Energy turnover of vascular endothelial cells.
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.
Culic
O
O
Institut für Herz- und Kreislaufphysiologie, Heinrich Heine Universität Düsseldorf, Germany.
Gruwel
M L
ML
Schrader
J
J
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001011:Aorta; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006019:Glycolysis / Q000187:drug effects; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010758:Phosphorus; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D004791:Enzyme Inhibitors; C004717:diacetylmonoxime; D015638:Cytochalasin D; D010758:Phosphorus; D000968:Antimycin A; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D003847:Deoxyglucose; D000251:Adenosine Triphosphatases; D003931:Diacetyl; D000077191:Wortmannin