Energy turnover of vascular endothelial cells
Energy turnover of vascular endothelial cells
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DOI：
10.1152/ajpcell.1997.273.1.c205
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发表时间：
1997-07-01
影响因子：
5.5
通讯作者：
Schrader, J
中科院分区：
文献类型：
--
作者：
Culic, O;Gruwel, MLH;Schrader, J
两种无创方法，量热法和P-31核磁共振（NMR），用于进一步定义内皮细胞的能量消耗和能量提供反应。用P-31-NMR测定细胞ATP含量；用量热法测量ATP转换产生的热通量。为了进行这些测量，猪主动脉内皮细胞在微载体珠上培养，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，经核磁共振测定不含磷酸肌酸。在这样一个系统中，量热法测量的热通量反映了细胞ATP的周转速率。通过使用atp依赖过程的抑制剂，获得了基础热通量（231 +/- 65.5 mu W/mg蛋白）的以下变化：2,3-丁二酮单肟（肌动球蛋白- atp酶抑制剂）为18%，wortmannin（肌球蛋白轻链激酶抑制剂）为17%，细胞松弛素D（肌动蛋白聚合抑制剂）为10%，环己亚胺（蛋白质合成抑制剂）为23%，thapsigargin（内质网Ca2+- atp酶抑制剂）为11%，巴菲霉素A(1)为6%（溶酶体H+- atp酶抑制剂）。rhodamine-phalloidin细胞化学结果显示，细胞松弛素D、2,3-丁二酮单肟、wortmannin和thapsigargin引起F-actin分布的变化。在一个单独的实验系列中，当细胞灌注不含葡萄糖的培养基时，热通量下降了40%，而细胞ATP保持不变。抑制2-脱氧-d -葡萄糖糖酵解使热通量降低了73%，并且P-31-NMR不再可见ATP。尽管大量的ATP消耗持续了3小时，但当2-脱氧-d -葡萄糖被去除时，细胞完全恢复了热通量和ATP。结果，连同先前发表的Na+-K+- atp酶[M。L. H. Gruwel， C. Alves， J. Schrader。点。[j] .中国生物医学工程学报，2016,(6):559 - 558 . [j] .中国生物医学工程学报。肌动球蛋白- atp酶（18%）和蛋白质合成（23%）构成了最大的部分。至少四分之三的ATP合成是由糖酵解提供的。当糖酵解受到抑制时，内皮细胞表现出显著的协调下调ATP合成和消耗的能力。
Two noninvasive methods, calorimetry and P-31 nuclear magnetic resonance (NMR), were used to further define energy-consuming and energy-providing reactions in endothelial cells. With P-31-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 mu W/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 Ca2+-ATPase), and 6% for bafilomycin A(1) (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 P-31-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 (Heal 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.