Respiratory defect as an early event in preservation-reoxygenation injury of endothelial cells.

Characterization of preservation injury in endothelial cells has been primarily accomplished by measurement of cell viability. To analyze early events and cellular mechanisms of preservation-reoxygenation injury, we developed high-resolution respirometry for the study of mitochondrial function in endothelial cells, to provide a quantitative marker for sublethal stress. Cultured human umbilical vein endothelial cells were stored for 4 and 8 hr at 4 degrees C under an atmosphere of 95% N2 and 5% CO2 in University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions. Respiration of suspended cells, measured after reoxygenation in growth medium at 37 degrees C, was significantly reduced in all treatments in comparison to controls not subjected to cold preservation. In contrast, trypan blue staining was unchanged after 4 hr of preservation and was significant only after 8 hr. After 8 hr of cold storage in UW and HTK solutions, respiration was 64+/-5% and 49+/-6%, respectively, of controls (46.5+/-3.3 pmol O2 x s(-1 x 10(-6) cells), indicating significantly better protection by UW solution than HTK solution. A titration regimen with substrate (succinate), uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and inhibitors of complexes I and III (rotenone and antimycin A) resulted in identical respiratory response patterns in all treatments. The plasma membrane remained impermeable to succinate. Inner mitochondrial membrane function was preserved as indicated by a constant relative increase of respiration after uncoupling. These results demonstrate that loss of catalytic capacity for respiration constitutes an early event in preservation-reoxygenation injury, whereas membrane damage is not a primary defect. Respirometric evaluation of sublethal cell injury and localization of cell damage may provide selective guidelines for further optimization of strategies in organ preservation.

Oxygen dependence of respiration in coupled and uncoupled endothelial cells.

We studied the oxygen dependence of respiration in cultured human umbilical vein endothelial cells by use of high-resolution respirometry. The rate of oxygen consumption varied from 30 to 50 pmol O2.s-1.(10(6) cells)-1 over a sixfold range of cell densities. Respiration was stimulated up to 3.5-fold by uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol, and the PO2 at half-maximal respiration (P50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of approximately 0.015 kPa in P50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P50 in coupled cells, as deduced from the shallow slope of the flux dependence of P50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.

Control of mitochondrial and cellular respiration by oxygen.

Control and regulation of mitochondrial and cellular respiration by oxygen is discussed with three aims: (1) A review of intracellular oxygen levels and gradients, particularly in heart, emphasizes the dominance of extracellular oxygen gradients. Intracellular oxygen pressure, pO2, is low, typically one to two orders of magnitude below incubation conditions used routinely for the study of respiratory control in isolated mitochondria. The pO2 range of respiratory control by oxygen overlaps with cellular oxygen profiles, indicating the significance of pO2 in actual metabolic regulation. (2) A methodologically detailed discussion of high-resolution respirometry is necessary for the controversial topic of respiratory control by oxygen, since the risk of methodological artefact is closely connected with far-reaching theoretical implications. Instrumental and analytical errors may mask effects of energetic state and partially explain the divergent views on the regulatory role of intracellular pO2. Oxygen pressure for half-maximum respiration, p50, in isolated mitochondria at state 4 was 0.025 kPa (0.2 Torr; 0.3 microM O2), whereas p50 in endothelial cells was 0.06-0.08 kPa (0.5 Torr). (3) A model derived from the thermodynamics of irreversible processes was developed which quantitatively accounts for near-hyperbolic flux/pO2 relations in isolated mitochondria. The apparent p50 is a function of redox potential and protonmotive force. The protonmotive force collapses after uncoupling and consequently causes a decrease in p50. Whereas it is becoming accepted that flux control is shared by several enzymes, insufficient attention is paid to the notion of complementary kinetic and thermodynamic flux control mechanisms.