School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Invest Ophthalmol Vis Sci. 2011 Oct 17;52(11):8086-93. doi: 10.1167/iovs.11-8086.
To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO(3)(-), sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux.
MCT1-4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pH(i) were measured in BCEC in HCO(3)(-)-free or HCO(3)(-)-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na(+)/H(+) exchange blocker), disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells.
MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pH(i) measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpH(i)/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO(3)(-) but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO(3)(-), was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA.
MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H(+) transport. Lactate dependent proton flux can activate Na(+)/H(+) exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO(3)(-), HCO(3)(-) transport, NHE and CA activity.
鉴定并定位在牛角膜内皮细胞（BCEC）中表达的单羧酸转运蛋白（MCTs），并验证假设：HCO（3）（-）、碳酸氢钠共转运蛋白（NBCe1）、钠氢交换器（NHE）和碳酸酐酶（CA）活性缓冲作用有利于乳酸通量。
通过 RT-PCR、Western blot 分析和免疫荧光筛选 MCT1-4 的表达。在无 HCO（3）（-）或富含 HCO（3）（-）的 Ringer 中，在有和没有尼氟酸（MCT 抑制剂）、乙酰唑胺（ACTZ，CA 抑制剂）、5-（N-乙基-N-异丙基）amiloride（EIPA）（Na（+）/H（+）交换阻断剂）、二磺酸钠 4,4'-二异硫氰酸基二苯乙烯-2,2'-二磺酸盐（DIDS；阴离子转运抑制剂）或用 NBCe1 特异性小干扰（si）RNA 处理的细胞中，测量 BCEC 中的内源性乳酸外排和/或 pH（i）。
MCT1、2 和 4 在 BCEC 中表达。MCT1 定位于侧膜，MCT2 定位于侧膜和顶膜，而 MCT4 定位于顶膜。pH（i）测量显示，乳酸脉冲 20 秒后，细胞发生明显的乳酸诱导酸化（LIA）。尼氟酸孵育显著降低 pH（i）变化率（dpH（i）/dt）和乳酸诱导的细胞酸化。乳酸去除后，EIPA 抑制碱化。在 HCO（3）（-）存在下，乳酸依赖性质子通量显著增加，但 ACTZ 可减少其通量。内源性产生的乳酸的外排速度在 HCO（3）（-）存在时明显更快，在顶膜上更大，在顶膜侧被 ACTZ 降低，在顶膜和基底外侧被 NBCe1 特异性 siRNA、DIDS 或 EIPA 降低。
MCT1、2 和 4 在 BCEC 的顶膜和基底外侧膜（BL）表面表达，与尼氟酸敏感的乳酸-H（+）转运一致。乳酸依赖的质子通量可以激活 Na（+）/H（+）交换，并通过存在 HCO（3）（-）、HCO（3）（-）转运、NHE 和 CA 活性来最大限度地增加细胞内缓冲能力来促进其转运。
