Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes.
[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]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 guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]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, [3H]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 [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-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 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-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 [3H]Ins as labeled precursor.
Claro
E
E
Department of Biochemistry, University of Tennessee, Memphis.
Wallace
M A
MA
Fain
J N
JN
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D003568:Cytidine Monophosphate / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism* / Q000502:physiology
D010716:Phosphatidylinositols; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D002217:Carbachol; D010738:Type C Phospholipases; D003568:Cytidine Monophosphate