Functional aspects of creatine kinase isoenzymes in endothelial cells
American Journal of Physiology - Cell Physiology
rgen Schrader. Functional aspects of creatine kinase isoenzymes in endothelial cells. Am J Physiol Cell Physiol 281: C320-C328, 2001.-To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, highenergy phosphates were continuously monitored by 31 P NMR spectroscopy in pig AECs attached to microcarrier beads. CK
... rier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 Ϯ 0.01 and 0.15 Ϯ 0.08 U/mg, respectively) was Ͻ3% of that of cardiomyocytes (6.46 Ϯ 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mia-CK was demonstrated by indirect immunofluorescence staining using Mia-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 Ϯ 0.6 nmol ⅐ h Ϫ1 ⅐ mg protein Ϫ1 ), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction ("shuttle hypothesis"). endothelium; transport; energy metabolism; phosphorus-31 nuclear magnetic resonance spectroscopy RESULTS 31 P NMR of porcine aortic ECs. Energy metabolism of perifused porcine macrovascular ECs from the aorta (AECs) was investigated by 31 P NMR spectroscopy. In fully relaxed spectra, peak areas represent the concentration of phosphorus nuclei of the respective compounds. A representative example is shown in Fig. 1 . Resonances of inorganic phosphate, PCr, NAD, and nucleotide triphosphates (␣-, ␤and ␥-phosphate reso-C322 CREATINE KINASE ISOENZYMES IN ENDOTHELIAL CELLS by 10.