Multiple G-Protein-dependent Pathways Mediate the Antisecretory Effects of Somatostatin and Clonidine in the HT29-19A Colonic Cell Line


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Abstract

Using the functionally differentiated colonic cell line, HT29-19A, we have examined sites at which inhibitory G-proteins mediate the antisecretory actions of somatostatin (SST) and the alpha2-adrenergic agonist, clonidine (CLON) at the epithelial level. Both agents caused a dose-dependent inhibition (EC50: SST 35 nM; CLON 225 nM) of Cl- secretion (assessed by changes in short circuit current) activated by cAMP-mediated agonists, PGE2 and cholera toxin. Inhibition was accompanied by a reduction in intracellular cAMP accumulation and could be blocked by pretreatment with pertussis toxin at a concentration (200 ng/ml) which activated ADP-ribosylation of a 41-kD inhibitory G protein in HT29-19A membranes. Secretion stimulated by the permeant cAMP analogue, dibutyryl cAMP, was also inhibited by SST and CLON (30-50%; P < 0.005), indicating additional inhibitory sites located distal to cAMP production. Both agents were effective inhibitors of secretion mediated through the Ca2+ signaling pathway. SST (1 micromolars) and CLON (10 micromolars) reduced the Isc response to the muscarinic agonist, carbachol, by 60-70%; inhibition was reversed in pertussis toxin-treated cells. These effects did not, however, involve inhibition of the carbachol-induced increase in cellular inositol 1,4,5-trisphosphate levels or the rise in cytosolic calcium, (Ca)i. Inhibition by SST of secretion induced by phorbol 12,13 dibutyrate but not by the calcium agonist, thapsigargin, suggests that SST may act at a distal inhibitory site in the Ca2+-dependent secretory process activated by protein kinase C. We conclude that SST and alpha2-adrenergic agonists can act directly on intestinal epithelial cells to exert a comprehensive inhibition of Cl- secretion mediated through both cAMP and Ca2+/protein kinase C signaling pathways. Inhibition is mediated via pertussis toxin-sensitive G-proteins at sites located both proximal and distal to the production of second messengers. (J. Clin. Invest. 1993. 92:603-611.) Key words: alpha2-adrenergic. cyclic adenosine monophosphate. inhibitory G-protein. intestinal epithelial cell. ion secretion. somatostatin

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