首页 | 本学科首页   官方微博 | 高级检索  
检索        


Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells
Authors:Kuang Kunyan  Li Yansui  Yiming Maimaiti  Sánchez José M  Iserovich Pavel  Cragoe E J  Diecke Friedrich P J  Fischbarg Jorge
Institution:Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, 630 West 168th St., New York, NY 10032, USA.
Abstract:The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.
Keywords:Na+ channel  EnaC  Na+ pump  fluorescent Na+ indicator  SBFI  stromal thickness  specular microscope  Na+-HCO3 cotransporters
本文献已被 ScienceDirect PubMed 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号