CONCERTED CMP-DEPENDENT [H-3] INOSITOL LABELING OF PHOSPHOINOSITIDES AND AGONIST ACTIVATION OF PHOSPHOLIPASE-C IN RAT-BRAIN CORTICAL MEMBRANES

[H-3]Inositol ([H-3]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [H-3]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 phosphatidylinositol (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 guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling. however, CMP-independent incorporation of [H-3]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [H-3]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [H-3]PtdIns was detected because production of myo-[H-3]inositol 1-monophosphate was minimal and myo-[H-3]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and H-3-polyphosphoinositide formation were unaffected by GTP-gamma-S and carbachol and had no or little lag period, GTP-gamma-S- and carbachol-stimulated appearance of H-3-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [H-3]Ins to H-3-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 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 [H-3]Ins as labeled precursor.