Echevarría M, Kuang K, Iserovich P, Li J, Preston G M, Agre P, Fischbarg J
Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Am J Physiol. 1993 Nov;265(5 Pt 1):C1349-55. doi: 10.1152/ajpcell.1993.265.5.C1349.
Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)
培养的牛角膜内皮细胞（CBCEC）将液体从基底表面运输至顶端表面。在本研究中，我们检测了这些细胞的质膜是否存在水通道。我们将BCEC培养在玻璃板上，并监测细胞散射光的强度（IS）。我们测定了在受到10%低渗刺激时IS变化的动力学常数（k），以计算质膜的渗透系数（Pf）。在37℃时，我们发现k值为0.68±0.07 s-1，Pf为93.3±33微米/秒（n = 13）。巯基试剂对氯汞苯磺酸盐（pCMBS；1 mM）使Pf降低了75%；5 mM二硫苏糖醇可逆转这种抑制作用。在10 - 37℃范围内Pf的活化能（Ea）为4.7±0.7千卡/摩尔（n = 5）。高Pf值、pCMBS的抑制作用以及低Ea强烈提示存在水通道。因此，我们测试了将从CBCEC制备的聚腺苷酸加尾RNA（poly(A)+ RNA）注射到非洲爪蟾卵母细胞中是否会导致水通道的表达。注射后四天，我们根据暴露于低渗培养基时体积增加的速率测量了卵母细胞的Pf值。在注射50 nl水的对照卵母细胞中，Pf为13.4±0.3微米/秒（n = 63）。在注射poly(A)+ RNA（50 ng/卵母细胞，溶于50 nl水中）的卵母细胞中，Pf为40.9±1.6微米/秒（n = 72）。（摘要截断于250字）
