Proceedings of The Physiological Society
University of Cambridge (2004) J Physiol 555P, C34
Release of nitric oxide (NO) from the hindlimb of the anaesthetized rat in response to systemic hypoxia and adenosine infusion
Clare J. Ray and Janice M. Marshall
Department of Physiology, Medical School, University of Birmingham, Birmingham B15 2TT, UK
The hindlimb vasodilatation evoked in the rat by systemic hypoxia is mediated by adenosine acting at the A1 receptors and is attenuated by the NO synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME; Bryan & Marshall, 1999a,b). Further studies showed the adenosine-mediated component of hypoxia-induced hindlimb dilatation and of proximal arterioles can be restored after NOS blockade providing a background level of NO is restored with an NO donor whereas the hypoxia-induced dilatation of terminal arterioles was not restored (Edmunds et al. 2003; Edmunds & Marshall, 2003). These studies raised the question of whether hypoxia-and adenosine-induced vasodilatation is simply NO-dependent or mediated by an increase in NO synthesis. We have now investigated the release of NO from the rat hindlimb by measuring NO concentration ([NO]) in the blood by reduction of plasma nitrate (NO3-) to nitrite (NO2-) followed by electrochemical measurement of [NO] following the reduction of NO2- by a 0.1M H2SO4/KI solution.
Rats were anaesthetized with Saffan (7-12 mgkg-1hr-1 I.V.) and humanely killed by anaesthetic overdose. Arterial and venous blood samples (100 µl) were taken from the femoral artery and vein before and in the 5th min of systemic hypoxia (8 % inspired O2) or infusion of adenosine (1.2 mgkg-1min-1, I.A.). In Group 1 (n = 8) and Group 2 (n = 6), systemic hypoxia and adenosine infusion evoked vasodilatation that was significantly attenuated by L-NAME (10 mgkg-1 I.V.) and by the adenosine A1-receptor antagonist DPCPX (0.1 mgkg-1 I.V.; see Bryan & Marshall, 1999a,b). In Group 1, systemic hypoxia and adenosine infusion evoked a significant increase in venous-arterial [NO] difference ([NO]v-a) from -1.4 ± 0.9 to 6.6 ± 1.6*** and from 2.3 ± 0.8 to 8.4 ± 1.8** nM respectively (mean ± S.E.M.; ***, **, *P < 0.0001, 0.001, 0.05 ANOVA for repeated measures with Fisher's post hoc test), which was abolished in the presence of L-NAME, from -0.72 ± 0.9 to -0.87 ± 0.7 and 0.72 ± 0.8 to -0.97 ± 1.1 nM respectively. In Group 2, DPCPX abolished the increase in [NO]v-a evoked by systemic hypoxia (from -4.2 ± 1.8 to 12.5 ± 3.7** to -0.63 ± 2.6 to 3.3 ± 2.9 nM) and reduced by ~50 % the increase in [NO]v-a evoked by adenosine infusion (from 1.1 ± 1.5 to 23.7 ± 13.7* to -0.43 ± 2.9 to 11.6 ± 5.9 nM).
Thus, we have provided the first direct evidence that the muscle vasodilatation of systemic hypoxia that is mediated by adenosine acting at A1 receptors, is accompanied by an increase in the release of NO.
Where applicable, experiments conform with Society ethical requirements