Nature of the neutral Na(+)-Cl- coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl-/HCO3- double exchange, hydrochlorothiazide-sensitive Na(+)-Cl- symport and Na(+)-K(+)-2Cl- cotransport are all involved.

Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 4-acetamido-4'-iso-thiocyanostilbene-2,2'-disulfonate (SITS) did not affect fluid absorption, 25 mM SCN- abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3 +/- 1.6 (n = 21) to 8.6 +/- 1.6 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN- or bumetanide. Cl- influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10(-4) M bumetanide, but was slowly inhibited by 10(-3) M HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10(-4) M bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl- influx eliminated. Intracellular Cl- accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10(-3) M HCTZ, reached a minimum within 15-30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent. The results obtained show that Na(+)-Cl- symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport. Thus, the three forms of neutral Na(+)-Cl(-)-coupled transport so far evidenced in epithelia, Na+/H+, Cl-/HCO3- double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na(+)-Cl- symport and bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport, are all present in the apical membrane of rabbit gallbladder.