Small-conductance Cl- channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP.
The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
Kunzelmann
K
K
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
Kubitz
R
R
Grolik
M
M
Warth
R
R
Greger
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D002277:Carcinoma / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism; D003470:Culture Media; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D007038:Hypotonic Solutions; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009496:Neurotensin / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D009994:Osmolar Concentration; D014407:Tumor Cells, Cultured
D002712:Chlorides; D003470:Culture Media; D007038:Hypotonic Solutions; D007473:Ion Channels; D009579:Nitrobenzoates; C016276:8-bromocyclic GMP; D009496:Neurotensin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D015759:Ionomycin; D000255:Adenosine Triphosphate; D002217:Carbachol; D006152:Cyclic GMP; D002118:Calcium