CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells

• Published in: Biochemical journal 1990-12, Vol.272 (3), p.813-816
• Main Authors: CUBITT, A. B, GERSHENGORN, M. C
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; Cell Line; Cytidine - pharmacology; Cytidine Diphosphate - pharmacology; Cytidine Monophosphate - pharmacology; Cytidine Triphosphate - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Inositol - metabolism; Kinetics; Magnesium - pharmacology; Membrane Proteins; Phosphorus Radioisotopes; Phosphotransferases - metabolism; Pituitary Neoplasms; Rats; Transferases; Transferases (Other Substituted Phosphate Groups); Tritium
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2720813

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.