Studies on phosphoinositide metabolism in retinal rod outer segments

The present work was undertaken to determine whether isolated mammalian photoreceptors (bovine rod outer segments, ROS) were capable of synthesizing and hydrolyzing phosphoinositides, and if so, how synthesis and hydrolysis might be regulated. Synthesis of phosphoinositides was demonstrated by the incorporation of radioactively labeled precursors. ROS incubated with (γ32P)ATP produced labeled phosphatidic acid (PA), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). When Mn2+, CTP and inositol were added, labeled phosphatidylinositol (PI) was also produced. (3H)Inositol was incorporated into PI, PIP and PIP2, although prolonged incubation was required for detectable incorporation into PIP2. Incorporation of (3H)inositol was dependent on CTP, indicating that labeling proceeded via synthesis, not base exchange. Incubation with (α-32P)CTP produced labeled CDP-diacylglycerol, an intermediate in PI synthesis. Incorporation of labeled precursors was stimulated by Mg2+, Mn2+ and spermine, but unaffected by light. Phosphoinositide hydrolysis was measured using exogenous 3H-labeled substrates. PI, PIP, and PIP2 were all hydrolyzed but most attention was devoted to PIP2. Crude enzyme preparations contained an endogenous inhibitor whose effects were Ca2+-dependently relieved by calmodulin antagonists. This inhibitor does not appear to be calmodulin but may be a novel Ca2+-binding regulatory protein. No evidence for regulation of PLC activity by light or G-proteins was obtained. The effects on PLC activity of Mg2+, Mn2+, spermine, pH and detergents were also characterized