Hormone-induced Protein Phosphorylation. I. Relationship between Secretagogue Action and Protein Phosphorylation in Intact Cells from the Exocrine Pancreas and Parotid Endogenous

Abstract

We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid Iobules were preincubated with 32p~ for 45 min at 37°C, washed, and then incubated at 37°C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic Iobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37°C; (b) an analogous phosphorylated polypeptide was observed in isoproterenolstimulated parotid Iobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic Iobules and propranolol to isoproterenol-stimulated parotid Iobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid. The mechanism whereby secretagogues are able to elicit a characteristic biological response in their respective target ceils is unclear at the moment. There is now increasing evidence that protein phosphorylation is involved in mediating the effects of a variety of the actions of hormones in many diverse enzymological and physiological processes (1). To gain further insight into the mechanism of secretagogue action in exocrine glands, we examined the relationship between protein phosphorylation and hormone action in the intact cell to determine whether this covalent modification mediates or modulates any of the biological actions of secretagogues. The acinar cells of the exocrine pancreas and parotid offer good systems for studying secretagogue effects on endogenous protein phosphorylation since homogeneous cell preparations can be prepared, several different secretagogues exist which elicit discharge through different intracellular messengers, and the effects of these hormones on calcium and cyclic nucleotide levels during secretion are well characterized (2, 3, 4). Since cAMP, cGMP, and Ca 2+ have been implicated in the secretion of exportable proteins from the exocrine pancreas, protein kinases are plausible targets for these putative secretagogue mediators and, in fact, both cAMP and cGMP-dependent protein kinases have been partially purified from homogenates of rat pancreas (5, 6). We examined the relationship between endogenous protein phosphorylation and secretagogue action in situ in gland lobules under physiological conditions using the rat exocrine pancreas and parotid as model systems. A preliminary note on this research has been published (7). MATERIALS AND METHODS An chemicals used were of reagent grade and were obtained from the following sources: carbamylcholine chloride (carbachol), isoproterenol hydrochloride, atropine, propranolol, TRIS, HEPES, and dibutyryl cAMP were from Sigma Chemical Co. (St. Louis, MO); N-methyl-N'-nitro-N-nitrosoguanidine and 12-O-tetradecanoyl-phorbol-13-acetate from Aldrich Chemical Co. (Milwaukee, WI); and THE IOURNAL OF CELL BIOLOGY-VOLUME 95 DECEMBER 1982 903-908 © The Rockefeller University Press • 0021-9525/82/12/0903/06 $1.(30 9 0 3 soybean trypsin inhibitor from Worthington Biochemical Co. (Freehold, N J). 4,5-'~H-L-leucine (5 mCi/ml, 45 Ci/mmol) was obtained from Schwartz/Mann (Orangeburg, NY); carrier-free a2Pi (Ha32PO4) (285 Ci/mg; 1 Ci = 3.7 x 10 m becquerels) was obtained from New England Nuclear (Boston, MA). Cholecystokinin octapeptide (CCK-8) and synthetic secretin were generous gifts from Dr. Miguel Ondetti (Squibb Institute for Medical Research, Princeton, N J). A23187 was a generous gift from Dr. Robert L. Hamill of Eli Lilly Research Laboratories

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Cite this paper

@inproceedings{Freedman2002HormoneinducedPP, title={Hormone-induced Protein Phosphorylation. I. Relationship between Secretagogue Action and Protein Phosphorylation in Intact Cells from the Exocrine Pancreas and Parotid Endogenous}, author={Steven D. Freedman and James D. Jamieson}, year={2002} }