Regulation of the Na+-K+(NH4+)-2Cl(-) cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to differen t concentrations of NH4Cl, and the variations of the intracellular concentr ation of calcium ([Ca2+](i)) and the intracellular pH (pH(i)) were measured using fura-2 and 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxy More than 5 mmol /l NH4Cl significantly increased the [Ca2+](i) without affecting the respon se to 100 mu mol/l carbachol. When exposed to 1 and 5 mmol/l NH4Cl, the cel ls acidified immediately. At 30 mmol/l, NH4Cl first alkalinized the cells a nd the pH(i) subsequently dropped. This drop reflects the uptake of NH4+ io ns that dissociate to NH3 and H+ in the cytosol. These protons are exchange d for extracellular sodium by the Na+/H+ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification ind uced by 1 mmol/l NH4Cl. Ouabain partly blocked the uptake of NH4+. In the c ombined presence of ouabain and bumetanide (an inhibitor of the Na+-K+-2Cl( -) cotransporter), 1 mmol/l NH4Cl alkalinized the cells. The contribution o f the Na/K ATPase and the Na+-K+-2Cl(-) cotransporter in the uptake of NH4 was independent of the presence of calcium in the medium. Isoproterenol in creased the uptake of NH4+ by the cotransporter. Conversely, 1 mmol/l extra cellular ATP blocked the basal uptake of NH4+ by the cotransporter. This in hibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADP beta S. ATP o nly slightly inhibited the increase of cyclic AMP (-22%) by isoproterenol b ut fully blocked the stimulation of the cotransporter by the beta-adrenergi c agonist. ATP increased the release of H-3-arachidonic acid from prelabele d cells but SK&F 96365, an imidazole-based cytochrome 9450 inhibitor, did n ot affect the inhibition by ATP. It is concluded that the activation of a p urinoceptor inhibits the basal and the cyclic AMP-stimulaied activity of th e Na+-K+ 2Cl(-) cotransporter. (C) 1999 Wiley-Liss, Inc.