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 (Jm, Js) 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, Jm and Js time courses were each described by two exponential decays (A, B); the rate constants, kA and kB, were 0.71 +/- 0.03 and 0.16 +/- 0.01 min-1, respectively, and correspondingly the half-times (tA1/2, tB1/2) were 1.01 +/- 0.05 and 5.00 +/- 0.44 min (n = 10); these parameters were not significantly different for Jm and Js time courses. Js was always greater than Jm (Js/Jm = 2.02 +/- 0.22 and 1.43 +/- 0.17 for A and B decays). Under SCN- treatment in steady-state conditions, both Jm and Js time courses were described by only one exponential decay, the component B being abolished. Moreover tA1/2 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- JBm at 0 min time of the washout experiment, the cell-lumen Cl- backflux in steady-state was calculated to be equal to about 2 mumol 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 x 10(-4) M) largely increased 36Cl- JBm. 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 Js/Jm of the cellular component, an indication of a reduced JBm. 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.(ABSTRACT TRUNCATED AT 400 WORDS)