Jentsch T J, Stahlknecht T R, Hollwede H, Fischer D G, Keller S K, Wiederholt M
J Biol Chem. 1985 Jan 25;260(2):795-801.
22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.
在哇巴因（10⁻⁴M）存在的情况下，研究了培养的牛角膜内皮细胞汇合单层对²²Na⁺的摄取，以抑制钠的主动排出。在碳酸氢盐盐溶液中，氨氯吡脒（10⁻³M）或4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸（SITS）（10⁻³M）均可使摄取减少到相似程度。在氨氯吡脒存在下，经SITS预处理的细胞摄取进一步减少。在含有哇巴因（10⁻⁴M）和氨氯吡脒（10⁻³M）的盐溶液中，对SITS敏感的摄取进行了进一步表征。它绝对依赖于碳酸氢盐，其他质膜可渗透缓冲剂（50 mM乙酸盐或25 mM甘氨二嗪）无法替代。它是碳酸氢盐和钠浓度的饱和函数。半数最大通量出现在3至7 mM HCO₃之间（在151 mM Na时）和35至60 mM Na之间（在28 mM HCO₃时）。与富含钠的细胞相比，钠缺乏细胞的摄取减少，并且²²Na⁺负载细胞中对SITS敏感的²²Na⁺流出到无钠培养基中的量少于流出到钠盐水的量，表明钠的反式刺激。在不存在碳酸氢盐的情况下研究了氨氯吡脒敏感途径，以抑制通过SITS敏感途径的摄取。²²Na⁺进入钠缺乏细胞的摄取在pH 6至8范围内随细胞外pH急剧增加，并且可被10⁻³M但不能被10⁻⁵M氨氯吡脒大量阻断。得出的结论是，牛角膜内皮细胞至少拥有两种不同的钠摄取途径，氨氯吡脒敏感的²²Na⁺通量由Na⁺/H⁺反向转运体介导，而SITS敏感过程可能与先前电生理研究推测的碳酸氢盐-钠共转运系统相同。
