Considerable phospholipase D (PLD) activity is localized in myocardial sarcoplasmic reticular (SR) membranes, where it may take part in the regulation of Ca2+ movements. In this study, we examined thiol group dependence as a possible regulatory mechanism for SR PLD. SR membranes isolated from rat heart were exposed to four types of thiol group modifiers, which all induced a decrease in SR PLD activity that was prevented by dithiothreitol. Furthermore, since abnormalities in thiol status and Ca2+ homeostasis are characteristic for the myocardial cell damage induced by oxidative stress, we also studied the effects of oxidants on the SR PLD activity. The enzyme was not affected by xanthine-xanthine oxidase, but was depressed by hydrogen peroxide and by hypochlorous acid. These inhibitory effects were prevented by catalase as well as by methionine and dithiothreitol, respectively. Furthermore, reduced glutathione protected against the hydrogen peroxide-induced depression, whereas oxidized glutathione inhibited SR PLD. The results indicate that SR PLD activity is inhibited by nonradical oxidants, hydrogen peroxide and hypochlorous acid, through reversible modification of associated thiol groups. Thus, the enzyme may be controlled by the glutathione redox status of the cardiac cell.

Cardiac sarcolemmal (SL) phospholipase C (PLC) is a key enzyme in the signal transduction of several cardiac receptors. Thus, the earlier described Ca(2+)-stimulated SL PLC activity may represent variously coupled enzymes. The present study was undertaken to delineate the alpha 1-adrenoceptor/G protein-stimulated PLC activity in purified cardiac SL vesicles. Although certain detergents and membrane pore formers enhanced SL PLC activity, measured as formation of 3H-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] from 3H-phosphatidylinositol 4,5-bisphosphate, alpha 1-adrenoceptor stimulated activity was not observed. When SL vesicles were preincubated (0-4 degrees C) with substrate in detergent-free buffer, subsequent incubation (37 degrees C; in mM: 100 NaCl, 2 EGTA, 1.8 CaCl2, 10 LiCl) resulted in a time-dependent production of 3H-Ins(1,4,5)P3, that was increased in the presence of 100 microM GTP gamma S. GTP gamma S stimulation of SL PLC activity required the presence of Mg2+ and Ca2+, but was lost at (sub)millimolar concentrations of these bivalent cations. Mg2+ (0.01-10 mM) promoted a 2,3-diphosphoglycerate-insensitive phosphatase activity. GTP gamma S enhanced the sensitivity of SL PLC to Ca2+, but did not increase the maximum Ca2+ (0.1-1 mM) stimulated SL PLC activity. At 5 microM Ca2+, GTP gamma S induced a concentration-dependent rise in inositol phosphates production, which was further elevated by the alpha 1-agonist, phenylephrine (PhE). The PhE-effect was inhibited by the alpha 1-antagonist prazosin, but not by the beta-antagonist atenolol. These results show that the components necessary for the alpha 1-adrenoceptor transmembrane signal are associated with the SL membrane and can be functionally coupled.