Regulation of the Na+-K+(NH +4)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH4Cl, and the variations of the intracellular concentration of calcium ([Ca2+]i) and the intracellular pH (pHi) were measured using fura‐2 and 2′,7′‐bis‐(2‐carboxy‐ethyl)‐5(6)‐carboxyfluorescein. More than 5 mmol/l NH4Cl significantly increased the [Ca2+]i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH4Cl, the cells acidified immediately. At 30 mmol/l, NH4Cl first alkalinized the cells and the pHi subsequently dropped. This drop reflects the uptake of NH +4 ions that dissociate to NH3 and H+ in the cytosol. These protons are exchanged for extracellular sodium by the Na+/H+ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH4Cl. Ouabain partly blocked the uptake of NH +4. In the combined presence of ouabain and bumetanide (an inhibitor of the Na+‐K+‐2Cl− cotransporter), 1 mmol/l NH4Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na+‐K+‐2Cl− cotransporter in the uptake of NH +4 was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH +4 by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH +4 by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl‐ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β‐adrenergic agonist. ATP increased the release of 3H‐arachidonic acid from prelabeled cells but SK&F 96365, an imidazole‐based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP‐stimulated activity of the Na+‐K+‐2Cl− cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley‐Liss, Inc.