水通道蛋白1表达水平对牛角膜内皮细胞中钴（Ⅱ）通透性的影响。

Sun X C, Allen K T, Xie Q, Stamer W D, Bonanno J A

School of Optometry, Indiana University, Bloomington, Indiana 47401, USA.

Invest Ophthalmol Vis Sci. 2001 Feb;42(2):417-23.

PURPOSE

Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2).

METHODS

BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1.

RESULTS

Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease.

CONCLUSIONS

AQP1 does not contribute to PCO(2) in corneal endothelial cells.

目的

角膜内皮细胞的液体转运依赖于HCO(3)(-)和CO(2)通量。在卵母细胞表达系统和重组蛋白脂质体中进行的CO(2)通透性（P:CO(2)）测量表明，水通道蛋白AQP1可以转运CO(2)。然而，一种AQP1基因敲除小鼠模型并未显示出红细胞或肺中存在通过AQP1转运CO(2)的证据。由于HCO(3)(-)和CO(2)通量对内皮功能至关重要，因此进行了本研究以确定牛角膜内皮细胞（BCEC）汇合培养物中AQP1的表达水平是否会影响膜PCO(2)。

方法

通过反义寡核苷酸（AO）转染或腺病毒反义-AQP1（AV）感染降低BCEC内源性AQP1的表达。通过腺病毒正义-AQP1（SV）感染使AQP1过表达，该感染可指导重组AQP1的表达。

结果

AO转染使AQP1的表达和渗透水通透性（对照P(f)=0.046±0.005 cm/秒）分别降低了45%和36.5%，AV感染使其分别降低了67%和49%。SV感染使AQP1的表达增加了三倍多，但P(f)仅增加了37%。腺病毒空病毒（EV）感染未改变AQP1的表达或P(f)。通过测量暴露于富含CO(2)/HCO(3)(-)的溶液后细胞内pH值下降的速率来测定PCO(2)，使用pH敏感荧光染料2',7'-双-(2-羧乙基)-5-(和-6)-羧基荧光素（BCECF）进行测量。对照、寡核苷酸转染和腺病毒感染的细胞之间，BCEC的表观PCO(2)（0.0036±0.00023 cm/秒）没有差异。将对照细胞暴露于0.5 mM对氯汞苯磺酸（pCMBS）3至5分钟也可使P(f)降低超过50%，但这对细胞内pH值下降速率没有影响。