Dual Regulatory Mechanisms of Proton Transport in Rat Papillary Collecting Duct Cells in Culture

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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 H⁺CO₃--free medium, pH⁺ 7.40. The resting intracellular pH⁺ (pH⁺_i) was 7.16±0.03 (n=20). When PCD cells had been acidified by pretreatment with NH⁺4Cl, pH⁺_i 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 pH⁺_i recovery from acid loading was inhibited by amiloride to about 55% of the control recovery (half-maximal effect at 100μM). The rate of pH⁺_i recovery after the readdition of Na⁺+ to a sodium-free medium exhibited saturation kinetics (half maximal rate at 28mM). Dicyclohexylcarbodiimide (DCCD), an inhibitor of a plasma membrane proton ATPase, and the depletion of cellular ATP induced by 2mM potassium cyanide (KCN) also partially inhibited the rate of pH⁺_i recovery after cell acidification with a NH⁺4Cl load. When PCD cells were treated with 1mM DCCD, amiloride almost completely inhibited pH⁺_i, recovery. Amiloride and the removal of external Na⁺+ had induced a gradual fall in pH⁺_i 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.