KC1 and 4 mM DTT, pH 7.0 at 4°C) with 10 strokes of a7 ml glass-glass douncer. SR membranes were then collected by centrifuging at23,000 x g (ray = 7.0 cm; 17,000 rpm JA2O rotor) for 30 mm. SR membrane pelletswere resuspended in Buffer 3 (0.25 M sucrose, 0.3 M KC1 and 0.1 M Tris-Ci, pH 7.2at 4°C), using 10 strokes of a 1 ml Potter-Elvehjem homogenizer, frozen in liquid N2and stored at -70°C.32When homogenates and SR membranes were prepared from 32P-labeledmyocytes subsequent to stimulation, alterations in protein-bound phosphate due tothe action of kinase, phosphatase or protease activities were minimized by carryingout all procedures at 4°C and by homogenizing the myocytes in Buffer 4 (50 mMNa2HPO4,10 mM Na2EDTA and 25 mM NaF). The SR pellets were washed inBuffer 4 with 0.6 M NaC1 added (Buffer 5). The final SR preparation wasresuspended in Buffer 3. Under these conditions,[32P]phosphoproteins have beenshown to be stable for several hours [Lindemann and Watanabe (1985); Lindemannet at. (1983)].2.2.5. Characterization of SR Membranes from Isolated Myocytes2.2.5.1. Biochemical Assays2.2.5.1.1. Determination ofCa2-Transport ActivityCa2-uptake activity was determined by a modification of the method byTada et at. (1974). This assay was linear with respect to protein concentration (0 -80 mg/ml) and incubation time (0 -15 mm; Mahey (1986)). Fractions (10 pg proteinin 50 tl) were added and pre-incubated for 140 s at 30°C in a reaction medium (400iii) containing (final concentrations): 40 mM histidine/HC1 buffer (pH 6.8 at 20°C),110 mM KC1, 5 mM MgCl2, 5 mM NaN3, 0.25 M sucrose, 1 mM DTT, 5 mM ATP(Tris salt) in the presence and absence of 2.5 mM oxalate (Tris salt) and 5 Mruthenium red. The reaction was initiated by the addition of 50 pi EGTA-buffered45Ca2 (126 j.tM CaC12 and 175 j.tM EGTA resulting in 2 pM free Ca2; 200,000dpmJ5O .d) and was terminated after 140 s by transferring an aliquot (400 iii) of thereaction mixture onto a Whatman GF/C filter. The filter was washed once with 15ml of 40 mM Tris-Ci, pH 7.2 at 20°C to remove non-specifically bound calcium. After33drying, the radioactivity on the filters was quantitated by liquid scintillationcounting in a Canberra Packard Tricarb scintillation spectrometer. All membranefractions (fresh or frozen) were assayed for Ca2-uptake activity within 24 hrs oftheir isolation. The Ca2-transport activity of each fraction was calculated asfollows:nmolCa2/mi&mg protein = average dpm (140 s)-1 (mg proteinY1specific activity of45Ca2(dpmlnmol)For the calculation of the total Ca2-transport activities of each SR or othermembrane-containing fraction, the yield of protein (mg) has been normalized sothat each was obtained from a starting myocyte population of 3 x 106 cells. Thesenormalized protein values were used in the calculation of the totalCa2+transportactivities in Tables 3 and 7 (sections 3.3.2.1. and 3.3.2.2.).2.2.5.1.2. Determination ofCa2/K and NafK ATPase ActivitiesPotassium-stimulated, calcium-dependent ATPase (Ca2fK-ATP se)activity was measured under the conditions described by Jones and Besch (1984).Inorganic phosphate was determined by the method of Raess and Vincenzi (1980).The reaction buffer contained (400 p1): 45 mM histidine, 1 mM EGTA, 5 mM MgC12,100 mM KC1, 1.84 mM ouabain, 11 ml\’I NaN3 and 1.15 mM DTT in the absence orpresence of 0.6 mM CaC12 (1.5 pM free Ca2). Tubes contained either 3 ig (SR) or10 .tg (other membrane fraction) of protein. Following a 10 mm pre-incubation at37°C, the reaction was initiated by the addition of 5 mM Tris-ATP and terminatedafter 30 mm by the addition of 200 p1 10% (wlv) SDS solution with rapid mixing.Following this, 200 p1 of 9% ascorbic acid (w/v) was added with rapid mixing.Subsequently, 200 p1 of 1.25% ammonium molybdate in 6.5% H2S04was added toeach tube every 30 s with rapid mixing. After a 30 mm incubation at room34temperature, the absorbance at 660 nm was measured at 30 s intervals on aHewlett Packard Diode Array Spectrophotometer (model 8452A). The Pj content ofeach sample was determined from a calibration curve ranging from 0 - 750 nmolP/ml. The Ca2/K-ATPase activity was taken as the difference between ATPhydrolysis measured in the presence and absence of 0.6 mM CaC12 (1.5 iM freeCa2jand was calculated as follows:nmollminJmg protein = (nmol Pi) (30 minY1 (mg protein)1For the calculation of total Ca21K-ATPase activities of each SR or othermembrane containing fraction, the yield of protein (mg) has been normalized so thateach was obtained from a starting myocyte population of 3 x i06 cells. Thesenormalized protein values were used in the calculation of the totalCa2+/K+ATPaseand Na7X-ATPase activities in Tables 4, 6, 8 and 9 (sections 3.3.2.1. and 3.3.2.2.).Sodium and potassium-dependent ATPase (Na/K-ATPase) activity wasmeasured as described above, except that Ca2+ was omitted and the reaction buffercontained 10 mM KC1 and 110 mM NaC1 and was determined in the absence orpresence of 1.5 mM ouabain. Tubes contained either 3 .tg (SR) or 10 g (othermembrane fraction) of protein. To unmask latent activity [Jones and Besch (1984)1,prior to the start of the reaction, protein samples were incubated for 10 mm in thepresence of alamethicin (0.1 mg/mg protein). NaiK-ATPase activity was taken asthe difference between ATP hydrolysis measured in the presence and absence of 1.5mM ouabain and the activity of each sample and fraction was determined as above.2.2.5.1.3. Determination of Cytochrome C Oxidase ActivityMitochondrial membrane cytochrome c oxidase activity of myocytehomogenates and SR membranes was assayed according to the method of Smith(1955) as described by Wharton and Tzagoloff (1967). A cuvette containing 30 mM35potassium phosphate buffer (pH 7.4 at 37°C) and 50 11 of 1% (w/w) reducedcytochrome c was preincubated at 37°C. The reaction was initiated by the additionof 10 t1 of membrane vesicles (10 tg protein) to the cuvette. Cytochrome c oxidaseactivity was determined by monitoring the decrease in absorbance at a wavelengthof 550 nm over a period of one minute. The rate of cytochrome c oxidase wasdetermined according to the following formula:Specific Activity = k [cytochrome ci / [protein]where:Specific Activity = nmoles cytochrome c/minute/mg proteink = ln[A0/A01 mini A0 = A550 at time 0 SA01 A550 attime60 sThe absorbance of each sample in the cuvette in the presence of KCN wassubtracted prior to the calculation.2.2.5.1.4. Determination of Protein ConcentrationProtein was determined by a modified Lowry procedure [Markwell et at.(1981)1 which included SDS to solubilize the protein samples. BSA (Fraction V,Boehringer Mannheim) was used for the standard curve (0 - 25 j.ig protein). Forprotein samples in SDS-sample buffer, this Lowry method was modified by addingSDS-sample buffer to the standard curve. Protein content was also determined bythe dye binding method of Bradford (1976) using the reagents and procedures forthe microassay supplied by Bio-Rad Laboratories. Bovine ‘y-globulin was used tostandardize each assay. For both methods, the protein concentration of the samplesolution was then determined from linear regression of the standard curve samples.362.2.5.1.5. Determination of Free Calcium ConcentrationsFree calcium concentrations were calculated using the FORTRAN program“CATIONS” written by Goldstein (1979). Equilibrium constants for cations andligands were obtained from Martell and Smith (1979-1982) except in the case ofmonoprotonated ligands which were calculated as described by Blinks et al. (1982).Constants were corrected for temperature using the BASIC program “LOGTEMP”based on the formula given by Tinoco et al. (1978) and using enthalpy valuestabulated by Martell and Smith (1979-1982). The constants were then adjusted forpH and ionic strength [Blinks et al. (1982); Martell and Smith (1979-1982)1.2.2.5.2. Electrophoretic Methods2.2.5.2.1. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis2.2.5.2.1.1. Polyacrylamide Gradient (5 -20% and 10- 20%) GelsSodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE),employing polyacrylamide gradients (5 - 20% and 10 - 20%), was performed usingthe discontinuous buffer system described by Laenimli (1970). Gloves were wornthroughout the SDS-PAGE procedure including the preparation of all buffers andstock solutions. All stock solutions were filtered to 0.22 I.tm. All components of thecasting apparatus were cleaned using lint-free paper towels. Glass plates weresoaked immediately prior to use and between uses in 0.1 M HC1. The 5% “resolving”gel solution consisted of 375 mM Tris/HC1 buffer (pH 8.8 at 20°C) containing: 5%(w/v) acrylamide; 0.14% (wlv) N,N’-methylene-bis-acrylamide (BIS); 0.1% (w/v)sodium dodecylsulfate (SDS); 1 mM Na2EDTA; 1.35% (v/v) glycerol; 0.025%tetraethylmethylene diamine (TEMED) and 0.3 mg/ml ammonium persulfate. The3710% “resolving” gel solution consisted of 375 mM Tris/HC1 buffer (pH 8.8 at 20°C)containing: 10% (w/v) acrylamide; 0.27% (w/v) BIS; 0.1% (wlv) SDS; 1 mMNa2EDTA; 1.35% (v/v) glycerol; 0.025% TEMED and 0.3 mg/mi ammoniumpersulfate. The 20% “resolving” gel solution consisted of 375 mM TrisfHCl buffer(pH 8.8 at 2000) containing: 20% (w/v) acrylaniide; 0.53% (w/v) BIS; 0.1% (w/v) SDS;1 mM Na2EDTA; 5.8% (vlv) glycerol; 0.05% TEMED and 0.23 mg/ml ammoniumpersulfate.Once prepared, the resolving gel solutions were transferred to a gradientformer and the gradient poured over a period of five minutes. The gel was thenoverlayered with 0.6 ml of isobutanol and allowed to polymerize for 12-18 hours atroom temperature. Prior to use, the isobutanol was decanted and the resolving gelflushed with distilled and deionized water and then overlaid with a “stacking” gelsolution.The “stacking” gel consisted of the following: 126 mM Tris/HC1 buffer (pH 6.8at 20°C); 5% acrylamide; 0.14% BIS; 1 mM Na2EDTA; 0.14% TEMED and 0.4mg/mi ammonium persulfate. A PTFE “comb” was inserted to facilitate formation ofsample wells. This solution was allowed to polymerize for 50 minutes at roomtemperature.Electrophoresis of gels was performed in a Bio-Rad Protean II Apparatuswith cooling core. The electrode buffers consisted of 25 mM Tris base, 192 mMglycine and 0.1% SDS in the upper reservoir and 25 mM Tris/HC1 buffer (pH 8.3 at20°C) in the lower reservoir. Gels were run at 25 mA (constant current) and 1000 Vuntil the dye front entered the slab gel, after which, the current was increased to 35mA. Gels were run until all dye fronts were off the bottom of the gels for at least 10mm or for a total of 1.5 volt-hours. The protein standards used for the estimation ofmolecular mass (in kilodaltons) were: myosin (200), 3-galactosidase (116.25),phosphorylase b (92.5), bovine serum albumin (66.2), ovalbumin (45), carbonicanhydrase (31), soybean trypsin inhibitor (21.5), lysozyme (14.4) and ubiquitin (8.3).38Samples were prepared for electrophoresis by solubilization in SDS-sampiebuffer containing: 5% SDS, 2.5% glycerol, 0.2 M Tris-Ci (pH 6.8 at 20°C), 0.5 M f3-mercaptoethanol and 0.025% bromophenol blue. Samples were then either boiled for3 mm or incubated at 37°C for 10 mm. Samples were allowed to cool, thencentrifuged at 14,000 rpm for 2 minutes in an Eppendorf microcentrifuge atmaximum speed and loaded into the sample wells.2.2.5.2.1.2. Polyacrylamide and Bis-acrylamide Gradient GelsSDS-PAGE was also carried out employing polyacrylamide and bisacrylamide gradients (10 - 20% polyacrylamide with 0.27 - 1.0% BIS). The 10%“resolving” gel solution consisted of 375 mM Tris/HC1 buffer (pH 8.8 at 20°C)containing: 10% (w/v) acrylamide; 0.27% (w/v) BIS; 0.1% (w/v) SDS; 1 mMNa2EDTA; 2.7% (v/v) glycerol; 0.025% TEMED and 0.3 mg/mi ammoniumpersulfate. The 20% “resolving” gel solution consisted of 375 mM Tris/HC1 buffer(pH 8.8 at 20°C) containing: 20% (w/v) acrylamide; 1.0% (w/v) BIS; 0.1% (w/v) SDS;1 mM Na2EDTA; 5.8% (v/v) glycerol; 0.05% TEMED and 0.23 mg/mi ammoniumpersulfate.The procedures for the formation of the gradient gel, the stacking gel, theelectrophoresis and the sample preparation for these gels was the same as insection 2.2.5.2.1.1.2.2.5.2.1.3. Polyacrylamide Non-Gradient GelsSDS-PAGE was also performed using polyacrylamide (15%) gels that did notcontain a gradient according to the method of Li et al. (1990). The 15% “resolving”gel solution consisted of 375 mM Tris/HC1 buffer (pH 8.8 at 20°C) containing: 15%39(w/v) acrylamide; 0.4% (w/v) BIS; 0.1% (w/v) SDS; 1 mM Na2EDTA; 1.35% (vfv)glycerol; 0.019% TEMED and 0.3 mg/mi ammoniuni persulfate.The procedures for the formation of the stacking gel, the electrophoresis runand the sample preparation for these gels was the same as in section 2.2.5.2.1.1.2.2.5.2.2. Staining of Sodium Dodecylsulfate Polyacrylamide Gels2.2.5.2.2.1. Coomassie Brilliant Blue StainThe gels were allowed to stain for 1 hr at room temperature, with gentleagitation, in 500 ml of 0.25% (w/v) Coomassie Brilliant Blue R250 inmethanollddH2O/acetic acid (5:5:1). The first destaining step was for 1 hr in 500 ml(3 changes) of methanollddH2O/acetic acid (5:5:1). This was followed by a seconddestaining step in ddHO/methanolJacetic acid (15:4:1) until the backgroundbecame transparent.2.2.5.2.2.2. Stainsall StainGlycoproteins and calcium binding proteins in myocyte SR membranes fromwhole rat heart and isolated ventricular myocytes were visualized using the cationiccarbocyanine dye “Stainsall” according to the method of King and Morrison (1976)as modified by Campbell et at. (1983). SDS-PAGE gels were fixed overnight in 25%(v/v) isopropanol. As SDS will cause the dye to precipitate, fixed gels were thenwashed with 5 changes of (25%; v/v) isopropanol (200 ml) over an 8 hour period. Theefficiency of the washing procedure was tested by adding a drop of the washsolution to 1 ml of the staining solution in a small (10 x 75 mm) test tube. If noprecipitate was observed, the gel was transferred into a solution of 30 mM TrisfHClbuffer (pH 8.8 at 20°C) containing 0.0025% (w/v) “Stainsall”, 25% (v/v) isopropanol40and 7.5% (v/v) formaniide. Gels were maintained in the dark at room temperaturewith constant gentle agitation for 48 hours during color development. Due to thephotosensitivity of this stain, gels were stored in staining solution in a dark place.2.2.5.2.3. Drying of SDS-PAGE GelsAfter staining with Coomassie blue, SDS-PAGE gels, were dried between twosheets of porous cellulose (BioGel Wrap, BioDesign mc). Gels were clamped into aPlexiglass gel drying frame and were allowed to dry in a fume hood, at roomtemperature, for 12 hrs.2.2.5.2.4. Western Blotting2.2.5.2.4.1. Antibody LocalizationPolypeptides from each sample were first separated by SDS-PAGE and thenimmunoblotted using the buffer system of Towbin et al. (1979). Gels wereequilibrated for 30 minutes in buffer containing 25 mM Tris and 192 mM glycineand then electrophoretically transferred onto Zeta-Probe membranes. Transfer wasfor 60 minutes at 20 volts using a Bio-Rad Trans Blot Semi-Dry blotting apparatus.All of the following steps were carried out at room temperature with gentle rotation.Non-specific binding sites on the Zeta-Probe membrane were blocked by incubationovernight in 5% (w/v) skimmed milk powder (Carnation) in Tris-buffered saline (20mM Tris/HC1 buffer (pH 7.5 at 20°C) containing 0.5 M NaCl; TBS). Following a 20mm rinse with Tris-buffered saline containing 0.2% Tween-20 (TTBS), themembrane was incubated with the primary antibody in TTBS and 1% (w/v) skimmilk powder for 3 hrs. Subsequent to two 10 mm rinses with TTBS, membraneswere incubated with secondary antibody (alkaline phosphatase or horse-radish41peroxidase conjugated Goat F(ab’)2 immunoglobulins; 1:2,000 v/v dilution) in TTBSand 1% (w/v) skim milk powder for 3 hrs. Membranes were then rinsed twice for 10mm in TTBS and then once for 15 mm in TBS. Immunoreactive bands were thenvisualized by reaction with alkaline phosphatase development reagent (45 ml 100mM Tris-HC1 (pH 9.5 at 20°C) and 5 mM MgC12; 5 ml 0.1% (w/v) p-nitro bluetetrazolium chloride and 0.5 ml 0.5% 5-bromo-4-chloro-3-indolyl phosphate ptoluidine salt (BCIP) in 70% N,N-dimethylformamide) or horse-radish peroxidasedevelopment reagent (60 mg 4-chloro-1-napthol in 20 ml methanol mixed into 100ml TBS with 60 .tl ice cold 30% H20). The molecular masses of theinimunoreactive bands were determined relative to biotinylated SDS-PAGEstandards, which had also been transferred to the membrane and visualized byincluding their aviclin alkaline phosphatase- or horse radish peroxidase-conjugatesin the secondary antibody incubation.Primary antibodies were obtained from the following sources: A monoclonalantibody to phospholamban, antibody Al [Suzuki and Wang (1986)1, was a generousgift from Dr. Jerry. H. Wang, University of Calgary, Calgary, Alta., Canada. Thisantibody was used at a dilution of 1:2,735 v/v. Polyclonal antibodies to PKCI32 (TypeII) and PKCx (Type III) were provided by Dr. Christopher Wilson (Department ofPediatrics, University of Washington, Seattle, WA) and were raised againstsynthetic peptides which appear in the amino acid sequences of the subspecies asdeduced from their cDNA sequence {Kikkawa et al. (1987)1. These polyclonalantibodies against PKC Type II and Type III were prepared against peptidesSFVNSEFLKPEVKS (Type II sequence, amino acid residues 660-673) andAGNKVISPSEDRRQ (Type III sequence, amino acid residues 3 13-326),respectively. In the latter peptide, the conservative substitution of an arginine forlysine-325 was made [Makowski et al. (1988)]. These antibodies were used at adilution of 1:250 v/v.422.2.5.2.4.2. Ruthenium Red StainingFor the detection of Ca2-binding proteins, the method of Charuk et al.(1990) was utilized. SR membrane samples were separated by SDS-PAGE andtransferred to 0.45 jim nitrocellulose using the semi-dry apparatus and the buffersystem of Towbin et al. (1979) as described in the previous section (2.2.5.2.4.1.).After transfer, the nitrocellulose was stained in 60 mM KC1, 5 mM MgC12, 10 mMTris-HC1 (pH 7.5 at 20°C) with 25 jiM ruthenium red and in the presence andabsence of 50 mM CaC12.2.2.5.2.5. Analysis of SDS-PAGE Gels, Autoradiographs and Western BlotsElectrophoretic gels, autoradiographs and western blots were analyzed usingcomputer image analysis with a Visage 110 Bio Image Analyzer (Bio Image, AnnArbor, MI) consisting of a high resolution camera (1024 x 1024 pixels) and a SunMicrosystems work station with Whole Band Analysis software to determine themolecular mass of the protein bands. This software calculates the molecular mass ofeach band by logarithmic interpolation using an average of the per pixel differencesamong all the defined standard molecular mass markers.2.2.5.2.6. AutoradiographyDried SDS-PAGE gels were exposed to X-ray film (Kodak X-Omat AR, X-OmatRP or MR-i) with an intensifying screen (Cronex Lightning Plus, Dupont) for 24 -96 hours at -70°C. Following this, films were developed by incubating in Developer(Kodak) for 3 minutes, a 60 s rinse in 3% acetic acid, followed by a 3 minuteincubation in Fixer (Kodak) and washing for 10 minutes under running water.432.2.6. Isolation and Quantitation of Protein Kinase C2.2.6.1. Preparation of Myocyte Cytosol and Membrane FractionsIsolated adult rat ventricular myocytes were washed and resuspended inKrebs-Henseleit buffer containing (mM): NaC1, 134; KC1, 4.7; CaC12, 1.0; glucose,10; ascorbic acid, 0.56; and HEPES, 15. Myocytes (1 x 106 rod shaped cells/mi) wereincubated for 1, 5 or 10 mm at 37°C in the presence and absence of 1% ethanol, 0.2mM OAG (in 1% ethanol) or 30 jiM R59022 and 0.2 mM OAG (in 1% ethanol).Immediately after the incubation, the cells were quickly centrifuged at 300 rpm for60 s, the supernatant removed and the cells were frozen in liquid N2. Frozenmyocyte pellets were homogenized in a wiggle bug apparatus in a buffer containing(mM): MOPS, 20; EGTA, 15; Na2EDTA, 2; Na3VO4, 1; DTT, 1 and 13-glycerophosphate, 75. The frozen pellet was placed into a precooled (with liquid N2)capsule. The capsule was agitated for 15 s, 250 pi homogenization buffer was addedand the capsule was agitated for another 15 s. The homogenate was removed to acentrifuge tube, another 250 jil homogenization buffer was added and the capsuleagitated for 5 s. This rinse was also added to the centrifuge tube. The capsule wasrinsed twice more with 250 jil aliquots of homogenization buffer and these rinseswere also added to the centrifuge tube.The myocyte homogenates were centrifuged at 100,000 rpm for 11 mm(240,000 x g) in a Beckman Optima TLA-100.2 Ultracentrifuge or at 50,000 rpm for30 mm (240,000 x g) in a Beckman L-8 Ultracentrifuge. The supernatant wasreserved for the cytosolic fraction and the pellet was resuspended in 1 ml of thehomogenization buffer with a 1 ml PTFE-glass douncer. The resuspended pellet wascentrifuged at 100,000 rpm for 25 mm (240,000 x g) in a Beckman Optima TLA100.2 Ultracentrifuge or at 50,000 rpm for 30 mm (240,000 x g) in a Beckman L-8Ultracentrifuge. This supernatant was combined with the first supernatant and44constitutes the myocyte cyt,osolic fraction. This fraction was immediately frozen inliquid N2 and stored at -70°C. The pellet was resuspended for 15 mm on ice with aPTFE-glass douncer in 1 ml of homogenization buffer containing 1% Triton X-100.The resuspended pellet was centrifuged as before. The supernatant was reserved asthe detergent-solubilized membrane fraction and immediately frozen in liquid N2and stored at -70°C.2.2.6.2. Preparation of Cytosol from Rat Brain and Cytosol and Membrane Fractionsfrom Bovine TracheaRat brain pieces, frozen in liquid N2 immediately after dissection, werehomogenized with 12 strokes of a motorized Potter-Elvehjem (50 ml) in 10 volumesof a buffer containing (mM): Tris-HC1, 20 (pH 7.5 at 20°C); EGTA, 10; Na2EDTA, 5;sucrose, 330; PMSF, 2; DTT, 2; 25 pg/ml leupeptin and 0.5% Triton X-100. Thebrain homogenate was centrifuged at 60,000 rpm for 35 mm (240,000 x g) in aBeckman L-8 Ultracentrifuge. The supernatant was reserved for the cytosol fractionand the pellet was resuspended in 10 ml of homogenization buffer with themotorized Potter-Elvehjem. The resuspended pellet was centrifuged as described forthe homogenate. The resulting supernatant was combined with the firstsupernatant and referred to as the cytosolic fraction.Bovine trachea sections, frozen in liquid N2, were homogenized in a buffercontaining (mM): Tris-HC1, 20 (pH 7.5 20°C); EGTA, 10; Na2EDTA, 5; sucrose, 330;PMSF, 2; DTT, 2; and 25 ig/m1 leupeptin. Homogenization was carried out with awiggle bug apparatus as described in section 2.2.6.1. Cytosol and membranefractions were isolated as described in section 2.2.6.1.452.2.6.3. DEAE-Cellulose ChromatographyDEAE-cellulose chromatography was utilized to fractionate myocyte, ratbrain and bovine trachea cytosol and detergent-solubilized membrane preparationsto remove endogenous compounds and excess detergent which may interfere withthe assay of PK C activity. DE-52 cellulose was prepared by washing with 0.1 NHC1, removing the fines 3 times by washing with ddH2O, washing with 0.1 NNaOH and then ddH2O before resuspending in 0.1 N HC1 and bringing the pH to7.4 (at 20°C) with 2.0 M Tris base. Columns were prepared as described by Thomaset al. (1987): DE-52 cellulose (2 ml) (1:1 v/v) was pipetted with a Pasteur pipetteinto disposable polypropylene columns (11 ml). The packed column (1 ml bedvolume) was equilibrated with and stored in 20 mM Tris-HC1 (pH 7.5) at 4°C.Before use, columns were washed with 10 ml of buffer containing (miVi): Tris-HC1(pH 7.5 at 4°C), 20; EGTA, 10; Na2EDTA, 5; sucrose, 330; PMSF, 2; DTT, 2; and 25pg/ml leupeptin. Cytosol and detergent-solubilized membrane fractions (2 ml each)were slowly applied to the columns with pasteur pipettes and the columns washedtwice with 3 ml of the above buffer. PK C was then eluted with 1 ml of buffercontaining (mM): Tris-HC1 (pH 7.5 at 4°C), 20; EGTA, 0.5; Na2EDTA, 0.5; NaCl,300; PMSF, 2; DTT, 2; and 25 jig/mi leupeptin. Leupeptin (25 jig/mi) was addedimmediately to the eluted fractions and PK C activity was assayed directly or thefractions frozen in liquid N2 and stored at -70°C for assay the next day. Onoccasion, the protein in the eluted fractions was concentrated to a volume of 100 p.1by centrifugation (5,000 x g) for 30 - 60 mm in Centricon-lO microconcentrators(Amicon). Leupeptin (25 jig/mi) was added immediately to the concentratedfractions and PK C activity was assayed directly or the fractions frozen in liquid N2and stored at -70°C for assay the next day.462.2.6.4. Fast Protein Liquid ChromatographyFast protein liquid chromatography (FPLC) was utilized to fractionatemyocyte cytosol and detergent-solubilized membrane preparations to removeendogenous compounds and excess detergent which interferes with the assay of PKC activity. FPLC was carried out using a 1 ml Mono-Q column (anion-exchange,Pharmacia) on a Pharmacia FPLC system according to the method of Pelech et al.(1991). Myocyte cytosol and detergent-solubilized membrane samples (4 mg in 2 ml)were loaded onto the Mono-Q column washed with (mM): MOPS (pH 7.2 at 20°C),10; EGTA, 5; Na2EDTA, 2; Na3VO4,1; DTT, 1 and 3-glycerophosphate, 25. PK Cwas eluted at a flow rate of 0.8 m]Jmin into 250 .t1 fractions with a 15 ml lineargradient of 0 - 0.8 M NaC1 in the above buffer. PK C activity was assayed eitherimmediately after elution or the fractions were frozen in liquid N2 and stored at -70°C until assayed.2.2.6.5. Determination of Protein Kinase C ActivityProtein kinase C activity was determined by a modification of the methoddescribed by Kikkawa et al. (1982). The reaction mixture (55 jil) contained (finalconcentration) 20 mM HEPES/KOH buffer (pH 7.5 at 20°C), 1 mg/ml histone TypeIII-S, 0.1 mM[7-32P]ATP (1 x i06 dpmltube; 0.08 Cilmmol), 5 mM DTT, 10 mMMgCl2,500 nM protein kinase inhibitor peptide, 20 jig/mi leupeptin, 1 mM EGTA,0.875 mM CaCl2 (1 M free Ca2j, 80 jig/mI phosphatidylserine (PS), 8 jig/mi 1-stearoyl-2-arachidonylglycerol (SAG) and 10 p1 of FPLC or other cytosol andmembrane fraction. The phospholipid-independent protein kinase activity wasmeasured under the same conditions without the addition of calcium orphospholipid and in the presence of 3 mM EGTA. All assays were performed inpolypropylene tubes. Following a 200 s preincubation, phosphorylation was47initiated by adding [y-32P]ATP and quenched after an additional 140 s bytransferring a 45 p1 aliquot of the mixture onto 2 cm2 pieces of Whatman P-81(cellulose-phosphate) ion exchange chromatography paper. The P-81 paper squareswere then individually washed 3 times for 10 mm with 10 ml of 70 mM phosphoricacid and then each was washed overnight with 10 ml of 70 mM phosphoric acid. Thecontent of each square was determined by liquid scintillation counting. Lipidswere stored at -20°C in either chloroformlmethanol (95:5) (phospholipids) or hexane(neutral lipids, fatty acids) containing 0.05% (w/v) butylated hydroxytoluene toprevent oxidation of double bonds. Lipid vesicles were prepared immediately priorto use by evaporation to dryness under a gentle stream of nitrogen gas followed bysonication under nitrogen, until clear, in 20 mM HEPES/KOH (pH 7.5 at 20°C)using a bath-type sonicator (Branson 1200). The sonicating bath was maintained ata constant temperature by running cold water through a cooling coil.PK C activity was calculated as phosphate incorporated into histone asfollows:pmol Pilmin/ml of fraction = (cpmIlO tl sample - blank cpm) (100)(140 s) (specific activity ofATP; cpmlpmol)The area under the curve (AUC) for each major PK C activity peak wascalculated, from the FPLC profiles, using trapezoidal approximation. Prior to thecalculation, the specific enzyme activity measured in the absence of Ca2 and lipidswas subtracted from the specific activity measured in the presence of Ca2+ andlipids.nAUC = E (vol. fraction) (c + ci+1) (fj÷1 -1=1_______248where: vol. fraction = volume of fraction iq = activity in fraction ifj = fraction # of fraction iFor these experiments, where the fraction volume was constant (0.25 ml) the aboveformula simplifies to:AUC (pmoL’min) = 0.25 ml (C1/2 + C2 + C3 + .... + Cf/2)where: was pmol/min/ml of the first fraction of the peakC2,C3, etc. were pmol/min!ml of the fractions in the body of the peakCf was pmollminlml of the last fraction of the peakAnalysis of variance (Two-way ANOVA) was performed on the means of the AUCusing the statistical computer program SYSTAT. The level of significance was a =0.05.2.2.6.6. Protein Concentration of PK C Activity PeaksThe proteins in the peaks of PK C activity from the FPLC profile wereconcentrated by combining the FPLC fractions of the peaks and centrifuging each inCentricon-lO microconcentrators (Amicon). Subsequent to centrifugation at 8,000rpm for 60 mm, the samples were washed with 1 ml of 20 mM Tris-HC1 (pH 7.5 at20°C), 0.5 mM Na2EDTA and 0.5 mM EGTA and centrifuged at 8,000 rpm for 90mm. After concentration, SDS-sample buffer (4-fold concentrate) was added tosolubilize the proteins. Samples were then boiled for 90 s and centrifuged for 2 mmin an Eppendorf microcentrifuge at maximum speed. Samples were separated bySDS-PAGE using 5 -20% gradient gels as described in section 2.2.5.2.1.1.49Subsequent to electrophoresis, gels were western blotted as described in section2.2.5.2.4.1.2.2.7. Phosphorylation of Proteins2.2.7.1. Phosphorylation of Proteins in Isolated MyocytesIsolated adult rat ventricular myocytes (3 - 6 x i06 rod shaped myocytes)were incubated in a Krebs-Henseleit buffer (pH 7.4 at 20°C) containing (mM): NaC1,134; KC1, 4.7; CaC12, 1.0; glucose, 10; ascorbic acid, 0.56; and HEPES, 15; with andwithout[32P]orthophosphate (0.5 mCi/nil) for 60 mm at 37°C. After incubation,myocytes (1 x 106 rod shaped myocytes) were stimulated (from 1 to 30 mm at 37°C)with one or more of the following: 0.01 or 1 iM isoproterenol, 0.1 or 1 mIVI HA1004,1 mM H-8, 0.1 mM forskolin, 120 jtM CGS 9343 B, 5% DMSO, 4 iM o-PDD or PMA,10% ethanol, 0.2, 0.25 or 0.4 iM OAG. The reaction was terminated by rapidcentrifugation at 300 rpm for 60 s and the supernatant discarded. Myocytes werehomogenized to prepare homogenates and purified SR fractions in buffers toprevent the dephosphorylation of proteins as described in section 2.2.3.2. and2.2.4.2. The proteins in the homogenate samples and final SR pellets wereprecipitated by the addition of cold 10% TCA. Samples were kept on ice for 15 mmand then centrifuged at maximum speed in an Eppendorf microcentrifuge for 30mm. The TCA was removed and the pellets resuspended in SDS-sample buffer,vigorously mixed and neutralized with 2 M Tris-base. Aliquots of each sample wereremoved for the determination of protein. Subsequently, one-half of each samplewas boiled and gel electrophoresis carried out using 5 - 20% polyacrylamidegradient gels as described in section 2.2.5.2.1.1. Gels were stained as described insection 2.2.5.2.2.1. and dried as described in section 2.2.5.2.3. Autoradliography wasperformed as described in section 2.2.5.2.6. For the non-radioactive phosphorylation50of proteins in isolated myocytes, SDS-PAGE was carried out as described in section2.2.5.2.1.2. and 2.2.5.2.1.3., followed by western blotting as described in section2.2.5.2.4.1.To quantitate the radioactivity incorporated into specific protein bands,phosphorylated bands were localized on the dried gel by comparison with theautoradiograph. The protein bands were then excised from the gel with scissors andthe gel pieces soaked for 12 hrs in liquid scintillation fluid in the dark, prior toquantitation in the scintillation spectrometer.2.2.7.2. Phosphorylation and Dephosphorylation of Proteins in Isolated SR VesiclesWhen isolated canine cardiac ventricular (a kind gift from Dr. B. Allen; Allen(1992)) or rat myocyte SR membrane vesicles were phosphorylated, the reactionmedium (100 jJ) consisted of 20 mM Histidine/HC1 buffer (pH 7.0 at 30°C), SRvesicles (75 pg), 5 mM MgC12, 0.1 mM EGTA, 1 mM DTT, 1 mM Na3VO4,5 mMNaN3, 5 mM NaF and 15 jig catalytic subunit of PK A. Phosphorylation wasinitiated by the addition of 5 p1 of 0.15 mM ATP (Tris salt). After 5 nun at 30°C, thereaction was terminated by the addition of 100 p1 of cold 10% TCA to the reactionmedium. The sample was mixed and kept on ice for 15 mm prior to centrifugation inan Eppendorf microcentrifuge for 30 mm at maximum speed. The TCA was removedand 90 p.1 of SDS-sample buffer was added. The pellet was resuspended by vigorousmixing and then neutralized with 2 M Tris base.When isolated canine cardiac ventricular or rat myocyte SR membranevesicles were dephosphorylated after isolation, the reaction medium (100 p.1)consisted of 40 mM Histidine/HC1 buffer (pH 6.8 at 30°C), SR vesicles (75 jig), 10mM EGTA, 5 mM NaN3 and 120 mM KC1. Dephosphorylation was initiated by theaddition of 1 p.1(25 units) of alkaline phosphatase. After 15 - 30 mm at 30°C, thereaction was terminated and the sample processed as described previously.513. RESULTS3.1. Characterization of Isolated Adult Rat Ventricular Myocytes3.1.1. General Viability StudiesTwo methods for the isolation of myocytes were used throughout thesestudies (section 2.2.1.1. and 2.2.1.2.). Both methods yielded 3 - 5 x i06 rod-shaped,Ca2 tolerant (1.0 mM), quiescent (non-beating) myocytes/rat heart. The majorcriteria utilized in the demonstration of myocyte viability were an elongatedstriated morphology, the exclusion from the cell of trypan blue and the ability of thecells to contract upon electrical stimulation. The elongated and striated morphologyof these viable myocytes was maintained for at least 4 hours (Figure 1). Utilizingtrypan blue exclusion staining (Figure 2), it was demonstrated that the rod-shapedcells in the preparation did not take up the dye and thus, had intact sarcolenunalmembranes. However, the round, blebbed cells (non-viable myocytes) present, werestained intracellularly with the dye, indicating breaks in the sarcolemmalmembrane.The current literature [Jacobson and Piper (1986)] indicates that adultventricular myocytes which are physically and biochemically intact and functionaldo not contract spontaneously in culture. Previous studies [Dani et al. (1979)] haveshown that adult myocytes which contract spontaneously after isolation areelectrochemically shunted and undergo the Ca2+ paradox. Intact myocytes retainthe morphological and functional ability to contract in response to electricalstimulation [Pelzer et al. (1984)]. When myocytes were isolated and thenstimulated as described by Haworth et al. (1980), all of the rod-shaped cellscontracted synchronously. The non-viable myocytes (round cells) did not respond toelectrical stimulation.52Figure 1. Photograph of a crude preparation of adult rat ventricular myocytes.(magnification: x 400).53Figure 2. Trypan blue staining of a crude preparation of adult rat ventricularmyocytes. (magnification: x 100)543.1.2. Maintenance of Hormone ResponsivenessThe retention of cell surface receptors on the isolated myocytes was assessedby probing the integrity and coupling of the (3-adrenergic system. This criterion wasutilized since the ability of the myocytes to produce cAMP intracellularly requiresintact 3-receptors on the cell surface and the coupled sarcolemmal intramembranecomponents of the adenylate cyclase system (G proteins and adenylate cyclase).Isolated myocytes in suspension were incubated with isoproterenol (a nonspecific f3-receptor agonist) and forskolin (direct activator of adenylate cyclase) to stimulatethe production of cAMP. As can be seen from Table 1, isoproterenol and forskolinelevate the intracellular level of cAMP in the myocytes 3.7- and 11.7-fold,respectively, over the resting basal level.3.2. Homogenization of Isolated Adult Rat Ventricular MyocytesTwo different myocyte homogenization methods were developed in order toaccommodate the different types of experiments performed. Completehomogenization of all of the myocytes isolated was carried out using a ‘Zero’-clearance Potter-Elvehjem homogenizer. This method was suitable for experimentswhere thorough homogenization was necessary. It was found that SR membranesisolated from myocytes homogenized with the ‘Zero’-clearance Potter-Elvehjem orwith other harsh methods of homogenization (sonication, French press, Polytronhomogenizer and wiggle bug) resulted in preparations that had very low Ca2-uptake andCa2IK-ATPase activities (Table 2). Also, homogenization with theFrench Press, wiggle bug and sonication effectively broke the cells open but theresulting SR cell membranes isolated were highly contaminated with mitochondrialand sarcolemmal membranes. Polytron homogenization did not break very many ofthe cells as well as resulting in isolated cell membranes that had very little55TABLE 1. cAMP accumulation in isolated adult rat ventricular myocytes.Isolated myocytes were incubated at 37°C with 0.5 mM IBMX and were stimulatedwith 1 pM isoproterenol or 100 jiM forskolin for 2 mm. Results are the mean ±S.E.M. (n) denotes the # of experiments.cAMP (pmoIJlO4rod-shaped cells)Basal 16.2 ± 1.4 (6)Isoproterenol 60.4 ± 4.2 (5)Forskolin 190.1 ± 52.5 (3)56TABLE 2. CafK-ATPase activities of several preparations obtained bydifferent myocyte homogenization and SR centrifugationprotocols.Ca2fK-ATPase activity was determined as described in section 2.2.5.1.2. Only thecalcium-stimulated values are shown. Ca2+upt ke activity was determined asdescribed in 2.2.5.1.1. and only the oxalate-stimulated values are shown. Specificenzyme activities are expressed as nmollmirilmg protein. Each result represents thedata from a single experiment. N.D. - not determined.Specific ActivityFraction Method Ca2-ATP se Ca2-Upt keHomogenate French Press N.D. N.D.SR ModifiedC&Fa 369.61 N.D.Homogenate Sonication 16.19 N.D.SR Modified C & F 34.13 N.D.Homogenate Wiggle Bug 30.32 N.D.SR Modified C & F 36.82 N.D.Homogenate Polytron N.D. N.D.SR Modified C & F 14.36 N.D.Homogenate Zero-clearance 18.79 N.D.SR Modified C & F 40.11 N.D.Homogenate Zeroc]bearance 19.59 N.D.SR Jones 86.94 N.D.Homogenate Zero-clearance 36.24 5.50SR Original C & F c 50.77 3.30Homogenate Dounce 46.32 3.01SR Original C & F 85.01 18.57Homogenate Dounce 76.82 8.59SR Original C & F 95.48 13.64a - modified from Chamberlain and Fleischer (1988)b - from Jones et al. (1979)c - from Chamberlain and Fleischer (1988)57enzymatic activity.Thus, when SR membranes were to be isolated from the myocytes a differenthomogenization procedure was required. In previous attempts to homogenize thesecells with a close fitting (20 .t) glass douncer and a loose-fitting teflon-glass PotterElvehjem homogenizer it was noted that only approximately 1/3 of the myocyteswere broken. However, when SR membranes were isolated from such a myocytehomogenate they contained high Ca2-uptake and Ca2iK-ATPase activities.This procedure was therefore used whenever SR membranes were isolated from themyocytes (see section 2.2.3.2. and 2.2.4.2.).3.3. Characteristics of SR Membranes Isolated From Adult Rat Ventricular Myocytes3.3.1. Preparation of SR Membranes from Isolated MyocytesA method was developed for the isolation of SR membrane vesicles from adultrat ventricular myocytes prepared from a single rat heart (see section 2.2.4.2.).Utilizing this method, SR membranes were prepared from myocytes within 3 h.When using the control buffers, this SR isolation method yielded 542.7 ± 50.1 pg ofSR protein (mean ± S.D.) from 3 x 106 rod-shaped myocytes (n = 5). The yield of SRprotein was less when buffers were used which prevented the dephosphorylation ofproteins to prepare SR membranes (194.5 ± 45.8 pg SR protein; mean ± S.D., from 3x i6 rod-shaped myocytes; n = 5).3.3.2. Marker Enzyme Activities583.3.2.1. SR Membranes Prepared in Control BuffersThe SR membranes isolated were characterized for the presence of SRmarker enzymes and other enzymatic activities indicative of contamination withmembranes from other cellular organelles. Oxalate-stimulated Ca2+upt keactivity was used as a specific marker for the SR since it had previously been shownthat mitochondrial and sarcolemmal membranes do not support oxalate-facilitatedCa2-transport [Jones et al. (1979); Solaro and Briggs (1974)]. Table 3 shows Ca2-uptake activities in the presence and absence of oxalate in homogenates and SRmembrane vesicles from isolated adult rat ventricular myocytes. Ca2+upt keactivity was found to be 98% ATP-dependent in the myocyte SR membranes. Thefinal SR fraction transported Ca2+ in the presence of oxalate at a rate of 107.3nmollmin/mg protein. When compared to the specific activity of the homogenate,this result suggests that the SR was enriched in this activity approximately 18-fold(Table 3). In the absence of oxalate, the specific activity ofCa2-uptake in the SRand homogenate preparations were 4.0 and 1.7 nmollminlmg protein, respectively.This indicates only a 2.4-fold enrichment of oxalate-independent Ca2-uptakeactivity and suggests that the preparation of SR membranes did not result in the copurification of oxalate-independent Ca2+transporting membranes. Uponcomparing the total Ca2-uptake activities in the SR and homogenates preparedfrom myocytes, approximately 60% of the oxalate-stimulated Ca2-uptake activityof the homogenate was recovered in the SR membrane preparation (Table 3). Ascan be seen from Table 3, when the Ca2+rele se channels in the junctional SRwere blocked by ruthenium red (5 M; Nagasaki and Fleischer (1988)) the Ca2-uptake activity in the presence of oxalate increased by 93% and the oxalatestimulated Ca2-uptake increased by 97%. Ruthenium red had no effect on thespecific activity of oxalate-independentCa2-uptake in myocyte SR membranes.TABLE3.Ca2-uptakeactivitiesof fractionsobtainedduringtheisolationof SRmembranesfromadultratventricularmyocytes.Ca2-transportactivitywasdeterminedasdescribedinsection2.2.5.1.1.Resultsarereportedasmean±S.E.M.Specificactivitiesareexpressedasnmollmin/mgprotein.Totalactivitieswerecalculatedbymultiplyingthespecificactivitymeasuredbythetotalamountofproteininthespecificfractionandareexpressedasnmollmin. Thenumber inbrackets()indicatesthenumberoffractionsassayed.Pellet1and2-from330xgand5000xgcentrifugations,respectively;RR-Rutheniumred.Ca2-UptakeCa2-UptakeCa2-Uptake(+oxalate)(-oxalate)Oxalate-StimulatedSpecificTotalSpecificTotalSpecificTotalActivityActivityActivityActivityActivityActivityHomogenate5.9±2.4(6)135.0±28.8(5)1.7±0.4(6)55.1±3.9(5)5.1±2.8(5)83.0±38.0(4)Pellet12.4±0.4(6)59.9±12.5(6)1.1±0.2(6)25.1±5.4(5)1.5±0.3(5)39.8±10.1(5)Pellet29.9±4.6(6)38.3±15.5(6)1.3±0.1(6)5.6±0.7(6)8.6±4.6(6)32.7±15.6(6)SR107.3±3.8(6)52.1±5.6(6)4.0±0.3(6)2.0±0.3(6)103.3±3.8(6)50.1±5.4(6)SR+RR207.5±9.7(4)110.0±8.8(4)3.8±0.3(4)2.0±0.2(4)203.7±11.8(4)108.0±8.7(4)60The specific and total activities of the SR Ca2fK-ATPase were alsomeasured in the SR membranes isolated from adult ventricular myocytes. Asshown in Table 4, during the isolation procedure the specific activity of Ca2/K-ATPase in the SR fraction increased 5.6-fold over the activity found in thehomogenate. However, only 9% of the total Ca2LK-ATPase activity in thehomogenate was recovered in the final SR fraction and 91% of the total activity waslost during the isolation. After the first centrifugation, 76% of the activity was inthe first pellet and after the second centrifugation, 9% was in the second pellet.In each of the tables (Table 3 and 4), the yield of protein (mg) for eachfraction has been normalized so that each was obtained from a starting myocytepopulation of 3 x 106 cells. These normalized protein values were used in thecalculation of the total activities in Tables 3 and 4.Comparing the Ca2iK-ATPase and Ca2-uptake activities of the SRmembrane vesicles obtained from isolated adult rat ventricular myocytes to theactivities from SR preparations isolated from whole rat heart(s) (Table 5) showsthat the activities of the myocyte SR preparation obtained to be similar to or higherthan the activities of SR preparations from whole rat heart.Since the most likely membranes to contaminate the SR membranepreparation were sarcolemmal and mitochondrial in origin, the following markerswere chosen as a measure of contamination: ouabain-sensitive Na7K+ATPase(sarcolemma) and cytochrome c oxidase (inner mitochondrial membrane). Thelatent activity of the Na/K-ATPase was exposed by pre-treating the membranesamples with alamethicin. In Table 6, the specific ouabain-sensitive Na/KATPase activity in the myocyte SR preparation was increased 6.5-fold over thatfound in the homogenate. However, the total ouabain-sensitive NaiK+ATPaseactivity was decreased by 93% during the purification of the SR membranes fromthe homogenate and only 7% of the total activity was recovered in the SRmembranes. The specific activity of cytochrome c oxidase in the myocyte SR61TABLE 4. Ca2fK-ATPase activities of fractions obtained during theisolation of SR membranes from adult rat ventricularmyocytes.Ca2fK-ATPase activity was determined as described in section 2.2.5.1.2. Only thecalcium-stimulated values are shown. Total activities (nmollmin) were calculated bymultiplying the specific activity (nmollminlmg protein) measured by the totalamount of protein (mg) in the fraction. Results are reported as mean ± S.E.M. Thenumber in brackets ( ) indicates the number of fractions assayed. Pellet 1 and Pellet2 are the same as defined in Table 3.Specific Activity Total ActivityHomogenate 74.1 ± 19.6 (6) 2,248. ± 345. (5)Pellet 1 68.1 ± 14.9 (6) 1,699. ± 378. (6)Pellet 2 50.0 ± 13.4 (6) 210.0 ± 60.5 (6)SR 414.0 ± 61.8 (6) 201.7 ± 37.3 (6)62TABLE 5. Ca2-ATP se and Ca2-uptake activities of SR membranesisolated from whole adult rat hearts compared to those fromisolated adult ventricular myocytes.Ca2-ATP se activities are theCa2-stimul ted data only. Ca2-uptake activitiesare the oxalate-stimulated uptake data only. N.A. - not available.nmollminlmg proteinCa2-ATP se Ca2-Upt ke Reference414 ± 62a 107 ± 4a Wientzek and Katz(1991)d38 ± 14a 74 ± 26a Limas (1978)e80 166 ± 13a Penpargkul et al. (1980)e153 ± 1a,c 31 ± 35ac Wei et al. (1976)e213a,c 8.1 ± 06a Lamers and Stinis(1980)e274 ± 16a,c 23.8 ± 16a,c Ganguly et al. (1983)e360 ± 50a,c 68 ± 5a Narayanan (1983)e437 ± 488b Lopaschuk et al. (1983)e510 ± 70b 263c Barker et al. (1988)eN.A. DeFoor et al. (1980)eN.A. 131.6 ± 13a,c Naylor et al. (1975)ea Mean ± S.E.M.b Mean ± S.D.c Value was recalculated from referenced Data from isolated myocytese Data from whole rat hearts63TABLE 6. Marker enzyme activities of homogenate and SR membranesisolated from adult rat ventricular myocytes.NafK-ATPase activity (latent and patent) was determined by pre-incubating themembrane fractions with alamethicin for 10 mm at 37°C. Only the ouabainsensitive values are shown. Total marker activities (tmo1Jmin) were calculated bymultiplying the specific activity (.tmoIJminJmg protein) measured by the totalamount of protein (mg) in the rat myocyte homogenate or SR membrane vesiclepreparation. Results are reported as mean ± S.E.M. The number in brackets ( )indicates the number of myocyte homogenates or SR preparations assayed.Na+fK+ATPase Cytochrome c OxidaseSpecific Activity Total Activity Specific Activity Total ActivityHomogenate 0.04 ± 0.01 (6) 1.70 ± 0.18 (5) 0.21 ± 0.05 (5) 12.28 ± 3.18 (6)SR 0.26 ± 0.05 (6) 0.12 ± 0.02 (6) 2.42 ± 0.31 (5) 0.95 ± 0.12 (4)64membranes increased 11.5-fold from the homogenate values. Again, as with theNa/K-ATPase activity, the total cytochrome c oxidase activity was decreased by92% during the isolation so that only 8% of the total mitochoncirial activity of thehomogenate was recovered in the SR fraction.3.3.2.2. SR Membranes Prepared in Buffers That Prevent DephosphorylationTable 7 shows Ca2-uptake activities from isolated adult rat ventricularmyocytes, in the presence and absence of oxalate, in homogenates and SRmembrane vesicles prepared using methods to prevent dephosphorylation. Thefinal SR fraction transported Ca2+ in the presence of oxalate at a rate of 116.1nmollminlmg protein. When compared to the specific activity of the homogenate,this result suggests that the SR was enriched in this activity approximately 33-foldover the homogenate. In the absence of oxalate, the specific activity ofCa2+upt kein the SR and homogenate preparations were 6.3 and 1.2 nmollminJmg protein,respectively. This indicates only a 5.3-fold enrichment of oxalate-independentCa2-uptake activity in the final SR membranes and suggests that the preparationof SR membranes did not result in the co-purification of oxalate-independent Ca2-transporting membranes. Upon comparison of the total Ca2-uptake activities ofthe SR to that in homogenates prepared from myocytes, 22% of the oxalatestimulated Ca2-uptake activity of the homogenate was recovered in the SRmembrane preparation. As can be seen from Table 7, when the Ca2+rele sechannels were blocked by ruthenium red (5 iiM) the Ca2-uptake activity of themyocyte SR preparation, in the presence of oxalate, increased by 34% and whencorrected for oxalate-independentCa2-uptake, increased by 35%. Ruthenium redincreased the oxalate-independentCa+upt ke in myocyte SR membranes by 11%.Table 8 shows that the SRCa27K-ATPase specific activity increased 2.0-fold during the purification of SR membranes from the myocyte homogenate.TABLE7.Ca2-uptakeactivitiesoffractionsobtainedduringtheisolationof SRmembranesfromadultratventricularmyocytesusingbufferstopreventdephosphorylation.Ca2-transportactivitywasdeterminedasdescribedinsection2.2.5.1.1.Resultsarereportedasmean±S.E.M.Specificactivitiesareexpressedasnmol/min!mgprotein.Totalactivitieswerecalculatedbymultiplyingthespecificactivitymeasuredbythetotalamountofproteininthespecificfractionandareexpressedasnmollmin. Thenumberinbrackets()indicatesthenumberoffractionsassayed.Pellet1and2-from330xgand5000xgcentrifugations,respectively;RR-Rutheniumred.Ca2-UptakeCa2-UptakeCa2-Uptake(+oxalate)(-oxalate)Oxalate-StimulatedSpecificTotalSpecificTotalSpecificTotalActivityActivityActivityActivityActivityActivityHomogenate3.5±0.6(5)148.2±27.0(5)1.2±0.1(5)49.8±7.4(5)2.3±0.4(5)98.4±20.0(5)Pellet12.3±0.2(5)70.0±11.1(5)1.0±0.1(5)30.4±2.3(5)1.2±0.3(5)39.6±10.3(5)Pellet 211.0±0.9(5)36.0±4.0(5)1.4±0.3(5)4.8±1.3(5)9.6±0.8(5)31.2±2.8(5)SR116.1±20.8(5)22.6±3.8(5)6.3±0.5(5)1.2±0.2(5)109.9±20.4(5)21.4±3.7(5)SR+RR155.3±34.4(5)30.0±6.7(5)7.0±0.9(5)1.4±0.2(5)148.3±33.6(5)28.5±5.7(5)66TABLE 8. Ca2fK-ATPase activities of fractions obtained during theisolation of SR membranes from adult rat ventricularmyocytes using buffers to prevent dephosphorylation.Ca27K-ATPase activity was determined as described in section 2.2.5.1.2. Only thecalcium-stimulated values are shown. Total activities (nmollmin) were calculated bymultiplying the specific activity (nmollminlmg protein) measured by the totalamount of protein (mg) in the fraction. Results are reported as mean ± S.E.M. Thenumber in brackets ( ) indicates the number of fractions assayed. Pellet 1 and Pellet2 are the same as defined in Table 7.Specific Activity Total ActivityHomogenate 122.2 ± 20.2 (5) 5,227. ± 147. (3)Pellet 1 63.9 ± 14.1 (5) 1,993. ± 528. (5)Pellet 2 36.5 ± 10.3 (5) 131.9 ± 45.9 (5)SR 250.8 ± 72.9 (3) 74.6 ± 26.0 (4)67However, the total Ca2/K-ATPase activity decreased by 98.6% during thepurification so that only 1.4% of the activity in the homogenate was recovered in thefinal SR membrane fraction.The sarcolemmal and mitochondrial marker enzyme activities were alsomeasured in this SR preparation. Table 9 shows that although the specific activityof Na+fK+ATPase and cytochrome c oxidase increased by 3.8- and 9.7-foldrespectively, the total Na+/K+ATPase and cytochrome c oxidase activity decreasedby 97.8 and 95.3%, respectively. Therefore, only 2.2% of the total Na7X-ATPaseactivity and 4.7% of the total cytochrome c oxidase activity in the homogenate wasrecovered in the SR membrane fraction.3.3.3. Protein Profile of SR Membranes3.3.3.1. SR Membranes Prepared in Control BuffersOne-dimensional SDS-PAGE analysis using Coomassie blue stainingdemonstrated that myocyte and rat heart homogenate and SR preparations hadproteins of similar molecular mass and staining intensity (Figure 3). Thecharacteristic SR protein band at 110 kfla has been shown to be the Ca2/K-ATPase [Brandi et al. (1986)1. Several protein bands in close proximity to eachother at 53 to 55 kDa were also detected. This molecular mass region of the gel wasexpected to contain at least three known SR proteins; calsequestrin, caireticulin,and the 53 kDa glycoprotein. A Stainsall stained gel (Figure 4) of myocyte and ratheart SR preparations demonstrated several pink stained bands as glycoproteinsand a blue stained band at 55 kDa, which may be calsequestrin.A new method for the detection of various types of Ca2-binding proteinsfollowing electrophoresis is staining with ruthenium red. In a previous study[Charuk et al. (1990)], this inorganic dye was found to bind to the same proteins as68TABLE 9. Marker enzyme activities of homogenate and SR membranesisolated from adult rat ventricular myocytes usingbuffers to prevent dephosphorylation.Na/K-ATPase activity (latent and patent) was determined by pre-incubating themembrane fractions with alamethicin for 10 mm at 37°C. Only the ouabainsensitive values are shown. Total marker enzyme activities (tmolImin) werecalculated by multiplying the specific enzyme activity (moUmin!mg protein)measured by the total amount of protein (mg) in the rat myocyte homogenate or SRmembrane vesicle preparation. Results are reported as mean ± S.E.M. The numberin brackets ( ) indicates the number of myocyte homogenate or SR preparationsassayed.Na+/K+ATPase Cytochrome c OxidaseSpecific Activity Total Activity Specific Activity Total ActivityHomogenate 0.09 ± 0.01 (4) 3.61 ± 0.47 (4) 0.48 ± 0.05 (5) 20.18 ± 1.67 (5)SR 0.34 ± 0.01 (3) 0.08 ± 0.01 (3) 4.67 ± 0.85 (3) 0.95 ± 0.05 (3)69kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -AB CDFigure 3. Coomassie blue-stained SDS-PAGE gel of homogenates and SRmembranes from whole rat heart (A & B) and isolated rat ventricularmyocytes (C & D). Lanes A and C contain homogenates and B and Dcontain SR membranes. Lanes contain 20 jig protein each and sampleswere heated at 37°C for 10 mm prior to electrophoresis. Molecular massmarkers are in the left, right and center lane of the gel. This gel was arepresentative result from at least 10 separate experiments. kDa-kilodaltons.kDa200. -116. -97.4 -66.2 -42.7 -31.0 -14.4 -E—Calsequestrn70Figure 4. Stainsall stained SDS-PAGE gel of whole rat heart (A & B) and isolatedrat ventricular myocytes (C & D). Lanes A and C contain homogenate andB and D contain SR membrane samples. Lanes contain 20 g proteineach and samples were heated at 37°C for 10 mm prior toelectrophoresis. Molecular mass markers are in the left, right and centerlane of the gel and are indicated in kilodaltons (kDa). This gel was arepresentative result from 3 separate experiments.AB CDr-’-: 1•-/71detected by the45Ca2-overlay technique. Figure 5 shows that in both myocyteand rat heart SR preparations, ruthenium red in the absence of CaC12 (lanes A &B), bound mainly to 2 wide bands of protein of molecular mass 53-55 kDa(calsequestrin and/or caireticulin and glycoprotein) and 97-100 kDa (Ca2fK-ATPase). Another protein band which became stained, but not as intensely, wasdetected at 16 kfla in both SR preparations. The presence of 50 mM CaC12 in thestaining buffer greatly reduced the binding of ruthenium red to all of these proteins(Figure 5, lanes C & D).Using SDS-PAGE of isolated SR membrane vesicles, it was generally notpossible to visualize phospholamban with either Coomassie blue or silver stainingtechniques. Therefore, visualization of phospholamban in SR membranepreparations was usually accomplished by autoradiography (after radioactivelabelling) or immunoblotting techniques. Figure 6 illustrates a Western blot ofmyocyte and rat heart SR preparations separated by SDS-PAGE and probed withthe monoclonal antibody Al, which is specific for phospholamban [Suzuki and Wang(1986)1. This blot shows that in unboiled samples there were 2 major mobilityforms of phospholamban, visualized at 8 and 25 kDa. A third fainter band atapproximately 27 kDa was also visualized. This band may represent aphosphorylated species of oligomeric phospholamban [Wegener and Jones (1984)1.When the sample was boiled prior to SDS-PAGE electrophoresis, the majority ofphospholamban dissociated into the 8 kDa form with a small amount dissociating toa 15 kDa form.3.3.3.2. SR Membranes Prepared in Buffers That Prevent DephosphorylationThe protein proffle of myocyte homogenate and SR membranes prepared inbuffers that prevent the dephosphorylation of proteins was carried out using onedimensional SDS-PAGE analysis with Coomassie blue staining. Figure 7AB CD 72kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -Figure 5. Ca2-binding proteins of SR membranes purified from isolated ratventricular myocytes (A & C) and whole rat heart (B & D), separatedby SDS-PAGE and transferred to nitrocellulose. The blot was stainedwith ruthenium red in the absence (A & B) and presence (C & D) of 50mM CaC12.Lanes contain 50 ig protein each and samples were boiledfor 90 s prior to electrophoresis. Molecular mass is indicated inkilodaltons (kDa). This blot was a representative result from 3 separateexperiments.Identification of phospholamban in SR membranes purified fromisolated rat ventricular myocytes (A & B) and whole rat heart (C & D),separated by SDS-PAGE and transferred to Zeta-Probe membranes.The blot was probed with a monoclonal antibody to phospholamban.Each lane contains 50 .tg protein. Lanes B & D were boiled for 3 mmprior to electrophoresis. HPL and LPL are the high and low molecularmass forms of phospholamban, respectively. Molecular mass markersare in the left lane of the blot. kDa - kiodaltons. This blot was arepresentative result from at least 3 separate experiments.73DkDa97-66-43-A B C31-21- I*h14- =.-aHPLLPL+Figure 6.+ BOILEDAB C 74kDa200.- LiH116.-__ __974 -H —---gigs c - L1100.41-__31.0--HPL21.5-14.4-___Figure 7. Coomassie blue-stained SDS-PAGE gel (A & B) and western blot (C) ofmyocyte homogenates and SR membranes prepared in buffers toprevent dephosphorylation. Lane A contains myocyte homogenate andB and C contain SR membranes. Lane C contains 25 jig protein and allsamples were heated at 37°C for 10 mm prior to electrophoresis. Theblot was probed with a monoclonal antibody to phospholamban. HPLis the high molecular mass form of phospholamban, respectively. Thegel and blot are a representative result from at least 10 separateexperiments for the gel and 2 separate experiments for the blot.Molecular mass markers are in the left lane of the blot. kDa -kilodaltons.75demonstrates that the protein profile of this preparation was very similar to themyocyte and rat heart homogenate and SR (isolated in control buffers) preparationsshown in Figure 3, containing proteins of similar molecular mass and stainingintensity. A Western blot of unboiled myocyte SR membranes prepared in buffersthat prevent dephosphorylation (Figure 7) demonstrates a single band at 26 kDa,which is the high mobility form of phospholamban.3.4. Phosphorylation ofProteins in Isolated Intact Adult Rat Ventricular Myocytes3.4.1. Proteins Phosphorylated in Response to PK A ActivationProtein kinase A (cAMP-dependent protein kinase; PK A) was stimulated inintact myocytes by treatment of the cells with isoproterenol, a non-specific 13-receptor agonist. Treatment of intact myocytes with isoproterenol leads to theintracellular accumulation of cAMP as shown previously in Table 1. A rise inmyocyte intracellular cAMP has previously been shown to activate PK A [Tsien(1977); Tada et al. (1976)1. In the present phosphorylation studies, SR membranevesicles were prepared from intact isolated adult rat ventricular myocytes whichwere radioactively labelled with[32P]orthophosphate and then stimulated withisoproterenol. Afterwhich the SR membranes were isolated using buffers whichminimized the dephosphorylation of proteins (see section 2.2.4.2.).Incubation of[32Pj-labelled myocytes with 1 p.M isoproterenol for 1 mmspecifically increased the phosphorylation of 4 protein bands (8.5, 27, 31 and 152kDa) in myocyte homogenates (Figure 8.A.). Upon boiling this sample, thephosphorylated band at 27 kiJa was lost and the phosphorylated band at 8.5 kDabecame more intense, suggesting the dissociation of phospholamban from theoligomeric to the monomeric form. In the SR fraction isolated from myocytesstimulated for 1 mm with 1 p.M isoproterenol, the phosphorylation of 3 protein76Autoradiograph of homogenates (A, B, E, & F) and SR membranes (C,D, G, & H) from control and isoproterenol-stimulated rat ventricularmyocytes. Myocytes were labelled with32P-orthophosphate and thenincubated as control (A, B, C & D) or stimulated with isoproterenol (E,F, G & H). Lanes B, D, F & H were boiled for 3 mm prior toelectrophoresis. HPL and LPL are the high and low molecular massforms of phospholamban, respectively. Molecular mass is indicated inkilodaltons (kDa). This autoradiograph was a representative resultfrom at least 10 separate experiments. Iso. - isoproterenol.A B C DE FG H_kDa200-116 -97 -66 -43 -31 -21 -14-8-- ÷ -— HPLLPL+ BOILED+ ISO.Figure 8.A.+— + -- + + +,177bands was increased (8.5, 15 and 27 kDa). Boiling of the SR preparation prior toelectrophoresis also caused the loss of the phosphorylated band at 27 kDa and anincrease in phosphorylation of the bands at 8.5 and 15 kDa. Boiling did not affectthe phosphorylation profile of any of the other proteins in the homogenate or SRpreparation in either control or isoproterenol-stimulated myocytes. Figure 8.B.shows the SDS-PAGE gel of the experiment in Figure 8.A. Each correspondingsample lane of the gel has a similar protein profile. In the SR samples frommyocytes treated with isoproterenol, more protein may have been recovered duringthe isolation procedure as these lanes seem to contain more protein, as judged bythe intensity of the Coomassie blue staining.Figure 9 illustrates the Western blot of homogenate and SR fractions isolatedfrom myocytes stimulated with 1 pM isoproterenol for 1 mm and probed with anantibody to phospholamban. In SDS-PAGE gels of myocyte homogenates, it was nopossible to detect phospholamban by Coomassie blue staining since this proteindoes not stain well with this dye. However, as shown in Figure 9, the monoclonalantibody Al was able to detect phospholamban in the homogenate fraction. Boththe low (8.5 kDa) and high (27 kDa) molecular mass forms of phospholamban, inhomogenate and SR membrane preparations from control and isoproterenolstimulated myocytes, bound the antibody. Together with the boiling-induced shiftin molecular weight, antibody studies confirm that the phosphorylated bandsstimulated by isoproterenol were, in fact, phospholamban.Myocytes pre-labelled with{32P]orthophosphate were treated with 0.1 miViforskolin in DMSO (final DMSO was 5% v/v) to activate PK A via the directactivation of adenylate cyclase (Figure l0.A.). Forskolin increased thephosphorylation of 4 protein bands (8.5, 27, 31 and 152 kDa) in a myocyte crudeparticulate fraction. In this figure, the protein phosphorylation pattern obtainedusing forskolin can be compared to that found when the myocytes were stimulatedwith 1 M isoproterenol. The number and types of protein bandsAB CDEFGH 78kDa200.-116.-97.4 - — — — _;;..——•*S&.-AOI7-31.0-01_I_ .J —14.4-- +- + -+ - + BOILED- - -- ++ + + ISO.Figure 8.B. SDS-PAGE gel of homogenate (A, B, E, & F) and SR membranes (C,D, G, & H) from control and isoproterenol-stimulated rat ventricularmyocytes. Myocytes were labelled with32P-orthophosphate and thenincubated as control (A, B, C & D) or stimulated with isoproterenol (E,F, G & H). Lanes B, D, F & H were boiled for 3 mm prior toelectrophoresis. Molecular mass is indicated in kilodaltons (kDa). Thisgel was a representative result from at least 10 separate experiments.Iso. - isoproterenol.79-LPLA BC D E F G HkDa66- —43-31-—HPL2114-- + - + BOILED- -. - - ÷ ÷ + +IsO.Figure 9. Western blot of homogenates (A, B, E & F) and purified SR membranes(C, D, G & H) from control (A, B, C & D) and isoproterenol-stimu[ated(E, F, G & H) rat ventricular myocytes. The blot was probed with themonoclonal antibody to phospholamban, Al. Lanes B, D, F & H wereboiled for 3 mm prior to electrophoresis. HPL and LPL are the high andlow molecular mass forms of phospholamban, respectively. This blot wasa representative result from 3 separate experiments. Molecular massmarkers are in the left lane of the blot. kDa - kilodaltons. Iso.-isoproterenol.÷kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -BOILEDISO. -FORSK.-øFigure 10.A. Autoradiograph of crude membrane fractions from control (A, B, G &H), isoproterenol (C, D, I & J) and forskolin-stimulated (E, F, K & L)myocytes. Lanes A to F were stimulated for 1 mm and lanes G to L for2 mm. Lanes B, D, F, H, J & L were boiled for 90 sec prior toelectrophoresis. HPL and LPL are the high and low molecular massforms of phospholamban, respectively. Molecular mass is indicated inkilodaltons (kDa). This autoradiograph was a representative resultfrom 3 separate experiments. ISO - isoproterenol, FORSK - forskolin.ABCDE FG HI JKL 80<—HPL-LPL- + - + --- ++ -+ - + - + - ++ + --- + + -- ++81phosphorylated by PK A in these crude membrane fractions isolated from thetreated myocytes were the same whether PK A was activated by either forskolin orisoproterenol. Figure 10.B. shows the SDS-PAGE gel of the experiment illustratedin Figure 10.A. This gel shows that the corresponding sample lanes all contained asimilar protein profile and that the proteins were stained to the same intensity.3.4.1.1. Effect of PK A Inhibitors on PK A Stimulated Protein PhosphorylationTo block the phosphorylation of proteins by PK A the intact 32P-labelledmyocytes were pretreated with inhibitors of PK A, prior to stimulation withisoproterenol. Two isoquinolinesulfonamide derivatives were used as specificinhibitors of PK A, HA1004 and H-8. As can be seen from Figure 11.A.,pretreatment of the myocytes for 30 mm with 100 tM HA1004 did not inhibit the.ability of isoproterenol (1 .tM) to increase the phosphorylation of 4 protein bands(8.5, 27, 31 and 152 kDa) in myocyte homogenates and of 3 protein bands (8.5, 15and 27 kDa) in myocyte SR membranes. Figure 11.B. illustrates the Coomassieblue-stained gel of the experiment shown in Figure 11.A. This gel shows that thecorresponding sample lanes all contained a similar protein profile. However, onthis gel there appears to be more protein in lanes containing SR samples fromHA1004 treated myocytes judging by the intensity of the Coomassie blue staining;this may be due to increased protein recovery during the SR isolation procedure. InFigure 12.A., myocytes were pretreated for 15 mm with 1 mM HA1004 or H-8 andthen stimulated with 0.01 .tM isoproterenol. Isoproterenol increased thephosphorylation of 4 protein bands (8.5, 27, 31 and 152 kDa) in myocytehomogenates and 2 protein bands (8.5 and 27 kDa) in myocyte SR membranes. Itappears that HA1004 and H-8 did not inhibit the activation of PK A. Figure 12.B.shows the SDS-PAGE gel of the experiment in Figure 12.A. This gel shows that thecorresponding sample lanes all contained a similar protein profile.82ABCDEFG HIJKLkDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -= = = =- — —0 — —=-+-+- +- +-+-+ BOILED-- ++ - - ++ - - ISO.- - - + + FORSK.Figure 10.B.- + + -SDS-PAGE gel of crude membrane fractions from control (A, B, G &H), isoproterenol (C, D, I & J) and forskolin-stimulated (E, F, K & L)myocytes. Lanes A to F were stimulated for 1 mm and lanes G toL for 2 mm. Lanes B, D, F, H, J & L were boiled for 90 sec prior toelectrophoresis. Molecular mass markers are in the left lane and thesixth lane from the right and are indicated in kilodaltons (kDa). Thisgel was a representative result from 3 separate experiments. PSO -isoproterenol, FORSK - forskolin.83kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -ABCD EFGHBOILEDISO.11A1004Figure 11.A. Autoradiograph of homogenates (A, B, E & F) and SR membranes (C,D, G & H) isolated from isoproterenol-stimulated myocytes in theabsence (A, B, C & D) and presence (E, F, G & H) of HA1004. Lanes B,D, F & H were boiled for 3 mm prior to electrophoresis. HPL and LPLare the high and low molecular mass forms of phospholamban,respectively. Molecular mass is indicated in kilodaltons (kDa). Thisautoradiograph was a representative result from 3 separateexperiments. ISO - isoproterenol.-HPL—LPL- + - ++ + + +- + - ++ + + ++ + +kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -ABCD EFGH- + BQ1LED‘so.HA100484Figure 11.B. SDS-PAGE gel of homogenates (A, B, E & F) and SR membranes (C,D, G & H) isolated from isoproterenol-stimulated myocytes in theabsence (A, B, C & D) and presence (B, F, G & H) of HA1004. Lanes B,D, F & H were boiled for 3 nun prior to electrophoresis. Molecularmass markers are in the left lane and the fifth lane from the right andare indicated in kiodaltons (kDa). This gel was a representativeresult from 3 separate experiments. ISO - isoproterenol.- + - + - ++ + + + + ++ +- - -- + + + +85kfla200. -116. -97.4 -66.2 -42.7 -ABCD EFGH IJKLFigure 12.A. Autoradiograph of homogenates (A, B, E, F, I & J) and SRmembranes (C, D, G, H, K & L) isolated from isoproterenol-stimulated myocytes in the absence (A, B, C, D, I, J, K & L) andpresence (E, F, G & H) of HA1004 and in the absence (A- H) andpresence (I, J, K & L) of H-8. Lanes B, D, F, H, J & L were boiled for3 mm prior to electrophoresis. HPL and LPL are the high and lowmolecular mass forms of phospholamban, respectively. Molecularmass is indicated in kilodaltons (kDa). This autoracliograph was arepresentative result of 3 separate experiments. ISO - isoproterenol.31.0-21.5 -14.4 -<—HPL-LPL- + - + - + - + + - + BOILED+ + + + + + + + + + + + ISO.- - - - ++++ - -- HA1004- - - - +‘ + + H-886ABCD EFGHI JKL- + - + - + - + - + -+ + + + + + + ÷ + + + ++ + + + -- - -- + + + ++ BOILEDISO.- 11A100411-8Figure 12.B. SDS-PAGE gel of homogenates (A, B, E, F, I & J) and SRmembranes (C, D, G, H, K & L) isolated fromsoproterenol-stimulated myocytes in the absence (A, B, C, D, I, J, K & L) andpresence (E, F, G & H) of HA1004 and in the absence (A - H) andpresence (I, J, K & L) of H-8. Lanes B, D, F, H, J & L were boiled for3 mm prior to electrophoresis. Molecular mass markers are in theleft lane and the sixth lane from the left and are indicated inkilodaltons (kDa). This gel was a representative result from 3separate experiments. ISO - isoproterenol.kfla200. -116. -97.4 -66.2 -42.7 -31.0 -21.5-14.4 -I______-‘— 4-87More protein appears to have been recovered in the SR membranes isolated from H-8 and HA1004 treated myocytes, since the lanes on the gel seem to contain moreprotein, as judged by the intensity of the Coomassie blue staining.3.4.2. Proteins Phosphorylated in Response to PK C ActivationIn preliminary studies, intact 32P-labelled myocytes were treated with anactive phorbol ester, PMA (phorbol 12-myristate, 13-acetate) or an inactive phorbolester, cL-PDD (4x-phorbol 12, 13-didecanoate), to modulate the activity ofCa2/phospholipid-dependent protein kinase (PK C). Myocytes were treated for 3mm either with 4 j.tM PMA or 4 pM x-PDD (in 0.4% DMSO). As shown in Figure 13and previously, isoproterenol increased the phosphorylation of 4 protein bands (8.5,27, 31 and 152 kDa) in myocyte homogenates and 2 protein bands (8.5 and 27 kDa)in myocyte SR membranes. Both phorbol esters stimulated the phosphorylation ofthe 2 protein bands of phospholamban (8.5 and 27 kiJa) in myocyte homogenatesand SR membranes isolated from phorbol ester-treated myocytes. In this particularexperiment, the amount of SR membranes isolated from isoproterenol-treatedmyocytes was lower than that isolated from cells treated with the phorbol esters,resulting in a faint signal on the autoradiograph.Myocytes were also stimulated with OAG (1-oleoyl-2-acetyl-glycerol), a cellpermeant, synthetic, diacylglycerol analog known to activate PK C in other cellsand tissues [Witters and Blackshear (1987)1. As shown in Figure 14, when 32P-labelled myocytes were treated with isoproterenol (1 .iM for 1 mm), 5% DMSO orOAG (0.25 mM in 5% DMSO for 5 mm), 2 protein bands of phospholamban (8.5 and27 kiJa) in myocyte homogenates and SR membranes were phosphorylated. Also,with isoproterenol treatment a 31 kDa protein band was phosphorylated.Stimulation of intact 32P-labelled myocytes with ethanol (10%) and 0.2 mMOAG (in 10% ethanol) for 10 mm to activate PK C, did not result in the stimulationAB CD EFGH IJKL 88+ BOILED- - - - - - -- ISO.- - -- + + + + - - - - x-PDD- - - - - - -- + + + + PMAAutoradiograph of homogenates (A, B, E, F, I & J) and SR membranes(C, D, G, H, K & L) isolated from myocytes stimulated withisoproterenol (A, B, C & D), ct-phorbo1 didecanoate (E, F, G & H) andphorbol myristate acetate (I, J, K & L). Lanes B, D, F, H, J & L wereboiled for 3 mm prior to electrophoresis. HPL and LPL are the high andlow molecular mass forms of phospholamban, respectively. Molecularmass is indicated in kilodaltons (kDa). This autoradiograph was arepresentative result from 2 separate experiments. ISO - isoproterenol,o-PDD -x-phorbol didecanoate, PMA - phorbol myristate acetate.rkfla200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4.-Figure 13.<—HPL<-LPL.- ++ + + +- + - + - + - + -89De—HPL14-LPL— + — + — + — + — + —+ BOILED+ + + + — - — .- — — — — ISO.— ———+ + + + + + + + DMSO—— -—- —- — + + + -i-OAGFigure 14. Autoradiograph of homogenates (A, B, E, F, I & J) and SR membranes(C, D, G, H, K & L) isolated from myocytes stimulated withisoproterenol (A, B, C & D), DM50 (E, F, G & H) and GAG (in DM50)(I, J, K & L). Lanes B, D, F, H, J & L were boiled for 3 mm prior toelectrophoresis. HPL and LPL are the high and low molecular massforms of phospholamban, respectively. Molecular mass is indicated inkilodaltons (kDa). This autoradiograph was a representative resultfrom 2 separate experiments. ISO - isoproterenol, DMSO - dimethylsulfoxide, GAG - 1-oleoyl-2-acetylglycerol.kDa FGH KL90of phosphorylation of any proteins in either case (Figure 15.A.). For comparison,the stimulation of protein phosphorylation (bands at 8.5, 27, 31 and 152 kDa inmyocyte homogenates and 8.5 and 27 kDa in myocyte SR membranes) byisoproterenol treatment of the myocytes (1 jiM for 1 mm) is also shown. The SDSPAGE gel of this experiment (Figure 15.B.) shows that each corresponding samplelane has a similar protein profile. However, as illustrated on the gel by theintensity of the Coomassie blue staining, the protein recovered in the homogenatesamples from ethanol and OAG in ethanol-treated myocytes appears to be less thanthat recovered in the homogenate from isoproterenol-treated cells. Less protein alsoappeared to be recovered in the SR membrane samples from OAG in ethanol-treated myocytes than from isoproterenol or ethanol-treated myocytes.Figure 16 shows an autoradiograph of control myocytes and myocytesstimulated with 0.4 mM OAG (in 2% ethanol) for 10 mm. Treatment of myocyteswith OAG resulted in the phosphorylation of bands at approximately 18 kDa in SRmembranes and 300 kDa in the homogenate fraction however, the stimulation ofphosphorylation of these proteins was not a consistent result. Therefore, it appearsthat OAG treatment of myocytes did not result in the stimulation ofphosphorylation of protein bands above the control level.3.4.3. Concerted Kinase Regulation of Protein Phosphorylation3.4.3.1. Possible Involvement of Calcium/Calmodulin-Dependeñt Protein KinaseThe possible contribution of calciumlcalmodulin-dependent protein kinase(CAM PK) to the stimulation of protein phosphorylation upon the activation of PK Awas also examined by treating the myocytes with an inhibitor of CAM PK.Myocytes were pretreated with 120 jiM CGS 9343 B (in 1.2% DMSO) for 5 mm andthen stimulated with 1 jiM isoproterenol (in 1.2% DMSO) for 1 mm. As shown in9ikDaABCD EFGH IJKL116. -97.4 -66.2 -42.7 --HPLFigure 15.A. Autoradiograph of homogenates (A, B, E, F, I & J) and SRmembranes (C, D, G, H, K & L) isolated from myocytes stimulatedwith isoproterenol (A, B, C & D), ethanol (E, F, G & H) and OAG (inethanol) (I, J, K & L). Lanes B, D, F, H, J & L were boiled for 3 mmprior to electrophoresis. HPL and LPL are the high and lowmolecular mass forms of phospholamban, respectively. Molecularmass is indicated in kilodaltons (kDa). This autoradiograph was arepresentative result from 2 separate experiments. ISO -isoproterenol, ETOR - ethanol, OAG - 1-oleoyl-2-acetylglycerol.31.0 -21.5-14.4-II_i- **:<-LPL- + -+ -+-+ - +- + BOILED+ + + + - - - - - - - - ISO.+ ÷ + ÷ ÷ + + + ETOH-- -- ++++ OAGABCD EFGHIJKL 92I1:I_________. .———-- +- + -+- +-+-+ BOILED- - - - - - -- ISO.+ ÷ + + + + + + ETOH- ++++ OAGSDS-PAGE gel of homogenates (A, B, E, F, I & J) and SRmembranes (C, D, G, H, K & L) isolated from myocytes stimulatedwith isoproterenol (A, B, C & D), ethanol (E, F, G & H) and OAG (inethanol) (I, J, K & L). Lanes B, D, F, H, J & L were boiled for 3 mmprior to electrophoresis. Molecular mass markers are in the left laneand in the sixth lane from the left and are indicated in kilodaltons(kDa). This gel was a representative result from 2 separateexperiments. ISO - isoproterenol, ETOH -ethanol, OAG - 1-oleoyl-2-acetylglycerol.kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -Figure 15.B.+ + + ÷93kDaABCDEFGH— — —— — — —Figure 16. Autoradiograph of homogenates (A, B, E & F) and SR membranes (C,D, G & H) isolated from control (A, B, C & D) myocytes and myocytesstimulated with OAG (E, F, G & H). Lanes B, D, F & H were boiled for3 mm prior to electrophoresis. HPL and LPL are the high and lowmolecular mass forms of phospholamban, respectively. Molecularmass is indicated in kilodaltons (kfla). This autoradiograph was arepresentative result from 3 separate experiments. OAG- 1-oleoyl-2-acetyiglycerol.200. -116. -97.4 -66.2 -42.7 -31.0.-21.5 -14.4 -144—b- + - + - + - ++ + + +-HPL—LPLBOILEDOAG94Figure 17, isoproterenol in the presence or absence of CGS 9343B, increased thephosphorylation of 4 protein bands (8.5, 27, 31 and 152 kDa) in myocytehomogenates and 2 protein bands (8.5 and 27 kDa) in myocyte SR membranes.Inhibition of CAM PK did not alter the pattern of stimulated proteinphosphorylation upon activation of PK A in intact myocytes.3.4.3.2. Possible Involvement of Protein Kinase CPhosphorylation of proteins by PK C was further investigated by treating themyocytes simultaneously with 1 i.IM isoproterenol and 0.4 mM OAG (in 2% ethanol).Intact 32P-labelled myocytes were pretreated for 10 mm with OAG and thenstimulated with isoproterenol for 1 mm. Figure 18 shows that treatment ofmyocytes with isoproterenol alone resulted in the stimulation of phosphorylation of5 protein bands (8.5, 15, 27, 31 and 152 kDa) in myocyte homogenates and 3 proteinbands (8.5, 15 and 27 kiJa) in myocyte SR membranes. Myocytes treatedsimultaneously with isoproterenol and OAG show the same phosphorylated bandsas with isoproterenol alone. Upon comparison of the samples from myocy-tesstimulated with isoproterenol, in the absence and presence of OAG, there do notappear to be any differences in the number of protein bands phosphorylated.The protein bands whose phosphorylation were stimulated by isoproterenol(8.5, 27, 31 and 152 kiJa) were excised from the gels and the radioactivityincorporated into these bands quantitated using liquid scintillation counting.Figure 19 shows the degree of incorporation of radioactivity into the 8.5 and 27 kiJabands of phospholamban from homogenates and SR membranes isolated fromcontrol, isoproterenol, OAG and isoproterenol plus OAG-treated myocytes. Thereare no apparent differences in the incorporation of radioactivity into the 8.5 and 27kDa bands (unboiled and boiled) of the homogenate samples between the fourdifferent myocyte treatments. In contrast, there appears to be an increase in theABCDEFGH 95kDa200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -<—HPLE—LPL- + - ÷ - + - + BOILED+ + ÷ + + + + + ISO.+ + + + + + + + DMSO- - - - + + + + CGS9343BFigure 17. Autoradiograph of homogenates (A, B, E & F) and SR membranes (C,D, G & H) isolated from DMSO and isoproterenol-stimulated myocytesin the absence (A, B, C & D) and presence (B, F, G & H) of CGS 9343 B.Lanes B, D, F & H were boiled for 3 mm prior to electrophoresis. HPLand LPL are the high and low molecular mass forms of phospholamban,respectively. Molecular mass is indicated in kilodaltons (kfla). Thisautoradiograph was a representative result from 2 separateexperiments. ISO- isoproterenol, DMSO - dimethyl sulfoxide.-wABCD EFGH 96Figure 18. Autoradiograph of homogenates (A, B, E & F) and SR membranes (C,D, G & H) isolated from myocytes stimulated with isoproterenol (A, B, C& D) and with OAG and isoproterenol (E, F, G & H). Lanes B, D, F & Hwere boiled for 3 mm prior to electrophoresis. HPL and LPL are thehigh and low molecular mass forms of phospholamban, respectively.Molecular mass is indicated in kilodaltons (kDa). This autoradiographwas a representative result from 3 separate experiments. ISO -isoproterenol, OAG - 1-oleoyl-2-acetylglycerol.— — — e — —kDa200. -116. -97.4 -66.2 -42.7 -31.0-21.5 -14.4. ——.11___- + - + - + - ++ + + +-HPL-LPLBOILEDISO.OAG+ + + +- + + + +97Unboiled BoiledSR MembranesFigure 19. Incorporation of radioactivity into 27 (A) and 8.5 (B) kDa protein bandsfrom SDS-PAGE gels. Results are reported as mean ± S.E.M., exceptwhere noted in the figure. Numbers in brackets ( ) represent ranges. ISOA — Control.tII I I — iso.C— Iso. + QAGT— QAGnCDL II— it CI IC’4r.-.,t1- ICDcIC Ii • •‘ -NC ‘ C’4 II —II II C I i LI IC4.cxzI II.DI n N C4II ii II III C ccIHM I.N__J91ci)CaCC0La-a)CEa)aC0C-)a)C0CCa)-4.-04—a-0)CE4--a)aU,-4-C0C-)3020100-60 -40 -200B IIC_IIo CN ttd.._.T—.. .-.o I—I LIII C CD N Lf)NC N T... .cN cNii II r—i-,— -CC•NUnboiled BoiledHomogenates- isoproterenol, OAG - 1-oleoyl-2-acetylglycerol.98incorporation of radioactivity into the 8.5 and 27 kiJa bands (unboiled and boiled) inthe SR membranes from isoproterenol-treated myocytes as compared to the control,OAG and isoproterenol plus OAG-treated myocytes. There seems to be nodifference in the incorporation of radioactivity into the 8.5 and 27 kDa bands(unboiled and boiled) between the control, OAG and isoproterenol plus OAG-treatedmyocytes. In Figure 20, the incorporation of radioactivity into the 31 and 152 kDaprotein bands in homogenates from control, isoproterenol, OAG and isoproterenolplus OAG-treated myocytes is shown. There are apparently no differences in theincorporation of radioactivity into these two bands between unboiled and boiled orbetween the four different myocyte treatments. However, these results (Figure 19and 20) must be viewed with caution since the data varies over a wide range andwere obtained from a small number of experiments.Figure 21 shows an autoradiograph of cytosolic fractions isolated from controlmyocytes and myocytes stimulated with isoproterenol (1 tM), OAG (0.4 mM in 2%ethanol) and isoproterenol (1 jiM) plus OAG (0.4 mM in 2% ethanol). Cytosolicfractions isolated from isoproterenol and OAG plus isoproterenol-treated myocytesshow an increase in the phosphorylation of the 21, 31 and 152 kDa proteins over thephosphorylation levels of these proteins noted in cytosolic fractions isolated fromcontrol and OAG-treated myocytes. The phosphorylation of a 24 kfla proteinappears to decrease in the cytosolic fractions isolated from isoproterenol or OAGplus isoproterenol-treated myocytes as compared to the phosphorylation of thisprotein in the cytosolic fractions from control or OAG-treated myocytes. Thereappears to be no apparent difference in the proteins phosphorylated in the cytosolicfraction from isoproterenol or OAG plus isoproterenol-treated myocytes.Several protein bands from the myocyte cytosolic fractions (152, 31, 24 and 21kDa), whose state of phosphorylation seemed to have changed with the fourdifferent myocyte treatments, were excised from the gels and the radioactivityincorporated into these bands quantitated using liquid scintillation counting. TheC0CC-4-000)CEa)0(I)-IC0C)A____— ControlI I—Iso.— Iso. + OAGIS25x1 — QAGB99G) 300C0C__0 200L 1001)a.4-CD0C)0200‘150 -100 -50-0--‘--Unboiled BoiledHomogenatesFigure 20. Incorporation of radioactivity into 31 (A) and 152 (B) kDa proteinbands from SDS-PAGE gels. Results are reported as mean ± S.E.M. (n =3), except for the controls (n = 2). Ranges for the controls are: A)unboiled (4.2 -14.8) and boiled (3.8 - 14.2); B) unboiled (4.7 - 9.6) andboiled (4.8 -11.0). Iso - isoproterenol, OAG - 1-oleoyl-2-acetylglycerol.-n ‘In100kDaABC D200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 -Figure 21.‘so.- - + + OAG+ - +Autoradiograph of myocyte cytosolic fraction isolated from control (A),isoproterenol (B), OAG (C) and isoproterenol and OAG-treated (D)myocytes. Samples were heated at 37°C for 10 mm prior toelectrophoresis. Molecular mass is indicated in kilodaltons (kDa). Thisautoradiograph is a representative result from 2 separateexperiments. ISO - isoproterenol, OAG - 1-oleoyl-2-acetylglycerol.101incorporation of radioactivity into the 31 kDa band appeared to be increased in thecytosolic fraction from isoproterenol and OAG plus isoproterenol-treated myocytesas compared to control and OAG-treated myocytes [control (n = 2): 3.41 (1.25- 5.57),isoproterenol (n = 2): 6.72 (1.07 - 12.36), OAG (n = 2): 2.28 (0.96 - 3.59),isoproterenol plus OAG (n = 2): 4.30 (1.61- 6.98) cpm4tg protein in the lane; mean(range)]. Also, the incorporation of radioactivity into the 152 kfla protein bandappeared to be increased in the cytosolic fraction from isoproterenol and OAG plusisoproterenol-treated myocytes as compared to control and OAG-treated myocytes[control (n = 2): 2.68 (1.12 - 4.24), isoproterenol (n = 2): 5.32 (1.24 - 9.40), OAG (n =2): 1.60 (0.78 - 2.41), isoproterenol plus OAG (n = 2): 3.61 (1.88 - 5.34) cpmlp.gprotein in the lane; mean (range)]. The radioactivity incorporated into the 24 kDaband appeared to be the highest from the cytosolic fractions from control myocyteswith incorporation decreasing to the same level in myocytes treated withisoproterenol, OAG and isoproterenol plus OAG [control (n 2): 9.76 (3.51 - 16.01),isoproterenol (n = 2): 5.45 (1.49- 9.41), OAG (n = 2): 4.3 (1.91- 6.69), isoproterenolplus OAG (n = 2): 2.81 (1.31 - 4.31) cpm4tg protein in the lane; mean (range)]. Theradioactivity incorporated into the 21 kDa band appeared to be similar betweencontrol, isoproterenol and OAG plus isoproterenol-treated myocytes but appeared tobe decreased in OAG-treated myocytes. Since the above results were obtained froma small number of experiments they must be viewed with caution.3.5. Phosphorylated Species of PhospholambanUsing 15% (non-gradient) SDS-PAGE gels and immunoblotting it waspossible to separate distinct species of oligomeric phospholamban from control,dephosphorylated and phosphorylated canine cardiac ventricle SR membranes(Figure 22) as described by Li et al. (1990). However, using this methodology it wasABC DEF 102kDa200. -116.-97466.2-42.7-31.0-—HPL21.5-14.4— 1IFigure 22. Western blot (A- C) and SDS-PAGE gel (D - F) of canine cardiacventricular SR membranes probed with a monoclonal antibody tophospholamban. Lanes A & D contain SR membranesdephosphorylated with alkaline phosphatase, lanes B & E containcontrol SR and lanes C & F contain SR phosphorylated by catalyticsubunit of PK A. Samples were heated at 37°C for 10 mm prior toelectrophoresis and each lane of the gel contained 25 .ig protein. HPLare the high molecular mass forms of phospholamban. The gel wasstained subsequent to transfer. Molecular mass markers are in theleft lane of the blot and are indicated in kilodaltons (kDa). The blotand gel are a representative result from 2 separate experiments.103not possible to separate distinct oligomeric species of phospholamban from ratmyocyte SR membranes. Utilizing 10 20% double gradient (10 - 20%polyacrylamide and 0.27 - 1.0% bisacrylamide) SDS-PAGE gels andimmunoblotting, however, it was possible to achieve some separation of thedifferent oligomeric species of phospholamban from control, dephosphorylated andphosphorylated myocyte SR membranes (Figure 23). A sample of phosphorylatedcanine SR membranes separated by this system is also shown for comparison.Homogenate and SR membrane samples from control myocytes and myocytesstimulated with isoproterenol (1 j.iM) alone, OAG (0.4 mM in 2% ethanol) alone andisoproterenol (1 .tM) and OAG (0.4 mM in 2% ethanol), together, were separatedwith the 10 - 20% double gradient gel and then western blotted and probed with amonoclonal antibody to phospholamban (Figure 24). This blot shows that inhomogenates and SR membranes from myocytes treated with isoproterenol orisoproterenol plus OAG, phospholamban was phosphorylated to a similar, higherdegree (as evidenced by the increase in number of the species . of oligomericphospholamban separated) than in the presence of OAG alone or from controlmyocytes. There seem to be no differences between the number of oligomericspecies of phospholamban formed in the homogenates and SR membranes ofmyocytes treated with isoproterenol or isoproterenol plus OAG. The number ofoligomeric species of phospholamban formed was not different betweenhomogenates and SR membranes from control and OAG-treated myocytes.3.6. Stimulation ofProtein Kinase C in Adult Rat Ventricular Myocytes3.6.1. Activation of Protein Kinase C in Adult Rat Ventricular MyocytesFrom these foregoing studies, it was not evident that PK C had beenactivated since results show that there was no protein whose phosphorylation waskDaABCD EFGH 104IILa..- --___-...:Figure 23. Western blot (A - D) and SDS-PAGE gel (E - H) of rat myocyte (A - C &E- G) and canine cardiac ventricle (D & H) SR membranes probed witha monoclonal antibody to phospholamban. Lanes A & E contain SRmembranes dephosphorylated with alkaline phosphatase, lanes B & Fcontain control SR and lanes C, D, G & H contain SR phosphorylated bycatalytic subunit of PK A. Samples were heated at 37°C for 10 mm priorto electrophoresis and each lane of the gel contained 25.‘g protein. HPLand LPL are the high and low molecular mass forms of phospholamban,respectively. The gel was stained subsequent to transfer. Molecularmass markers are in the left lane of the blot and are indicated inkilodaltons (kDa). The blot and gel are a representative result from 4separate experiments.200. -116. -97.4 -66.2 -42.7 -31.0 -21.5 -14.4 --HPL-LPL105A B CD E F GilHpLLPL-Figure 24.-——______-- + + - + +- - -- + + + +kDa- 31.0- 215- 14.4Western blot of homogenates (A, C, E & G) and SR membranes (B, D,F & H) from control (A & B), isoproterenol (C & D), OAG (E & F) andisoproterenol and OAG-stimulated (G & H) myocytes. The blot wasprobed with a monoclonal antibody to phospholamban. Samples wereheated at 37°C for 10 mm prior to electrophoresis and each lane of thegel contained approximately 4 g protein. HPL and LPL are the highand low molecular mass forms of phospholamban, respectively.Molecular mass is indicated in kilodaltons (kDa). This blot was arepresentative result from 3 separate experiments. ISO -isoproterenol, OAG - 1-oleoyl-2-acetylglycerol.‘so.OAG106stimulated by PK C activators. Therefore, we undertook a closer examination of theactivation of PK C in isolated myocytes.3.6.1.1. Isolation and Quantitation of PK C After DEAE-Cellulose ChromatographyIn a series of preliminary experiments, it was attempted to isolate PK C fromcytosolic fractions and membranes prepared from control and OAG-treatedmyocytes by DEAE-cellulose chromatography. Table 10 shows that in the presenceof the PK C activators, Ca2 and lipids (phosphatidylserine and diacyiglycerol), theactivity of PK C from the rat brain and bovine trachea cytosolic preparations wasmuch higher than in the absence of the activators. The PK C activity from the ratmyocyte preparations was similar to that found in the rat brain and bovine tracheain the absence of the activators, Ca2+ and lipids. However, in the presence of Ca2+and lipids, the PK C activity from the rat myocyte cytosol was not increased. TheDEAE separation of rat myocyte cytosol was repeated in six separate experimentsand all had similar results to those shown here. When the myocyte cytosol wasseparated by FPLC, a large increase in the activity of PK C in the presence ofactivators was demonstrated. Therefore, future studies on the isolation andquantitation of PK C from myocyte cytosolic fractions and membranes utilizedFPLC to partially purify these preparations.3.6.1.2. Isolation and Quantitation of PK C After Fast Protein LiquidChromatographyTo investigate the possible movement of PK C from the cytosol to the crudemembrane fraction in intact ventricular myocytes, FPLC (fast protein liquidchromatography) was used. Fractions obtained using FPLC were assayed for PK Cactivity with a standard PK C assay which utilized histone as the substrate. ToTABLE 10. Protein Kinase C Activities. 107Protein kinase C activity was determined for all preparations as described insection 2.2.7. Only the values from the cytosolic fraction are shown from eachpreparation. Ca2fLipid stimulated activities were calculated by subtracting thespecific enzyme activity (pmollminlmg protein) measured in the absence ofCa2fLipids from that measured in the presence ofCa2/Lipids. Results shown areof a representative experiment in each case. (DEAE) indicates that the cytosolswere separated by DEAE-cellulose column chromatography. (FPLC) indicates thatthe cytosol was separated by fast protein liquid chromatography.pmollminJmg proteinCa2fLipid—Ca27Lipids +Ca2LLipids Stimulated ActivityRat Brain PK C (DEAE) 186.0 2,030.0 1,844.0Bovine Trachea (DEAE) 254.0 1,361.0 1,107.0Rat Myocyte (DEAE) 188.9 190.5 1.6Rat Myocyte (FPLC) 42.1 768.3 726.2108determine the approximate fractions where PK C would elute from the FPLCcolumn, a sample of pure rat brain PK C was fractionated on the column. Figure 25shows that the activity of rat brain PK C eluted from the FPLC column fromfractions 27-35. Cytosolic and membrane fractions obtained from control myocytesand separated by FPLC (Figure 26) show that a large part of the protein in thesefractions does not bind very tightly to the Mono Q colmnn and eluted in the initialfractions collected. From the cytosol, the next largest amount of protein eluted fromfractions 27-35 and from the membrane fraction the next largest amount of proteineluted from fractions 47-50. Figure 27 shows the PK C activity profile of controlmyocyte cytosolic and membrane fractions assayed in the absence and presence ofthe peptide inhibitor of cAMP-dependent protein kinase. The inhibition of PK Aduring the assay of PK C activity greatly decreased the variability of thephosphorylating activity. Subsequently, all assays for PK C activity contained thePK A inhibitor.PK C activity was measured in cytosolic and membrane fractions isolatedfrom control myocytes incubated for 1, 5 and 10 mm (Figure 28) at 37°C. In thecytosol, three peaks of Ca2 and lipid-dependent PK C activity were eluted.Fraction 27-3 1 contained the peak with the highest activity. Two smaller peaks ofactivity occurred at fractions 32-34 and 39-42. In the membrane fraction, two majorpeaks of PK C activity were eluted. Eluting in the initial fractions, from fractions 6-15 was a peak of PK C activity that contained a large proportion of Ca2 and lipid-independent activity. Fractions 27-3 1 contained the major peak of Ca2 and lipid-dependent PK C activity from the membrane fraction. Calculation of the areaunder the curve (AUC) of the major peaks of Ca2 and lipid-dependent PK Cactivity (fractions 27-31) for the cytosolic and membrane preparations (Figure 29)show that there were no significant differences (Two-way ANOVA, p > 0.05)between the peaks obtained from control myocytes that had been incubated for 1, 5or 10 mm. The PK C activities in the presence of 3 mM EGTA (without Ca2 orC0E0.4Fraction NumberFigure 25. FPLC profile of purified rat brain PK C. PK C was assayed either inthe presence of 1 pM Ca2 (free), 80 ig/ml phosphatidylserine and8 .tg/rn1 1-stearoyl-2-arachidonylglycerol (•) or in the presence of noadded Ca2 or lipids and 3 mM EGTA (El). The data presented here wasfrom a single experiment.1090.80.62500020000 -150001 00005000 -0///0 10 200.20.030 40 50 60CDECa)0U-1 .51100.80.60.49C)az0.20.0 0.0Fraction NumberFigure 26. Protein elution FPLC profile of control myocyte cytosol (D) andmembrane (R) fractions. The data presented here was from a singleexperiment.I I I I I I I0 10 20 30 40 50 60 700.80.60.40.20.00.8 —Uaz0.60.40.20.0111C///I I II I I I I800600400-oU)t’ 200ao %%O c0a-a)-I-—(I)0-c04002000Figure 27.D///I I I I I••0 10 20 30 40 50 600 10 20 30 40 50 60Fraction Number Fraction NumberPK C activity of FPLC profiles of control myocyte cytosols (A & C) andmembranes (B & D) assayed in the absence (A & B) and presence (C &D) of cAMP-dependent protein kinase inhibitor. PK C was assayedeither in the presence of 1 tM Ca2 (free), 80 ig/ml phosphatidylserineand 8 pg/ml 1-stearoyl-2-arachidonylglycerol (•) or no added Ca2 orlipids and 3 mM EGTA (D). The data presented are from two separateexperiments. The experiment shown in C & D was repeated for n = 3.300C1_I DB-————-I——I—III___________________________________Figure28.PKCactivitiesofFPLCprofilesofcontrolmyocytecytosols(A,C&E)andmembranes(B,D&F)incubatedfor1(A&B),5(C&D)and10mm(E&F).PKCwasassayedeitherinthepresenceof1tMCa2(free),80.tg/mlphosphatidylserineand8pg/m11-stearoyl-2-arachidonylglycerol()ornoaddedCa2orlipidsand3mMEGTA(EJ).Thesedataareeacharepresentativeplotfromexperimentsthathaveeachbeenrepeatedthreetimes.AI A—It——‘I—.i—eLrirniiiii1.......IIIII800600400E2000 0 LC 8•E0C0-200o-c 0100 0EI—I—,,—I‘I—I______________I— /1—II’——.•••••—IIIF———————0.80.60.40.2-‘0.00.80 00.6z0.40.20.00102030405060FractionNumber—0102030405060FractionNumber0102030405060FractionNumber113C00C-)C)a)0aCa)4-0L0Figure 29. Area under the curve (AUC) of PK C activity from FPLC fractions 27 -31 of myocyte cytosols (A) and membranes (B). Results are expressed asmean ± S.E.M. (n = 3), except for ethanol cytosol (1 & 5 mm), ethanolmembrane (5 mm) and OAG (1 mm) where n = 4 and ethanol cytosol (5mm) where n = 5. Results for myocytes treated with R59 and OAG aremean (n = 2), ranges are: cytosols - 1 mm (45 - 131); 5 mm (17 - 59); 10mm (16- 38) and membranes - 1 mm (4 - 18); 5 mm (0.4 - 5.2); 10 mm (0- 7.4). * - denotes significantly different from control (Two-way ANOVA,p <0.05).4003002001 0001007550 -250BCE0E0C)D4C-)a)Cl)aca)00**1 mm 5 mm 10 mmTime (mm)114lipid) were subtracted from the activity in the presence of Ca2 and lipids, prior tothe AUC calculation.The protein from the PK C activity peak fractions obtained from the controlcytosol and membrane fractions were concentrated, separated by SDS-PAGE andimmunoblotted. These blots were then probed with antibodies to the PK C isozymestype III and type II (Figure 30). The cytosolic peak at fractions 27-3 1 demonstratedtwo irnmunoreactive bands of PK C; the band which demonstrated a highermolecular weight may be an autophosphorylated species of PK C. These fractionsalso contained both type II and type III PK C. The second cytosolic peak atfractions 32-34 contained type III PK C and the third cytosolic peak at fractions 39-42 did not contain type III PK C. The membrane peak that contained a largeproportion of Ca2 and lipid-independent PK C activity (fractions 6-15) contained 2bands of type III but not type II PK C. The band of type III PK C at approximately50 kfla may be the proteolytically active form of PK C, PK M. Fractions 27-31 fromthe membrane preparation contained two bands of type III PK C; the banddemonstrating a higher molecular weight may be an autophosphorylated species ofPK C. The kinase peaks at fractions 39 and 42 from the membrane preparation didnot contain type III PK C.To stimulate PK C in intact myocytes, the cells were treated with OAG.Since OAG was dissolved in ethanol, the effect of ethanol alone on the PK C activityof myocytes was determined. Cytosolic and membrane fractions were preparedfrom myocytes treated with 1% ethanol for 1, 5 and 10 mm (Figure 31). Ascompared to the PK C activity profiles from control cytosol and membrane fractions,ethanol did not affect the number or position of the peaks found in the cytosol ormembrane preparations from ethanol-treated myocytes. The PK C activity in themembrane peak that contained a large proportion of Ca2 and lipid-independentPK C activity (fractions 6-15) was also of the same magnitude as that found in thecontrol. The major peak of Ca2 and lipid-dependent PK C activity (fractions 27A1151 000800600400200a b C d kDai:66—— —— —— ———0 —. iu • — —0.80.6fl A --J.rC-)z0.2. 0.0600.8-oa)-4-00aL.0C.)Ca)-4-0aLI)0-c0-c-4-aL.0L0C-)Ca)-4-C-caC/)0-C0EE00E0ECEa0Ea0 10 20 30 40 505004003002001 000Ba b c d e kDa -9766-—————————0.6A A -‘‘-•C-)az0.20.00Figure 30.I I I I I10 20 30 40 50 60FraciTon NumberWestern blots of PK C activity peaks from control (1 mm) myocytecytosol (A) and membrane (B) fractions, probed with antibodies to Type11(a) and Type III PK C (b - e). The right lane of the blot containsmolecular mass markers expressed in kilodaltons (kDa). This blot wasthe result of 1 experiment.Figure31.PKCactivitiesofFPLCprofilesofethanol-treatedmyocytecytosols(A,C&E)andmembranes(B,D&F)incubatedfor1(A&B),5(C&D)and10mmCE&F).PKCwasassayedeitherinthepresenceof1.tMCa2(free),80jig/miphosphatidylserineand8j.ig/rril1-stearoyl-2-arachidonylglycerol()ornoaddedCa2orlipidsand3mMEGTA(IJ).Thesedataareeacharepresentativeplotfromexperimentsthathaveeachbeenrepeatedatleastthreetimes.a,-4-h—...0—a LC0 C., C Do-c 0 (I, o-C 0800600400200 0300200100 0C—————.—I—iv./——._iiiiWIiii.ii‘i.-ii-*A—/————.mIlWS‘IIIIIB——-—.4”;E—————I——I— II,—.fl—It—I.——.RaIIIUp—III0.80.60.40.20.00.8() 00.6z0.40.20.0DF//-////—/——.////60060010203040506001020304050FractionNumberFractionNumber1020304050FractionNumber117-31) from the membrane fraction appeared to contain less PK C activity than thesame peak from the control profiles. Calculation of the AUC (Figure 29) of thesepeaks from both the cytosol and membrane fractions isolated from ethanol-treatedmyocytes revealed that, when compared to the AUC from control peaks there wassignificantly less PK C activity (Two-way ANOVA, p < 0.05). As shown in thecontrol profiles, there were no significant differences (Two-way ANOVA, p > 0.05)found in the AUC of the major Ca2 and lipid-dependent PK C activity peaksobtained from either the cytosol or membrane fractions prepared from ethanol-treated myocytes that had been incubated for 1, 5 or 10 mm.PK C activity profiles from cytosolic and membrane fractions isolated frommyocytes treated with 0.2 mM OAG (in 1% ethanol) for 1, 5 and 10 mm are shownin Figure 32. As compared to the control and ethanol PK C activity profiles, OAGhas not affected the number or position of the peaks found in the cytosol ormembrane preparations from OAG-treated myocytes. The PK C activity in themembrane peak that contained a large proportion of Ca2 and lipid-independentPK C activity (fractions 6-15) was also of the same magnitude as that found in thecontrol and ethanol profiles. Just as in the ethanol profile, the major peak of Ca2and lipid-dependent PK C activity (fractions 27-3 1) from the membrane fractionappeared to contain less PK C activity than the same peak from the control profiles.As before, calculation of the AUC of these peaks from both cytosol and membranefractions isolated from OAG-treated myocytes was carried out to determine whetherthis reduction of PK C activity was of statistical significance. Figure 29 shows thatwhen compared to the AUC from control peaks there was significantly less PK Cactivity in these peaks (Two-way ANOVA, p < 0.05). However, when the AUC ofthese OAG-treated peaks was compared to the AUC of the ethanol-treated peaks,from both cytosol and membrane fractions, there was no significant difference found(Two-way ANOVA, p> 0.05). As shown in the control and ethanol profiles, therewere no significant differences found (Two-way ANOVA, p > 0.05) in the AUC of the0 0)-I L—LELC0 C) Do-c 0 U) o 0 Figure32.0.0—0.8C) 00.6zPKCactivitiesofFPLCprofilesofOAG-treatedmyocytecytosols(A,C&E)andmembranes(B,D&F)incubatedfor1(A&B),5(C&D)and10mm(E&F).PKCwasassayedeitherinthepresenceof1i.LMCa2(free),80ig/mlphosphatidylserineand8i.g/rril1-stearoyl-2-arachidonylglycerol()ornoaddedCa2orlipidsand3mMEGTA(11).Thesedataareeacharepresentativeplotfromexperimentsthathaveeachbeenrepeatedatleast threetimes.800600400200 0IIIII0.80.60.40.2.—..IIIII300200100 0IIIIIIIIIIIIIIIII010203040506001020304050600102030405060FractionNumberFractionNumberFractionNumber0.40.20.0119major Ca2 and lipid-dependent PK C activity peaks obtained from either thecytosol or membrane fractions prepared from OAG-treated myocytes that had beenincubated for 1, 5 or 10 mm. Taken together, the above results suggest that therewas no movement of PK C from the cytosolic to the crude membrane fraction upontreatment of the myocytes with OAG to indicate the activation of PK C.The protein from the PK C activity peak fractions obtained from the FPLC ofthe OAG-treated cytosol and membrane fractions were concentrated, separated bySDS-PAGE and western blotted. These blots were then probed with antibodies tothe PK C isozymes type III and type II (Figure 33). The results obtained weresimilar to those obtained from the blots of peaks from control myocytes. Thecytosolic peak at fractions 27-3 1 demonstrated two immunoreactive bands of PK C;the band demonstrating a higher molecular weight may be an autophosphorylatedspecies of PK C. These fractions also contained both type II and type III PK C. Thesecond cytosolic peak at fractions 32-34 contained both type III and type II PK Cand the third cytosolic peak at fractions 39-42 did not contain type III PK C. Themembrane peak that contained a large proportion of Ca2 and lipid-independentPK C activity (fractions 6-15) contained 2 bands of type III but not type II PK C.Again, the PK C type III band at 50 kDa may be the proteolytically activated form,PK M. Fractions 27-3 1 from the membrane preparation contained type III PK C.To investigate further the activation of PK C by OAG, intact myocytes werepretreated for 10 mm with 30 tM R59022, a compound known to inhibitdiacylglycerol kinase in intact cells and tissues [de Chaffoy de Courcelles et al.(1985)]. Diacyiglycerol kinase metabolizes diacyiglycerols in the cell to phosphatidicacid, which does not activate PK C. Inhibition of DAG kinase, therefore, wouldlengthen the amount of time that OAG remained unmetabolized by the kinase ordecrease the amount of OAG that was metabolized by the kinase. Afterpretreatment, the myocytes were again stimulated by OAG (0.2 mM in 1% ethanol)for 1, 5 and 10 mm (Figure 34). These profiles show results similar to those1200.80.60.4C)az00.210 20 30 40 50 60.000.8-D 1200a)-4-a09000a) 0-.-4-a—- 0oEC,)o- 3000-.0600a)oa—... 400—S— .S 0.4-4-C)E 200U-) Q 0.2-0-.0 0.0I I I I I I I0 10 20 30 40 50 60Fraction NumberFigure 33. Western blots of PK C activity peaks from OAG-treated (10 mm)myocyte cytosol (A) and membrane (B) fractions, probed with antibodiesto Type II (in A: a & C; in B: a) and Type III PK C (in A: b, d & e; in B: b& c). The right lane of the blot contains molecular mass markersexpressed in kilodaltons (kDa). This blot was the result of 1 experiment.0.6Figure34.PKCactivitiesofFPLCprofilesofR59022andOAG-treatedmyocytecytosols(A,C&E)andmembranes(B, D&F)incubatedfor1(A&B), 5(C&D) and10mm(E&F).PKCwasassayedeitherinthepresenceof1jtMCa2(free),80jig/miphosphatidylserineand8jig/mi1-stearoyl-2-arachidonylglycerol(ornoaddedCa2orlipidsand3mMEGTA(ED.Thesedataareeacharepresentativeplotfromexperimentsthathaveeachbeenrepeatedtwice.A.——IVF.1WII.._.cc&prfl,II.••-o a).4-L—..0—0 0 LC0-0 C,)Qo-c 0800600400200 0300200100 0EF—F—FFFFFFFFFFFF•F.•... IIIIC—FFFFFFFFFFFFF•F-IFL—IIIIIDFF—FF\FFFFFFF)•F‘FI‘•-‘.‘—-‘\._I.__•....IIIIIBFF-FFFFF—F01020304050FractionNumber0.80.60.40.20.00.8C)0.60.40.20.0F—A Ii.\/.1IIIIII102030405060FractionNumber6001020304050FractionNumber600122obtained with OAG treatment alone. As above, calculation of the AUC of the majorpeak of Ca2 and lipid-dependent PK C activity (fractions 27-3 1) from both cytosoland membrane fractions was carried out to determine whether there were anydifferences between these PK C activity peaks and the AUC of the PK C activitypeaks of the control, ethanol or OAG-treated myocytes. Figure 29 shows that theAUC of the peaks from both cytosol and membrane fractions isolated from R59022and OAG-treated myocytes were similar to those obtained from ethanol or OAGtreated myocytes at 1 mm. However, at 5 and 10 mm the AUC was apparentlylower than from either the ethanol or OAG-treated myocytes. These differences arereflected in both cytosol and membrane fractions. These results were not testedstatistically due to the low number of experiments performed.1234. DISCUSSION4.1. Isolated Adult Rat Ventricular MyocytesIntact adult rat ventricular cardiac muscle cells (myocytes) were utilized forthis study since they have several distinct advantages over isolated SR membranevesicles and intact heart muscle preparations (see section 1.3.). Unlike isolated SRvesicles, myocytes contain the various regulatory mechanisms and structuresnecessary for contraction. Intact heart muscle preparations suffer fromcomplications such as a heterogeneous cell population, intercellular interactionsand multicompartmentation [Jacobson and Piper (1986)]. Isolated myocytes lackdiffusional barriers and have the advantage of being a homogeneous cellpreparation. As well, regulatory mechanisms can be studied without the complexsyncytial, neuronal and hormonal effects that exist in the intact myocardium[Jacobson and Piper (1986)]. Finally, results obtained from isolated myocytes allowthe specific localization of a particular activity to the muscle cell. In this study, theadvantages of the isolated myocyte preparation experimentally, enabledinvestigations into protein kinase activities inside these cells. As well, by utilizingsubcellular fractions from the myocytes, phosphorylated proteins and kinaseactivities were localized intracellularly. However, a disadvantage of the isolatedmyocyte preparation used in the present studies was unlike working myocytes orwhole heart tissue these cells were in the quiescent state. The results of thesestudies therefore may not reflect the situation found in working myocytes duringthe excitation-contraction-relaxation cycle.In order to successfully carry out the present studies, the isolated adult ratventricular myocyte preparation was required to have a high yield/rat heart, a highproportion of viable cells and inter-preparation reproducibility. As well, the viablemyocytes obtained were to possess the following qualities: Tolerance to124physiological temperature (37°C) and calcium concentrations (0.5-1.0 mM),quiescence (i.e. myocytes do not contract spontaneously), the ability to contract uponelectrical stimulation and maintenance of characteristics such as rod-shapedmorphology and SL membrane integrity for the duration of the experimentalprocedure. Several different methods of myocyte isolation were tried [Powell et at.(1980); Wittenberg and Robinson (1981); Farmer et at. (1983); Lundgren et at.(1984); Vander Heide et at. (1986); Langer et at. (1987); Haworth et at. (1989)1 in anattempt to find a reproducible preparation, with the aforementioned cellularproperties. However, many of these methods did not yield myocyte preparationswith the above characteristics. The most difficult parameter to satisfr was theprocurement of a high viable myocyte yield. It was found that this was greatlydependent on the type and lot of collagenase enzyme used for the cell dissociationsteps as had been noted by other investigators [Jacobson (1989)]. The isolation ofadult rat ventricular myocytes with the above characteristics was established usingthe methods outlined in section 2.2.1. The modified method of myocyte isolation byLi et at. (1988) and Wimsatt et at. (1990) (see section 2.2.1.2.) was found to producepreparations of viable myocytes on a more consistent basis than the modifiedmethod of Piper et at. (1982).In addition to morphology, viability and contractility in response to electricalstimulation, isolated myocytes were characterized for their ability to produce cAMP.The results presented (see section 3.2.1. and Table 1) showed that the isolatedmyocytes retained the 3-adrenergic receptor on the cell surface and the sarcolemmalintramembrane components of the adenylate cyclase system (G proteins andadenylate cyclase). As well, these results demonstrated that these componentswere still functionally coupled. In the isolated myocytes, the basal and stimulatedlevels of cAMP were higher than cAMP levels found in myocytes by otherinvestigators [Buxton and Brunton (1983); Claycomb et at. (1984)1. There may betwo reasons for this: 1) the incubation buffer contained 0.5 mM IBMX (a cAMP-125dependent phosphodiesterase inhibitor) which would have increased the amount ofcAMP produced by inhibiting cAMP breakdown and 2) in Table 1, cAMP resultswere reported as pmol/104viable rod-shaped cells. Many other studies [West et al.(1986); lVIilIar et al. (1988); Bode and Brunton (1988); Pauwels et al. (1989)] reportcA1VIP levels using units of pmollmg protein, which are difficult to interpret, sincethese investigators do not state how many viable myocytes their preparationcontains. Therefore, pmollmg protein represents non-viable as well as viablemyocytes.4.2. Sarcoplasmic Reticulum Membranes Isolated from Adult Rat VentricularMyocytes4.2.1. Homogenization of Isolated Adult Rat Ventricular MyocytesDuring these studies, it was noted that isolated myocytes were relativelyrobust to disruption by homogenization as compared to other cell types. Twodifferent procedures were developed (see section 2.2.3.2.): One method (utilizing the‘Zero’-clearance homogenizer) succeeded in breaking 100% of the cells and releasingtheir cellular contents but the SR membranes isolated by this procedure had lowCa2-uptake andCa2/K-ATPase activities (Table 2) and were found to be highlycontaminated with enzymatic activities from other cellular membranes (SL andmitochondrial). The second procedure (see section 2.2.3.2., using a glass douncerand a motorized Potter-Elvehjem) only broke 30% of the cells, but the SRmembranes subsequently isolated retained enzymatic activities associated with theSR and contamination was held to a reasonably low level (< 10%). This method wasused for the preparation of SR membranes for the studies involving proteinphosphorylation.1264.2.2. Preparation of Sarcoplasmic Reticulum Membranes from Isolated MyocytesThere have been no previous reports of the isolation of SR membranes fromisolated adult rat ventricular myocytes. In preliminary studies, an attempt wasmade to prepare SR membranes from frozen myocyte homogenates. However, itwas not possible to obtain any SR membranes from these homogenates.Consequently, only fresh myocyte homogenates from freshly isolated cells were usedto prepare SR membranes. Several methods for the preparation of SR membraneswere used (see Table 2). With many of the methods, it was found that most of thecellular material (nuclei, myofibrils, mitochondria, SL, SR, etc.) was sedimentedafter the first centrifugation, leaving no material from which to purify SRmembranes. It was concluded that upon homogenization, large complexes ofcellular material formed, entrapping the membranes. Thus, attempts at furtherpurification were unsuccessful. It was found that it was necessary to dilutehomogenates (1 : 35 v/v, cells : Buffer) to prevent aggregation of membranes andproteins during the SR isolation procedure. In contrast, methods used for thehomogenization of whole rat hearts prior to preparation of SR membranes onlydilute the tissue 1 : 5 with homogenization buffer [Barker et al. (1988); Lamers andStinis (1980); Feher and LeBolt (1990)1.Many studies of SR function and regulation utilize SR membrane vesiclesprepared from canine heart [Wegener and Jones (1984); Chamberlain and Fleischer(1988)]. This tissue provides abundant starting material for the isolation of SRmembranes and pure preparations of either network or junctional SR [Chamberlainand Fleischer (1988)]. However, due to the limited access to canine tissue and theunavailability of canine disease models, many investigators have preferred to userat or guinea pig models. Utilizing whole rat heart(s), SR isolation methods nowavailable yield preparations which contain both network and junctional SRmembranes [Barker et at. (1988); Lopaschuk et at. (1983); Veleema and Zaagsma127(1981)]. An attempt was made to isolate SR membranes from myocytes by themethod of Chamberlain and Fleischer (1988) since, with this method, purifiednetwork and junctional membranes can be separated. However, the Chamberlainand Fleischer method was optimized for canine heart and it was not possible toreproduce this preparation using isolated rat heart myocytes. Even when thebuffers from this procedure were used together with the centrifugation protocoldevised, the recovery of SR membranes was very low. This may be due to thepresence of sucrose in the buffers which may have reduced the pelleting efficiency ofthe membranes to the point that SR vesicles could not be recovered from the smallamount of starting material typically obtained from a myocyte isolation.Subsequently, a method to isolate purified SR membranes from isolated adultrat ventricular myocytes was developed (see section 2.2.4.2.) based on the SRpreparation of Harigaya and Schwartz (1969) as modified by Jones et al. (1979). Forthe isolation of SR membranes from myocytes during the phosphorylation studies, itwas also necessary to use methods which prevented the dephosphorylation ofproteins. Alterations in protein-bound phosphate due to the action of kinase,phosphatase or protease activities were minimized during membrane isolationthrough the use of a phosphate buffer system combined with the presence of NaF (aphosphatase inhibitor) and EDTA (to chelate Mg2 and Ca2, thereby, inactivatingCa2-dependent proteases and phosphatases) [Lindemann et al. (1983)].4.2.3. Enzymatic Activities and Protein Profile of Myocyte Sarcoplasmic ReticulumThe myocyte SR preparation developed contained the two major structuraltypes of SR, network and junctional SR. The presence of phospholamban and theCa2+fK+ATPase (Figures 3, 5 and 6) was evidence for the existence of network SR.The presence of junctional SR in our preparation was illustrated by the occurrenceof calsequestrin (Figure 4) and the presence of the Ca2+re1e se channel as128indicated by an increase in Ca2-uptake activity upon the addition of rutheniumred (Table 3), a known blocker of the channel [Nagasaki and Fleischer (1988)].Oxalate-stimulated Ca2+upt ke activity was used as a specific marker forSR since it had previously been shown that mitochondrial and sarcolemmalmembranes did not support oxalate-facilitatedCa2-transport [Jones et al. (1979);Solaro and Briggs (1974)1. In the presence of oxalate, the specific Ca2-uptakeactivity of the myocyte SR preparation prepared in control buffers increased 18-foldupon isolation from the myocyte homogenate (Table 3). Approximately 60% of theoxalate-stimulated Ca2+upt ke activity present in the homogenate was recoveredin the final SR preparation. In addition, oxalate-stimulated Ca2-uptake activitywas increased in the SR preparation by 97% in the presence of ruthenium red. Thespecific activity ofCa2/K-ATPase of the myocyte SR preparation prepared incontrol buffers increased 5.6-fold upon isolation from the myocyte homogenate andonly 9% of the activity in the homogenate was recovered in the final SR preparation(Table 4). It is unclear why, during the purification of SR membranes, the specificactivity ofCa2Lupt ke increased by 18-fold and 60% of this activity was recovered,while theCaiK-ATPase specific activity only increased 5.6-fold and only 9% ofthis activity was recovered. Since these activities are functions of the same enzyme,theCa2+/K+.ATPase, it would be reasonable to expect that the same percentage ofthese activities would have been recovered. Further, it would seem unlikely thatthe ATPase activity of the enzyme could have deteriorated during the SR isolationprocedure while the uptake activity remained intact, since the ATPase activity isrequired for uptake activity. Several different factors may contribute to thisapparently paradoxical result: There are slight differences in the buffers andmethods used for the determination ofCa2iK-ATPase from those utilized forCa2-uptake activity. As well,Ca2fK-ATPase activity was determined at steadystate andCa2+upt ke activity was determined at initial rates. The measurementofCa2/K-ATPase activity is not as specific an activity of the SR as is the Ca2-129uptake activity, since there are otherCa2+stimulated ATPases and processes inthe cell that utilize ATP while only the SR membrane can support oxalatestimulatedCa2-uptake activity [Jones et al. (1979); Solaro and Briggs (1974)]. Inthe assay forCafK-ATPase activity, ouabain and NaN3 were included to inhibitthe other two major ATPases in heart tissue, the Na+tK+ATPase and themitochondrial ATPase, respectively. However, it is not unlikely that there may beanotherCa2-stimul ted ATP-utilizing activity that may have interfered with themeasurement of SRCa2/K-ATPase activity, especially in the homogenate. If thevalue obtained for SR CaiK-ATPase activity in the myocyte homogenate wasartifactually increased by other ATP-utilizing activities, this may account for thelow recovery of specific activity calculated in the final SR fraction.The “coupling ratio” refers to the stoichiometry ofCa2+transport relative toATP hydrolysis and is defined as the net Ca2-flux divided by the net ATPase ratemediated by theCa2iK-ATPase [Feher and Fabiato (1990)]. The stoichiometry ofthe Ca2+pump reaction is 2 Ca2+ ions transported for each ATP moleculehydrolyzed [Yamada et al. (1970)]. Typical “coupling ratio” values found in canineSR by other authors range from 0.3 to 0.8 [Chamberlain et al. (1983); Jones andCala (1981); Feher and Lipford (1985)]. Values for rat SR show a greater variation,ranging from 0.03 to 2.0 (calculated from Table 5). In the myocyte SR preparationused, the coupling ratio found (in the presence of ruthenium red), 0.5, is withinthese two ranges. Most often, the coupling ratio is approximated by simultaneouslymeasuring the rate of oxalate-stimulated Ca2-uptake and ATP hydrolysis.However, most of the above authors (including the present studies) did not measureCa2-uptake and ATPase activity simultaneously. Nevertheless, this calculationdemonstrates that the specific activities ofCa2-uptake and ATPase obtained in themyocyte SR membranes used, appear to be coupled in a similar manner topreviously published SR preparations.130The Ca2-uptake activity of the myocyte SR preparation developed wassimilar to SR preparations from whole rat heart reported by DeFoor et at. (1980)and Naylor et at.(1975) but higher than that found in other studies in whole ratheart SR preparations [Lopaschuk et at. (1983); Ganguly et at. (1983); Limas (1978);Wei et at. (1976); Lamers and Stinis (1980); Narayanan (1983); Table 51. Two SRpreparations from whole rat heart had higherCa2-uptake activity [Penpargkul etat. (1980); Barker et at. (1988)]. Recently, Wimsatt et at. (1990), using digitoninlysed adult rat ventricular myocytes, measured Ca2-uptake into the SR ofmyocytes at a rate of 160 nmo]Jmin!mg protein in the presence of ruthenium red.This compares favorably with the Ca2-uptake activity found in this study in thepresence of ruthenium red (208 nmollmin/mg protein). The myocyte SR Ca2fK-ATPase activity was similar to that found by Lopaschuk et at. (1983) andNarayanan (1983) and was higher than that obtained using the SR preparationsfrom whole rat heart [Ganguly et at. (1983); Limas (1978); Wei et at. (1976);Penpargkul et at. (1980); Lamers and Stinis (1980)]. Only one SR preparation fromwhole rat heart [Barker et at. (1988)] possessedCa2/K-ATPase and Ca2-uptakeactivities which were both higher than the SR preparation from myocytes reportedhere. However, this preparation [Barker et at. (1988)] consisted mainly of networkSR and required a minimum of 5 rat hearts and 18 hours to prepare. Recently,Feher and LeBolt (1990) have developed an SR preparation from whole rat heart,stabilized by sodium metabisuffite, which exhibited very highCa2/K-ATPase andCa2-uptake activities (1,070 and 360 nmo]JminJmg protein, respectively). Itremains to be determined whether these high values truly reflect the activitiespresent in the intact rat myocardium.To measure contamination of the isolated SR membranes with enzymeactivities from other organelles, determinations of ouabain-sensitive Na+fK+ATPase (SL marker) and cytochrome c oxidase (inner mitochondrial membrane)activity were carried out. Only 7% of the total sarcolemmal ouabain-sensitive131NafK-ATPase activity and 8% of the total mitochondrial cytochrome c oxidaseactivity of the homogenate were recovered in the final myocyte SR preparation(Table 6). There are relatively few reports in the literature on the contamination ofisolated SR membranes with enzyme activities from other organelles of the ratheart. Some reports present the specific activity of SL marker enzymes, but thisactivity is not a true quantitation of how much of the contaminating enzymeactivity appears in the SR preparation. The specific activity is rather a reflection ofthe ability to isolate this enzymatic activity in an intact form. Values ofcontamination of isolated canine SR by sarcolemmal and mitochoncirial enzymeactivities, expressed as total activity, have been published by Chamberlain andFleischer (1988). These authors found 7 - 10% contamination by SL enzymaticactivity and 2% contamination by mitochondrial enzyme activity. Therefore, thecontamination by SL and mitochondrial enzyme activity, by 7 and 8% respectively,of the myocyte SR preparation in this current study is comparable to that found inthe SR membrane prepared from canine heart by other investigators.4.2.4. Comparison of Myocyte Sarcoplasmic Reticulum Membranes Prepared inControl Buffers Versus Buffers Used to Prevent DephosphorylationFor the isolation of SR membranes from myocytes during the phosphorylationstudies, it was necessary to use methods which prevented the dephosphorylation ofproteins during the procedure. These methods employed the same centrifugationprotocol as was utilized during the isolation of SR in control buffers, but used adifferent buffer system. This change in buffer resulted in the isolation of SRmembranes with a similar protein profile (Figure 7) but with slightly differentenzymatic properties (Figures 7, 8 and 9) than SR isolated in control buffers. Whenbuffers to prevent dephosphorylation were used to prepare SR membranes the yieldof SR protein obtained was less (194 ± 45 g SR protein; mean ± S.D., from 3 x i06132rod-shaped myocytes; n=5) when compared to that obtained using control buffers(542±50 .tg SR protein; mean ± S.D., from 3 x 106 rod-shaped myocytes; n=5).There was no difference found between the oxalate-stimulated Ca2+upt kespecific activities (Tables 3 and 7) in these two SR preparations (107.3 ± 3.8, mean ±S.E.M., n = 6 (control buffers) us 116.1 ± 20.8, mean ± S.E.M., n = 5(dephosphorylation buffers) unpaired t-test p > 0.05). However, only 22% of thetotal oxalate-stimulated Ca2+upt ke activity was recovered in the SR membranesprepared in buffers to prevent dephosphorylation (Table 7), as compared to 60% incontrol buffers (Table 3). As well, while using buffers to prevent dephosphorylation,there was a 33-fold increase in the Ca2-uptake specific activity in the presence ofoxalate during the isolation of the SR fraction from the homogenate, whereas incontrol buffers this activity had increased only 18-fold in the SR fraction ascompared to the homogenate. Using buffers to prevent dephosphorylation (Table 7),oxalate-independent Ca2-uptake activity increased 5.3-fold in the SR membraneswhen compared with the activity found in the homogenate. In SR membranesprepared in control buffers, this activity was only increased by 2.4-fold over thatfound in the homogenate. Finally, ruthenium red increased oxalate-stimulatedCa2-uptake specific activity in the SR fraction prepared in control buffers by 97%(Table 3), whereas ruthenium red only increased this activity by 35% in SRpreparations prepared in buffers to prevent dephosphorylation (Table 7).The specific activity of Ca27K-ATPase of SR membranes prepared inbuffers to prevent dephosphorylation (Table 8) was decreased by 40% as comparedto the activity from SR membranes prepared in control buffers (Table 4 and 8; 414 ±61, mean ± S.E.M., n = 6 (control buffers) vs 250 ± 72, mean ± S.E.M., n = 3(dephosphorylation buffers)). The specific activity ofCaiK-ATPase (250 ± 72nmollmin/mg protein; mean ± S.E.M.; n = 3) found in the presence of buffers toprevent dephosphorylation in this study compares well to that found in anotherstudy where SR membranes were also isolated in the same buffers from whole rat133heart (333±5 nmollminlmg protein; mean ± S.E.; n = 148; Lindemann et al. (1983)).During the isolation procedure in buffers to prevent dephosphorylation, the specificactivity ofCa2/K-ATPase in the SR membranes increased only 2-fold over theactivity found in the homogenate, whereas in control buffers there was a 5.6-foldincrease; only 1.4% of the total Ca2/K-ATPase activity in the homogenate wasrecovered as compared to 9% recovery in SR membranes prepared in control buffers.SR membranes prepared in buffers to prevent dephosphorylation alsodemonstrated less contamination by SL and mitochondrial enzymatic activities(Table 9): Only 2.2% of the total sarcolemmal ouabain-sensitive Na/K-ATPaseactivity and 4.7% of the total mitochondrial cytochrome c oxidase activity in thehomogenate was recovered in the SR membranes, whereas 7% and 8% of theseactivities, respectively, were recovered in the SR membranes prepared in controlbuffers (Table 6). Ouabain-sensitive NaiK-ATPase specific activity in SRmembranes increased 3.8-fold over the activity in the myocyte homogenate inbuffers to prevent dephosphorylation as compared to an increase of 6.5-fold in theSR membranes prepared in control buffers. In SR membranes isolated in buffers toprevent dephosphorylation, cytochrome c oxidase specific activity increased 9.7-foldduring the purification of SR membrane vesicles from myocyte homogenates,whereas in SR membranes isolated in control buffers this activity was increased11.5-fold.These differences in enzymatic activities between SR membranes isolated incontrol buffers and in buffers to prevent the dephosphorylation of proteins may bedue to effects on the extraction and sedimentation of SR membrane vesicles duringthe isolation procedure by components of the dephosphorylation buffer. However,even though there were some enzymatic activity differences between these two SRpreparations, since their protein profiles were similar, these differences did notappear to affect the results obtained on the phosphorylation of proteins in intactventricular myocytes.1344.3. Protein Phosphorylation in Isolated Adult Rat Ventricular Myocytes Stimulatedwith PKA and PK CIn early studies on the nucleotide, adenosine triphosphate (ATP), it wasnoted that the terminal phosphate group participated in transfer reactions asreviewed by Barany and Barany (1981). Subsequently, the hydroxyl groups ofserine, threonine and tyrosine were shown to be acceptors for the y-phosphate groupof ATP. Now it is possible to phosphorylate all nucleophilic amino acid residues inproteins and that the phosphate transferred can originate from a variety of sources[Vener (1990)]. There are two major types of protein phosphorylation, catalytic andregulatory. Catalytic protein phosphorylation was found to include the formation ofphosphorylated intermediates in the active sites of enzymes catalyzing thephosphoryl group transfer [Vener (1990); Knowles (1980)1. Physiologically, the firstinterest in protein phosphorylation occurred when cAMP was found to activate aprotein kinase (PK A) isolated from rabbit skeletal muscle [Walsh et al. (1968)].Since that time, many other kinases and functions of protein phosphorylation havebeen and are still being elucidated. Regulatory protein phosphorylation is the mostcommon type of reversible post-translational modification of proteins. It controlsalmost all cellular processes by altering the catalytic activity or functionalproperties of other proteins [Cohen (1985); Krebs (1986)].4.3.1. Proteins Phosphorylated in Response to PK A ActivationResults presented in this study (see section 3.4.1.) indicate that stimulationof PK A by the 3-adrenergic receptor agonist, isoproterenol, specifically increasedthe phosphorylation of 5 protein bands (8.5, 15, 27, 31 and 152 kiJa; Figure 8.A.).The 152 kfla protein was tentatively identified as C-protein by its molecular mass.C-protein is a myofibril protein and a major substrate of PK A in the heart135[Robinson-Steiner and Corbin (1986)1. C-protein was previously found to bephosphorylated in isoproterenol-stimulated saponin-permeabilized [Miyakoda et at.(1987)] and intact adult rat myocytes [Onorato and Rudolph (1981); Blackshear etat. (1984); George et at. (1991)1. The phosphorylated protein band at 31 kDa wastentatively identified as troponin I by its molecular mass. Troponin I usuallydisplays a molecular mass of 28 kiJa, however, in isolated perfused rat hearts,troponin I has been found to be phosphorylated in response to 3-adrenergicstimulation and have a molecular mass ranging from 28 to 30 kDa [Karczweski etat. (1990); Le Peuch et at. (1980)]. Several other studies on the phosphorylation ofproteins in isolated adult rat myocytes [Onorato and Rudolph (1981); Blackshear etat. (1984); Miyakoda et at. (1987)1 have noted the phosphorylation of a 28 kDaprotein, which they tentatively identify as troporiin I. Recently, George et at. (1991)have found a 31 kDa protein phosphorylated in response to isoproterenol-treatmentof isolated adult rat ventricular myocytes and have identified this protein astroponin I using a monoclonal antibody to cardiac troponin I. The 8.5, 15 and 27kDa phosphorylated protein bands in this study were identified as the SR protein,phospholamban, on the basis of mobility on SDS-PAGE gels and by immunoblotting(Figures 6 and 8.A.). Phosphorylated phospholamban has also been identifiedfollowing isoproterenol-stimulation of both saponin-permeabilized [Miyakoda et at.(1987)] and intact isolated adult rat myocytes [Blackshear et at. (1984); George et at.(1991)]. Previously, a 15 kDa protein from the SL membrane has been found to bephosphorylated in response to isoproterenol in the perfused guinea pig heart [Prestiet at. (1985)]. In the present study, there was a phosphorylated band in the SRmembranes from control myocytes at 15 kDa, whose phosphorylation appeared toincrease in SR membranes isolated from isoproterenol-stimulated myocytes. The 15kDa protein band in the present study was probably not the result of a contaminantfrom the SL since this band was recognized by the phospholamban monoclonalantibody (Figure 6). In addition, boiling the SR membranes isolated from136isoproterenol-stimulated myocytes prior to SDS-PAGE appeared to increase theintensity of this band (Figure 8.A.) whereas the SL 15 kDa band was not affected byboiling [Presti et al. (1985)1. These results suggest that the 15 kDa band in myocytehomogenates and SR membranes was a form of phospholamban (di- or trimeric).Isolated myocytes were also treated with forskolin to activate PK A by directaction on adenylate cyclase (Figure 1O.A.). Forskolin treatment was found tostimulate the phosphorylation of 4 proteins (152, 31, 27 and 8.5 kDa) in a crudemembrane fraction isolated from the myocytes. Using isolated adult myocytes[George et al. (1991] and in isolated perfused rat heart [England and Shahid(1987)], forskolin was found to stimulate the phosphorylation of troponin I and C-protein. Also, Fliegel and Drummond (1985) using perfused guinea pig heart, foundthat forskolin stimulated the phosphorylation of a 25 kiJa protein, which wastentatively identified as phospholamban. Thus, regardless of whether the myocyteswere treated with isoproterenol or forskolin, PK A increased the phosphorylation ofthe same proteins: C-protein, troponin I and phospholamban.Several authors [Miyakoda et al. (1987); Blackshear et al. (1984); George etal. (1991); Onorato and Rudolph (1981)] have shown antagonism of the effects ofisoproterenol on protein phosphorylation in isolated ventricular myocytes bypropranolol (a 3-receptor antagonist). This competition would, however, occur atthe level of the receptor and not at the level of the kinase. In this study, an attemptwas made to inhibit the actions of PK A directly by utilizing twoisoquinolinesulfonamides, HA1004 and H-8. Both of these inhibitors displayselectivity for PK A (Kj (pM): 2.3 and 1.2, respectively) and inhibit PK A bycompeting with ATP for the free enzyme at the active site [Hidaka et al. (1984);Hagiwara et al. (1987)1. In the present study, neither of the two compounds (100iiM (HA1004) or 1 mM (HA1004 or H-8)) were able to inhibit the stimulation ofprotein phosphorylation in intact myocytes treated with isoproterenol (0.01 or 1.0iiM; Figures 11.A. and 12.A.). This lack of an inhibitory effect on PK A catalyzed137protein phosphorylation by these inhibitors may reflect an inability of thesecompounds to permeate the myocyte and to inhibit PK A in the compartmentswhere it is localized [Buxton and Brunton (1983)].4.3.2. Proteins Phosphorylated in Response to PK C ActivationIn experiments using isolated SR membranesCa2/phospholipid-dependentprotein kinase (PK C) was shown to phosphorylate phospholamban with aconcurrent increase inCa2+upt ke [Movsesian et al. (1984)]. PK C was also foundto phosphorylate isolated cardiac troponin I and troponin T, either in the free formor in the troponin-tropomyosin complex [Katoh et al. (1983); Risnik et al. (1987)].PK C has also been implicated in the decreased Ca2-sensitivity of themyofilaments due to the phosphorylation of troponin I and T [Gwathmey and Hajjar(1990)1. In SL membranes isolated from canine heart, PK C was found tophosphorylate 7 different proteins [Yuan and Sen (1986)]. In perfused adult rathearts, activators of PK C have been found to decrease the rate and force ofcontraction [Yuan et al. (1987)]. However, recently MacLeod and Harding (1991)found that treatment of isolated adult rat and guinea pig ventricular myocytes withphorbol esters results in a positive inotropic effect, which may be due to a PK C-induced increase in systolic Ca2. In addition, phospholamban, troponin I and C-protein were found not to be phosphorylated in response to PK C stimulation of theperfused heart but a 28 kDa cytosolic protein was phosphorylated [Edes andKranias (1990)1. No studies on the phosphorylation of proteins by PK C have beencarried out in isolated adult ventricular myocytes.Phorbol esters are tetracyclic diterpenes isolated from croton oil [Bohm et al.(1935)] which were found to be tumor promoting on mouse skin [Berenbium (1941)].Tumor-promoting phorbol esters have been shown to directly activate PK C[Castagna et al. (1982)] by substituting for naturally occurring diacylglycerols.138Non-tumor promoting phorbol esters have been shown not to affect PK C activity[Castagna (1987)]. In addition, PK C was found to act as a receptor for phorbolesters [Castagna et al. (1982); Driedger and Blumberg (1980)].Although phorbol esters can be used to activate PK C in cells and tissues, theactual physiological activator of PK C is diacyiglycerol (DAG). Because phorbolesters bypass the normal membrane signal transduction pathway, results withthese compounds may be affected by the nonparticipation of some regulatorysignals. It has also been observed that certain phorbol esters will only activatecertain PK C isozymes [Ryves et al. (1991); Robles-Flores et al. (1991)1. Somestudies have concluded that the effects of phorbol esters were independent of PK Cactivation [Murphy et al. (1991); Kraft et al. (1986); Motasim Billah et al. (1989);Tao et al. (1989); Pollock et al. (1986); Matsumoto et al. (1988)]. It has also beenfound that phorbol esters are not specific for PK C but can bind to another protein,neuronal chimaerin [Ahmed et al. (1990)]. Phorbol esters and cell-permeantdiacylglycerols have frequently been used to activate PK C in intact tissues.However, there are some differences with respect to their effects on cellularfunctions and in their activation of PK C. In GH3 pituitary cells, diacyiglycerolactivates a potential inhibitory pathway for PK C but phorbol esters did not[Kolesnik and Clegg (1988)1. In addition, in these cells phorbol ester inhibited Kinduced Ca2 influx while diacylglycerol did not [MacEwan and Mitchell (1991)1.Further, diacylglycerol inhibits cAMP production in granulosa cells but phorbolesters do not [Shinohara et al. (1985)1. In HL-60 cells, tumor promoting phorbolester caused 95% of the cells to adhere to culture dishes [Yamamoto et al. (1985)1and a 40% decrease in cellular ATP [Dawson et al. (1987)], whereas diacylglycerolhad no effect on either of these parameters. In GH4C5 pituitary cells, phorbolesters and diacylglycerols both activate PK C but their actions on PK C-mediatedfunctions such as prolactin release and synthesis and cell stretching differ[Ramsdell et al. (1986)]. In islet cells, diacyiglycerol induced an increase in cytosolic139Ca2 but phorbol ester did not [Thomas et al. (1991)1. In liver plasma membranes,phorbol esters were found to stimulate the phosphorylation of 10 proteins but DAGonly increased the phosphorylation of 6 proteins [Kiss and Luo (1986)]. In ratpancreatic acini, unlike phorbol ester, flAG had no effect on basal or stimulatedamylase release [Brockenbrough and Korc (1987)1. Finally, it must be kept in mindthat addition of flAG or phorbol esters to activate PK C are an attempt to mimic anintracellular function and that their activation or inhibition of cellular processesmay not reflect the actual situation inside the cell. An example of this is a studywhich suggests that OAG and phorbol esters depress the Ca2+ current in sensoryneurons independent of their effects as activators of PK C [Hockberger et al. (1989)].Since there are many reported differences between phorbol esters anddiacyiglycerols in the activation of PK C, studies were carried out with both phorbolesters and a synthetic diacyiglycerol. In this study, isolated adult rat ventricularmyocytes were treated with a tumor promoting and a non-tumor promoting phorbolester (Figure 13). Both phorbol esters (in 0.4% DMSO) stimulated thephosphorylation of two protein bands of phospholamban (8.5 and 27 kfla). Theseresults are interesting in that the active and inactive phorbol esters wereapparently able to phosphorylate phospholamban, in contrast to a previous study,in perfused guinea pig heart, where tumor promoting phorbol esters did notstimulate the phosphorylation of phospholamban [Edes and Kranias (1990)].Diacyiglycerols such as the cell permeant synthetic diacyiglycerol, OAG, havedetergent-like properties which allow them to activate PK C without damagingintact cell membranes. In this study, OAG (in 5% DMSO) treatment of isolatedadult myocytes was found to phosphorylate the two protein bands ofphospholamban at 8.5 and 27 kDa (Figure 14). In this experiment, it was alsofound that 5% DMSO, by itself, could phosphorylate these two bands ofphospholamban. There appeared to be no differences between DMSO, with andwithout OAG in the phosphorylation of phospholamban. It therefore seems140reasonable to conclude that OAG may not have contributed to the phosphorylationof phospholamban and that DMSO was solely responsible for this phosphorylation.This result shed a different light on the results obtained with tumor promoting andnon-tumor promoting phorbol esters which were also dissolved in DMSO (0.4%). Itnow appears highly likely that the phosphorylation of phospholamban obtainedwith both active and inactive phorbol esters may have been due to the DM50 andnot to the actions of the phorbol esters. It appears that the DMSO effect on proteinphosphorylation may also be concentration-independent since, at both 0.4 and 5%DM50, phospholamban became phosphorylated. To further investigate this solventeffect, isolated myocytes were treated with OAG dissolved in ethanol (10%; Figure15.A.). These results show that neither ethanol, by itself or with OAG, was able tostimulate the phosphorylation of proteins in intact myocytes. Treatment ofmyocytes with a higher concentration of OAG and a lower concentration of ethanol(Figure 16) also did not consistently result in the stimulation of proteinphosphorylation in isolated adult rat ventricular myocytes.4.3.3. CalciumlCalmodulin-dependent Protein Kinas e InvolvementPrevious studies have shown thatCa2+/calmodulindependent protein kinase(CAM PK) phosphorylates phospholamban in the perfused heart [Le Peuch et at.(1980); Wegener et at. (1989); Lindemann and Watanabe (1985); Karczewski et at.(1987); Vittone et at. (1990)] and in isolated SR vesicles [Davis et at. (1983); Molla etat. (1985); Plank et at. (1983); Katz and Remtulla (1978); Le Peuch et at. (1979)1with an associated increase in the rate ofCa2-uptake and in the affinity of theCa2-pump for Ca2 [Katz and Remtulla (1978); Davis et at. (1983); Plank et at.(1983)1. The physiological significance of CAM PK phosphorylation is notunderstood as, in the perfused heart, it does not occur in response to elevatedextracellular Ca2 [Lindemann and Watanabe (1985); Vittone et at. (1990)] but only141after 3-receptor activation and PK A phosphorylation of phospholamban [Wegeneret al. (1989); Karczewski et al. (1987)1. Recently, Colyer and Wang (1991) havefound that the additional phosphorylation of phospholamban by CAM PKsubsequent to phosphorylation by PK A, did not increase SR Ca2-pump activityabove the level achieved by PK A phosphorylation. It has also been suggested thatin perfused rat hearts, CAM PK may only be able to phosphorylate phospholambanwhen intracellular cAMP levels are high [1 pmo]Jmg wet wt.; Vittone et al. (1990)].In addition, Gasser et al. (1988) have shown that CAM PK may preferentiallyphosphorylate phospholamban in junctional SR and PK A may preferentiallyphosphorylate phospholamban in network SR.To investigate the possible effect of CAM PK on the protein phosphorylationobserved with PK A in isolated myocytes, CGS 9343 B was used to specificallyinhibit theCa2/calmodulin protein kinase (CAM PK). CGS 9343 B is four timesmore potent than trifluoperizine as an inhibitor of calmodulin activity and does notinhibit the activity of PK C at concentrations over 200 tM [Norman et al. (1987);Hill et al. (1988)]. This compound has been found to be effective in inhibiting 1P3-stimulated Ca2-re1e se in rat liver epithelial cells at a concentration of 110 pM[Hill et al. (1988)1 and at 10 j.tM, protected perfused pig hearts from reperfusioninjury subsequent to ischemia [Das et al. (1989)]. In the present study, pretreatment of isolated myocytes with CGS 9343 B had no effect on the stimulation ofprotein phosphorylation by isoproterenol (Figure 17). This result indicates that inisolated adult myocytes, CAM PK was probably not involved in the stimulation ofprotein phosphorylation by PK A.4.3.4. Protein Phosphorylation in Response to PK A and PK C ActivationTo date, there have been no studies on protein phosphorylation in anyexperimental model of the heart subsequent to activation of both PK A and PK C.142As described in section 4.3.3., in perfused hearts treated with isoproterenol, asynergistic phosphorylation of phospholamban occurs due to the activation of bothPK A and CAM PK. However, these kinase pathways appear to operate underdifferent conditions and, perhaps, in different areas of the SR (see section 4.3.3.).Since PK A and PK C have both been shown to phosphorylate phospholamban in SRvesicles and purified preparations of troponin I, the effect of the simultaneousactivation of both of these kinases on protein phosphorylation in isolated adultventricular myocytes was investigated. The results presented here show that in thepresence of OAG and isoproterenol, together, the same proteins werephosphorylated as when myocytes were treated with isoproterenol alone (Figure18). To further investigate the incorporation of radioactivity, the protein bandswhose phosphorylation was stimulated by isoproterenol (8.5 and 27 kDa forms ofphospholamban, troponin I and C-protein) were quantitated by liquid scintillationcounting (Figure 19 and 20). In myocyte homogenate samples, there were noapparent differences in the incorporation of radioactivity into phospholamban,troponin I or C-protein between control, isoproterenol, OAG or isoproterenol plusOAG-treated myocytes. However, in myocyte SR membranes, there was anapparent increase in the incorporation of radioactivity into the two bands ofphospholamban from isoproterenol-treated myocytes as compared with SRmembranes from control, OAG and isoproterenol plus OAG-treated myocytes.There appeared to be no apparent difference in the incorporation of radioactivityinto the two bands of phospholamban between SR membranes prepared fromcontrol, OAG or isoproterenol plus OAG-treated myocytes. The interpretation ofthese results is limited due to the wide variation of the data and by the smallnumber of experiments performed.Since Edes and Kranias (1990) have shown that PK C phosphorylated a 28kDa cytosolic protein and Katoh et al. (1981) have found that PK C phosphorylateda 38 and a 49 kDa cytosolic protein in guinea pig heart, the phosphorylation of143cytosolic proteins was examined in isolated adult rat ventricular myocytes. Theincorporation of radioactivity into cytosolic fractions isolated from control,isoproterenol, OAG and isoproterenol plus OAG-treated myocytes shows that thephosphorylation state of proteins at 21, 24, 31 and 152 kDa appeared to changewith the different treatments (Figure 21). The phosphorylation of the 21, 31(troponin I) and 152 (C-protein) kDa proteins were apparently increased in thecytosolic fractions from isoproterenol and OAG plus isoproterenol-treated myocytesas compared to that found in cytosolic fractions from control and OAG-treatedmyocytes. The results for troponin I and C-protein are in agreement with theresults obtained earlier with isoproterenol treatment of myocytes. Thephosphorylation of the 24 kDa protein appears to decrease in the cytosolic fractionsfrom isoproterenol and OAG plus isoproterenol-treated myocytes as compared tothat found in cytosolic fractions from control and OAG-treated myocytes. Theidentity of the 21 and 24 kDa proteins is unknown at this time. Quantitation of theradioactivity incorporated into these bands by liquid scintillation counting confirmsthe above result for troponin I and C-protein. The incorporation of radioactivityinto the 24 kDa protein was the highest in the cytosol from control myocytes withdecreased incorporation of radioactivity into this protein from cytosolic fractionsprepared from isoproterenol, OAG and OAG plus isoproterenol-treated myocytes.Regarding the 21 kDa protein, the incorporation of radioactivity was similarbetween the cytosolic fractions isolated from control, isoproterenol and OAG plusisoproterenol-treated myocytes but decreased in cytosolic fractions from OAGtreated myocytes. However, interpretation of these results is limited due to thewide variation in the data and the small number of experiments performed. Unlikethe previously reported studies [Edes and Kranias (1990); Katoh et al. (1981)], inthe present study, neither a 28, 38 or 49 kDa cytosolic protein was found to bephosphorylated in either OAG or OAG plus isoproterenol-treated myocytes.1444.4. Phosphorylated Species of Otigomeric PhospholambanA pentamer of phospholamban contains a total of 10 possible phosphorylationsites [Simmerman et at. (1986)]. Since the phosphorylation of phospholambanreduces its electrophoretic mobility on SDS-PAGE gels under certain conditions[Wegener and Jones (1984)] western immunoblots of these gels demonstratemultiple bands of the phospholamban pentamer as a result of filling one or bothphosphorylation sites in each monomer. Following phosphorylation by PK A, 6bands of phosphorylated pentameric phospholamban were observed, correspondingto the phosphorylation of monomers 0 to 5 at a single site [Li et at. (1990); Gasser etat. (1986); Imagawa et at. (1986); Wegener and Jones (1984); Wegener et al. (1989)).Following phosphorylation by both PK A and CAM PK, eleven bands ofphosphorylated pentameric phospholamban are observed, indicating thephosphorylation of monomers 0 to 5 at both phosphorylation sites.Several authors have described the separation of oligomeric species ofphosphorylated phospholamban from isolated canine SR [Li et at. (1990);Kasinathan et at. (1988); Gasser et at. (1988); Imagawa et at. (1986); Wegener andJones (1984)) and from perfused guinea pig heart [Wegener et at. (1989)]. Therehave been no reports on the separation of oligomeric species of phosphorylatedphospholamban from whole rat heart, rat SR preparations or from isolated adult ratventricular myocytes.In this study, four bands of phosphorylated pentameric phospholamban wereobtained from control and dephosphorylated (with alkaline phosphatase) caninecardiac ventricular SR vesicles as described by Li et at. (1990) (Figure 22). Sixbands of phosphorylated pentameric phospholamban were obtained from canine SRvesicles phosphorylated by the catalytic subunit of PK A (Figure 22). Using ratmyocyte SR membranes, this method was not able to separate the phosphorylatedpentameric species of phospholamban which all migrated as a single large band.145Another SDS-PAGE gel system was therefore developed (see section 2.2.5.2.1.2.)and on the immunoblot, four bands of phosphorylated pentameric phospholambanwere separated from dephosphorylated (with alkaline phosphatase), seven bandsfrom control and five bands from phosphorylated (with catalytic subunit of PK A)rat myocyte SR vesicles (Figure 23). A sample of canine SR vesicles phosphorylatedby the catalytic subunit of PK A was separated into nine bands using this SDSPAGE system (Figure 23). This was unusual since only six bands of phosphorylatedpentameric phospholamban were expected. Perhaps, as seen in previous studies, anendogenous kinase activity was associated with the SR [Molla and Demaille (1986)1and during the phosphorylation reaction it was able to add to the species ofphospholamban phosphorylated by the catalytic subunit of PK A. On inspection ofthese western blots (Figure 22 & 23), it is not clear why the phosphorylatedphospholamban bands from canine SR vesicles appear to separate much moredistinctly from one another than do the phosphorylated phospholamban bands frommyocyte SR vesicles. However, this was a consistent result and may be due to someinherent differences between these two types of SR. In both the canine and myocyteSR vesicles, the control and dephosphorylated samples contain bands ofphosphorylated phospholamban instead of only the dephosphorylated form. Again,it may be that under the control and dephosphorylation incubation conditions, anendogenous kinase activity associated with the SR was able to phosphorylatephospholamban into these phosphorylated forms or it may indicate that myocyte SRis in some way resistant to dephosphorylation.The SDS-PAGE system developed to separate the phosphorylated forms ofphospholamban in myocyte SR was used, together with inimunoblotting, todetermine the phosphorylated forms of pentameric phospholamban in SRmembranes isolated from control, isoproterenol, OAG and isoproterenol plus OAGtreated myocytes (Figure 24). In homogenate and SR membranes from control andOAG-treated myocytes only two bands of phosphorylated pentameric146phospholamban were obtained. In homogenate and SR membranes fromisoproterenol and OAG plus isoproterenol-treated myocytes, 5 bands ofphosphorylated pentameric phospholamban were separated. Thus, the treatment ofmyocytes with OAG did not result in the phosphorylation of phospholamban abovethat found in control myocytes and did not affect the number of species ofphosphorylated phospholamban formed in the presence of isoproterenol.In addition to its importance as a regulatory mechanism in myocardialrelaxation, the phosphorylation of phospholamban is modified in several diseasestates. In ischemic pig heart, with increasing duration of ischemia, phospholambanphosphorylation became increasingly reduced with a concurrent reduction in Ca2+pump activity [Schoutsen et al. (1989)]. In hyperthyroid hypertrophic rat hearts[Beekman et al. (1989)], there may be a decrease in the ratio of phospholamban toCa2h7K+ATPase in SR membranes. Endotoxin treatment of canine SR membranesresulted in an inhibition of phospholamban phosphorylation by CAM PK but not PKA and a stimulation in the rate of phospholamban dephosphorylation [Mohammedand Liu (1990)1. This last result may be significant to the mechanism of myocardialdysfunction in endotoxic shock. In idiopathic dilated cardiomyopathy studied inhuman failing hearts [Movsesian et al. (1990)], phospholamban mediatedstimulation ofCa2+upt ke was found to be normal. It remains to be determinedwhat other roles phospholamban phosphorylation may play in otherpathophysiological conditions of the heart. As well, the species of phosphorylatedpentameric phospholamban formed in the functioning myocardium under normaland diseased conditions remains to be determined.4.5. Activation ofPK C in Isolated Adult Ventricular MyocytesIn the results presented in this study on the stimulation of proteinphosphorylation by PK C in isolated adult rat ventricular myocytes (see section1473.4.2.), no proteins were specifically found to be phosphorylated by PK C, as hadbeen found previously in perfused hearts [Edes and Kranias (1990); Katoh et al.(1981)]. As well, a PK C activator (OAG) had no significant effect on thephosphorylation of myocyte proteins by PK A stimulation. These results can beexplained if PK C was found not to have been activated in the isolated myocytes.Thus, an examination into the activation of PK C in the intact adult ventricularmyocyte was undertaken. There are three methods used to determine whether PKC has become activated in a cellular system [Mitchell et al.(1989)]: 1) to documentthe movement (i.e. translocation) of the kinase from the cytosol of the cell to theplasma membrane (or particulate fraction), 2) to isolate the kinase and determinethe state of autophosphorylation and 3) to define possible protein targets for theenzyme and to determine whether they have been phosphorylated in response totreatments known to activate PK C in other cellular systems. Since results havealready been presented to show that PK C was not able to phosphorylate specificsubstrate(s) in isolated adult myocytes in response to OAG-treatment, method 3)will not be discussed further. Instead, the autophosphorylation and translocation ofPK C from myocyte cytosol to the membranes was investigated as a measure of PKC activation.4.5.1. Translocation of PK C in Isolated Adult Rat Ventricular MyocytesDue to the activation of PK C by phospholipids, diacyiglycerols and otherlipids, it was reasoned that the movement of the enzyme from its reservoir in thecytosol to the membrane must occur in order for the kinase to become activated[Gopalakrishna et at. (1986)]. Although, the mechanisms involved in this processare not fully known or understood, the following is a description of what is atpresent thought to occur [Nelsestuen and Bazzi (1991)]: PK C (inactive) in thecytosol, perhaps associated with microtubules [Ito et al.(1989)], becomes stimulated148(mechanism unknown) to travel to a membrane (for example: the plasmamembrane) whereupon it can form two active membrane-associated states[Nelsestuen and Bazzi (1991)]. One state is dependent on low Ca2+ concentrationsto bind to membrane phospholipids and is reversible [Nelsestuen and Bazzi (1991)].This form of PK C can be removed from the membrane with Ca2+ chelators[Kikkawa et al. (1982)] and can bind phorbol esters; this binding reaction is alsoreversible [Bazzi and Nelsestuen (1989)]. The formation of the second membranestate, which is an irreversible complex, is promoted by increased Ca2+concentrations and DAG or tumor-promoting phorbol esters. This irreversiblecomplex behaves as an integral membrane protein and no longer requires Ca2+,lipid or phorbol esters as cofactors for activity. In order to form this irreversiblecomplex, it is thought that PK C undergoes a conformational change which allows aportion of the molecule to become inserted into the membrane [Bazzi andNelsestuen (1988a)]. The irreversible membrane-bound form can be released bysolubilizing the membrane in detergent [Bazzi and Nelsestuen (1988b)]. Inaddition, the catalytic fragment of PK C (PK M) can be generated from intact PK CbyCa2-dependent protease and is active in the absence of phospholipid and Ca2in the cytosol [Kishimoto et al. (1983)]. The possibility that the distribution of PK Cbetween the cytosol and the membrane is in dynamic equilibrium controlled bylevels of free intracellular Ca2has recently been proposed [Phillips et al. (1989)1.Unlike samples from rat brain and bovine trachea, chromatography ofmyocyte cytosolic fractions and detergent-solubilized membranes using DEAEcellulose was found to be inadequate and did not allow detection of PK C activity(Table 10). In the present study, it was necessary to fractionate myocyte cytosolicfractions and detergent-solubilized membranes by FPLC prior to the assay of PK Cactivity, to partially purifSr the kinase and to remove the detergent from themembrane fraction. Fractionation of the myocyte cytosol and membrane samples byFPLC allowed the PK C activity to be subsequently stimulated by Ca2 and lipids.149The specific activity of PK C in myocyte cytosol was found to be 726 pmollminlmgprotein. This value is approximately 12-fold higher than that found previously incytosol from canine ventricles [Yuan and Sen (1986)], 17-fold higher than thatpreviously found in cytosolic fractions from whole rat heart [Tanaka et al. (199 1)1and 3-fold higher than that previously found in cytosolic fractions from perfusedguinea pig heart [Edes and Kranias (1990)1. The use of FPLC fractionation mayhave removed an inhibitory or other conflicting endogenous activity in the myocytecytosol and membrane fractions which interfered with the assay of PK C activity,since a large proportion of the protein in these samples did not bind very tightly tothe Mono Q column (Figure 26). This inhibitory or other conflicting activity wasapparently not removed by chromatography on DEAE-cellulose. In relation to this,Pearson et al. (1990) and Mozier et al. (1990) have purified two protein inhibitors(PKC inhibitors 1 and 2) of PK C from bovine brain. As well, there appeared to be avery large amount of PK A activity in the myocyte cytosol and membrane fractions(Figure 27). When the peptide inhibitor of PK A was included in the assay for PK Cactivity there was a substantial reduction in the number of interfering peaks,allowing the unmasking of kinase activity solely due to PK C.PK C activity was measured in cytosol and membrane fractions isolated fromcontrol, ethanol, OAG (in ethanol) and OAG plus R59022-treated myocytesincubated for 1, 5 and 10 mm. In each of the cytosol fractions isolated frommyocytes from these 12 treatment groups, three peaks of Ca2 and lipid-dependentPK C activity were eluted. The peak with the highest activity was always found infractions 27-31 (Figure 28, 31, 32 and 34). In the control membrane fractions, twopeaks of Ca2 and lipid-dependent PK C activity were eluted and again the peakwith the highest activity was usually found in fractions 27-31 (Figure 28, 31, 32 and34). In the initial FPLC fractions (6 - 15) from each membrane sample, a largeportion of the PK C activity peak contained Ca2 and lipid-independent activity. Itis not known at this time what other kinase activities may be included in this peak.150The major peak of Ca2 and lipid-dependent PK C activity (fractions 27-31) frommembrane fractions of ethanol and OAG-treated myocytes appeared to contain lessPK C activity than the same peak from the control profiles.Calculation of the area under the curve (AUC) of the major peaks of Ca2and lipid-dependent PK C activity (fractions 27-31) for the cytosolic and membranepreparations (Figure 29) show that in regards to incubation time, there were nosignificant differences (Two-way ANOVA, p > 0.05) between the peaks obtainedfrom control, ethanol, OAG and OAG plus R59022-treated myocytes that had beenincubated for 1, 5 or 10 mm. When compared to the AUC from control peaks, therewas significantly less PK C activity (Two-way ANOVA, p <0.05) in the peaks fromboth the cytosol and membrane fractions isolated from ethanol and OAG-treatedmyocytes. When the AUC of the OAG-treated peaks was compared to the AUC ofthe ethanol-treated peaks, from both cytosol and membrane fractions, no significantdifferences were found (Two-way ANOVA, p> 0.05). The AUC of the peaks fromboth cytosol and membrane fractions isolated from R59022 plus OAG-treatedmyocytes were similar to those obtained from ethanol or OAG-treated myocytes at 1mm. However, at 5 and 10 mm, the AUC was apparently lower than from eitherethanol or OAG-treated myocytes, these differences are reflected in both cytosol andmembrane fractions. Therefore, it appears that ethanol has decreased PK Cactivity in both cytosol and membrane fractions, including those treated with OAG,which was dissolved in ethanol. In addition to possible physical effects on thesarcolemmal membrane [Polimeni et al. (1983)1, ethanol has several other effects onthe myocardium. Ethanol (2.5- 5%) has been shown to protect rat heart fromcalcium paradox injury [Auffermann et al. (1990)] and to increase the levels ofcAMP in the heart through multiple effects on adenylate cyclase, including anenhancement of the interaction between G5 and the catalytic unit in cardiacmembranes [Feldman et al. (1989)]. Ethanol has also been found to inhibitNa+/Ca2+exchange and to arrest hamster heart during systole [Auffermann et al.151(1988)]. In isolated adult rat ventricular myocytes, ethanol (1%) has been found tocause contractile depression, to reduce the magnitude of the Ca2+transient and tocause concentration-dependent depletion of SR Ca2 [Danziger et al. (1991)]. Atthis time, the effect(s) of ethanol on PK C and its activity are not known. It is notknown, as well, which of the above effects on the heart, if any, may havecontributed to the decrease in PK C activity observed in this study in ethanol-treated isolated myocytes.In the present study, treatment of myocytes with OAG did not produceactivation of PK C as determined by the lack of movement from the cytosol to themembrane fraction. Previously, PK C was found to be translocated from the cytosolto the membrane fraction in perfused rat and guinea pig hearts in response totumor-promoting phorbol esters [Edes and Kranias (1990); Yuan et al. (1987)].However, in a perfused guinea pig heart study [Edes and Kranias (1990)], there wasan incongruity between the decrease of PK C activity in the cytosol (from 232 to 180pmollminlmg protein; a decrease of 22%) and the increase in activity associatedwith the membrane (from 119 to 392 pmollminlmg protein; an increase of 230%). Inisolated adult rat ventricular myocytes, both tumor-promoting phorbol esters and1,2-dioctanoylglycerol (a synthetic cell-permeant diacyiglycerol; DiC8) increased themembrane association of PK C [Capogrossi et al. (1990)].As discussed previously (see section 4.3.2.), there are many studies whichindicate that phorbol esters do not act in the same way as diacyiglycerols and thismay be due to their binding to proteins other than PK C or to their sustainedactivation of PK C. Movement of PK C from the cytosol to the membrane fraction inexperimental cardiac models has not been shown with the diacyiglycerol, OAG.There has been a report which demonstrates that synthetic lipids, OAG and DiC8,do not cause the same biological effects in platelets indicating that they are notequal activators of PK C [Krishnamurthi et al. (1987)]. It has been suggested thatthe lack of effects with OAG could be the result of its weak ability to cross152membranes, other studies [Ebeling et al. (1985); Lapetina et al. (1985)] have foundthat it is a much less potent activator of PK C than DiC8. Since Capogrossi et al.(1990) have shown that DiC8 causes PK C translocation in isolated adult ratventricular myocytes and in the present study, OAG did not cause translocation ofPK C, there is some evidence in these cells that OAG and DiC8 are not equalactivators of PK C. In the present study, an attempt was made to increase theconcentration and persistence of OAG in the myocytes by inhibiting one of theenzymes responsible for diacylglycerol metabolism, cliacyiglycerol kinase, withR59022 [de Chaffoy de Courcelles et al. (1985)1. Elevated levels of OAG resulted inthe augmentation of PK C activity in intact platelets [de Chaffoy de Courcelles et al.(1985)]. However, there are several limitations to the use of R59022. There are twoisozymes of DAG kinase in mammalian tissues and R59022 only inhibits one of theisoforms [Sakane et al. (1989)]. As well, several studies [Mahadevappa and Sicilia(1988); Nasmith and Grinstein (1989)] have shown that R59022 exerts an effect oncellular functions that are independent of its action on DAG kinase. The mostsignificant limitation to the use of R59022 is that in isolated adult rat ventricularmyocytes, DAG kinase is not the major pathway for the metabolism ofdiacylglycerols, rather dliacylglycerols are metabolized through a pathway involvingDAG lipase [Hee-Cheong and Severson (1989)1. Taking into account theselimitations of R59022, and the possibility that OAG may not be a potent activator ofPK C in myocytes, it is perhaps not surprising that in this study, neither OAG norOAG plus R59022 was seen to cause the translocation of PK C from the cytosol tothe membrane fraction.In some studies, the concept that translocation of PK C indicates activation ofthe phosphorylating activity of this enzyme has come into question [see review byWoodgett et al. (1987); Bosca et al.(1989)]. In astrocytoma cells, the redistributionof PK C was found not to correlate with either the extent or duration of thephosphorylation of PK C substrates [Trilivas et al. (1991)]. Another recent study153has found intracellular receptor proteins, in the cytoskeletal components of themembrane fraction of neonatal rat heart, that bind PK C (RACKS: receptors forctivated -kinase; Mochly-Rosen et al. (1991)). Binding of PK C to these proteinswas specific, saturable and concentration-dependent. At this time, it is difficult toascertain the ramifications of these results on the pathway of activation of PK C bytranslocation to membranes in the adult rat ventricular myocyte.4.5.2. Autophosphorylation of PK C in Isolated Adult Rat Ventricular MyocytesAnother method used to determine the activation of PK C in cellular systemsis evidence for the autophosphorylation of the enzyme [Mitchell et al. (1989)]. Theautophosphorylation reaction of PK C involves an intrapeptide mechanism wherebya single polypeptide chain phosphorylates itself [Newton and Koshland (1987)].This reaction occurs on both the catalytic and regulatory domains of PK C and atotal of 6 autophosphorylation sites have been found in the sequence of type II PK C[Flint et al. (1990)]. The autophosphorylation of PK C can be monitored by twomethods: 1) the incorporation and quantitation of radioactive phosphate [Mitchell etal. (1989)] and 2) the separation of PK C on SDS-PAGE gels and visualization of theincrease in molecular weight upon autophosphorylation [Huang et al. (1986a & b)1.In this study, the protein in the PK C activity peak fractions obtained from cytosolicand membrane fractions isolated from control and OAG—treated myocytes wasconcentrated, separated by SIJS-PAGE and western blotted. These blots were thenprobed with antibodies to the PK C isozymes type III and type II (Figure 30 and 33).The major cytosolic peak at fractions 27-3 1 from both control and OAG-treatedmyocytes demonstrated two immunoreactive bands of PK C for both type II andtype III at approximately 80 kDa. The less intense band, demonstrating a highermolecular weight, may represent an autophosphorylated species of PK C and thelower, more intense band may represent the nonphosphorylated form. The second154cytosolic peak at fractions 32-34 from control myocytes contains a single band oftype III PK C at approximately 80 kDa and this peak from OAG-treated myocytescontains a single band of type III and also a single band of type II PK C. Both ofthese single bands migrate at the position of what may representnonphosphorylated PK C. The third cytosolic peak at fractions 39-42 from bothcontrol and OAG-treated myocytes was found not to contain type III PK C. Themembrane peak that contains a large proportion of Ca2 and lipid-independent PKC activity (fractions 6-15) from both control and OAG-treated myocytes contains 2bands of type III but not type II PK C. The band of type III PK C at approximately80 kDa may represent the nonphosphorylated form of PK C and the band at 50 kiJamay represent the proteolytically active form of PK C, PK M. Fractions 27-31 of themembrane preparations from both control and OAG-treated myocytes contains 2bands of type III PK C, which may represent the autophosphorylated andnonphosphorylated forms.Since these western blots may have indicated that a portion of the PK C incytosol and membrane fractions from control myocytes may already be in theautophosphorylated state, and since autophosphorylation of PK C is a prerequisiteto its phosphorylation of substrate proteins, this may indicate that PK C wasactivated, prior to the control incubation. During the isolation of myocytes, insulinand several other amino acids and vitamins are used in the perfusion and washingbuffers. Perhaps one of these buffer components caused the partial activation of PKC, as reflected in the production of the autophosphorylated form. However, theseblots also show a similar large proportion of PK C from control and OAG-treatedmyocytes which may be in the nonphosphorylated form. Thus, using the increase inmolecular mass upon autophosphorylation as an indication of PK C activationdemonstrates that OAG was not able to activate a substantial portion of this kinasein isolated adult rat ventricular myocytes.155Previous studies have shown that autophosphorylation occurs with a rise inmolecular weight for type II of PK C [Huang et al. (1986a & b); Flint et al. (1990);Pelech et al. (1991)]. However, type III PK C is known to becomeautophosphorylated but an upshift in the molecular weight upon theautophosphorylation of type III PK C has not been documented before.In addition to type II and type III PK C, the heart also contains a largeamount of the PK C isozyrne e [Schaap et al. (1989)]. The activity of this isozyme isCa2-independent but it is activated by phospholipids, DAG and phorbol esters.Unlike PK C types II and III, PK does not phosphorylate the substrate histoneIllS very well [Schaap and Parker (1990)]. Therefore, in the present studies, theactivity of PK c was not measured since histone IllS was used as the substrate.Insulin has been shown to activate the activity of PK e in cytosol and membranefractions from fetal chick neurons but did not cause translocation. As well, insulinand tumor-promoting phorbol ester were found to upshift the molecular weight ofPK C on SDS gels [Heidenreich et al. (1990). Although no studies have been carriedout as yet on PK in experimental models of the myocardium, it will be of greatinterest in the future to determine whether this isozyme contributes functionally tothe effects of PK C activation in the heart.5. SUMMARY AND CONCLUSIONSIn summary, a method for the isolation of a high number of viable adult ratventricular myocytes was established. The availability of these myocytes enabledthe development of methods for myocyte homogenization and isolation of SRmembranes from these homogenates. The myocyte SR membrane preparationexhibited similar Ca2-transport and Ca2-ATP se activity as well as a similarprotein profile to SR membranes isolated from intact rat heart tissue. Also, these156SR membranes exhibited low levels of contamination by enzymatic activities fromother cellular membranes.The availability of purified SR membranes from adult rat ventricularmyocytes provided a useful model for the study of the regulation of SR function byprotein phosphorylation. For these studies, myocyte SR membranes were isolatedand characterized in buffers developed to prevent the dephosphorylation of proteins.These SR membranes exhibited a protein profile similar to those isolated in controlbuffers, less contamination by enzymatic activities from other cellular membranesand lower recovery ofCa2-uptake andCa2/K-ATPase activities. Three distinctproteins (phospholamban, a 31 and a 152 kDa protein) were phosphorylated by PKA in homogenates and SR membranes from adult rat myocytes stimulated withisoproterenol and forskolin. The stimulation of protein phosphorylation in myocytehomogenates and SR membranes by isoproterenol could not be inhibited by twodifferent inhibitors of PK A. Also, an inhibitor of CAM PK did not affect thestimulation of protein phosphorylation in myocyte homogenates and SR membranesby isoproterenol. Treatment of isolated adult rat myocytes with DMSO or phorbolesters dissolved in DMSO resulted in the phosphorylation of phospholamban inmyocyte homogenates and SR membranes. When OAG and isoproterenol were usedtogether to stimulate protein phosphorylation in isolated adult rat myocytes, thesame proteins were phosphorylated to similar degrees as observed in homogenatesand SR membranes treated with isoproterenol alone. In cytosolic fractions isolatedfrom isoproterenol and OAG plus isoproterenol-treated myocytes, thephosphorylation of protein bands at 21, 31 and 152 kfla was stimulated. Thephosphorylation of a 24 kiJa protein appeared to be decreased in myocytes treatedwith isoproterenol, OAG and isoproterenol plus OAG.The separation of phosphorylated pentameric species of phospholamban fromrat myocyte SR was found to be more difficult to achieve than from SR membranesprepared from canine heart. In control and OAG-treated myocytes, two species of157phosphorylated pentameric phospholamban were obtained. In myocytes treatedwith isoproterenol or OAG plus isoproterenol, 5 species of phosphorylatedpentameric phospholamban were obtained.To assay the activity of PR C from myocyte cytosol and membrane fractions,FPLC fractionation and the inclusion of the peptide inhibitor of PK A were requiredin order to remove an inhibitory or interfering activity and to inhibit the largeamount of PK A activity found in these fractions. The specific activity of PK C inmyocyte cytosol was found to be much higher than that previously found present incytosolic fractions from canine, rat or guinea pig heart. Three peaks of Ca2+ andlipid-dependent PR C activity were found in cytosolic fractions isolated fromcontrol, isoproterenol, OAG and isoproterenol plus OAG-treated myocytes. Themain peak of activity contained type II and type III isozymes of PR C, in perhapsautophosphorylated and nonphosphorylated states. The second major peakcontained perhaps only nonphosphorylated forms of type III PR C in controlmyocytes and type II and III PK C from OAG-treated myocytes. The third peak ofPK C activity in the cytosol did not contain type III PK C protein. Two peaks ofCa2 and lipid-dependent PK C activity were found in membranes isolated fromcontrol, isoproterenol, OAG and isoproterenol plus OAG-treated myocytes. Themain peak of activity contained type III PR C, which may be present in bothautophosphorylated and nonphosphorylated states. The second peak, whichcontained a large Ca2 and lipid-independent kinase activity contained type III PRC perhaps only in the nonphosphorylated and proteolyzed PK M form. There wereno differences seen in the number or types of peaks of PR C activity formed withrespect to incubation time. There was significantly less PK C activity in membranefractions from myocytes that had been treated with ethanol.In conclusion, the results presented in this work have demonstrated:1) For the first time, the isolation of SR membranes from isolated adult ratventricular myocytes.1582) Stimulation of PK A in adult ventricular myocytes results in thephosphorylation of phospholamban, a 31 and a 152 kDa protein. 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