CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells.
CMP is known to activate phosphatidylinositol (PtdIns)/inositol (Ins) base exchange and has been reported to activate reversal of PtdIns synthase also. Because it is possible that PtdIns synthase acting in the reverse direction, followed by re-incorporation of ambient Ins, could be responsible for base-exchange activity, we characterized these processes in rat pituitary GH3 cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl but without added Ins, CMP stimulated rapid accumulation of [3H]Ins and decreases in [3H]PtdIns; the Km for CMP was 1.7 mM. CDP and CTP were less effective, whereas 2'-CMP, 3'-CMP, other nucleoside monophosphates and cytidine did not influence this process. In permeabilized cells prelabelled to isotopic equilibrium with [3H]Ins and [32P]Pi, CMP stimulated decreases in both the 32P and 3H labelling of PtdIns, but did not increase that of [32P]phosphatidic acid. These findings demonstrate that in the absence of added Ins the effect of CMP is not via activation of base exchange nor via a phospholipase D, but by reversal of PtdIns synthase. In permeabilized cells prelabelled with [3H]Ins and [32P]Pi, unlabelled Ins inhibited loss of 32P labelling of PtdIns caused by CMP while markedly stimulating loss of 3H labelling of PtdIns and release of [3H]Ins. These data demonstrate that Ins inhibits reversal of PtdIns synthase, but stimulates base exchange. We conclude that in GH3 cells reversal of PtdIns synthase and PtdIns/Ins base exchange are both stimulated by CMP, but are distinct processes.
Cubitt
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AB
Department of Medicine, Cornell University Medical College, New York, NY.
Gershengorn
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MC
D000818:Animals; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002460:Cell Line; D003562:Cytidine / Q000494:pharmacology; D003565:Cytidine Diphosphate / Q000494:pharmacology; D003568:Cytidine Monophosphate / Q000494:pharmacology*; D003570:Cytidine Triphosphate / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D010911:Pituitary Neoplasms; D051381:Rats; D017855:Transferases (Other Substituted Phosphate Groups); D014316:Tritium
D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D014316:Tritium; D007294:Inositol; D003562:Cytidine; D003565:Cytidine Diphosphate; D003570:Cytidine Triphosphate; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; C496767:Cdipt protein, rat; D003568:Cytidine Monophosphate; D008274:Magnesium