Activation of the Na+-K+(NH4+)-2Cl(-)-cotransporter from rat submandibularglands in response to VIP

A cellular suspension from rat submandibular glands was prepared with colla genase. The intracellular pH (pH(i)) was estimated with 2',7'-bis-(2-carbox y-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH4Cl, the pH(i ) transiently increased (diffusion of NH3) and then dropped (influx of NH4). Isoproterenol increased 2.5-fold the rate of NH4+ influx; bumetanide, an inhibitor of the Na+-K+-2Cl(-)-cotransporter blocked the response to isopr oterenol, confirming that the beta-adrenergic agonist stimulated the cotran sporter. Forskolin (1 mu mol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 mu mol/L) also increased the activity of the cotransporter. Cy clic AMP rather than calcium was the mediator of this activation since 1) c arbachol which increased the [Ca2+](i) fivefold increased the uptake of NH4 + by only 50%; 2) only high concentrations of VIP significantly increased t he [Ca2+](i); 3) incubation in the presence of EGTA had no effect on the re sponse to VTP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransport er by VIP, forskolin, and isoproterenol was inhibited by H8, n inhibitor of cAMP-dependent protein kinase. It is concluded that the Na+-K+-2Cl(-)-cotr ansporter of rat submandibular glands is activated by isoproterenol, forsko lin, and neuropeptides of the VIP family by a mechanism involving cAMP-depe ndent processes. The activation of the cotransporter by VIP could partly ex plain the potentiating effect of VIP on the response to sialagogues like su bstance P or muscarinic agonists. (C) 1998 Elsevier Science Inc.