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 2A , t 1 2B ) 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 2A 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 m B at 0 min time of the washout experiment, the cell-lumen Cl– backflux in steady-state was calculated to be equal to about 2 mgrmol 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 m B . 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 m B . 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.