Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.
The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.
D001254:Astrocytoma / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis*; D037342:Pertussis Toxin; D011976:Receptors, Muscarinic / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D013268:Stimulation, Chemical; D013403:Sugar Phosphates / Q000096:biosynthesis*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D011976:Receptors, Muscarinic; D013403:Sugar Phosphates; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D002217:Carbachol; D012969:Sodium Fluoride; D037342:Pertussis Toxin