We have recently demonstrated that moderate hypoxia causes a substantial increase in NAD(P)H-related fluorescence in pulmonary arteries (Leach et al. 2001), and others have suggested that ROS generation is increased. It is, however, unclear whether this phenomenon also occurs in systemic arteries (which do not constrict to hypoxia), and the subcellular origins are not known. We therefore examined NAD(P)H-related autofluorescence in isolated rat pulmonary and mesenteric arteries (taken from humanely killed rats) using a 340/380 nm ratiometric technique, and in freshly isolated VSM cells using imaging of autofluorescence at an excitation wavelength of 340 nm. ROS generation was estimated using the rate of oxidation of the fluorescent dye dichlorodihydro-fluorescein diacetate (DCF). Preparations were subjected to moderate (1 % oxygen, ~20 mmHg) or severe hypoxia (0 %, < 5 mmHg); addition of rotenone (100 nM) at the end of each experiment was used to estimate complete reduction to NAD(P)H. Localisation of the hypoxia-induced rise in NAD(P)H fluorescence was performed using imaging and the mitochondrial marker Mitotracker green. Changes in NAD(P)H-related fluorescence are expressed as a percentage of the rotenone-induced response. In pulmonary arteries 1 % oxygen caused a rapid rise in fluorescence to 69.7 ± 6.1 % (mean ± S.E.M.; n = 8) of that induced by rotenone; as would be predicted, this reached 99.5 ± 1.0 % (n = 9) with 0 % oxygen. In contrast, 1 % oxygen caused a significantly smaller rise in NAD(P)H in mesenteric arteries than in pulmonary (17.1 ± 4.6% n = 6; P < 0.001, paired t test), although 0 % oxygen was similar (101.3 ± 5.5% n = 5). Imaging of pulmonary and mesenteric VSM cells demonstrated a reticular pattern of autofluorescence that strongly co-localised with MitoTracker Green. During hypoxia the increase in autofluorescence was strongly correlated with the mitochondria (> 85 % co-localisation, n = 9), whereas in cytosolic areas the increase was weak or absent. Hypoxia (1 %) caused a ~500 % increase in DCF oxidation rate in isolated pulmonary arteries and VSM cells (n = 3 and 10). A similar increase was observed in mesenteric VSM cells (n = 8), but relative quantification was difficult due to differences in loading. Our results suggest that mitochondria from pulmonary VSM are more sensitive to hypoxia than those from mesenteric VSM in terms of reducing NAD(P)H consumption by the electron transport chain.

This work was supported by The Wellcome Trust.

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