title: Muscarinic receptor-mediated translocation of calmodulin in SK-N-SH human neuroblastoma cells.
abstract: The role of muscarinic receptor-mediated polyphosphoinositide hydrolysis and subsequent calcium signals in altering the subcellular localization of calmodulin (CaM) was examined in SK-N-SH human neuroblastoma cells. Upon incubation of the cells with the full agonist carbachol, a 4.5- to 5-fold increase in CaM in the cytosol was observed, from 126 ng of CaM to 629 ng of CaM. There was an accompanying 68% decrease in membrane-bound CaM. The increase in the cytosol was maximal by 15 min, as was a corresponding decrease in membrane-associated CaM. The redistribution of CaM was maintained for at least 2 hr, before returning toward control levels by 4 hr. The EC50 values for carbachol in eliciting the translocation were 3.7 microM for the increase in cytosol and 1.3 microM for the decrease in membranes. The maximal changes in CaM concentration in both membranes and cytosol occurred with 10 microM carbachol. Incubation of the SK-N-SH cells with the partial muscarinic agonists bethanechol and arecoline resulted in 27 and 26% decreases in membrane-associated CaM, respectively, and 28 and 35% increases in cytosolic CaM, respectively. Thus, the partial agonists were less efficacious than carbachol in eliciting changes in CaM localization. Atropine completely blocked the carbachol-stimulated translocation, whereas the nicotinic agonist 1,1-dimethyl 4-phenylpiperazinium had no effect on the localization of CaM. Activation of receptors coupled to adenylate cyclase did not affect distribution of CaM. CaM content in membranes and cytosol of cells incubated with prostaglandin E1 or the alpha 2-adrenergic agonist UK 14,304 was not different from control values. The ionophore ionomycin (10 microM) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 nM) were both able to elicit changes in CaM distribution. Ionomycin caused a 64% increase in CaM in the cytosol, with no significant change in membrane CaM. TPA elicited a decrease in membrane-associated CaM, with a corresponding increase in CaM in the cytosol. When TPA and ionomycin were incubated together, the translocation was equal in magnitude to that observed with carbachol alone. The protein kinase C inhibitor H-7 blocked the TPA-stimulated response and partially blocked the carbachol-stimulated response. The CaM-binding protein neuromodulin, which demonstrates a decreased affinity for CaM in the presence of Ca2+ and when phosphorylated by protein kinase C, was present in both membranes and cytosol of SK-N-SH cells.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
authors.forename: L A
authors.initials: LA
authors.lastname: Mangels
authors.forename: M E
authors.initials: ME
authors.lastname: Gnegy
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000262:Adenylyl Cyclases / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D007546:Isoquinolines; D010879:Piperazines; D011976:Receptors, Muscarinic; D015759:Ionomycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium