Muscarinic-agonist and guanine nucleotide activation of polyphosphoinositide phosphodiesterase in isolated islet-cell membranes.
Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5'-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.
D037361:Adenylate Cyclase Toxin; D000818:Animals; D018723:Bethanechol; D001626:Bethanechol Compounds / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006150:Guanine Nucleotides / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology*; D009355:Neomycin / Q000494:pharmacology; D037342:Pertussis Toxin; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D001626:Bethanechol Compounds; D006150:Guanine Nucleotides; D010566:Virulence Factors, Bordetella; D018723:Bethanechol; D002217:Carbachol; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D009355:Neomycin