Carbachol-stimulated phosphorylation of the Na-K-Cl cotransporter of avian salt gland. Requirement for Ca2+ and PKC Activation.

The Na-K-Cl cotransporter of avian salt gland is a membrane-bound 170-kDa protein that is phosphorylated in response to cAMP- and Ca(2+)-dependent secretogogues and is homologous to the Na-K-Cl cotransporter in another Cl-secreting epithelia; the shark rectal gland (Torchia, J., Lytle, C., Pon, D. J., Forbush, B., and Sen, A. K. (1992) J. Biol. Chem. 267, 25444-25450). In the present study we assess the role of Ca2+ and protein kinase C (PKC) activation on the phosphorylation of the Na-K-Cl cotransporter. Although the addition of ionomycin alone did not significantly stimulate cotransporter phosphorylation, concurrent addition of ionomycin plus the tumor promoter phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent increase in phosphorylation. Immunoprecipitation experiments, using a monoclonal antibody which specifically recognizes the cotransporter, suggested that the response to CCh or ionomycin plus PMA was quantitatively similar (5-fold) and was localized exclusively on serine residues. In contrast, when 4 alpha-phorbol was added in the presence of ionomycin, no stimulation was observed. To further assess the involvement of PKC on cotransporter phosphorylation the effects of protein kinase inhibitors were tested. Both staurosporine and calphostin C inhibited phosphorylation of the cotransporter at concentrations known to inhibit PKC, whereas the calmodulin antagonist W-7 had no significant effect. The requirement for Ca2+ was tested further by removing Ca2+ from the incubation medium and stimulating with CCh. Under these conditions, the CCh-stimulated phosphorylation was transient and, furthermore, could be completely inhibited by preloading the cells with the Ca2+ chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) prior to stimulation. The involvement of protein phosphatases on the phosphorylation of the Na-K-Cl cotransporter was also tested. The addition of okadaic acid stimulated phosphorylation by approximately 3-fold. Taken together these results suggest that the phosphorylation state of the cotransporter involves a dynamic interplay between changes in intracellular Ca2+, PKC, and protein phosphatase activities.