title: Regulation of the Na+-K+(NH4+)-2Cl- cotransporter of rat submandibular glands.
abstract: 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 micromol/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 NH4+ 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 NH4+. 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 NH4+ was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH4+ by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH4+ 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 ADPbetaS. ATP only slightly inhibited the increase of cyclic AMP (-22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the beta-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.
authors.affiliation: Laboratoire de Biochimie générale et humaine, Institut de Pharmacie, Université libre de Bruxelles, Brussels, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Chaïb
authors.forename: E
authors.initials: E
authors.lastname: Kabré
authors.forename: M
authors.initials: M
authors.lastname: Métioui
authors.forename: M C
authors.initials: MC
authors.lastname: Franco
authors.forename: J P
authors.initials: JP
authors.lastname: Dehaye
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002021:Buffers; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003470:Culture Media / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D007545:Isoproterenol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology; D013363:Submandibular Gland / Q000166:cytology / Q000378:metabolism*
chemical_list: D002021:Buffers; D002352:Carrier Proteins; D003470:Culture Media; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D000643:Ammonium Chloride; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol; D002118:Calcium