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TRIACYLGLYCEROL LIPASES AND THE STIMULATION OF LIPOLYSIS BY ISOPROTERENOL IN RAT HEART MYOCYTES. A. Kryski, I. Ram~rez, and D.L. Severson. Department of Pharma- cology, University of Calgary, Calgary, Alberta, Canada. T2N 1N4.

Isoproterenol (ISO, i0 ~M) stimulates the hydrolysis of endogenous triacyl- glyeerols (TG) in isolated rat heart myocytes. An acid TG lipase with a pH optimum of 4.5 has been characterized in myocytes. The specific activity of the acid TG lipase was 9.62 • 0.95 (n = 16) nmol/hr/mg protein in myocyte homogenates compared to 7.21 • 0.44 (n = 8) nmol/hr/mg protein in 400 x g supernatants from whole heart homogenates. Since incubation of myocytes with the lysosomotropic inhibitor methylamine (5 mM) did not influence basal or hormone-stimulated rates of lipo- lysis then presumably the acid lipase is not involved in the lipolytic process. A neutral lipase in myocytes was characterized as having a pH optimum of 7.5 and was stimulated by serum (3%). The serum stimulation was enhanced by low concentrations of NaCI (200 mM), CaCI 2 (50 mM), and MgCI 2 (50 mM); higher salt concentrations were inhibitory. The neutral enzyme was stimulated 5-fold (n = 6) by Apo C-II, and both serum and Apo C-If stimulations were inhibited 80% (n = 3) and 76% (n = 2), respect- ively, by Apo C-Ill. Incubation of myocytes with ISO resulted in no consistent change in the neutral TG lipase activity in the myocytes. Supported by MRC of Canada, and CIRIT (Catalonia, Spain).

Ca2+-ACTIVATED PROTEASE (CAP) ACTIVITIES IN HYPERTENSIVE CARDIOMYOPATHY IN THE RAT. T. Kuo, M. Spalla, W. Tsang, F. Giacomelli, J. Wiener. Department of Pathology, Wayne State University Medical School, Detroit, MI 48201.

We have shown previously that there is elevation of the CAP activities in the heart of rats with two-kidney, one clip Goldblatt hypertension (J. Mol. Cell. Cardiol. 15, 173, 1983). It appears that there are three forms of CAP activities in hyper- tensive hearts. Two of these activities (peak IA and peak 2) may be detected by the initial DE-52 column fractionation whereas the third activity (peak I) is revealed only after subsequent chromatography to remove the endogenous inhibitor. In order to determine the nature of the increase in CAP activities and the relation among these three peaks, purification of CAP from hearts of control as well as hypertensive rats has been carried out. Purified CAP I (low-Ca 2+ form) requires only 0.3~M Ca 2+, whereas purified CAP II (high-Ca 2+ form) requires 0.5mM Ca 2+ for half-maximal activi- ty. Both proteases consist of a single peptide with M r = 93K and M r = 83K for CAP I and CAP II respectively. CAP I and CAP II also show different behavior regarding autoproteolysis in the presence of Ca 2+. While peak i and peak 2 from hypertensive hearts have been indentified with CAP I and CAP II respectively, peak IA appears re- lated to CAP II by autolysis. Whether antolysis of CAP occurs in vivo after induc- tion of hypertension is currently being investigated. (Supported by USPHS HL23603.)

ELECTROGENESIS IN HYPOXIC MYOCARDIUM: VARIATIONS WITH EXTERNAL K. N. Leblanc, E. Ruiz- Ceretti. Dept. of Biophysics, Univ. of Sherbrooke, CHUS, Sherbrooke, Que. JIH 5N4.

In contrast to fast action potentials, the amplitude of the slow depolarizing phase of depressed fast responses (high external K) is not depressed byhypoxia (Ruiz-Ceretti et al. JMCC, 15, 845, 1983). To further analyze this phenomenon the maximum rate of rise (Vmax) of the slow depolarization at 2.5, 5, 7.5 and I0 mM K was measured in normoxia and hypoxia~ We used TTX to depress the fast initial component and thus obtain a good resolution of the two phases in the V signal. Rabbit hearts were perfused with Krebs at 33~ and 15 to 18 ml/min. The Vma x of the slow rising phase did not vary with K in normoxia (7.3 to 8~2 V/s). In contrast, Vmax decreased to. about 6V/swith hypoxia at 2.5 and 5 mM and increased to 9-10 V/s at 7.5 and IOmMK o. Vma x is a measure of the net inward current determined by a mixture of the slow inward current and the outward currents coexisting during that phase of the action potential~ The insensitivity of Vma x to K in normoxia suggests that outward K currents do not play a major role during the slow rising phase which leaves the slow inward current Isias the major determinant

o

of Vma x, If thus interpreted, our results suggest that the Isf-dependent depolarization becomes insensitive to metabolic inhibition wren contributing to the generation of a depressed fast response instead of a fast action potential. The role of endogenous catecholamines on these effects remains to be established. Supported by MRC, and the Quebec Heart Fund. N~ Leblancis a predoctoral fellow of the Canadian Heart Foundation.