Nucleotide receptors regulate membrane ion transport in renal epithelial cells

Regulation of plasma membrane ion transport by endogenous purinergic receptors was assessed in a distal renal (A6) cell line. Nucleotide analogues stimulated Na-K-Cl cotransport activity with relative potencies of ATP > UTP > ATPγS > 2-methylthio-ATP = α,β-methylene ATP. Activation of nucleotide receptors with extracellular ATP and nucleotide analogues increased intracellular calcium concentration ([Ca²⁺](i)) primarily by release of intracellular calcium stores, with relative potency of agonists similar to that seen for stimulation of Na-K-Cl cotransport. Neither the change in [Ca²⁺](i) nor the stimulation of cotransport was abolished by the adenosine receptor antagonist 8-{4-[N-(2-aminoethyl)carbamoylmethoxy]-phenyl}-1,3- dipropylxanthine (XAC). In contrast to the adenosine A₂ receptor agonist 5'- N-ethylcarboxamidoadenosine, nucleotide analogues had no discernible effect on cytosolic adenosine 3',5'-cyclic monophosphate levels or adenylyl cyclase activity. To address possible mechanisms for stimulation of Na-K-Cl cotransport by the nucleotide receptor, ¹²⁵I efflux and patch-clamp studies were used to measure chloride secretion. ATP and ionomycin markedly enhanced ¹²⁵I efflux and whole cell currents, consistent with activation of chloride conductance pathways. Diphenylamine-2-carboxylate, a chloride channel blocker, eliminated the effects of ionomycin, forskolin, adenosine, and ATP on Na-K-Cl cotransport. This study demonstrates that nucleotide receptors in this model of renal epithelium initiate distinct regulation of Na-K-Cl cotransport. Nucleotide receptors may effect their responses through primary activation of membrane chloride channels.