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 O-2 . 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 Po-2 at half-maximal respiration (P-50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P-50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of similar to 0.015 kPa in P-50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P-50 in coupled cells, as deduced from the shallow slope of the flux dependence of P-50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P-50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.