Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit Cl- secretion, their effects on Cl- transport and intracellular Ca(2+) concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl- sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca(2+)-sensitive fluorescent probe, fura-2AM were used to assess Cl- transport and [Ca2+]i, respectively. Basal Cl- transport (0.274 +/- 0.09 mM/sec) was inhibited significantly by the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 50 microM, 0.068 +/- 0.02 mM/sec; P < 0.001) and the Na+/K+/ 2Cl- cotransport inhibitor furosemide (1 microM, 0.137 +/- 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be l- > F- > or = Cl- > Br-. DPC inhibited l- influx by approximately 50%, F- influx by 80%, Cl-influx by 85%, and Br- influx by 90%. Furosemide significantly inhibited influx of Br- (84%) and Cl- (81%) but not of F- and l-. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl- transport. Cl- influx in mM/second was stimulated by 1 microM histamine (0.58 +/- 0.05), 10 microM neurotensin (2.07 +/- 0.32), 1 microM serotonin (1.63 +/- 0.28), and 0.1 microM of the Ca2+ ionophore A23187 (2.05 +/- 0.40). The Cl- permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl- influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl- permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl- transport by activating the Na+/K+/2Cl- cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl- channels and the cotransporter, and their actions are cyclooxygenase-independent.