Nature of the neutral Na⁺-Cl⁻ coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na⁺/H⁺, Cl⁻/HCO ⁻₃ double exchange, hydrochlorothiazide-sensitive Na⁺-Cl⁻ symport and Na⁺-K⁺-2Cl⁻ cotransport are all involved

Summary

Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na⁺ transport was calculated from it), at 27°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 μl cm⁻² hr⁻¹ (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10⁻³ m concentration; the apparent IC₅₁₀ was about 2×10⁻⁴ 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⁻/HCO ⁻₃ 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.