Oxidants Depress the Synthesis of Phosphatidylinositol 4,5-Bisphosphate in Heart Sarcolemma

Abstract 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. PtdIns 4,5-P 2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P 2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P 2 . Exposure to the xanthine–xanthine oxidase–ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine–xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H 2 O 2 ) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an • OH scavenger) did not modify the H 2 O 2 -induced depression of the kinases, eliminating any role of • OH. Furthermore, the IC 50 of H 2 O 2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 μM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H 2 O 2 did not affect the activities of the kinases; however, oxidized glutathione 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 redox ratio was observed. Thiol modifiers ( N -ethylmaleimide, methyl methanethiosulfonate, or p -chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with 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.