title: Stimulation of Na+,K+,Cl- cotransport by forskolin-activated adenylyl cyclase in fetal human nonpigmented epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, University of California, San Francisco 94143.
authors.forename: R B
authors.initials: RB
authors.lastname: Crook
authors.forename: J R
authors.initials: JR
authors.lastname: Polansky
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D002924:Ciliary Body / Q000166:cytology / Q000196:embryology; D005576:Colforsin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D005333:Fetus; D006801:Humans; D007473:Ion Channels / Q000037:antagonists & inhibitors; D008565:Membrane Proteins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000196:embryology; D011188:Potassium / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D005576:Colforsin; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D012413:Rubidium; D011188:Potassium