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.
Warhurst
G
G
Department of Medicine, University of Manchester, Hope Hospital, Salford, UK.
Fogg
K E
KE
Higgs
N B
NB
Tonge
A
A
Grundy
J
J
D000230:Adenocarcinoma / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000737:chemistry; D000242:Cyclic AMP / Q000096:biosynthesis*; D004357:Drug Synergism; D006801:Humans; D010727:Phosphoric Diester Hydrolases; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*; D014970:Xanthines / Q000494:pharmacology
D002147:Calmodulin; D014970:Xanthines; D005576:Colforsin; C008514:methylxanthine; D014660:Vasoactive Intestinal Peptide; D002217:Carbachol; D000242:Cyclic AMP; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D002118:Calcium