Involvement of calmodulin in Ca(2+)-activated K+ efflux in human colonic cell line, HT29-19A.

The receptor-mediated agonist, neurotensin (NT) stimulated Ba(2+)- and charybdotoxin-sensitive 86Rb (K+) efflux in the HT29-19A colonic cell line. Efflux was also stimulated by ionomycin and thapsigargin and could be abolished by incubation with the intracellular Ca2+ chelator, BAPTA. Together, these data suggest a rise in [Ca2+]i is prerequisite for activation of K+ efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca2+]i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca2+]i was transient, returning to baseline within 4-5 min, while efflux was sustained over a much longer period (> 12 min). Ca(2+)-activated 86Rb efflux was inhibited by pretreatment with calmodulin (CaM) antagonist, W7. W7 had no effect on basal efflux, but reduced both NT- and IM-activated efflux up to 80%, with a Ki of 38 microM. Other CaM antagonist inhibited efflux with an order of potency (TFP approximately W8 > W7 >> W5) consistent with inhibition of a CaM-dependent process. Inhibition by W7 was not abolished by ouabain or bumetanide, indicating its effects are not mediated by action upon K+ uptake processes. W7 did not inhibit NT-stimulated 125I efflux but significantly reduced efflux stimulated by the Ca2+ ionophore, ionomycin. NT-stimulated 86Rb+ efflux was localized to the basolateral membrane of HT29-19A monolayers grown on permeable supports. These data are consistent with the involvement of CaM in mediating Ca(2+)-dependent activation of K+ conductance in HT29-19A colonocytes.

Ca(2+)-mobilising agonists potentiate forskolin- and VIP-stimulated cAMP production in human colonic cell line, HT29-cl.19A: role of [Ca2+]i and protein kinase C.

This study has examined the involvement of the Ca(2+)-signalling pathway in the regulation of agonist-stimulated cAMP responses in the human colonic adenocarcinoma cell line, HT29-cl.19A. The muscarinic agonist, carbachol (CCh) stimualted rapid increases in cellular IP3 and cytosolic Ca2+, [Ca2+]i in HT29-cl.19A cells. These were accompanied by a small but significant increase in basal cAMP levels and a marked (3-4-fold) potentiation of both forskolin- (FSK) and VIP-stimulated cAMP generation. Similar effects were observed with two other Ca(2+)-mobilising agonists, neurotensin and ATP. The failure of CCh to elicit potentiation of adenylate cyclase in broken cell preparations indicated an indirect action. Potentiation could be mimicked by the calcium ionophore, ionomycin, and thapsigargin and inhibited 70-90% by depleting intracellular Ca2+ stores suggesting that a rise in [Ca2+]i is the primary mediator of this response. In contrast, increasing [Ca2+]i levels to > 500 nM caused a significant inhibition of FSK-stimulated cAMP generation. The involvement of protein kinase C (PKC) was also assessed. PKC activators phorbol 12,13 dibutyrate (PDB) and 1-oleoyl-2-acetyl glycerol (OAG) potentiated FSK-stimulated cAMP production by 50-70% though PDB markedly inhibited the cAMP response to the receptor-mediated cAMP agonist, VIP. Neither effect could be elicited by the inactive phorbol ester, 4 alpha-phorbol, 12,13 didecanoate (PDD). PKC inhibitors staurosporine and H7 reduced by approximately 25% the CCh-induced potentiation of FSK-stimulated cAMP generation. In conclusion, these results suggest that stimulation of the phosphoinositidase C pathway in HT29-cl.19A colonocytes induces a 'sensitisation' of the adenylate cyclase system resulting in a dramatic amplification of agonist-stimulated cAMP generation. Increases in [Ca2+]i appear to be an important mediator of potentiation though activation of PKC may also play a significant role.

Multiple G-protein-dependent pathways mediate the antisecretory effects of somatostatin and clonidine in the HT29-19A colonic cell line.

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 alpha 2-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 microM) and CLON (10 microM) 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 Ca(2+)-dependent secretory process activated by protein kinase C. We conclude that SST and alpha 2-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.