Wolosin J M, Alvarez L J, Candia O A
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029.
Am J Physiol. 1988 Nov;255(5 Pt 1):C595-602. doi: 10.1152/ajpcell.1988.255.5.C595.
Incubation of lenses with the acetoxymethyl ester of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) led to selective, prolonged entrapment of the deesterified, pH-sensitive, membrane-impermeable form of the dye (BCECF) in the lens epithelial cells allowing, thereby, for the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. Under anaerobic conditions in physiological solution, the epithelium maintained a pHi of 7.36 +/- 0.14 (mean +/- SD, n = 6 lenses). In the absence of CO2 and HCO3-, the pHi increased to 7.83 +/- 0.38 (n = 55). In this condition, either the removal of Na+ or the addition of amiloride to the medium resulted in a decrease of pHi. This acidification was increased in amiloride-inhibitable fashion when lenses were loaded with Na+ before extracellular Na+ removal. The extent of artificially generated cellular acidosis (induced by the "NH4+ loading" technique) was markedly decreased by the presence of extracellular Na+ or reverted on its addition when the ion was initially absent; the latter reversal was inhibited by amiloride. This inhibition was by-passed by monensin addition. The existence of a Na+-H+ antiport in the epithelial cells is concluded.
将晶状体与2,7 - 双羧乙基 - 5(6) - 羧基荧光素的乙酰氧基甲酯（BCECF - AM）一起孵育，导致脱酯后的、对pH敏感的、膜不可渗透形式的染料（BCECF）选择性且长时间地滞留在晶状体上皮细胞中，从而能够对完整晶状体上皮细胞内pH（pHi）进行连续荧光监测。在生理溶液中的厌氧条件下，上皮细胞维持的pHi为7.36±0.14（平均值±标准差，n = 6个晶状体）。在没有CO2和HCO3-的情况下，pHi升高至7.83±0.38（n = 55）。在这种条件下，去除Na+或向培养基中添加氨氯吡咪都会导致pHi降低。当在细胞外Na+去除之前晶状体被加载Na+时，这种酸化以氨氯吡咪可抑制的方式增加。人工产生的细胞酸中毒（由“NH4+加载”技术诱导）的程度在细胞外存在Na+时显著降低，或者当最初不存在该离子时，在添加Na+后恢复；后一种恢复被氨氯吡咪抑制。添加莫能菌素可绕过这种抑制作用。由此得出上皮细胞中存在Na+-H+反向转运体的结论。
