Staub F, Baethmann A, Peters J, Weigt H, Kempski O
Institute for Surgical Research, Ludwig Maximilians University, Munich, F.R.G.
J Cereb Blood Flow Metab. 1990 Nov;10(6):866-76. doi: 10.1038/jcbfm.1990.143.
Effects of severe lactacidosis were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium, which was rendered acidotic by addition of lactic acid in rising concentrations. A pH range of 7.4-4.2 was studied under maintenance of isotonicity and a normal electrolyte concentration of the medium. Cell swelling was quantified by flow cytometry using an advanced Coulter system with hydrodynamic focusing. The method was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide. The volume of C6 glioma cells was found to increase if the pH was titrated to pH 6.8 or below. From this level downward, the extent of cell swelling depended on the degree of acidosis and the duration of exposure. For example, lactacidosis of pH 6.2 for 60 min led to an increase in cell size to 124.5% of normal, while pH 5.0 or 4.2 led to a cell size of 151.1 or 190.9%, respectively. A comparative analysis of the acidosis-induced cell swelling was made by using sulfuric acid. Swelling of C6 glioma at a given pH was only half of what was found when using lactic acid. This indicates specific swelling-inducing properties of lactic acid, while cell viability was not differently affected by both acids. Of the C6 glioma cells, 89.1% were viable under control conditions at pH 7.4. The viability remained unchanged down to pH 6.2. At pH 5.6, viability remained normal for 30 min, but it decreased to 73.4% after 60 min. Further lowering of pH to 5.0 or 4.6 respectively, decreased the number of viable cells to 47.8 or 40.3%. At pH 4.2 only 21.1% of the cells were surviving 1 h of lactacidosis. Cell swelling from lactacidosis could be largely inhibited by replacement of Na+ and bicarbonate ions in the medium by choline chloride and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, suggesting an involvement of the Na+/H+ and Cl-/HCO3- antiporters in the swelling process. Omission of Na+ and bicarbonate was, however, associated with reduced viability of the glial cells in acidosis. The swelling response of astrocytes obtained from primary culture was similar to that of C6 glioma. Lactic acid was also more effective in inducing cell swelling than sulfuric acid at the same level of acidosis. In astrocytes, viability at, e.g., pH 5.6 appeared to be more affected by lactic than by sulfuric acid.(ABSTRACT TRUNCATED AT 400 WORDS)
通过使用原代培养的C6胶质瘤细胞和星形胶质细胞在体外分析严重乳酸性酸中毒的影响。将细胞悬浮在生理培养基中，通过添加浓度不断升高的乳酸使其变为酸中毒状态。在维持等渗性和培养基正常电解质浓度的情况下，研究了pH值范围为7.4 - 4.2的情况。使用具有流体动力学聚焦功能的先进库尔特系统通过流式细胞术对细胞肿胀进行定量分析。该方法还用于通过排除荧光染料碘化丙啶来评估细胞活力。发现如果将pH值滴定至6.8或更低，C6胶质瘤细胞的体积会增加。从这个水平向下，细胞肿胀的程度取决于酸中毒的程度和暴露时间。例如，pH值为6.2持续60分钟的乳酸性酸中毒会导致细胞大小增加至正常大小的124.5%，而pH值为5.0或4.2分别导致细胞大小为151.1%或190.9%。通过使用硫酸对酸中毒诱导的细胞肿胀进行了比较分析。在给定pH值下，C6胶质瘤细胞因硫酸引起的肿胀仅为乳酸引起肿胀的一半。这表明乳酸具有特定的肿胀诱导特性，而两种酸对细胞活力的影响没有差异。在pH值为7.4的对照条件下，89.1%的C6胶质瘤细胞具有活力。在pH值降至6.2之前，活力保持不变。在pH值为5.6时，30分钟内活力保持正常，但60分钟后降至73.4%。进一步将pH值分别降至5.0或4.6，活细胞数量分别降至47.8%或40.3%。在pH值为4.2时，仅21.1%的细胞在1小时的乳酸性酸中毒后仍存活。通过用氯化胆碱和N - 2 - 羟乙基哌嗪 - N'- 2 - 乙磺酸缓冲液替代培养基中的Na⁺和碳酸氢根离子，可在很大程度上抑制乳酸性酸中毒引起的细胞肿胀，这表明Na⁺/H⁺和Cl⁻/HCO₃⁻反向转运体参与了肿胀过程。然而，在酸中毒情况下，省略Na⁺和碳酸氢根与神经胶质细胞活力降低有关。从原代培养获得的星形胶质细胞的肿胀反应与C6胶质瘤细胞相似。在相同程度的酸中毒情况下，乳酸在诱导细胞肿胀方面也比硫酸更有效。例如，在pH值为5.6时，星形胶质细胞的活力似乎受乳酸的影响比受硫酸的影响更大。（摘要截断于400字）
