OXIDATIVE STRESS MODIFIES THE ACTIVITY OF CARDIAC SARCOLEMMAL PHOSPHOLIPASE-C

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels . The enzyme activity in rat cardiac sarcolemmal membranes that had be en preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts s uperoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Su ch decrease was fully prevented by catalase. Preincubation of sarcolem ma with hypochlorous acid also gave a significant inhibition of phosph olipase C, which was counteracted by the synthetic thiol reducer dithi othreitol. H2O2-pretreatment induced a concentration-dependent inhibit ion of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H,Oz effects. These da ta suggest that superoxide anions and hydroxyl radicals did not interf ere with phospholipase C activity, and that the nonradical oxidants, H 2O2 and hypochlorous acid, may have acted through oxidation of thiol ( SH) groups. The existence of reactive SH groups associated with the en zyme was confirmed by the inhibitory effects of SH modifiers (p-chloro mercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-et hylmaleimide and methyl methanethiosulfonate), which were prevented an d in some cases also reversed by dithiothreithol. The biological reduc er glutathione (CSH) was not able to recover the H2O2-induced inhibiti on of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enz yme was active in a wide range of GSH/GSSG redox states, but H2O2 pret reatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deac tivated the enzyme. The oxidants' concentrations that significantly im paired phospholipase C in this study were compatible with those occurr ing in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235 , 1991]. This supports the possibility that alteration of the receptor -associated phospholipase C may be a factor in the oxidant-related dys function of the ischemic-reperfused heart.