Oxidants depress the synthesis of phosphatidylinositol 4,5-bisphosphate inheart sarcolemma

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P-2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. Pt-dIns 4, 5-P-2 is synthesized in the SL membrane by coordinated and successive actio ns of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and th e generation of PtdIns 4,5-P-2 may be a factor in the cardiac dysfunction d uring pathophysiological conditions of oxidative stress. Therefore, we exam ined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P-2. Exposure to the xa nthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; h owever, catalase significantly prevented the xanthine-xanthine oxidase indu ced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in i nhibition of both the kinases, which was prevented by catalase and dithioth reitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine tan iron chelator) and mannitol tan . OH sca venger) did not modify the H2O2-induced depression of the kinases, eliminat ing any role of (OH)-O-.. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 mu M, respectively. In addition, incl usion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutat hione induced a significant depression, Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the r edox ratio was observed. Thiol modifiers UV-ethylmaleimide, methyl methanet hiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depre ss the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated w ith PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction. (C) 2000 Academic Press.