Crook R B, Polansky J R
Department of Ophthalmology, University of California, San Francisco 94143.
Invest Ophthalmol Vis Sci. 1994 Aug;35(9):3374-83.
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.
86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured human nonpigmented epithelial (NPE) monolayers.
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.
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.
确定人胎儿无色素睫状上皮细胞中Na⁺、K⁺、Cl⁻共转运的化学计量关系，以及腺苷酸环化酶激活剂福斯高林对Na⁺、K⁺、Cl⁻共转运的影响。
使用⁸⁶Rb⁺作为K⁺的标志物，研究哇巴因不敏感、布美他尼敏感的⁸⁶Rb⁺在培养的人无色素上皮（NPE）单层中的摄取。
确定了哇巴因不敏感、布美他尼敏感的⁸⁶Rb⁺摄取对Na⁺、K⁺和Cl⁻浓度的依赖性。在这些离子浓度分别约为12 mM、20 mM和120 mM时观察到最大摄取。通过希尔图分析表明，Na⁺、K⁺、Cl⁻共转运的化学计量关系为1:1:2，这是一个电中性过程。发现用1 μM福斯高林孵育细胞15分钟后，Na⁺、K⁺、Cl⁻共转运受到约1.5至2倍的刺激。哇巴因敏感的⁸⁶Rb⁺摄取和布美他尼不敏感、哇巴因不敏感的摄取均未受影响。1 mM的8-溴腺苷cAMP和8-氯苯硫基cAMP刺激Na⁺、K⁺、Cl⁻共转运约30%至40%，而福斯高林的非腺苷酸环化酶激活类似物1,9-二脱氧福斯高林作用很小。福斯高林对Na⁺、K⁺、Cl⁻共转运的刺激作用被细胞预先暴露于10 μM H-89（一种蛋白激酶A抑制剂）所阻断。在存在1 mM DIDS、30 μM NPPB、3 mM DPC或5 mM BaCl₂的情况下也观察到福斯高林的刺激作用，尽管这四种通道阻滞剂均不同程度地抑制了Na⁺、K⁺、Cl⁻共转运。
数据表明人NPE的Na⁺、K⁺、Cl⁻共转运体以1:1:2的比例转运Na⁺、K⁺和Cl⁻。腺苷酸环化酶的激活通过涉及蛋白激酶A的机制刺激Na⁺、K⁺、Cl⁻共转运。氯化物通道阻滞剂对Na⁺、K⁺、Cl⁻共转运的抑制增加了某些离子通道的活性可以影响通过Na⁺、K⁺、Cl⁻共转运的离子内流速率的可能性。
