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 Cl-36(-) on the luminal side; mucosal and serosal Cl-36(-) 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)(A), t(1/2)(B)) 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)(m)/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)(A) 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 Cl-36(-) 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 mu mol cm(-2)hr(-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 Cl-36(-) 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.