Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium
Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium
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DOI：
10.1152/ajpcell.00405.2004
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发表时间：
2005-03-01
影响因子：
5.5
通讯作者：
Bonanno, JA
中科院分区：
文献类型：
--
作者：
Li, JH;Sun, XC;Bonanno, JA
角膜透明度和水合控制依赖于角膜内皮的HCO 3-转运特性。最近的工作（13）表明，除了基底外侧1 Na（+）-2HCO（3）（-）协同转运蛋白外，还存在顶端1 Na（+）-3HCO（3）（-）协同转运蛋白（NBC 1）。我们研究了NBC 1协同转运蛋白是否显著促进基底侧或顶端HCO 3渗透性，以及协同转运蛋白是否参与培养的牛角膜内皮细胞的跨内皮净HCO 3通量。使用小干扰RNA（siRNA）降低NBC 1蛋白表达。免疫印迹分析显示，转染后4天，5 - 15 nM siRNA使NBC 1表达降低80 - 95%。通过测量在恒定pH或恒定CO2条件下从浴中除去HCO 3-时的pH（i）变化速率来确定顶侧和基底侧HCO 3-渗透性。使用任一方案，我们发现用NBC 1 siRNA处理的培养物的基底外侧HCO 3渗透性比未处理或siCONTROL siRNA处理的细胞低六倍。根尖HCO 3渗透性不受NBC 1 siRNA处理的影响。净非稳态HCO 3通量为0.707 +/- 0.009 mM。min（-1）。cm（2），当细胞用2 μ M毛喉素刺激时，增加至1.74 +/- 0.15。用5 nM siRNA处理使基底外侧到顶端的通量降低67%，而顶端到基底外侧的通量不受影响，使净HCO 3通量显著降低至0.236 +/- 0.002。NBC 1 siRNA处理或100 μ M哇巴因也消除了稳态HCO 3-通量，通过顶端室碱化测量。总的来说，减少基底外侧HCO 3渗透性，基底外侧到顶端流量，和净HCO 3通量作为NBC 1表达减少的结果表明，NBC 1在跨内皮HCO 3通量中起着关键作用，并且仅在基底外侧膜起作用。
Corneal transparency and hydration control are dependent on HCO3- transport properties of the corneal endothelium. Recent work ( 13) suggested the presence of an apical 1Na(+)-3HCO(3)(-) cotransporter (NBC1) in addition to a basolateral 1Na(+)-2HCO(3)(-) cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO3- permeability and whether the cotransporter participates in transendothelial net HCO3- flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA ( siRNA). Immunoblot analysis showed that 5 - 15 nM siRNA decreased NBC1 expression by 80 - 95%, 4 days posttransfection. Apical and basolateral HCO3- permeabilities were determined by measuring the rate of pH(i) change when HCO3- was removed from the bath under constant pH or constant CO2 conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO3- permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO3- permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO3- flux was 0.707 +/- 0.009 mM . min(-1) . cm(2) in the basolateral-to-apical direction and increased to 1.74 +/- 0.15 when cells were stimulated with 2 muM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO3- flux to 0.236 +/- 0.002. NBC1 siRNA treatment or 100 muM ouabain also eliminated steady-state HCO3- flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO3- permeability, basolateral-to-apical fluxes, and net HCO3- flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO3- flux and is functional only at the basolateral membrane.