Culic O, Gruwel M L, Schrader J
Institut für Herz- und Kreislaufphysiologie, Heinrich Heine Universität Düsseldorf, Germany.
Am J Physiol. 1997 Jul;273(1 Pt 1):C205-13. doi: 10.1152/ajpcell.1997.273.1.C205.
Two noninvasive methods, calorimetry and 31P nuclear magnetic resonance (NMR), were used to further define energy-consuming and energy-providing reactions in endothelial cells. With 31P-NMR, cellular ATP content was measured; with calorimetry, heat flux as a result of ATP turnover was measured. For these measurements, pig aortic endothelial cells were cultured on microcarrier beads and perfused in a column at constant flow rate. Pig aortic endothelial cells synthesize ATP mainly through glycolysis and, as determined by NMR, contain no phosphocreatine. In such a system, calorimetry-measured heat flux reflects rate of cellular ATP turnover. By use of inhibitors of ATP-dependent processes, the following changes in basal heat flux (231 +/- 65.5 microW/mg protein) were obtained: 18% for 2,3-butanedione monoxime (inhibitor of actomyosin-ATPase), 17% for wortmannin (inhibitor of myosin light chain kinase), 10% for cytochalasin D (inhibitor of actin polymerization), 23% for cycloheximide (inhibitor of protein synthesis), 11% for thapsigargin (inhibitor of endoplasmic reticulum Ca(2+)-ATPase), and 6% for bafilomycin A1 (inhibitor of lysosomal H(+)-ATPase). Cytochalasin D, 2,3-butanedione monoxime, wortmannin, and thapsigargin caused changes in F-actin distribution, as revealed by rhodamine-phalloidin cytochemistry. In a separate experimental series, when cells were perfused with a medium containing no glucose, heat flux decreased by 40% while cellular ATP remained unchanged. Inhibition of glycolysis with 2-deoxy-D-glucose decreased heat flux by 73%, and ATP was no longer visible with 31P-NMR. Despite this massive ATP depletion, which was maintained for 3 h, cells fully recovered heat flux and ATP when 2-deoxy-D-glucose was removed. The results, together with previously published data for Na(+)-K(+)-ATPase [M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H351-H358, 1995], demonstrate that > 70% of total ATP-consuming processes of endothelial cells can be attributed to specific cellular processes. Actomyosin-ATPase (18%) and protein synthesis (23%) comprise the largest fraction. At least three-fourths of ATP synthesized is provided by glycolysis. Endothelial cells exhibit the remarkable ability to coordinate downregulation of ATP synthesis and consumption when glycolysis is inhibited.
采用两种非侵入性方法，即量热法和31P核磁共振（NMR）法，进一步明确内皮细胞中能量消耗和能量供应反应。通过31P-NMR测量细胞ATP含量；通过量热法测量ATP周转产生的热通量。为进行这些测量，将猪主动脉内皮细胞培养在微载体珠上，并在柱中以恒定流速灌注。猪主动脉内皮细胞主要通过糖酵解合成ATP，并且如NMR所测定，其不含磷酸肌酸。在这样的系统中，量热法测量的热通量反映细胞ATP周转速率。通过使用ATP依赖性过程的抑制剂，获得了基础热通量（231±65.5微瓦/毫克蛋白质）的以下变化：2,3-丁二酮单肟（肌动球蛋白-ATP酶抑制剂）导致变化18%，渥曼青霉素（肌球蛋白轻链激酶抑制剂）导致变化17%，细胞松弛素D（肌动蛋白聚合抑制剂）导致变化10%，放线菌酮（蛋白质合成抑制剂）导致变化23%，毒胡萝卜素（内质网Ca(2+)-ATP酶抑制剂）导致变化11%，巴弗洛霉素A1（溶酶体H(+)-ATP酶抑制剂）导致变化6%。如罗丹明-鬼笔环肽细胞化学所显示，细胞松弛素D、2,3-丁二酮单肟、渥曼青霉素和毒胡萝卜素引起F-肌动蛋白分布的变化。在另一个实验系列中，当用不含葡萄糖的培养基灌注细胞时，热通量下降40%，而细胞ATP保持不变。用2-脱氧-D-葡萄糖抑制糖酵解使热通量下降73%，并且31P-NMR不再能检测到ATP。尽管这种大量的ATP消耗持续了3小时，但当去除2-脱氧-D-葡萄糖时，细胞完全恢复了热通量和ATP。这些结果与先前发表的关于Na(+)-K(+)-ATP酶的数据[M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H351-H358, 1995]一起表明内皮细胞总ATP消耗过程的>70%可归因于特定的细胞过程。肌动球蛋白-ATP酶（18%）和蛋白质合成（23%）占最大比例。合成的ATP至少四分之三由糖酵解提供。当糖酵解受到抑制时，内皮细胞表现出协调ATP合成和消耗下调的显著能力。
