Biphasic increase of apical Cl- conductance by muscarinic stimulation of HT-29cl.19A human colon carcinoma cell line: evidence for activation of different Cl- conductances by carbachol and forskolin.
The modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10(-4) M carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15 +/- 1 mV from a resting value of -52 +/- 1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22 +/- 1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11 +/- 1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6 +/- 1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4 +/- 0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl- conductance in the apical and basolateral membrane, the second phase by increased K+ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl- conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl- channels are involved in the carbachol response: one Ca2+ dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl- conductance. It is concluded that the short-lasting carbachol/Ca(2+)-dependent Cl- conductance is different from the forskolin-activated conductance. The increase of the Cl- conductance in the presence of forskolin by carbachol may be due to activation of different Cl- channels or to modulation of the PKA-activated Cl- channels by activated PKC.
Bajnath
R B
RB
Department of Experimental Zoology, University of Amsterdam, The Netherlands.
Dekker
K
K
Vaandrager
A B
AB
de Jonge
H R
HR
Groot
J A
JA
D002118:Calcium / Q000502:physiology; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D018118:Chloride Channels; D002712:Chlorides / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology*; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology; D014407:Tumor Cells, Cultured
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D011976:Receptors, Muscarinic; D005576:Colforsin; D002217:Carbachol; D000242:Cyclic AMP; D002118:Calcium