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 (G(Cl)), the symport inhibition might be merely due to a short circuit of the symport by G(Cl) 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 (a(i,Cl) and a(i,Na)) with selective theta microelectrodes and molecular biology methods were used. The 13Cl- 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 G(Cl) activation or abolition. After HCTZ treatment the decrease in a(i,Cl) (measured as the initial rate or in 3 min) was larger than the decrease in a(i,Na). The difference was reduced to one third in a group of epithelia in which the elicited G(Cl) was reduced to one third; moreover it was abolished in any case when G(Cl) was abolished with 10(-4) M SITS. The SITS-insensitive rate of a(i,Cl) decrease was equal to that of the a(i,Na) decrease in any case. Thus the a(i,Cl) decrease displays a component dependent on G(Cl) 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 G(Cl) 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.