Sodium‐Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit

The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na + ‐dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO 3 − plus 95% O 2 /5% CO 2 . Intracellular pH (pH i ) was determined from the steady‐state distribution of [ 14 C]benzoate. Recovery after acidification with NH 4 Cl was rapid ( t 1/2 = 5 min) and was dependent on external Na + (EC 50 = 12 m M ). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na + /H + antiport, blocked 80% of recovery when [Na + ] was 5 m M with IC 50 values of 100 n M . However, neither drug blocked recovery in normal [Na + ]. 4,4′‐Diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), an inhibitor of Cl − /HCO 3 − antiports, blocked recovery of pH i in a dose‐related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na + channel, nor with dimethylbenzamil, an inhibitor of Na + /Ca 2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH‐clamped with nigericin and KCl, the initial rate of 22 Na + uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na + ] with an EC 50 value of 8 m M , and was inversely related to the pH of the medium. The maximal inhibition of 22 Na + uptake by hexamethyleneamiloride was 60% with an IC 50 value of 76 n M . We conclude that both the Na + /H + antiport and a DIDS‐sensitive bicarbonate‐dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na + /H + exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.