Hydrochlorothiazide enhances the apical Cl− backflux in rabbit gallbladder epithelium: Radiochemical analysis

Hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na+-Cl− symport and to depolarize the apical membrane potential in the rabbit gallbladder epithelium. The depolarization was likely related to the opening of a Cl− conductance. To better understand whether an apical Cl− leak is involved in the mechanism of action of HCTZ, the transapical Cl− backflux was measured radiochemically by the washout technique. The gallbladder wall, pretreated with pronase on the serosal side to homogenize the subepithelium, was loaded with 36Cl− on the luminal side; mucosal and serosal 36Cl− effluxes (J m , J s ) were then measured every 2 min. The pretreatment with pronase did not alter the membrane potentials and the selectivity of the epithelium. Under control conditions and the tissue in steady-state, J m and J s time courses were each described by two exponential decays (A,B); the rate constants, k A and k B , were 0.71 ±0.03 and 0.16±0.01 min−1, respectively, and correspondingly the half-times (t 1 2 , t 1 2 ) were 1.01±0.05 and 5.00±0.44 min (n=10); these parameters were not significantly different for J m and J s time courses. J s was always greater than J m (J s /J m =2.02±0.22 and 1.43 ±0.17 for A and B decays). Under SCN− treatment in steady-state conditions, both J m and J s time courses were described by only one exponential decay, the component B being abolished. Moreover t 1 2 was similar to that predictable for the subepithelium. It follows that it is the component B which exits the epithelial compartment. Based on the intracellular specific activity and 36Cl− J at 0 min time of the washout experiment, the cell-lumen Cl− backflux in steady-state was calculated to be equal to about 2 μmol cm−2hr−1, in agreement with the value indirectly computable by other techniques. The experimental model was well responsive to different external challenges (increases in media osmolalities; luminal treatment with nystatin). HCTZ (2.5 · 10−4 m) largely increased 36Cl− J . The increase was abolished by luminal treatment with 10−4 m SITS, which not only brought back the efflux time courses to the ones observed under control conditions but even increased J s /J m of the cellular component, an indication of a reduced J . It is concluded that HCTZ opens an apical, SITS-sensitive Cl− leak, which contributes to dissipate the intracellular Cl− accumulation and to inhibit the NaCl transepithelial transport. Moreover, the drug is likely to reduce the basal electroneutral Cl− backflux supported by Na+-Cl− cotransport, in agreement with the inhibition of the cotransport itself.