Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels.
We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.
Devor
D C
DC
Department of Cell Biology and Physiology, University of Pittsburgh, Pennsylvania 15261, USA. dd2+@pitt.edu
Singh
A K
AK
Gerlach
A C
AC
Frizzell
R A
RA
Bridges
R J
RJ
D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000037:antagonists & inhibitors* / Q000502:physiology; D002994:Clofibrate / Q000494:pharmacology; D003022:Clotrimazole / Q000494:pharmacology*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004553:Electric Conductivity; D007413:Intestinal Mucosa / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D011188:Potassium / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism*; D012413:Rubidium / Q000493:pharmacokinetics
D001562:Benzimidazoles; D002120:Calcium Channel Agonists; D002712:Chlorides; D015221:Potassium Channels; D003022:Clotrimazole; D002994:Clofibrate; C102635:1-ethyl-2-benzimidazolinone; D012413:Rubidium; D011188:Potassium; D002118:Calcium