Putney L K, Brandt J D, O'Donnell M E
Department of Human Physiology, University of California, Davis 95616-8644, USA.
Invest Ophthalmol Vis Sci. 1999 Feb;40(2):425-34.
Previous results from this laboratory showed that intracellular volume of trabecular meshwork (TM) cells is regulated by the Na-K-Cl cotransport system. Other studies suggest that TM cell volume, in turn, is a determinant of permeability across the TM. Given that a decrease in outflow facility across the TM is thought to be the primary cause of elevated intraocular pressure in primary open-angle glaucoma, the present study was conducted to investigate the possibility that Na-K-Cl cotransport function may be altered in glaucomatous TM cells compared with normal TM cells.
Normal and glaucomatous human TM cells were cultured from donor eyes and trabeculectomy specimens, respectively. Trabecular meshwork cell monolayers were evaluated for Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx using 86Rb as a tracer for K. Cotransporter protein expression was determined by western blot analysis, and intracellular volume was determined radioisotopically using [14C]urea and [14C]sucrose as markers of total and extracellular water space, respectively.
Na-K-Cl cotransport activity of glaucomatous TM cells was found to be reduced by 32% +/- 2% compared with that of normal TM cells, whereas western blot analyses showed that cotransporter protein expression in glaucomatous TM cells was reduced by 64% +/- 14% compared with expression in normal TM cells. Also, exposure of normal TM cells to 10 microM norepinephrine or 50 microM 8-bromo-3',5'-cyclic adenosine monophosphate was found to diminish Na-K-Cl cotransport activity, whereas these agents were without effect on glaucomatous TM cell cotransport. Finally, resting cell volume of glaucomatous TM cells was found to be increased compared with that of normal TM cells, whereas intracellular volume of both cell types was reduced after exposure to 10 microM benzmetanide or 10 microM bumetanide.
These findings indicate that Na-K-Cl cotransport function and regulation are altered in glaucomatous TM cells compared with that of normal TM cells. However, the observation that cell volume of glaucomatous TM cells is greater than that of normal TM cells, despite reduced Na-K-Cl cotransport activity, suggests that other volume-regulatory ion flux pathways may be involved in the reduced outflow of glaucoma.
本实验室先前的研究结果表明，小梁网（TM）细胞的细胞内体积受钠-钾-氯共转运系统调节。其他研究表明，TM细胞体积反过来又是跨TM通透性的决定因素。鉴于跨TM的房水流出易度降低被认为是原发性开角型青光眼眼压升高的主要原因，本研究旨在探讨与正常TM细胞相比，青光眼TM细胞中钠-钾-氯共转运功能是否可能发生改变。
分别从供体眼和小梁切除术标本中培养正常和青光眼患者的人TM细胞。评估小梁网细胞单层的钠-钾-氯共转运活性，以哇巴因不敏感、布美他尼敏感的钾流入量来评估，使用86Rb作为钾的示踪剂。通过蛋白质印迹分析确定共转运蛋白表达，并使用[14C]尿素和[14C]蔗糖分别作为总体积和细胞外水空间的标志物，通过放射性同位素法测定细胞内体积。
发现青光眼TM细胞的钠-钾-氯共转运活性比正常TM细胞降低了32%±2%，而蛋白质印迹分析表明，青光眼TM细胞中共转运蛋白的表达比正常TM细胞降低了64%±14%。此外，发现正常TM细胞暴露于10μM去甲肾上腺素或50μM 8-溴-3',5'-环磷酸腺苷会降低钠-钾-氯共转运活性，而这些药物对青光眼TM细胞的共转运没有影响。最后，发现青光眼TM细胞的静息细胞体积比正常TM细胞增加，而两种细胞类型在暴露于10μM苄甲噻嗪或10μM布美他尼后细胞内体积均减小。
这些发现表明，与正常TM细胞相比，青光眼TM细胞中的钠-钾-氯共转运功能和调节发生了改变。然而，尽管钠-钾-氯共转运活性降低，但青光眼TM细胞的体积大于正常TM细胞，这一观察结果表明，其他体积调节离子通量途径可能参与了青光眼房水流出减少的过程。
