title: The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.
abstract: The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.
authors.affiliation: Neuroscience Laboratory, University of Michigan, Ann Arbor 48104-1687.
authors.forename: A K
authors.initials: AK
authors.lastname: Thompson
authors.forename: S P
authors.initials: SP
authors.lastname: Mostafapour
authors.forename: L C
authors.initials: LC
authors.lastname: Denlinger
authors.forename: J E
authors.initials: JE
authors.lastname: Bleasdale
authors.forename: S K
authors.initials: SK
authors.lastname: Fisher
mesh_terms: D000527:Alprostadil / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D009447:Neuroblastoma; D010095:Oxotremorine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D012601:Scopolamine / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
chemical_list: D004745:Enkephalins; D004963:Estrenes; D010716:Phosphatidylinositols; D011760:Pyrrolidinones; D011976:Receptors, Muscarinic; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D005576:Colforsin; D007294:Inositol; D010095:Oxotremorine; D012601:Scopolamine; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000527:Alprostadil