Phosphatidylinositol availability and polyphosphoinositide synthesis in pancreatic islet cell membranes.
Polyphosphoinositide synthesis in isolated islets of the rat was determined by the phosphorylation of endogenous phosphatidylinositol (PtdIns) by PtdIns kinase and [gamma-32P]ATP to form [32P]-phosphatidylinositol 4-phosphate (PtdInsP) in cell homogenates. Glucose stimulation of intact islets resulted in a time- and concentration-dependent reduction in PtdInsP synthesis. Similarly, the stimulation of intact islets with carbachol (CCh), cholecystokinin (CCK-8S), or tolbutamide for 15 min reduced PtdInsP production in a concentration-dependent manner. The effects of glucose, tolbutamide and CCh were reversible. PtdInsP hydrolysis did not account for the reduction in PtdInsP recovery. The addition of exogenous PtdIns to the PtdIns kinase assay significantly increased basal PtdInsP levels. In addition, exogenous PtdIns completely reversed the inhibitory effects of glucose and increased PtdIns kinase activity in homogenates of glucose-stimulated islets to levels found in control homogenate with PtdIns. Exogenous PtdIns also increased PtdIns kinase activity in CCK-8S-treated islets, although exogenous PtdIns did not overcome the tolbutamide-induced inhibition of PtdIns kinase. The Vmax of PtdIns kinase in homogenates of islets treated with tolbutamide was reduced significantly, although glucose did not affect the Vmax. In addition, the Km values for ATP and PtdIns were not altered by exposure of the islets to cell stimuli. The results suggest that the level of PtdIns in islet cell membranes is rate limiting for PtdInsP synthesis, and that tolbutamide is a noncompetitive inhibitor of PtdIns kinase.
Khalaf
L J
LJ
Department of Pharmacology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
Laychock
S G
SG
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism; D008297:Male; D010179:Pancreas / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000096:biosynthesis* / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012844:Sincalide / Q000494:pharmacology; D014044:Tolbutamide / Q000494:pharmacology
D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D002217:Carbachol; D014044:Tolbutamide; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D005947:Glucose; D012844:Sincalide