Lipopolysaccharide-induced alterations in oxygen consumption and radical generation in endothelial cells

Oxygen consumption rate (OCR) and generation of superoxide and nitric oxide (NO) in mouse aortic endothelial cells (MAECs) treated with lipopolysaccharide (LPS) were studied. The OCR was determined in cell suspensions at 37 degrees C by electron paramagnetic resonance (EPR) spectroscopy. LPS significantly altered the OCR in a dose and time-dependent fashion. The OCR was significantly elevated immediately following the treatment of MAECs with LPS (5 and 10 microg/ml) and NADPH (100 microM) whereas the same was depressed 1 h after exposure to similar conditions of incubation. Under similar experimental conditions, superoxide generation was also determined by EPR spectroscopy and cytochrome c reduction assays. A marginal increase in the superoxide production was observed when the cells were treated with LPS and NADPH alone whereas the same was further enhanced significantly when the cells were treated with LPS and NADPH together. The increase in oxygen consumption and superoxide production caused by LPS was inhibited by diphenyleneiodonium (DPI), suggesting the involvement of NAD(P)H oxidase. A significant increase in the NO production by MAECs was noticed 1 h after treatment with LPS and was inhibited by L-NAME, further suggesting the involvement of nitric oxide synthase (NOS). Thus, on a temporal scale, LPS-induced alterations in oxygen consumption by MAECs may be under the control of dual regulation by NAD(P)H oxidase and NOS.