Reduction in sensitivity to Cl^- channel blockers by HCO₃^--CO₂ in rabbit cortical collecting duct

We examined the ability of HCO₃^--CO₂ to modify the potency of Cl^- channel blockers in the renal cortical collecting duct (CCD) for the following two reasons. 1) From a practical point of view, there is, to our knowledge, no information regarding the effect of the HCO₃^--CO₂ buffer system on the potency of Cl^- channel blockers. 2) We showed in the companion manuscript [Am. J. Physiol. 257 (Cell Physiol. 26): C94-C101, 1989] that HCO₃^--CO₂ stimulates transepithelial anion exchange in the CCD. Based on precedent in the literature, we postulated that HCO₃^- stimulates the basolateral membrane Cl^- channel conductance. Here, we demonstrate that several Cl^- channel blockers can reduce CCD Cl^- self exchange when the solutions are buffered in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Concentrations of blockers producing 80% inhibition in HEPES, pH 7.4, produced only 20% inhibition in 25 mM HCO₃^--CO₂, pH 7.4. The ability of HCO₃^--CO₂ to reduce blocker potency had an IC₅₀ of only 2 mM. We also examined interactions of HCO₃^--CO₂ and blockers with regard to the principal cell basolateral Cl^- conductance. Blockers did not alter the Rb⁺ flux, a marker of K⁺ transport, but did reduce transepithelial conductance (G(T)), i.e., the blockers inhibited the principal cell basolateral Cl^- conductance. As was the case with intercalated cell anion exchange, G(T) measurements indicated that HCO₃^--CO₂ impaired the ability of Cl^- channel blockers to inhibit the principal cell Cl^- conductance. Two lines of evidence indicated that the effect is specific for the HCO₃^- (or CO₂) molecule: 1) a series of other small anions did not reproduce the HCO₃^- effect; and 2) isohydric elevation of HCO₃^- in high-K⁺ nigericin-treated cells reproduced the effect, indicating that an intracellular pH shift is not necessary to desensitize the Cl^- transport system to the blockers. These results imply that HCO₃^--CO₂ sensitivity represents an important characteristic of some Cl^- channels.