INHIBITION OF INTESTINAL CL- SECRETION BY CLOTRIMAZOLE - DIRECT EFFECT ON BASOLATERAL MEMBRANE K+ CHANNELS

We evaluated the effects of clotrimazole and clofibrate on Ca2+- and a denosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in t he colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO ) to activate the Ca2+-dependent K+ channel (K-Ca) in these cells to i nduce a sustained Cl- secretory current (I-sc). Clotrimazole potently inhibited the K-Ca-dependent I-sc, with an inhibition constant (K-i) o f 0.27 +/- 0.02 mu M. Clofibrate also inhibited the 1-EBIO-induced I-s c, albeit with lower affinity (K-i = 6.5 +/- 1.2 mu M). Clotrimazole ( 10 mu M) inhibited the I-sc response to the Ca2+-mediated agonist, car bachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with K-i values of 5.2 +/- 1.0 and 6.7 +/ - 1.1 mu M, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and c lofibrate on the basolateral K+ (I-K) and apical Cl- (I-Cl) currents f ollowing stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced I-K without affecting I-Cl. We determined the effects of clotrimazole and clofibr ate on K-Ca using Rb-86(+) uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced Rb-86(+) upt ake, with K-i values of 0.31 +/- 0.08 and 10.8 +/- 5.5 mu M, respectiv ely. Similarly, clotrimazole inhibited the Ca2+-induced Rb-86(+) uptak e with a K-i of 0.51 +/- 0.15 mu M. Charybdotoxin inhibited both the 1 -EBIO- and Ca2+-induced Rb-86(+) uptakes with similar affinities (K-i values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggestin g 1-EBIO and Ca2+ activate the same channel (K-Ca) in this assay. In e xcised, single-channel recordings both clotrimazole and clofibrate inh ibited K-Ca, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal K-C a, thereby inhibiting Cl- secretion. These results suggest that clotri mazole may be useful as an antidiarrheal.