ENERGY TURNOVER OF VASCULAR ENDOTHELIAL-CELLS

Two noninvasive methods, calorimetry and P-31 nuclear magnetic resonan ce (NMR), were used to further define energy-consuming and energy-prov iding reactions in endothelial cells. With P-31-NMR, cellular ATP cont ent was measured; with calorimetry, heat flux as a result of ATP turno ver was measured. For these measurements, pig aortic endothelial cells were cultured on microcarrier beads and perfused in a column at const ant flow rate. Pig aortic endothelial cells synthesize ATP mainly thro ugh glycolysis and, as determined by NMR, contain no phosphocreatine. In such a system, calorimetry-measured heat flux reflects rate of cell ular ATP turnover. By use of inhibitors of ATP-dependent processes, th e following changes in basal heat flux (231 +/- 65.5 mu W/mg protein) were obtained: 18% for 2,3-butanedione monoxime (inhibitor of actomyos in-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 bafilomyc in A(1) (inhibitor of lysosomal H+-ATPase). Cytochalasin D, 2,3-butane dione 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 r emained unchanged. Inhibition of glycolysis with 2-deoxy-D-glucose dec reased 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, cell s fully recovered heat flux and ATP when 2-deoxy-D-glucose was removed . The results, together with previously published data for Na+-K+-ATPa se [M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (H eal Circ. Physiol. 37): H351-H358, 1995], demonstrate that >70% of tot al ATP-consuming processes of endothelial cells can be attributed to s pecific cellular processes. Actomyosin-ATPase (18%) and protein synthe sis (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 c onsumption when glycolysis is inhibited.