ISSN:
1432-2013
Keywords:
Epithelial monolayers
;
HT-29/B6 cells
;
Single-channel patch clamp
;
Ussing chamber
;
Noise analysis
;
Fura-2 imaging
;
NPPB
;
DNDS
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
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 ORDIC. 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 μmol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl− secretion unless high concentrations were used (IC50=240±10 μmol/l). In order to exclude effects of NPPB other than on the apical Cl− channel, trans-epithelial 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 (f c=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 Cl− secretion occurred in the absence of changes in intracellular Ca2+. Thus, we conclude that there is an apical Cl− channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl− secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl− channel was calculated as the conductive site in the apical membrane during forskolin stimulation.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00370415
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