CA2-MOBILIZING AGONISTS POTENTIATE FORSKOLIN- AND VIP-STIMULATED CAMPPRODUCTION IN HUMAN COLONIC CELL-LINE, HT29-CL.19A - ROLE OF [CA2+](I) AND PROTEIN-KINASE-C()

This study has examined the involvement of the Ca2+-signalling pathway in the regulation of agonist-stimulated cAMP responses in the human c olonic adenocarcinoma cell line, HT29-cl.19A. The muscarinic agonist, carbachol (CCh) stimulated rapid increases in cellular IP3 and cytosol ic Ca2+, [Ca2+](i) in HT29-c1.19A cells. These were accompanied by a s mall but significant increase in basal cAMP levels and a marked (3-4-f old) potentiation of both forskolin- (FSK) and VIP-stimulated cAMP gen eration. Similar effects were observed with two other Ca2+-mobilising agonists, neurotensin and ATP. The failure of CCh to elicit potentiati on of adenylate cyclase in broken cell preparations indicated an indir ect action. Potentiation could be mimicked by the calcium ionophore, i onomycin, and thapsigargin and inhibited 70-90% by depleting intracell ular Ca2+ stores suggesting that a rise in [Ca2+](i) is the primary me diator of this response. In contrast, increasing [Ca2+](i) levels to > 500 nM caused a significant inhibition of FSK-stimulated cAMP generat ion. The involvement of protein kinase C (PKC) was also assessed. PKC activators phorbol 12,13 dibutyrate (PDB) and 1-oleoyl-2-acetyl glycer ol (GAG) potentiated FSK-stimulated cAMP production by 50-70% though P DB 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 staur osporine and H7 reduced by approximate to 25% the CCh-induced potentia tion of FSK-stimulated cAMP generation. In conclusion, these results s uggest that stimulation of the phosphoinositidase C pathway in HT29-c1 .19A colonocytes induces a 'sensitisation' of the adenylate cyclase sy stem 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 r ole.