Menconi M J, Salzman A L, Unno N, Ezzell R M, Casey D M, Brown D A, Tsuji Y, Fink M P
Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
Am J Physiol. 1997 May;272(5 Pt 1):G1007-21. doi: 10.1152/ajpgi.1997.272.5.G1007.
We previously demonstrated that ileal mucosal acidosis in pigs reversibly increases intestinal permeability to hydrophilic macromolecules, even in the absence of tissue hypoxia [A.L. Salzman, H. Wang, P.S. Wollert, T.J. Vandermeer, C.C. Compton, A.G. Denenberg, and M.P. Fink. Am. J. Physiol. 266 (Gastrointest, Liver Physiol, 29): G633-G646, 1994]. In an effort to further explore the mechanism(s) underlying this phenomenon, we examined the effect of acidic pH on the permeability characteristics of cultured Caco-2BBe (human intestinal epithelial) cells grown as monolayers on permeable supports. Permeability was determined by measuring the mucosal-to-basolateral flux of fluorescein disulfonic acid (FS; mol wt 478 Dal, fluorescein isothiocyanate-labeled dextran (FD4; average mol wt 4 kDa), or [3H]mannitol. Incubation of monolayers under hypercarbic conditions or with acidific bicarbonate-free medium significantly increased permeability to FS, FD4, and mannitol in a manner dependent on both time and pH. Incubation in medium at pH 5.43 for 24 h increased the release of lactate dehydrogenase and decreased the intensity of staining with calcein-acetoxymethyl ester, findings that are indicative of plasma membrane injury; nevertheless, the percentage of nonviable cells did not increase. Ultrastructural analyses revealed evidence of increased paracellular trafficking of horseradish peroxidase after incubation of monolayers under acidic conditions. Fluorescence confocal microscopy and temperature studies demonstrated that incubation at pH 5.43 induced an increase in both the intracellular uptake of FD4 and the activation energy for FS permeation across Caco-2BBe monolayers, respectively, suggesting increased transcellular permeation. Exposure to acidic conditions also decreased cellular levels of ATP. We conclude that acidosis increases both paracellular and transcellular permeability to hydrophilic macromolecules and leads to depletion of ATP.
我们之前证明，即使在没有组织缺氧的情况下，猪的回肠黏膜酸中毒也会可逆地增加肠道对亲水性大分子的通透性[A.L. 萨尔兹曼、H. 王、P.S. 沃勒特、T.J. 范德米尔、C.C. 康普顿、A.G. 登嫩伯格和M.P. 芬克。《美国生理学杂志》266卷（胃肠、肝脏生理学，第29期）：G633 - G646，1994年]。为了进一步探究这一现象背后的机制，我们研究了酸性pH对在可渗透支持物上以单层形式生长的培养Caco - 2BBe（人肠上皮）细胞通透性特征的影响。通透性通过测量荧光素二磺酸（FS；分子量478道尔顿）、异硫氰酸荧光素标记的葡聚糖（FD4；平均分子量4 kDa）或[³H]甘露醇从黏膜到基底外侧的通量来确定。在高碳酸血症条件下或用无碳酸氢盐的酸化培养基孵育单层细胞，会以时间和pH依赖的方式显著增加对FS、FD4和甘露醇的通透性。在pH 5.43的培养基中孵育24小时会增加乳酸脱氢酶的释放，并降低钙黄绿素乙酰氧甲酯的染色强度，这些发现表明质膜受到损伤；然而，非存活细胞的百分比并未增加。超微结构分析显示，在酸性条件下孵育单层细胞后，辣根过氧化物酶的细胞旁转运增加。荧光共聚焦显微镜和温度研究表明，在pH 5.43下孵育分别诱导了FD4的细胞内摄取增加以及FS透过Caco - 2BBe单层细胞的活化能增加，提示跨细胞通透性增加。暴露于酸性条件还会降低细胞内ATP水平。我们得出结论，酸中毒会增加对亲水性大分子的细胞旁和跨细胞通透性，并导致ATP耗竭。
