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1 Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
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Abstract |
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(Received 18 July 2005;
accepted after revision 22 August 2005;
first published online 25 August 2005)
Corresponding author C. J. Ray: Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, UK. Email: c.j.ray{at}bham.ac.uk
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Introduction |
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However, after addition of L-NAME, which blocks tonic synthesis of NO and so reduces baseline FVC, the increase in FVC evoked by systemic hypoxia was restored when baseline FVC was restored by infusion of an NO donor. This ‘restored’ hypoxia-induced dilatation was also attenuated by DPCPX (Edmunds & Marshall, 2001a; Edmunds et al. 2003). Thus, even in the absence of NO synthesis, hypoxia still evokes adenosine-mediated muscle dilatation providing that a background level of NO is present. Exogenous adenosine was also able to evoke an increase in FVC after NOS inhibition, when baseline FVC was restored by infusion of an NO donor, or by cell permeant cGMP. By contrast, the hypoxia-induced increase in FVC was not restored by cGMP (Edmunds et al. 2003). Indeed, further experiments led to the proposal that a background level of cGMP is needed for the action of adenosine, but a background level of NO is required for the hypoxia-induced release of adenosine (Edmunds et al. 2003).
Direct observations on muscle microcirculation extended these ideas. Proximal and terminal arterioles dilated in response to systemic hypoxia and L-NAME attenuated these dilator responses. However, restoration of a tonic level of NO by infusion of an NO donor restored the dilatation of proximal, but not terminal arterioles; the ‘restored’ response of primary arterioles was attenuated by DPCPX (Edmunds & Marshall, 2003). Given that changes in gross vascular conductance in skeletal muscle can be attributed to behaviour of proximal arterioles, the major resistance vessels (Hebert & Marshall, 1988; Mian & Marshall, 1991a), these results raised the possibility that under normal conditions when NO synthesis is possible, hypoxia-induced dilatation of proximal arterioles is not actually mediated by NO, but is simply dependent on a background level of NO for adenosine to be released and to act on the vascular smooth muscle. Alternatively, when NO synthesis is possible, hypoxia-induced dilatation of proximal arterioles is mediated by NO generated in response to adenosine, and when it is not possible, this dilatation is partly mediated by other mechanisms, providing that NO is present. The characteristics of the hypoxia-induced dilatation of terminal arterioles could be explained if endogenous adenosine can only dilate them by increasing the synthesis and release of NO (Edmunds & Marshall, 2001b, 2003).
In view of these findings, the present study was performed to test more directly whether systemic hypoxia does evoke significant release of NO from skeletal muscle vasculature by stimulating NOS, and whether this is achieved partly by locally released adenosine acting on A1 receptors. To this end, NO released into plasma of hindlimb muscle was assayed by using a NO-sensitive electrode to measure NO generated from plasma nitrite and nitrate (NOx) during acute hypoxia, before and after NOS inhibition with L-NAME or A1 receptor blockade with DPCPX. Complementary experiments were performed for changes evoked by infusion of adenosine.
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Methods |
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The femoral arterial and venous cannulae also allowed the removal of blood samples (100 µl) for analysis of arterial and venous plasma NOx as [NO]a and [NO]v, respectively (see below). Whole blood was taken into a microtube containing 5 µl heparinized saline (25 U ml–1). After rapid mixing, the microtubes were placed directly into a microcentrifuge and spun at 10 000 g for 5 min. Then 50 µl plasma was removed from each tube and rapidly frozen in liquid N2 before being stored at –80°C for the later determination of [NOx]. Next 50 µl of the plasma substitute hetastarch (Sigma, UK) was added to the remaining plasma and red blood cells in the tube and was re-infused into the animal.
A MacLab/8s (AD Instruments Ltd, Hastings, West Sussex, UK) data acquisition system sampling at 100 Hz was used to collect all cardiovascular variables onto a Power Mac G4 computer. FVC was derived on-line by dividing FBF by ABP. Following surgery, and before the experimental protocol began, an equilibration period of at least 45 min was allowed for the stabilization of all cardiovascular variables.
Experimental protocols
Group 1. In eight rats (body weight, 356 ± 21.2 g), the cardiovascular changes evoked by a 5-min period of systemic hypoxia (breathing 8% O2) and a 5-min infusion of adenosine (1.2 mg kg–1 min–1 I.A.), were recorded in random order before and 30 min after the administration of L-NAME (10 mg kg–1 I.V.). This dose and infusion rate of adenosine was chosen to evoke an increase in FVC similar to that induced by hypoxia (Bryan & Marshall, 1999a,b). A period of at least 10 min was allowed to elapse between stimuli so that cardiovascular variables could stabilize. During air breathing and in the fifth minute of hypoxia, arterial blood samples were taken for analysis of blood gases. In addition, in the minute before, and in the fifth minute of hypoxia or adenosine infusion, arterial and venous blood samples (see above) were taken for the later determination of plasma [NO] (see below).
Group 2. In a further group of six rats (body weight, 255 ± 13.0 g), a protocol similar to that described for Group 1 was performed, except that L-NAME was replaced by DPCPX (0.1 mg kg–1 I.V.). DPCPX is highly selective for A1 adenosine receptors (Daly et al. 1985), being 700-fold more selective for A1 compared with A2A receptors in binding studies (Coates et al. 1994). This dose of DPCPX reversed the increase in FVC evoked by a selective A1 receptor agonist (see Bryan & Marshall, 1999a and references therein).
Group 3. In a final group of eight rats (body weight, 245 ± 12.1 g), the protocol used in Groups 1 and 2 was repeated, with the vehicle for L-NAME and DPCPX (0.1 ml saline) instead of the administration of the drugs. This group of experiments served as a vehicle and time control.
Assay of NOX levels in arterial and venous plasma
The assay of NO generated from NOx in the arterial and venous plasma samples collected during Group 1, 2 and 3 protocols was a two-stage process which firstly involved the conversion of nitrate (NO3–) in the plasma to nitrite (NO2–), and secondly involved the electrochemical measurement of [NO] by use of an NO-sensitive electrode, following the reduction of NO2– by a 0.1 mol l–1 H2SO4/KI solution.
The conversion of NO3– to NO2– was performed using the Nitralyser II nitrate to nitrite reduction kit (WPI, FL, USA) and the protocol provided with the kit. Each 100-µl sample of supernatant produced by this protocol was then added to 10 ml 0.1 mol l–1 H2SO4/KI and the NO generated from NO2– was measured using an NO-sensitive electrode with a 2-mm diameter tip (ISO-NOP sensor, WPI) connected to an NO meter (ISO-NO Mark II, WPI). We have previously described the use of this electrode to measure NO released from the endothelial cells of freshly excised arterial vessels (Ray et al. 2002). Briefly, NO is produced according to the following equation:
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NOx assay controls
The NO that could be released from NO2– present in arterial and venous blood samples in the absence of chemical conversion of NO3– to NO2– as described above, was assessed by adding 12 µl plasma (the volume of plasma used in the NO3– to NO2– conversion) to 0.1 mol l–1 H2SO4/KI solution. Any change in NO output was recorded using the NO-sensitive electrode. This was carried out for each arterial and venous plasma sample collected in the protocols described above.
To assess the effectiveness of the NO3– to NO2– conversion, controls were carried out at the same time as the experimental plasma samples for each individual animal, by using NO3– standards. These assays were performed according to the protocol described above, but the plasma samples were replaced by NO3– standards at concentrations of 0, 20, 40, 60, 80, 100 and 120 nmol l–1.
Statistical analyses
All data are expressed as means ± S.E.M. Effects of hypoxia and adenosine infusion on cardiovascular variables (ABP, FBF and FVC), blood gas data and [NO]a, [NO]v and [NO]v-a before and after infusion of L-NAME, DPCPX or vehicle were analysed using ANOVA for repeated measures. In addition, because L-NAME caused a decrease in baseline FVC (see below), the muscle vasodilator responses were also analysed as the integral of the FVC over the 5-min period of hypoxia or adenosine infusion relative to the integral of FVC during the preceding 5 min. These comparisons were made before and after infusion of L-NAME, DPCPX and vehicle by using ANOVA. The conclusions that can be drawn from the analyses of FVC were the same whether the absolute values or the integrals of the changes were used: the text below and the figures show the outcome of the analyses on the absolute values. Between-group comparisons of baselines and control responses to hypoxia and adenosine infusion (ABP, FBF, FVC, blood gas data, [NO]a, [NO]v and [NO]v-a) were made by factorial ANOVA. In all cases, the level of statistical significance was set at P < 0.05. Scheffe's post hoc test was used to determine significant differences between measurements taken under different conditions in a given protocol.
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Results |
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Cardiovascular responses to systemic hypoxia and adenosine infusion. A 5-min period of systemic hypoxia (breathing 8% O2) resulted in a substantial fall in PaO2 and PaCO2 (Table 1), a fall in ABP and no change in FBF (Table 2), but an increase in FVC, indicating hindlimb vasodilatation (Fig. 1A; Bryan & Marshall, 1999a; Ray et al. 2002). A similar cardiovascular response was induced by adenosine infusion (1.2 mg kg–1 min–1 I.A.; Fig. 1A and Table 2), but there were no blood gas changes (data not shown; Bryan & Marshall, 1999a). L-NAME (10 mg kg–1 I.V.) caused a significant increase in baseline ABP and PaO2, and a significant decrease in baseline FBF and FVC (Tables 1 and 2 and Fig. 1A). However, L-NAME had no effect on baseline levels of PaCO2 and pH, or the fall in PaO2 and PaCO2 and the increase in pHa evoked by hypoxia (Table 1). Nevertheless, L-NAME significantly attenuated the increase in FVC evoked by systemic hypoxia and by adenosine infusion (Fig. 1A).
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Group 2: effects of DPCPX
Cardiovascular responses to systemic hypoxia and adenosine infusion. The responses evoked by systemic hypoxia and adenosine infusion were qualitatively similar to those of Group 1; although in Group 2, the increase in FVC evoked by adenosine was smaller than that evoked by systemic hypoxia (cf. Fig. 2A and Fig. 1A). DPCPX (0.1 mg kg–1 I.V.) had no significant effect on baseline cardiovascular variables (Table 2), or baseline PaCO2 or pHa, but significantly increased baseline PaO2 (Table 1). DPCPX did not affect the fall in PaO2 and PaCO2, nor the increase in pHa evoked by systemic hypoxia (Table 1), but greatly reduced the increase in FVC evoked by both systemic hypoxia and adenosine infusion (Fig. 2A).
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Group 3: vehicle
Cardiovascular responses to systemic hypoxia and adenosine infusion. The responses evoked by systemic hypoxia and adenosine infusion were similar to those of Group 1 and 2. The vehicle for the administration of L-NAME and DPCPX had no significant effect on baseline cardiovascular variables (Table 2) or the responses to hypoxia or adenosine infusion. In addition, the changes in blood gases and pHa evoked by hypoxia were unaffected by the administration of the vehicle for L-NAME and DPCPX (Table 1).
Plasma [NO]x during systemic hypoxia and adenosine infusion. As in Group 1 and 2, systemic hypoxia evoked a significant increase in [NO]a and [NO]v (Fig. 3B), and this response was unchanged after administration of vehicle. Adenosine infusion increased [NO]a before administration of vehicle and showed a strong tendency to increase [NO]a after administration of vehicle (P = 0.0564), [NO]v was significantly increased before and after vehicle. Vehicle had no effect on baseline [NO]a and [NO]v (Fig. 3B). As was also the case with Group 1 and 2, [NO]v-a was significantly increased during the fifth minute of hypoxia and adenosine infusion, this response was unchanged by vehicle (Fig. 3C). Vehicle also had no effect on baseline [NO]v-a.
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Baseline blood gas data varied slightly between experimental groups, PaO2 was significantly higher in Group 1 than Group 3, PaCO2 was higher in Group 1 and 2 than Group 3 and pHa was lower in Group 1 and 2 than Group 3 (Table 1). There were no differences between groups for baseline values of ABP, FBF or FVC or for magnitude of increases in FVC evoked by hypoxia under control conditions, but the increase in FVC evoked by adenosine was smaller (P < 0.05) in Groups 2 and 3 than in Group 1. Baseline levels of [NO]a, [NO]v and [NO]v-a did not differ between groups; neither did increases in [NO]v-a evoked by hypoxia or adenosine (see Discussion for further comment).
NOx assay controls
Addition of 12-µl plasma samples to 10 ml 0.1 mol l–1 H2SO4/KI evoked no change in the output from the NO meter. Moreover, control samples containing no NO3– standard caused no detectable increase in the output of the NO meter, demonstrating that the reagents used in the assay had no effect on the NO-sensitive electrode. The assay of the NO3– standards that was performed over the same time period as each set of experimental samples demonstrated a linear relationship between [NO3–] and NO produced. The percentage conversion for each set of experimental samples was calculated using the NO3– standards and was factored into the calculation of plasma [NO] in the experimental samples. Mean percentage conversion of NO3– to NO2– for all experiments, calculated from the regression of NO released from various concentrations of a NO3– standard, was 54.7 ± 5.68%.
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Discussion |
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The new findings of the present study are that both systemic hypoxia and adenosine infusion produced an increase in the NO generated from NOx in arterial and venous plasma of skeletal muscle ([NO]a and [NO]v, respectively) and an increase in [NO]v-a across muscle, and these changes were attenuated by L-NAME and by DPCPX. Our interpretation of these findings is that systemic hypoxia and adenosine increased the release of NO into the venous efflux of skeletal muscle. Before discussing this interpretation, it is important to consider the validity of the NO measurements.
Validity of the NO measurements
We have shown previously, as have others (Guo et al. 1996; Simonsen et al. 1999; Ray et al. 2002), that the type of NO electrode used in the present study allows reproducible NO measurements upon calibration within and between experimental days. The protocol used here to evaluate NO release into plasma from changes in [NOx] is novel; that is, reduction of plasma NO3– and NO2–, with electrochemical measurement of NO generated from NO2–. However, in previous studies, similar methods were used to measure NO released into human skin in microdialysis samples (Clough, 1999), and NO released from cultures of porcine coronary and aortic endothelial cells in vitro (Berkels et al. 2001). The assay of a range of NO3– standards performed along with each set of plasma samples allowed the effectiveness of the protocol to be assessed. In all cases, the conversion of NO3– standards demonstrated a linear relationship for NO released and the percentage conversion of NO3– to NO2– and thence to NO showed very little variation between experimental groups: we deduced that we measured 
55% of the NO held as NOx. In fact, because addition of plasma samples directly to the 0.1 mol l–1 H2SO4/KI reducing solution produced a negligible change in the NO measured, we surmise that the concentration of NO2– in the plasma was not significant and that NO released into plasma was rapidly converted via NO2– to NO3–, or was combined in some other form. Moreover, in Group 3, changes in [NO]a, [NO]v and thus [NO]v-a evoked by hypoxia and adenosine infusion were unaffected by drug vehicle over the same time course as the effect of L-NAME and DPCPX seen in Group 1 and 2, respectively, demonstrating that the protocol was highly reproducible.
The relative oxygenation of blood may affect plasma [NO3–]. In arterial blood, oxygenated haemoglobin (HbO2) predominates, and the major route of NO metabolism is reaction with HbO2 to form methaemoglobin (metHb) and NO3–. In venous blood, more deoxygenated haemoglobin (deoxyHb) is present and a higher proportion of NO generated reacts with deoxyHb to form nitrosylated haemoglobin (HbNO; Jia et al. 1996; Mateo & de Artiñano, 2000). Consequently, the measurement of NO generated from NO3– in venous plasma and therefore the calculation of [NO]v-a is likely to underestimate the amount of NO present under conditions of systemic normoxia. There is likely to be a further underestimation of NO released in systemic hypoxia when there is more deoxyHb in both arterial and venous blood. The reaction of NO with superoxide anions, with haem moieties of molecules other than Hb and with thiol groups on proteins, including Hb, to form S-nitrosothiols (SNO-Hb) (Jia et al. 1996; Mateo & de Artiñano, 2000) will also have contributed to the underestimation of the NO released when [NO3–] in plasma is used as an index.
The absolute levels of NOx measured in arterial and venous blood under baseline experimental conditions did not differ significantly between groups. However, Groups 1 and 2 tended to show lower levels than Group 3. Dietary intake of nitrites and nitrates has a major effect on plasma NOx levels, as does urinary excretion of NOx (Green et al. 1981a,b; Wagner et al. 1985). However, all rats used in the present study had been fed on standard rat chow prior to the acute experiments and there is no reason to suppose that the urinary excretion of NOx varied between the groups. There is also no reason to suggest that the balance between absorption of dietary nitrogen metabolites and urinary excretion differed in an age- or weight-related way, as Group 3 had the highest plasma levels of NOx and yet they were similar in age and weight to Group 2; Group 1 were older and heavier (see Methods). Further, there were no differences between the groups in the baselines of the cardiovascular variables (Table 2 and Figs 1–3) that might either explain, or reflect the differences in NOx levels. However, if it is accepted that NO is released into plasma when PaO2 falls, then differences between the groups in their baseline levels of PaO2 (see Table 1) may contribute to differences in their NOx levels: Group 3 had the lowest PaO2 and the highest NOx levels, while Group 1 had the highest PaO2 and the lowest NOx levels.
As far as the aims of the present study are concerned, what matters is not the absolute level of NOx, but the changes in [NOx] evoked by hypoxia and adenosine. Changes in [NOx] measured in samples taken only 5 min apart over a period of hypoxia or adenosine infusion could not have reflected changes in urinary excretion of NOx: the half-life of NO3– in plasma has been calculated at 1.5 h in mice (Veszelovszky et al. 1995) and as much as 3.8 h in dogs (Zeballos et al. 1995). Rather, they are more likely to have reflected changes in the NO released into the blood. The fact that both systemic hypoxia and adenosine infusion caused an increase in [NO]a, [NO]v and in [NO]v-a across hindlimb muscle demonstrates that both stimuli release NO from skeletal muscle. The measurements made in Group 3 clearly showed that within a given group of rats, the release of NO caused by both stimuli is highly reproducible over time.
Thus, taking all these factors into consideration, we conclude that the changes in plasma [NO] presented here may have underestimated the total amount of NO released and available to act on vascular smooth muscle to evoke vasodilatation, but they do give a reliable indication of the amount of NO released from muscle by systemic hypoxia and adenosine.
NO release during hypoxia- and adenosine-induced muscle vasodilatation
With these provisos, the fact that the increases in [NO]a, [NO]v and [NO]v-a evoked by hypoxia and adenosine in Group 1 were attenuated by L-NAME, demonstrates that these changes reflected the new synthesis of NO by NOS rather than the release of NO into plasma from HbNO, or from nitrosothiol compounds including SNO-Hb (see Jia et al. 1996). It might be argued that the increases in [NO]v-a were partly due to an increase in shear stress associated with the muscle vasodilator responses evoked by these stimuli. This seems very unlikely for in response to both stimuli, the evoked increase in FVC balanced the associated fall in ABP such that there was no change in FBF (see Table 2). Under these circumstances, it is unlikely that shear stress increased sufficiently to cause NO synthesis (see also Ray et al. 2002).
As L-NAME inhibits the tonic synthesis of NO by NOS as evidenced by the decrease in baseline FVC, a decrease in baseline NOx levels might have been expected. The fact that L-NAME had no significant influence on [NO]a, [NO]v or [NO]v-a during air breathing suggests that plasma [NOx] is maintained even in the absence of NO synthesis, as has been noted before (Sawada et al. 1994). This could be explained by liberation of NO and formation of NO2– and NO3– from the various NO stores. In particular, because L-NAME and DPCPX both increased baseline PaO2 by 10 mmHg, presumably by removing inhibitory effects of NO and adenosine on ventilation (Thomas et al. 1994; Gozal et al. 1997), this may have allowed a greater transfer of NO already held as HbNO, to the cysteine group on Hb, so forming SNO-Hb at the lungs during oxygenation of Hb and allowing more NO to be released during arterial–venous transit through the circulation to form NOx (Jia et al. 1996).
Taken together with the results discussed above, the finding that the increases in [NO]a, [NO]v and [NO]v-a evoked by systemic hypoxia and by adenosine infusion in Group 2 were attenuated by DPCPX, indicates that during systemic hypoxia, adenosine acting at A1 receptors is responsible for increased synthesis and release of NO from skeletal muscle. These findings do not exclude the possibility that systemic hypoxia and/or adenosine infusion also caused an increase in the synthesis and release of NO that was not mediated by A1 receptor stimulation: given the variance of the [NO] data, we cannot deduce with any certainty whether the apparent, small increase in [NO]v-a in response to both stimuli after administration of DPCPX (see Fig. 2C) was real.
Our hypothesis is that the NO released by these stimuli contributes to the accompanying increases in FVC. Thus, some consideration must be given to the fact that the magnitude of the vasodilator responses evoked by hypoxia and adenosine infusion were not directly related to the magnitude of the accompanying increases in [NO]v-a (see Figs 1–3). First, it should be noted that in Group 1, the increase in FVC evoked by hypoxia and adenosine were similar as we intended, whereas the increases in FVC evoked by adenosine infusion in Groups 2 and 3 were substantially smaller than those evoked by hypoxia, even though we infused adenosine at the same dose per kilogram body weight. This may reflect the fact that the rats of Groups 2 and 3 were substantially younger than those of Group 1: there is evidence that dilator responsiveness to adenosine is weak or absent in neonates and increases with maturity (Laudignon et al. 1990; Elnazir et al. 1996). Second, in Group 2, the selective A1 receptor antagonist DPCPX, decreased the muscle vasodilator response evoked by hypoxia by only 30%, whereas in most of our previous studies, 50% of the hypoxia-induced muscle dilatation was mediated by adenosine and/or by A1 receptor stimulation (e.g. Skinner & Marshall, 1996; Bryan & Marshall, 1999a). The explanation for this disparity may also lie in the size and age of the animals. In most of our previous studies, the rats weighed > 300 g (Skinner & Marshall, 1996; Bryan & Marshall, 1999a) and so were comparable to those of Group 1. By contrast, in our recent study (Ray et al. 2002), the mean weight of the rats was comparable to Groups 2 and 3 and adenosine receptor blockade reduced the increase in FVC evoked by breathing 8% O2 by only 23%.
Thus, there is clearly variation, which seems age-related, in the magnitude of the muscle vasodilatation evoked by hypoxia and adenosine infusion and in the contribution of A1 receptor stimulation to these responses, even though our previous studies (Bryan & Marshall, 1999a) and the results of Group 3, show that within a group of animals of similar age, the muscle vasodilator responses evoked by hypoxia and by adenosine infusion are reproducible.
Seen in this context, the apparent disparity between the evoked increases in FVC and in [NO]v-a may be explained by differences in the balance of the various contributors to the dilator responses. The responses evoked by hypoxia and exogenous adenosine not only reflect the local actions of these stimuli, but the magnitude of these responses are also affected by the vasoconstrictor influences of the increase in sympathetic nerve activity evoked by peripheral chemoreceptor stimulation and/or baroreceptor unloading and by the vasoconstrictor and dilator influences of hormones such as vasopressin and adrenaline (see Marshall, 1994). It should also be noted that exogenous adenosine can evoke vasodilatation in hindlimb muscle in an NO-independent, as well as in a NO-dependent manner (Bryan & Marshall, 1999b). Further, we do not know the extent to which the NO collected in venous plasma originates from the vessels that contribute to changes in FVC (for further discussion, see below). In other words, there is no reason to expect a simple direct relationship within or between groups for the increase in [NO]v-a evoked by the stimuli we used and the accompanying increase in FVC.
Origin of the NO
The increase in NO output evoked by adenosine infusion was probably the result of generation of NO by endothelial NOS (eNOS) rather than by neuronal NOS (nNOS) present in skeletal muscle or sympathetic neurones (Michel & Feron, 1997), as adenosine is rapidly taken up and metabolized by endothelial cells and is unlikely to have reached extravascular tissues (see Ray et al. 2002). Further, the evidence available suggests that the adenosine released during systemic hypoxia is of endothelial, rather than skeletal muscle origin (see Mian & Marshall, 1991b; Mo & Ballard, 2001; Edmunds et al. 2003). Thus, it is likely that this endogenous adenosine acts back on the endothelial cells via A1 receptors to stimulate eNOS and release NO. Consistent with this, in in vitro studies on rat aorta and iliac artery in which NO was measured using an NO-sensitive electrode, adenosine stimulated the synthesis and release of NO from the endothelium (Ray et al. 2002).
Given that both L-NAME and DPCPX reduced the hypoxia- and adenosine-evoked increases in FVC and the accompanying increases in [NO]v-a, and because changes in FVC largely reflect behaviour of proximal arterioles (see Introduction), it is likely that some of the NO released came from the endothelium of proximal arterioles. If so, then the dilatation of proximal arterioles is NO-mediated as well as NO-dependent, providing that eNOS activity is present (see Introduction; Edmunds & Marshall, 2003; Edmunds et al. 2003). A further portion of the NO release probably originated from the terminal arterioles, which we have already deduced show adenosine A1-, NO-mediated dilatation during systemic hypoxia (Edmunds et al. 2003). Finally, capillaries of skeletal muscle show NO-dependent dilatation in response to bradykinin (Mitchell & Tyml, 1996), while venous vessels show NO-dependent dilatation in response to agonists (Blackman et al. 2000) and adenosine-mediated dilatation in response to systemic hypoxia (Mian & Marshall, 1991b), and venous endothelial cells generate NO in response to a range of agonists (Vedernikov et al. 1988; Vallance et al. 1989; Pawloski & Chapnick, 1993; Hamilton et al. 1997; Sobrevia et al. 1997). This raises the possibility that the capillaries and venous vessels of skeletal muscle also contributed to NO released in response to systemic hypoxia and adenosine.
In summary, the results of the present study show for the first time, that NO is released into venous effluent of skeletal muscle by acute systemic hypoxia and by exogenous adenosine. They indicate that the NO most probably originated from the vascular endothelium and that it is newly synthesized by NOS activity arising, at least in part, from the action of adenosine on A1 receptors. The NO released makes a major contribution to hypoxia- and adenosine-induced muscle vasodilatation.
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