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  • Title: Stimulation of Na+,K+,Cl- cotransport by forskolin-activated adenylyl cyclase in fetal human nonpigmented epithelial cells.
    Author: Crook RB, Polansky JR.
    Journal: Invest Ophthalmol Vis Sci; 1994 Aug; 35(9):3374-83. PubMed ID: 7520028.
    Abstract:
    PURPOSE: To determine the stoichiometry of Na+,K+,Cl- cotransport in fetal human nonpigmented ciliary epithelial cells and the effect of forskolin, an adenylyl cyclase activator, on Na+,K+,Cl- cotransport. METHODS: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured human nonpigmented epithelial (NPE) monolayers. RESULTS: The dependence of ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake upon Na+, K+, and Cl- concentrations was determined. Maximal uptake was observed at about 12 mM, 20 mM, and 120 mM of these ions, respectively. Analysis by Hill plot suggested that the stoichiometry of Na+,K+,Cl- cotransport is 1:1:2, making this an electroneutral process. Na+,K+,Cl- cotransport was found to be stimulated approximately 1.5- to 2-fold after incubation of cells for 15 minutes with 1 microM forskolin. neither ouabain-sensitive 86Rb+ uptake nor bumetanide-insensitive, ouabain-insensitive uptake was affected. 8-Bromoadenosine cAMP and 8-chlorophenylthio cAMP at 1 mM stimulated Na+,K+,Cl- cotransport approximately 30% to 40%, whereas 1,9 dideoxyforskolin, a non-adenylyl cyclase-activating analogue of forskolin, had little effect. Stimulation of Na+,K+,Cl- cotransport by forskolin was blocked by prior exposure of cells to 10 microM H-89, a protein kinase A inhibitor. Stimulation by forskolin was also observed in the presence of either 1 mM DIDS, 30 microM NPPB, 3 mM DPC, or 5 mM BaCl2, although all four channel blockers inhibited Na+,K+,Cl- cotransport to various degrees. CONCLUSIONS: The data suggest that the human NPE Na+,K+,Cl- cotransporter transports Na+, K+, and Cl- in the ratio of 1:1:2. Activation of adenylyl cyclase stimulates Na+,K+,Cl(-)-cotransport via a mechanism involving protein kinase A. Reduction of Na+,K+,Cl- cotransport by chloride channel blockers raises the possibility that activities of some ion channels can influence the rate of ion influx via Na+,K+,Cl- cotransport.
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