Effect of phosphocreatine and related compounds on the phospholipid metabolism of ischemic heart.

Changes in the content of lysophosphoglycerides in a crude plasmalemmal fraction of canine heart during short-term ischemia (occlusion of the left descending coronary artery for 8 min) have been studied in the presence and in the absence of phosphocreatine and phosphocreatinine. In the control experiments without PCr or PCr-nine ischemia caused significant elevation of the content of LPG: that of lysophosphatidylcholine was increased by 83% and that of lysophosphatidylethanolamine by 168%. Intravenous administration of PCr and PCr-nine in doses of 300 mg/kg completely prevented accumulation of LPG in the ischemic zone. Because of the well-known arrhythmogenic properties of LPG, the inhibitory effect of PCr and PCr-nine on the elevation of their concentration in the ischemic zone may be closely related to the antiarrhythmic action of PCr and PCr-nine in acute myocardial ischemia.

Creatine kinase in regulation of heart function and metabolism. I. Further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems.

In isolated and purified cardiac myofibrillar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyruvate production from phosphoenol pyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmal preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at PK/CK = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts.

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).

The cardiac sarcolemmal protein kinase reaction. Kinetic characteristics and sensitivity to cyclic AMP.

Isolated and carefully purified preparations of the sarcolemmal membrane from rat cardiac muscle were used to study the membrane-bound protein kinase (PrK) reaction. Contamination of the membrane preparation by mitochondria and sarcoplasmic reticulum was practically absent, and the sarcolemmal membrane vesicles were mostly oriented inside out. These membrane vesicles contained protein kinase tightly bound to the membrane and able to phosphorylate both the endogenous membrane protein with molecular mass of 11,500 daltons and the exogenous added protein (histone, type II). Endogenous protein phosphorylation was completely independent of cAMP which only stimulated the phosphorylation of histone in the membrane-bound PrK reaction. The kinetics of the membrane-bound PrK reaction with MgATP and histone as substrates was studied, and the kinetic mechanism was found to be of sequential Bi-Bi type. Kinetic characteristics of the enzyme were very close to those found earlier for the soluble muscle enzyme. On the basis of data obtained, it is suggested that membrane-bound PrK may be of the same origin as the soluble enzyme and that cAMP may influence the binding of PrK to the surface membrane of cardiac cells.

Is the sarcolemmal Na+ -K+ ATPase involved in active calcium transport?

Sarcolemmal preparations from rat and guinea pig cardiac muscle consisting mostly of inside-out vesicles were found to accumulate Ca2+ in the presence of ATP. The normalized rate of calcium uptake (the rate of calcium accumulation divided by its initial content in vesicles) correlated with the Na+ -K+ ATPase activity of the preparation. ATP-dependent calcium uptake by sarcolemmal vesicles was inhibited by 40-50% by cardenolids, digitoxigenin and ouabain, when the latter was included inside the vesicles. The Ca2+ gradient formed in the presence of ATP was dissipated by the addition of external sodium; Li+ was found to be ineffective in this process. External sodium also caused calcium release from vesicles pre-equilibrated with calcium in the medium. In the energy-dependent calcium uptake, the membrane-bound creatine kinase ATP-regenerating system was found to be the most effective energy source for active calcium transport. The results obtained may be interpreted to show that either the cardiac sarcolemma contains an energy-dependent calcium transport system or the sarcolemmal Na+ -K+ ATPase is able to transport calcium instead of sodium.