Apical Na+-Cl- symport in rabbit gallbladder epithelium: a thiazide-sensitive cotransporter (TSC).

Cl− apically enters the epithelium of rabbit gallbladder by a Na+-Cl− symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl− conductance (GCl), the symport inhibition might be merely due to a short circuit of the symport by GCl rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl− uptake, electrophysiological determinations of intracellular Cl− and Na+ activities (ai,Cl and ai,Na) with selective theta microelectrodes and molecular biology methods were used. The 36Cl− uptake proved to be a measurement of the apical unidirectional Cl− influx (Jmc) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by GCl activation or abolition. After HCTZ treatment the decrease in ai,Cl (measured as the initial rate or in 3 min) was larger than the decrease in ai,Na. The difference was reduced to one third in a group of epithelia in which the elicited GCl was reduced to one third; moreover it was abolished in any case when GCl was abolished with 10−4m SITS. The SITS-insensitive rate of ai,Cl decrease was equal to that of the ai,Na decrease in any case. Thus the ai,Cl decrease displays a component dependent on GCl activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely GCl activation and Na+-Cl− symport inhibition. This Na+-Cl− symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.