Lauf Peter K, Misri Sandeep, Chimote Ameet A, Adragna Norma C
Cell Biophysics Group, 054 Biological Sciences Bldg., Wright State Univ. Boonshoft School of Medicine, Dayton, OH 45435, USA.
Am J Physiol Cell Physiol. 2008 Mar;294(3):C820-32. doi: 10.1152/ajpcell.00375.2007. Epub 2008 Jan 9.
This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
本研究探讨了低渗溶液中人类晶状体上皮细胞（LECs）钾离子通量的性质。通过85Rb摄取法测定总离子通量、钠钾泵、氯离子依赖的钠钾-2氯（NKCC）、钾氯（KCC）协同转运以及钾通道，通过原子吸收分光光度法测定细胞内钾（Kc），并在暴露于哇巴因±布美他尼（钠钾泵和NKCC抑制剂）以及不同渗透压下的离子通道抑制剂（分别含有钠、钾或甲基-d-葡糖胺以及氯、氨基磺酸盐或硝酸盐）后通过重量法测定细胞内水分。还进行了逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹分析和免疫化学实验。在等渗（300 mosM）培养基中，约90%的总铷离子内流通过钠钾泵和NKCC发生，约10%通过KCC以及残余的泄漏途径发生。低渗培养基（150 mosM）使Kc降低，钾通透性和细胞内水分增加了16倍，但未能使NKCC失活以及激活KCC。在低渗培养基中，用蔗糖替代或使细胞外钾离子浓度升高至>57 mM，但铷离子或铯离子则不能，可防止Kc和水分流失。铷离子内流等于Kc的流失，二者均被克霉唑（IC50约为25 microM）阻断，部分被IK通道KCa3.1的抑制剂1-[(2-氯苯基)二苯基甲基]-1H-吡唑（TRAM-34）阻断，但不被其他钾通道或连接蛋白半通道阻滞剂阻断。在几种阴离子通道阻滞剂中，[二氢茚基]氧基]链烷酸（DIOA）、4-2(丁基-6,7-二氯-2-环戊基茚满-1-酮-5-基)氧基丁酸（DCPIB）和根皮素分别完全或部分抑制了Kc的流失和铷离子内流。RT-PCR和免疫化学证实除了KCC1、KCC2、KCC3和KCC4亚型外，还存在KCa3.1通道。显然，IK通道可能与容积敏感性外向整流氯离子通道并行，在LECs中发挥调节性容积减小作用。
