STIMULATION OF NA- COTRANSPORT BY FORSKOLIN-ACTIVATED ADENYLYL-CYCLASE IN FETAL HUMAN NONPIGMENTED EPITHELIAL-CELLS(,K+,CL)

Purpose. To determine the stoichiometry of Na+,K+,Cl(-)cotransport in fetal human nonpigmented ciliary epithelial cells and the effect of fo rskolin, an adenylyl cyclase activator, on Na+,K+,Cl(-)cotransport. Me thods. Rb-86(+) as a marker for K+ was used to study ouabain-insensiti ve, bumetanide-sensitive Rb-86(+) uptake in cultured human nonpigmente d epithelial (NPE) monolayers. Results. The dependence of ouabain-inse nsitive, bumetanide-sensitive Rb-86(+) uptake upon Na+, K+, and Cl- co ncentrations was determined. Maximal uptake was observed at about 12 m M, 20 mM, and 120 mM of these ions, respectively. Analysis by Hill plo t suggested that the stoichiometry of Na+,K+,Cl(-)cotransport is 1:1:2 , making this an electroneutral process. Na+,K+,Cl(-)cotransport was f ound to be stimulated similar to 1.5- to 2-fold after incubation of ce lls for 15 minutes with 1 mu M forskolin. Neither ouabain-sensitive Rb -86(+) uptake nor bumetanide-insensitive, ouabain-insensitive uptake w as affected. 8-Bromoadenosine cAMP and 8-chloro-phenylthio cAMP at 1 m M stimulated Na+,K+,Cl(-)cotransport similar to 30% to 40%, whereas 1, 9 dideoxyforskolin, a non-adenylyl cyclase-activating analogue of fors kolin, had little effect. Stimulation of Na+,K+,Cl(-)cotransport by fo rskolin was blocked by prior exposure of cells to 10 mu M H-89, a prot ein kinase A inhibitor. Stimulation by forskolin was also observed in the presence of either 1 mM DIDS, 30 mu M NPPB, 3 mM DPC, or 5 mM BaCl 2, although all four channel blockers inhibited Na+,K+,Cl(-)cotranspor t 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(-)cotransp ort via a mechanism involving protein kinase A. Reduction of Na+,K+,Cl (-)cotransport by chloride channel blockers raises the possibility tha t activities of some ion channels can influence the rate of ion influx via Na+,K+,Cl-cotransport.