Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture.

Abstract

The regulation of proton transport and cytosolic pH was studied in rat papillary collecting duct (PCD) cells in culture using a pH-sensitive fluorescence probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on glass coverslips and dipped in a HC03 --free medium, pH 7.40. The resting intracellular pH (pHi) was 7.16 + 0.03 (n = 20). When PCD cells had been acidified by pretreatment with NH4C1, pHi immediately recovered toward the resting value. Two mechanisms participated in this recovery: a Na + -dependent mechanism which could be inhibited by amiloride (indicative of Na+-H+ exchanger) and a Na+-independent process (a proton ATPase). The pHi recovery from acid loading was inhibited by amiloride to about 55% of the control recovery (half maximal effect at 100 µM). The rate of pHi recovery after the readdition of Na+ to a sodium-free medium exhibited saturation kinetics (half maximal rate at 28 mM). Dicyclohexylcarbodiimide (DCCD), an inhibitor of a plasma membrane proton ATPase, and the depletion of cellular ATP induced by 2 mM potassium cyanide (KCN) also partially inhibited the rate of pHi recovery after cell acidification with a NH4C1 load. When PCD cells were treated with 1 mM DCCD, amiloride almost completely inhibited pH, recovery. Amiloride and the removal of external Na+ had induced a gradual fall in pHi to a new resting value and rapidly recovered when Na+ was added. We conclude that PCD cells grown in culture have at least two proton transport mechanisms: a Na+-H+ exchanger and a plasma membrane proton ATPase. The kinetics of these processes can be reliably assessed by the pH-sensitive fluorescent probe, BCECF. Both the Na+-H+ exchanger and the plasma membrane proton ATPase may contribute to urinary acidification.

Differences in regulation of pHi in large (≥10 nuclei) and small (≤5 nuclei) osteoclasts

“…In the Na ϩ -dependent category of large osteoclasts, pH i recovery was only partially inhibited by amiloride and bafilomycin A 1 , but when amiloride and bafilomycin A 1 were added together, pH i recovery was completely inhibited. The presence and activity of both a V-ATPase and Na ϩ /H ϩ exchanger in the same cell has also been reported in other cell types, i.e., alveolar macrophages, rabbit outer medullary collecting duct cells, rat papillary collecting duct cells, and guinea pig pancreatic duct cells (5,12,23,48). The findings of De Ondarza and Hootman (12) in guinea pig pancreatic duct cells were similar to ours in that neither amiloride nor bafilomycin A 1 alone significantly inhibited pH i recovery from an acid load, whereas both inhibitors added together completely abolished this recovery.…”