Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells.
The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.
Torchia
J
J
Department of Pharmacology, University of Toronto, Ontario, Canada.
Qu
Y
Y
Francis
J
J
Pon
D J
DJ
Sen
A K
AK
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D004372:Ducks; D015759:Ionomycin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000378:metabolism*; D012491:Salt Gland / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D007546:Isoquinolines; D010710:Phosphates; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011506:Proteins; D015759:Ionomycin; D001285:Atropine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate