Concerted CMP‐Dependent [ 3 H]Inositol Labeling of Phosphoinositides and Agonist Activation of Phospholipase C in Rat Brain Cortical Membranes

[ 3 H]Inositol ([ 3 H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [ 3 H]Ins was incorporated into a common lipid pool through both CMP‐dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl‐inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP‐dependent or independent fashions were hydrolyzed by guan‐osine 5′‐ O ‐(3‐thiotriphosphate) (GTPγS)‐ and carbachol‐stimulated phospholipase C. Unlike CMP‐dependent labeling, however, CMP‐independent incorporation of [ 3 H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxy‐cholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [ 3 H]Ins was incorporated into lipids primarily through the Ptdlns synthase‐catalyzed reaction because of the presence of deoxy‐cholate required to observe carbachol‐stimulation of phospholipase C. Little direct breakdown of [ 3 H]PtdIns was detected because production of myo ‐[ 3 H]inositol 1‐mono‐ phosphate was minimal and myo‐[ 3 H]inositol 1,4‐bisphos‐phate was the predominant product. Although PtdIns labeling and 3 Hpolyphosphoinositide formation were unaffected by GTPγS and carbachol and had no or little lag period, GTPγS‐ and carbachol‐stimulated appearance of 3 H‐Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [ 3 H]Ins to 3 H‐Ins phosphates was restricted to a narrow range of free calcium concentrations (10–300 nM). These results show the concerted activities of Ptdlns synthase, Ptdlns 4‐kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide‐dependent agonist stimulation of phospholipase C in a brain membrane system using [ 3 H]Ins as labeled precursor.