8978708

pubmed:Citation

1

Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 nM TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 microM. In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the alpha-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the epsilon-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.

eng

IM

MEDLINE

0022-3042

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40-6

pubmed-meshheading:8978708-Calmodulin, pubmed-meshheading:8978708-Carbachol, pubmed-meshheading:8978708-Cytosol, pubmed-meshheading:8978708-Enzyme Inhibitors, pubmed-meshheading:8978708-Humans, pubmed-meshheading:8978708-Immunoblotting, pubmed-meshheading:8978708-Indoles, pubmed-meshheading:8978708-Intracellular Signaling Peptides and Proteins, pubmed-meshheading:8978708-Isoenzymes, pubmed-meshheading:8978708-Membrane Proteins, pubmed-meshheading:8978708-Phosphorylation, pubmed-meshheading:8978708-Protein Kinase C, pubmed-meshheading:8978708-Proteins, pubmed-meshheading:8978708-Receptors, Muscarinic, pubmed-meshheading:8978708-Tetradecanoylphorbol Acetate, pubmed-meshheading:8978708-Tissue Distribution, pubmed-meshheading:8978708-Tretinoin, pubmed-meshheading:8978708-Tumor Cells, Cultured

Effect of continuous phorbol ester treatment on muscarinic receptor-mediated calmodulin redistribution in SK-N-SH neuroblastoma cells.

Journal Article, Research Support, U.S. Gov't, P.H.S.