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Effect of a-Adrenergic Stimulation on Activation of Protein Kinase C and Phosphorylation of Proteins in Intact Rabbit Hearts Laszlo Talosi and Evangelia G. Kranias The intracellular events and specifically the role of protein kinase C-mediated protein phosphorylation, after a-adrenergic receptor stimulation of the heart, are not well understood. We examined the phosphorylation of sarcolemmal, sarcoplasmic reticular, myofibrillar, and cytosolic proteins in perfused beating rabbit hearts on activation of protein kinase C by phenylephrine. Perfusion of rabbit hearts with phenylephrine was associated with a positive inotropic response, which was dose and time dependent. Maximal stimulation (1.54-fold increase in +dP/dt) was obtained with 10 gM phenylephrine at 4 minutes. Examination of the activity levels of protein kinase C in these hearts revealed a redistribution of this activity from the cytosolic to the membranous fraction, suggesting the activation of this enzyme in vivo. Prazosin, an al-adrenergic antagonist, prevented the increase in the inotropy and the redistribution of protein kinase C activity mediated by phenylephrine. Examination of the degree of phosphorylation of membranous, myofibrillar, and cytosolic proteins revealed that activation of protein kinase C in vivo was associated with increased phosphorylation of a 15-kd sarcolemmal protein and a 28-kd cytosolic protein. There were no increases in the degree of phosphorylation of phospholamban in the sarcoplasmic reticulum and of troponin I, troponin T, and C protein in the myofibrils, although these proteins were found to be substrates for protein kinase C in vitro. These findings provide evidence that protein kinase C is activated in response to a-adrenergic stimulation and that activation is associated with increased phosphorylation of a 15-kd sarcolemmal protein and a 28-kd cytosolic protein in the myocardium. (Circulation Research 1992;70:670-678) KEY WoRDs * a-adrenoceptors * protein kinase C * protein phosphorylation * heart

Stimulation of the heart by either a- or /3-adrenergic agonists is associated with increases in the force of contraction, although there are qualitative differences in the mechanical responses to these agents. /3-Adrenergic activation shortens both the time to peak tension and the relaxation time and increases contractility and chronotropy. ca-Adrenergic stimulation prolongs the time to peak tension, causes a slow and small increase in contractility, and does not affect relaxation or chronotropy.' The intracellular events after P-adrenergic stimulation have been well characterized, and the mechanism of action appears to involve increases in cAMP levels and cAMP-dependent phosphorylation of key regulatory proteins.2 However, the cellular mechanisms underlying a-adrenergic stimulation in the heart are not well understood, and it is not presently clear whether protein phosphorylation may be involved. a-Adrenergic stimulation occurs without increases in myocardial cAMP levels,3 but it is accompanied by increases in the activity of phospholipase C.45 The mechanism by which From the Department of Pharmacology and Cell Biophysics, University of Cincinnati (Ohio), College of Medicine. Supported by National Institutes of Health grants HL-26057

and HL-22619. Address for correspondence: Evangelia G. Kranias, Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0575. Received April 24, 1991; accepted November 20, 1991.

this enzyme is activated during receptor-agonist binding is thought to involve protein-protein coupling through a guanine nucleotide-binding regulatory protein.6 Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5 -diphosphate, with the resultant formation of myo-inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. 1P3 has been shown to mediate the release of Ca`+ from intracellular Ca2` stores in various tissues, although it is still controversial whether 1P3 can mobilize Ca21 from the sarcoplasmic reticulum (SR) in the myocardium.7,8 Recently, inositol trisphosphate and tetrakisphosphate have been reported to increase the sensitivity of the contractile proteins to calcium in porcine cardiac muscle fibers.9 Increased calcium sensitivity of the contractile proteins was also observed during a-adrenergic stimulation of rabbit hearts.10 1,2Diacylglycerol, the other derivative of phosphatidylinositol 4,5-diphosphate hydrolysis, which may act as the physiological activator of the Ca2-activated, phospholipid-dependent protein kinase (protein kinase C), was also reported to increase during a-adrenergic stimulation in the mammalian heart.'1 However, there is relatively little information available on the activation and the functional significance of protein kinase C in intact hearts. In one study, cultured neonatal rat heart myocytes were treated with the a-adrenergic agonist norepinephrine, and this was found to be associated with activation or translocation of protein kinase C from the cytosolic to the membranous fraction.'2 Activation of

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Talosi and Kranias a-Adrenergic Stimulation and Phosphorylation in Heart

protein kinase C may result in phosphorylation of key intracellular proteins, which may be linked to the inotropic response of the heart to a-adrenergic agonists. However, the role of protein kinase C in mediating the positive inotropic response of cardiac muscle to a-adrenergic stimulation presently appears controversial.13,14 In vitro studies have shown that several cardiac proteins may serve as substrates for protein kinase C. These include phospholamban"5 in the SR, a 15 -kd protein16 in sarcolemma, a 28-kd protein17 in the cytosol, and C protein,18 troponin I, and troponin T19 in the myofibrils. Actually troponin I and troponin T have each been reported to possess at least two phosphorylation sites for protein kinase C.19 Phosphorylation of phospholamban is associated with increased sensitivity of the Ca2 pump in SR,20 phosphorylation of the 15-kd sarcolemmal protein has been reported to increase the slow inward current,21 and phosphorylation of troponin I and troponin T has been suggested to modulate the Ca21 sensitivity of force production in cardiac muscle.22'23 The roles of phosphorylation of C protein in the myofibrils and the 28-kd cytosolic proteins are not presently known. Thus, several cardiac proteins can be phosphorylated by protein kinase C in vitro, but it is not presently known whether these cardiac proteins may also be phosphorylated in vivo and what role they play in mediating the inotropic response of the heart to a-adrenergic stimulation. The exception to this is the 15-kd sarcolemmal protein, which was shown to be phosphorylated in depolarized rat hearts stimulated by phenylephrine; this was associated with an increase in the slow inward current.21 However, activation of protein kinase C was not demonstrated, in that study, to clearly determine that the 15 -kd protein was a substrate for this enzyme in vivo. The aim of the present study was to determine whether a-adrenergic stimulation of intact beating rabbit hearts is associated with activation of protein kinase C and with increased phosphorylation of specific regulatory proteins, which may mediate the cardiac inotropic response. The experiments were carried out with rabbit hearts, because the a-adrenoceptor-mediated inotropic response is more pronounced in the rabbit than in other readily available mammals.10 The hearts were perfused with phenylephrine, and we examined changes in 1) hemodynamics, 2) phosphate labeling of inositol trisphosphates, 3) protein kinase C activity, and 4) phosphorylation of sarcolemmal, SR, myofibrillar, and cytosolic proteins. In parallel experiments, rabbit hearts were perfused with the ,3-adrenergic agonist isoproterenol, and its effects on protein phosphorylation were also determined. The differences detected in the phosphoproteins, obtained on activation of protein kinase C versus cAMP-dependent protein kinase, may provide an insight into the mechanisms involved in mediating the cardiac inotropic responses to a- versus ,B-adrenergic stimulation. Materials and Methods

Materials 32P-labeled orthophosphoric acid (Pi), [32p]IP3, ['H]IP3, and [py-32P]ATP were purchased from New England Nuclear, Boston. Phenylephrine, prazosin, propranolol, atropine, Dowex-1 or Dowex-50 ion exchange

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resins, phorbol 12-myristate 13-acetate, and phosphatidylserine were purchased from Sigma Chemical Co., St. Louis, Mo. P81 ion exchange chromatography paper was obtained from Whatman Biochemicals Ltd., UK. Bio-Gel P-6DG desalting gel and molecular weight standard protein solution were purchased from Bio-Rad Laboratories, Richmond, Calif. Thin-layer cellulose sheets and X-Omat films were purchased from Eastman Kodak Co. ScintiVerse scintillation cocktail was purchased from Fisher Scientific Co. All other chemicals were reagent grade. Heart Perfusion Hearts from anesthetized (30 mg/kg sodium pentobarbital) and heparinized (500 units/kg) rabbits (1.5 kg) were excised and perfused according to the method of Langendorff with a modified Krebs' buffer solution containing (mM) NaCI 118, KCl 4.7, CaCl2 2.5, KH2PO4 0.26, MgSO4 1.2, EDTA 0.5, NaHCO3 25.0, and glucose 5.5. The solution was saturated with 95% 02-5% CO2 (pH 7.4) at 37°C. Each heart was perfused with a constant aortic pressure of 65 mm Hg for 20 minutes in a drip-through mode. The perfusion circuit was then switched to a recirculating system containing 150 ml of the same buffer to which 2.0 mCi [32p]pi (New England Nuclear) was added. After 45 minutes of reperfusion with the isotope-labeled buffer, when the labeling of [y-32P]ATP reached equilibrium, the circuit was returned to the drip-through system using nonradioactive Krebs' solution. The drugs of interest were introduced into the buffer flow line. Phenylephrine was used as an a-adrenergic agonist, and isoproterenol (0.1 ,M) was used as a p-adrenergic agonist. To ensure the blockade of the different receptor systems, phenylephrine was administered in the presence of 0.1 ,uM atropine and 1 ,uM propranolol; isoproterenol was administered in the presence of 0.1 ,uM atropine and 0.1 ,uM prazosin. Control hearts were perfused under identical conditions and either in the presence of 0.1 ,uM atropine and 1 ,M propranolol (phenylephrine controls) or in the presence of 0.1 ,uM atropine and 0.1 ,lM prazosin (isoproterenol controls). The heart rate, ventricular pressure, and its first derivative (dP/dt) were continuously monitored through a pressure transducer attached to a multichannel polygraph (Grass Instrument Co., Quincy, Mass.). For the biochemical parameters assayed in this study, hearts were perfused as described above and either in the presence of 10 ,uM phenylephrine for 4 minutes or 0.1 gM isoproterenol for 3 minutes. Perfusion with 10 ,uM phenylephrine for 4 minutes was associated with a decrease in the coronary flow of the perfused heart (control, 41 +±1.5 ml/min; phenylephrine, 36.5+±1.5 ml/ min); perfusion with 0.1 ,uM isoproterenol for 3 minutes was associated with an increase in the coronary flow of the perfused heart (control, 41 ± 1.5 ml/min; isoproterenol, 48±4 ml/min). These changes in the coronary flow became significant after 2 minutes of infusion with either drug, and at that time the infusion rate was decreased by 10% in the case of phenylephrine, whereas it was increased by 15% in the case of isoproterenol. At the peak of the positive inotropic response to these agonists, the hearts were freeze-clamped with precooled (- 196°C) Wollenberger clamps, powdered, and stored under liquid nitrogen as we previously described.24

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Circulation Research Vol 70, No 4 April 1992

Assay of Protein Kinase C Activity Cytosolic and membranous fractions were isolated from control and phenylephrine- and prazosin-treated hearts perfused with nonradioactive buffer, as previously described.25 The membranous fraction was enriched in sarcolemmal enzyme markers, as indicated by the levels of Na+K+-ATPase (19 ,umol Pi/mg/hr), sodium azide-inhibitable ATPase (34 gLmol Pi/mg/hr) and Ca2+-ATPase (20 ,umol P/mg/hr) activities, and 5'nucleotidase activity (4 ,gmol Pi/mg/hr). Protein kinase C activity was determined by a modification of the method described by Yuan and Sen.25 The cytosolic or membranous proteins were incubated on ice for 60 minutes with 20 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose, 5 mM EDTA, and Triton X-100 to unmask the protein kinase C activity. The optimal detergent to protein ratios (wt/wt) were determined, and they were found to be 1.3 for the cytosolic fraction and 2.2 for the membrane vesicles. After preincubation, an aliquot containing 15 ,ug protein was assayed for protein kinase C activity in the presence of 30 mM phosphate buffer (pH 7.4), 25 mM KCl, 5 mM MgCl2, 10 mM NaF, 0.5 mM EGTA, 0.46 mM CaCl2 (1 gM Ca2+), 44 mM /3-mercaptoethanol, 60 ,ug histone, and 0.15 mM [y-32P]ATP (100 cpm/pmol) in a final reaction volume of 100 ,l. The assays were performed either in the absence or the presence of 5 gtg phosphatidylserine and 1 rM phorbol 12-myristate 13-acetate. Under these conditions, optimal protein kinase C activity was observed, and Pi incorporation was linear for up to 3 minutes at 30°C. Thus, incubations were carried out at 30°C for 3 minutes, and the reactions were terminated by addition of 10 gl of 0.83 M phosphoric acid. An aliquot (20 ul) from each reaction mixture was spotted on P81 ion exchange chromatography paper, the spotted paper was washed four times with 75 mM phosphoric acid, and the radioactivity was determined by liquid scintillation counting. Protein kinase C activity represents the difference in kinase activity obtained in the presence of 1 ,uM Ca2' alone and the activity obtained in the presence of 1 ,uM Ca2+, 5 g.g phosphatidylserine, and 1 ,uM phorbol 12-myristate 13-acetate.

Preparation and Gel Electrophoresis of Membranous, Myofibrillar, and Cytosolic Proteins Microsomes were prepared as previously described.21 Myofibrils were prepared from the pellet of the first centrifugation of the heart homogenates as we previously described.24 The supernatant (30 ml) obtained during the first high-spin centrifugation in the preparation of microsomes was used for the isolation of cytosolic proteins. Proteins were precipitated by addition of solid ammonium sulfate to the supernatant. The saturated ammonium sulfate solution was centrifuged at 20,000g for 20 minutes. The pellet was resolubilized in 1.5 ml inhibiting buffer containing (mM) Na2HPO4 30, NaF 15, and EDTA 6 (pH 7.0) and desalted on a Bio-Gel P-6DG desalting column (Bio-Rad). The final recovered volume of the concentrated cytosol was 4.5 ml, representing 6.6-fold concentration of the original cytosol. Polyacrylamide gel electrophoresis under denaturing conditions was performed according to Laemmli,26 using 10-15% and 7.5-17.5% gradient gels. The 32p_

labeled proteins detected by autoradiography and identified by their mobility relative to pure standards, were cut from the gels and counted for radioactivity. Phosphate incorporation into proteins was quantified by dividing the 32p incorporation in each band by the specific activity of [y-32P]ATP for each heart and was expressed as picomoles phosphate per milligram of protein loaded onto the gel lane.

Preparation of Inositol Trisphosphates Inositol phosphates were extracted from freezeclamped and powdered heart tissue with 0.5 M trichloroacetic acid27 and washed with 20 vol water-saturated diethyl ether. The final fraction was directly applied onto a Dowex-1 column (formate form) and eluted with formic acid-ammonium formate buffers.28 The fraction containing the inositol trisphosphates, as identified by the comigration of [3H]1P3 standard applied to the column, was free from different sugar monophosphates and bisphosphates, but it was heavily contaminated with [32P]-labeled ATP. The final purification was performed by electrophoretic separation. The effluent from the Dowex-1 column, containing the inositol trisphosphates, was mixed with a slurry of an equal amount of Dowex-50 resin. After centrifugation, an aliquot from the supernatant was directly spotted on cellulose thin-layer sheets and electrophoretically separated using 0.06 M sodium oxalate buffer (pH 1.5). Three distinct radioactive spots were obtained, and they were clearly separated from each other. One was identified as ATP, by applying [y-32P]ATP on the same plate. Another spot was identified as the inositol trisphosphate fraction by applying [32P]1P3 on the same plate. The third was not identified. This procedure did not allow separation of 1P3 from other inositol trisphosphate isomers; therefore, the results reflect the combined radioactivity of inositol trisphosphate isomers. After electrophoresis, the plates were dried and placed in contact with Kodak X-Omat films. The identified spot corresponding to the inositol trisphosphate fraction was cut and counted for radioactivity. The recovery of inositol trisphosphate extraction (85%) was monitored by including [3H]-labeled standards.

Other Procedures The specific activity of [y-32P]ATP in the perfused hearts was determined by the method described by Kopp and Barany.29 Specific radioactivities (mean±SEM) were 6.6±0.5 cpm/pmol ATP (n=8) for controls, 6.1±0.3 cpm/ pmol ATP (n =6) for phenylephrine-treated hearts, 6.0±0.2 cpm/pmol ATP (n=3) for prazosin-treated hearts, and 6.1±0.3 cpn/pmol ATP (n=4) for isoproterenoltreated hearts. Protein concentration was determined either by the Lowry30 or the amido black31 method for the samples used for the protein kinase C assay or polyacrylamide gels, respectively, using bovine serum albumin as a standard. Results are expressed as mean±SEM. Statistical analysis was carried out using Student's t test for unpaired observations. Values of p

Effect of alpha-adrenergic stimulation on activation of protein kinase C and phosphorylation of proteins in intact rabbit hearts.

The intracellular events and specifically the role of protein kinase C-mediated protein phosphorylation, after alpha-adrenergic receptor stimulation o...
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