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

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pH(i)) was estimated with 2',7'-bis-(2- carboxy-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 isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 μmol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 μmol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca²⁺](i) fivefold increased the uptake of NH₄⁺ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca²⁺](i);3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na⁺-K⁺-2Cl^--cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly, explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.