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.
Sahi
J
J
Department of Physiology and Biophysics, University of Illinois at Chicago 60612-7342, USA.
Wiggins
M P
MP
Gibori
G B
GB
Layden
T J
TJ
Rao
M C
MC
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D005665:Furosemide / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D009496:Neurotensin / Q000494:pharmacology; D010539:Permeability; D011806:Quinolinium Compounds / Q000378:metabolism; D011817:Rabbits; D012701:Serotonin / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D062367:ortho-Aminobenzoates; C085493:N-(ethoxycarbonylmethyl)-6-methoxyquinolinium; D012701:Serotonin; D000001:Calcimycin; D009496:Neurotensin; D005665:Furosemide; D006632:Histamine; C001209:fenamic acid; D002118:Calcium; D016257:Fura-2; D007213:Indomethacin