Regulation of muscarinic agonist-induced activation of phosphoinositidase C in electrically permeabilized SH-SY5Y human neuroblastoma cells by guanine nucleotides.
myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.
Wojcikiewicz
R J
RJ
Department of Pharmacology and Therapeutics, University of Leicester, England.
Lambert
D G
DG
Nahorski
S R
SR
D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability; D004558:Electric Stimulation; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006801:Humans; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010276:Parasympatholytics / Q000378:metabolism / Q000494:pharmacology; D010277:Parasympathomimetics / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006150:Guanine Nucleotides; D010276:Parasympatholytics; D010277:Parasympathomimetics; D012602:Scopolamine Derivatives; D001285:Atropine; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium; D019832:N-Methylscopolamine