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Binding [3H] GTP and GTP-ase activity were studied on the isolated preparation of myocardium sarcolemma. The Muscarinic acetylcholine receptors against carbocholine (10(-4) M) was shown to stimulate both the GTP binding by sarcolemma and its GTP-ase activity. The activating effect of carbocholine is blocked by the Muscarinic acetylcholine receptors antagonist atropine (10(-6) M). The GTP-ase activity of sarcolemma is reduced in presence of sodium fluoride which points out the enzyme activity of G-proteins. Thus, functioning of G-proteins and their Muscarinic acetylcholine receptors regulation were showed during in vitro experiments on preparation of myocardium sarcolemma.

The activating effect of a M-receptor agonist carbachol on phosphatidylinositol metabolism under perfusion of the isolated rabbit heart with 32Pi was shown. An increase of the inclusion of 32P in phosphatidylinositol-4-phosphate and phosphatidylinositol-4-diphosphate was found. Simultaneously there was detected an elevation of the levels of products of hydrolysis of inositol phospholipids--inositol-1,4-diphosphate and inositol-1,4,5-triphosphate. There was noted the blocking action of carbachol on the contractile activity of the heart that manifested itself in a lowering of the systolic pressure in the left ventricle and a slowing of the contraction rate.

Phosphorylation of cardiac sarcolemma proteins under stimulation of M-receptors by agonist carbacholine used to stimulate phosphatidylinositide cycle, was investigated in the isolated, rabbit heart perfused with 32Pi. Carbacholine (10(-7) stimulates the polyphosphoinositide metabolism which is expressed in the activated incorporation of 32P from [gamma-32P]ATP in polyphosphoinositide as well as in the increased content of the labelled inositol trisphosphate released through phosphatidylinositol-4,5-bisphosphate break-down by phospholipase C. The diacylglycerol produced simultaneously with inositol triphosphate as a second messenger activates the protein kinase C. This was confirmed by considerable activation of phosphorylation sarcolemma proteins-substrates of protein kinase C, with Mr 94, 87, 78, 51 and 46 kDa.

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.