title: The outwardly rectifying Cl- channel is not involved in cAMP-mediated Cl- secretion in HT-29 cells: evidence for a very-low-conductance Cl- channel.
abstract: The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl- channel (ORDIC) during cAMP-mediated Cl- secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl- channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-dinitro-2,2'-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ODIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl- secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50 = 0.5 +/- 0.3 mumol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl- secretion unless high concentrations were used (IC50 = 240 +/- 10 mumol/l). In order to exclude effects of NPPB other than on the apical Cl- channel, transepithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl- gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl- current showed a very-low-frequency Lorentzian noise component (fc = 1.4 +/- 0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D004594:Electrophysiology; D016257:Fura-2; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism* / Q000502:physiology; D009579:Nitrobenzoates / Q000494:pharmacology; D013267:Stilbenes / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D002712:Chlorides; D007473:Ion Channels; D009579:Nitrobenzoates; D013267:Stilbenes; D005576:Colforsin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D000242:Cyclic AMP; D002118:Calcium; D016257:Fura-2