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
✍ Scribed by Horst Fischer; Klaus -M. Kreusel; Beate Illek; Terry E. Machen; Ulrich Hegel; Wolfgang Clauss
- Book ID
- 104744891
- Publisher
- Springer
- Year
- 1992
- Tongue
- English
- Weight
- 990 KB
- Volume
- 422
- Category
- Article
- ISSN
- 0031-6768
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✦ Synopsis
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 C1-channel (ORDIC) during cAMP-mediated C1-secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured C1-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 ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced C1-secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to I retool/1. NPPB blocked the ORDIC at low concentrations (ICs0 = 0.5___0.3 gmol/1) by reducing its open probability, but NPPB did not block forskolin-induced C1-secretion unless high concentrations were used (ICs0 = 240_+ 10 gmol/l). In order to exclude effects of NPPB other than on the apical C1-channel, transepithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal CI-gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven C1-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. As revealed from fura-2 fluorescence measurements, forskolin-stimulated C1-secretion occurred in the absence of changes in intracellular Ca 2+ . Thus, we conclude that there is an api-