Unno N, Menconi M J, Smith M, Hagen S J, Brown D A, Aguirre D E, Fink M P
Department of Surgery, Beth Israel Hospital, Boston, Mass, USA.
Surgery. 1997 Jun;121(6):668-80. doi: 10.1016/s0039-6060(97)90056-8.
We recently reported that moderate degrees of adenosine triphosphate (ATP) depletion induced by chronic glycolytic inhibition or hypoxia increase the permeability of Caco-2BBe enterocytic monolayers. Interestingly, the development of lactic acidosis induced by anaerobic glycolysis ameliorates the development of hyperpermeability caused by chronic ATP depletion. We sought to further elucidate the mechanism(s) responsible for the apparent protection against epithelial hyperpermeability afforded by mild acidosis under conditions of metabolic inhibition.
Caco-2BBe monolayers growing on permeable supports in bicameral chambers were incubated with 2-deoxyglucose (2DOG) in a glucose-free (Glu-) environment to inhibit glycolysis. Permeability was determined by measuring the transepithelial flux of fluorescein sulfonic acid. Concentrations of intracellular calcium [Ca2+]i were determined fluorometrically by using fura-2.
When extracellular pH (pH0) was maintained at 7.4 or 5.5, incubation of monolayers for 24 hours with Glu-/2DOG increased permeability and depleted intracellular ATP levels. However, keeping pH0 at 7.0 to 6.0 ameliorated both the development of hyperpermeability and the depletion of ATP induced by Glu-/2DOG. These protective effects were observed under acidic conditions created either by addition to the medium of HCl or by incubation under an atmosphere containing 20% CO2. Incubation with Glu-/2DOG caused bulging of the apical membranes of cells (electron microscopy) and derangements in the perijunctional distribution of actin (fluorescence microscopy); however, these structural changes were ameliorated by mild acidosis. Acute chemical hypoxia at pH0 7.4 induced by Glu-/2DOG plus antimycin A decreased cellular ATP levels and elevated [Ca2+]i. Lowering pH0 to 6.8 ameliorated both the depletion of ATP and the increase in [Ca2+]i induced by Glu-/2DOG+antimycin A.
Moderate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.
我们最近报道，慢性糖酵解抑制或缺氧诱导的中度三磷酸腺苷（ATP）耗竭会增加Caco-2BBe肠上皮细胞单层的通透性。有趣的是，无氧糖酵解诱导的乳酸酸中毒可改善慢性ATP耗竭引起的高通透性的发展。我们试图进一步阐明在代谢抑制条件下轻度酸中毒对上皮高通透性提供明显保护作用的机制。
在双室培养箱中生长在可渗透支持物上的Caco-2BBe单层细胞，在无葡萄糖（Glu-）环境中与2-脱氧葡萄糖（2DOG）孵育以抑制糖酵解。通过测量荧光素磺酸的跨上皮通量来测定通透性。使用fura-2通过荧光法测定细胞内钙[Ca2+]i的浓度。
当细胞外pH（pH0）维持在7.4或5.5时，单层细胞与Glu-/2DOG孵育24小时会增加通透性并耗尽细胞内ATP水平。然而，将pH0保持在7.0至6.0可改善Glu-/2DOG诱导的高通透性的发展和ATP的耗竭。在通过向培养基中添加HCl或在含有20%二氧化碳的气氛中孵育所产生的酸性条件下观察到了这些保护作用。与Glu-/2DOG孵育导致细胞顶端膜膨出（电子显微镜观察）和肌动蛋白在连接周围分布紊乱（荧光显微镜观察）；然而，这些结构变化在轻度酸中毒时得到改善。Glu-/2DOG加抗霉素A在pH0 7.4诱导的急性化学性缺氧降低了细胞ATP水平并升高了[Ca2+]i。将pH0降至6.8可改善Glu-/2DOG +抗霉素A诱导的ATP耗竭和[Ca2+]i增加。
pH的适度降低可改善代谢抑制诱导的高通透性，可能是通过减少ATP耗竭和抑制[Ca2+]i增加来实现的。
