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INTEGRATIVE |
1 Department of Physiology, The Medical School, Birmingham B15 2TT, UK
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Abstract |
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(Received 3 March 2006;
accepted after revision 10 May 2006;
first published online 11 May 2006)
Corresponding author J. M. Marshall: Department of Physiology, The Medical School, Birmingham B15 2TT, UK. Email: j.m.marshall{at}bham.ac.uk
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Introduction |
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The adaptations that occur in ventilation and haematocrit (Hct) from the onset of chronic hypoxia have been extensively investigated (e.g. Olson & Dempsey, 1978; Dempsey & Forster, 1982; Ou et al. 1992). By contrast, relatively little is known of the cardiovascular adaptations. We have now confirmed (accompanying paper Walsh & Marshall, 2006) that resting hyperventilation is already present in rats exposed to chronic hypoxia (12% O2) for 1, 3 and 7 days (1, 3 and 7CH rats), while an increase in Hct is present in 3 and 7CH rats. However, despite the hyperventilation, there was no baseline tachycardia in the 1–7CH rats, suggesting dominance of the local effects of hypoxia on HR over the stimulatory effects of increased respiration. Further, in 1 and 3CH rats breathing 12% O2, ABP was decreased and FVC greatly increased relative to levels recorded in N rats acutely exposed to 12% O2; in 7CH rats, FVC had apparently returned to that recorded in N rats breathing 21% O2. Interpretation of these results is complicated by the effects of hypoxia-induced arteriogenesis and capillary angiogenesis in skeletal muscle on baseline and maximal FVC. These are present in 14CH and 3–4 week CH rats and already partly developed in 7CH rats (Price & Skalak, 1998; Smith & Marshall, 1999; Deveci et al. 2001). However, we argued that there is tonic muscle vasodilatation in 1CH rats, mainly caused by the local effects of hypoxia and that FVC falls again in 3–7CH rats when tissue O2 supply increases, due to the progressive increase in Hct and vascular remodelling. From the effects of the nitric oxide synthase (NOS) inhibitor L-NAME, we deduced that the decreased baseline ABP and increased FVC were mainly attributable to an accentuated, tonic vasodilator influence of NO which waned from the 1st to the 7th day of hypoxia (Walsh & Marshall, 2006).
In N rats, the component of the muscle vasodilatation evoked by acute hypoxia that is mediated by adenosine acting on A1 receptors is NO dependent (Skinner & Marshall, 1996; Bryan & Marshall, 1999b). Current evidence indicates this is because a tonic level of NO is required for hypoxia to release adenosine from endothelial cells and because A1 receptor stimulation activates NOS to release NO (Ray et al. 2002; Edmunds et al. 2003; Ray & Marshall, 2005). We therefore hypothesized that the raised FVC and decreased ABP in 1–7CH rats are sustained by the influence of tonically released adenosine on A1 receptors and therefore on NO synthesis.
The affinity of adenosine for A1 receptors is 80- to 1000-fold higher than for A2A receptors (Ueeda et al. 1991; Daly & Padgett, 1992). Stimulation of A2A receptors does not contribute to the muscle vasodilatation of acute hypoxia in N rats, but does contribute to the muscle vasodilatation evoked by exogenous adenosine (Bryan & Marshall, 1999a). This A2A receptor-mediated vasodilatation is NO dependent and adenosine can act on endothelial A2A receptors to increase NO synthesis and release (Bryan & Marshall, 1999b; Ray et al. 2002; Ray & Marshall, 2005). Thus, we hypothesized that adenosine is released in high enough concentrations in 1–7CH rats to stimulate vascular A2A receptors either tonically, or during an acute hypoxic challenge. Further, given the known influences of adenosine on HR and ventilation (see above), we hypothesized that in 1–7CH rats, adenosine exerts an inhibitory effect on HR via A1 receptors and inhibitory and stimulatory effects on ventilation via A1 and A2A receptors, respectively. Although the A2B and A3 receptors have even lower affinities for adenosine than A1 and A2A receptors (Zhou et al. 1992; Feoktistov & Biaggioni, 1997), their stimulation can produce vascular, cardiac and ventilatory responses (Ralevic & Burnstock, 1998). Thus, it was a reasonable hypothesis that their stimulation by endogenous adenosine may also influence the cardiovascular and ventilatory systems of 1–7CH rats.
We therefore compared the effects of the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) on the baseline levels of cardiovascular and ventilatory variables in N rats breathing 21% O2 and 1–7CH rats breathing 12% O2 and upon the responses evoked by acute hypoxia (breathing 8% O2). Baselines and responses evoked by acute hypoxia were then re-examined after 8-sulpho-phenyltheophylline (8-SPT), an adenosine receptor antagonist that is non-selective between the receptor subtypes and does not cross the blood–brain barrier (Evoniuk et al. 1987). Thus, we tested whether adenosine acting on peripheral adenosine receptors other than A1 receptors contributes to baselines or evoked responses in 1–7CH rats. Responses evoked by infusion of adenosine were also monitored to indicate the potential effects of adenosine and the efficacy of the receptor blockade.
We used the non-selective NOS inhibitor L-NAME in our companion study (Walsh & Marshall, 2006), and therefore did not test whether endothelial, inducible and/or neuronal NOS (eNOS, iNOS, nNOS, respectively), contribute/s to the generation of NO in 1–7CH rats. In pulmonary vasculature of 1–7CH rats, iNOS is up-regulated (Le Cras et al. 1996). Thus, in subgroups of N, 1 and 3CH rats, we tested the effect of the selective iNOS inhibitor aminoguanidine (Joly et al. 1994; Scott & McCormack, 1999) upon baseline levels and the muscle vasodilatation evoked by the NO-dependent dilator acetylcholine (ACh) (Gardiner et al. 1989).
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Methods |
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Protocol
From the time the tracheal cannula was inserted the CH rats breathed 12% O2 and the N rats breathed 21% O2 delivered from O2 and N2 gas cylinders via a rotometer mixing system, except when the gas mixture was switched to 8% O2 (see below). When all surgery had been completed, the Saffan infusion rate was reduced to 4–8 mg kg–1 h–1 until a stable level of anaesthesia was achieved (see Walsh & Marshall, 2006). All variables were allowed to stabilize for at least 30 min so that baseline values could be recorded. At the end of this period, arterial samples were removed for analysis of PaO2, PaCO2 and pHa. Adenosine was then infused via the tail artery at 1.2 mg kg–1 min–1 for 5 min. When the baseline levels were stable again, the inspirate was switched to 8% O2 for 5 min. Arterial samples were taken before and in the 5th minute of adenosine infusion and hypoxia for analysis of PaO2, PaCO2 and pHa.
When the baseline levels were again stable, the highly selective A1 receptor antagonist DPCPX (Kellet et al. 1989) was given at 0.1 mg kg–1
I.A. This dose produced selective blockade of the cardiovascular responses evoked by infusion of a selective A1 receptor agonist (Kellet et al. 1989; Bryan & Marshall, 1999a,b). New baseline levels were recorded when they had stabilized at 
10 min after DPCPX administration and then the responses evoked by adenosine and by hypoxia were re-tested as described above.
Each animal was then given the non-selective adenosine receptor antagonist 8-SPT at 20 mg kg–1
I.A. This dose virtually abolished the increase in FVC evoked by adenosine infusion (Skinner & Marshall, 1996). Baseline values were recorded at 10 min when they had stabilized and responses evoked by adenosine infusion and hypoxia were then re-tested as described above.
Subsequently, in a subgroup of six 1CH rats and six 3CH rats the effect of the iNOS inhibitor aminoguanidine and then L-NAME was tested upon the responses evoked by infusion of ACh. To this end, ACh was infused at 10 µg kg–1 min–1 for 5 min via the ventral tail artery, this dose being chosen to produce a substantial vasodilatation in hindlimb (Gardiner et al. 1989). When baseline levels had stabilized, aminoguanidine was given at 8.5 mg kg–1
I.A. Baseline levels were recorded at 10 min and then the response evoked by ACh was re-tested. This protocol was repeated with cumulative doses of aminoguanidine at 17.5, 35 and 70 mg kg–1
I.A. These doses of aminoguanidine were used on the basis that doses in this range abolished iNOS activity in rat lung and thoracic aorta in vivo and in vitro, respectively (Scott & McCormack, 1999). In fact, in rat lung in vivo, 17.5 mg kg–1 produced maximal selective inhibition of iNOS activity; 175 mg kg–1 did not produce any greater effect on iNOS, or an effect on eNOS activity (Scott & McCormack, 1999). Finally, L-NAME was given at 10 mg kg–1
I.A., a dose which produces maximal inhibition of tonic NO synthesis as judged from the effect on baseline ABP and FVC values (see Walsh & Marshall, 2006). Baselines were recorded after 10 min and the response evoked by ACh was re-tested. At the end of the experiments all animals were killed by an overdose of anaesthetic.
Analysis
All results are expressed as mean ±
S.E.M. Comparisons of baseline values and values recorded on the 5th minute of adenosine infusion or 8% O2 were compared within and between groups by using ANOVA followed by Bonferroni post hoc tests when appropriate. Similar comparisons were made of responses evoked by ACh before and after aminoguanidine and L-NAME. However, because the increase in FVC evoked by ACh tended to peak at 1 min of infusion and to decrease thereafter, whereas that evoked by hypoxia and adenosine reached a plateau that was more or less stable between the 2nd and 5th minute, the responses evoked by ACh were analysed at the end of the 1st minute of infusion. In all cases, P < 0.05 was considered to be significant.
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Results |
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In N rats, the A1 receptor antagonist DPCPX had no effect on the baseline values of the ventilatory or cardiovascular variables nor on blood gases or pHa (Fig. 1, Table 1). Similarly, DPCPX had no affect on baseline RF or VT in 1–7CH rats and there were no changes in blood gas or pH values (Fig. 1, Table 1). However, DPCPX increased baseline HR in 1CH rats but had no effect in 3 or 7CH rats (Fig. 1). Further, in 1 and 3CH rats, DPCPX decreased baseline FVC and increased baseline ABP, whereas there was no significant effect of DPCPX on baseline FVC or ABP in 7CH rats.
From these new baselines, in N rats, adenosine infusion still evoked an increase in RF, accompanied by an increase in FVC and fall in ABP, but FVC did not reach such a high level as before DPCPX. Further, HR reached a higher level in response to adenosine after DPCPX (Fig. 1). DPCPX had qualitatively similar effects on the responses evoked by adenosine in all CH rats. However, the evoked increase in RF reached significance only in 7CH rats and there was, correspondingly, an increase in PaO2 and pHa and a fall in PaCO2 only in 7CH rats (Fig. 1, Table 1). Adenosine still evoked increases in FVC and falls in ABP in all CH rats, but the FVC did not increase to such a high level and ABP did not fall to such a low level after DPCPX as before (Figs 1 and 2). Adenosine increased HR to a higher level than before DPCPX in all CH rats (Fig. 1).
The effects of DPCPX on the responses evoked by 8% O2 were similar in N and CH rats (Fig. 3). Hypoxia still evoked an increase in RF and VT in N and CH rats and these changes were apparently unaffected by DPCPX. On the other hand, although hypoxia still evoked an increase in FVC and fall in ABP in N and all CH rats, these changes were smaller after DPCPX, such that FVC did not reach such a high level and ABP did not reach such a low level after DPCPX as before (Figs 3 and 4). However, HR was significantly increased by the 5th minute of hypoxia in N rats and reached a higher level than before DPCPX, whereas there was no significant change in HR in CH rats.
Effects of 8-SPT
In N and all CH rats, the baseline values of the ventilatory and cardiovascular variables were not significantly different from those recorded after DPCPX, i.e. 8-SPT caused no further changes (see Fig. 1).
The effects of 8-SPT on the responses evoked by adenosine infusion were similar in N and all CH rats (Fig. 1). Adenosine continued to evoke an increase in RF in N rats, although any change in RF in the CH rats did not reach significance; there was no change in VT in N or CH rats. The increase in FVC that was still evoked by adenosine after DPCPX no longer occurred after 8-SPT in N or CH rats such that FVC was lower at the 5th minute of adenosine infusion after 8-SPT than after DPCPX (Figs 1 and 2). Adenosine still evoked a small fall in ABP after 8-SPT, but the level to which ABP fell in N rats was not as low after 8-SPT as before. The HR reached at the end of the 5th minute of adenosine infusion in N and all CH rats was lower after 8-SPT than after DPCPX (Figs 1 and 2).
By contrast, 8-SPT had very little effect on the responses evoked by 8% O2 in N or CH rats. However, the hypoxia-evoked increase in RF no longer occurred in any group of CH rats after 8-SPT, although there was still a significant increase in RF in N rats (Fig. 3). Concomitantly, hypoxia still evoked an increase in VT, an increase in FVC and a fall in ABP in N and all CH rats and these changes were similar after 8-SPT to those after DPCPX (Figs 3 and 4). In N rats, PaO2 and PaCO2 fell, while pHa increased, whereas amongst CH rats PaCO2 fell and pHa increased only in 1CH rats (Table 2).
Effects of aminoguanidine and L-NAME
ACh infusion given before the first dose of aminoguanidine evoked an increase in FVC and a fall in ABP in N rats, and in 1 and 3CH rats, that peaked at 1 min, the changes being similar in all groups (Fig. 5). Despite continued infusion for 5 min, FVC and ABP returned to baseline within 2 min; the other variables were not altered (see Fig. 6).
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Baseline values of PaO2, PaCO2 and pHa were not affected in N or 1 and 3CH rats even when aminoguanidine was given at 70 mg kg–1. In 1 and 3CH rats after L-NAME, there was an increase in PaO2 and in pHa (data not shown); the mechanisms that may underlie these changes are discussed in our companion paper (Walsh & Marshall, 2006).
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Discussion |
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Effects of DPCPX
ABP and FVC. That DPCPX attenuated the increase in FVC and ABP evoked by infusion of adenosine in N rats and all CH rats confirmed our previous results in N rats (Skinner & Marshall, 1996) and shows that in 1–7CH rats also, exogenous adenosine induces muscle vasodilatation and reduces ABP partly by stimulating A1 receptors. Thus, the finding that DPCPX increased baseline ABP and decreased baseline FVC in 1 and 3CH rats confirms our hypothesis that the tonic muscle vasodilatation and reduced ABP of 1–3CH rats is partly attributable to maintained release of adenosine acting on A1 receptors. In acute systemic hypoxia, the evidence suggests that: (i) adenosine is released from the endothelium of skeletal muscle blood vessels, rather than the skeletal muscle fibres (Mian & Marshall, 1991; Mo & Ballard, 2001); (ii) the release of adenosine from endothelium is dependent on a tonic level of NO (Edmunds & Marshall, 2001; Edmunds et al. 2003); and (iii) the dilator action of adenosine on muscle vasculature in acute hypoxia is due to A1-stimulated synthesis and release of NO (Ray et al. 2002; Ray & Marshall, 2005). Our companion study (Walsh & Marshall, 2006) indicated that the tonic muscle vasodilatation of 1 and 3CH rats is hypoxia dependent and due to an accentuated tonic influence of NO. Thus, we now propose that this tonic muscle vasodilatation is partly mediated by a hypoxia-dependent release of adenosine that acts on the endothelial A1 receptors to stimulate NO synthesis by eNOS.
Given DPCPX had no effect on baseline FVC or ABP in 7CH rats, it seems that the vascular remodelling in skeletal muscle of 7CH rats (Smith & Marshall, 1999), together with the increased Hct (Ou et al. 1992; Walsh & Marshall, 2006), has improved the O2 supply enough to offset the stimulus for adenosine release. If so, then the decrease in FVC that occurred in 7CH rats when they acutely breathed 21% O2 may be attributed to a hypoxia-dependent dilator other than adenosine, or more likely, to a hyperoxia-dependent vasoconstrictor (see Walsh & Marshall, 2006).
That the increase in FVC and fall in ABP evoked by acute hypoxia (breathing 8% O2) were reduced by DPCPX in N and all CH rats is consistent with our previous reports on N rats (e.g. Bryan & Marshall, 1999a). We showed that the increase in FVC evoked by acute hypoxia in 1–7CH rats was greatly attenuated by inhibition of NOS synthesis (Walsh & Marshall, 2006). Thus, we can propose that in 1, 3 and 7CH rats as well as in N rats, acute hypoxia caused additional release of adenosine in skeletal muscle that acts on A1 receptors to induce muscle vasodilatation, at least in part by increasing NO synthesis (Ray et al. 2002; Ray & Marshall, 2005).
The fact that we have shown a tonic dilator influence of adenosine on A1 receptors in 1–7CH rats and that the muscle vasodilator responses evoked by exogenous adenosine and adenosine released in acute hypoxia acting on A1 receptors were at least as large in 1–7CH rats as N rats is particularly significant. For, in many cell types, including cardiac myocytes and smooth muscle cells, prolonged exposure to adenosine leads to functional desensitization of A1 receptors that develops in minutes, lasts for hours and may involve uncoupling of A1 receptors from second messenger systems and internalization of A1 receptors (Ralevic & Burnstock, 1998; Saura et al. 1998). Given that the vasodilator effect of A1 receptors is mainly endothelium- and NO-dependent, it may be that endothelial A1 receptors do not desensitize and/or the functional consequence of desensitization is counteracted by increased release of NO (see Walsh & Marshall, 2006).
Heart rate. In a previous study (Bryan & Marshall, 1999a), infusion of a selective A1 receptor agonist induced a substantial decrease in HR in N rats that was reversed by the dose of DPCPX used in the present study. In the present study, infusion of adenosine had no significant effect on HR in N rats or 1–7CH rats before DCPX, but increased HR in N rats and CH rats after DPCPX. Thus, the ability of adenosine to cause bradycardia was apparently present in N and all CH rats and was revealed by DPCPX at the dose used.
On this basis, our finding that DPCPX had no effect on baseline HR in N rats, but increased baseline HR in 1CH rats, indicates that adenosine tonically released into the plasma or within the heart, reduces resting HR in CH rats by acting on cardiac A1 receptors. This is consistent with one of the hypotheses of the present study and a suggestion made in our companion study (Walsh & Marshall, 2006). Thus, we can propose that in 1CH rats, the explanation for baseline HR not being raised despite the resting hyperventilation, is that the local cardio-inhibitory effect of adenosine (Evans et al. 1982; Belardinelli et al. 1989) overcomes the sympathetically mediated tachycardia induced by hyperventilation (Thomas & Marshall, 1994). However, DPCPX did not affect baseline HR in 3 or 7CH rats. Moreover, whereas we confirmed DPCPX allowed better maintenance of HR during acute hypoxia in N rats (see Thomas & Marshall, 1994), this did not occur in 1–7CH rats (see Fig. 3).
Taken together, these findings suggest that the dominant influence on HR in 1–7CH rats is depression of the action of cardiac sympathetic nerves on cardiac ß-receptors (see Kacimi et al. 1992), and that accentuation of the cardio-inhibitory effect of adenosine wanes after the first day. This is consistent with the evidence that cardiac A1 receptors desensitize during prolonged exposure to adenosine and that 4 week CH rats show a decrease in cardiac A1 receptor density (Kacimi et al. 1993; Ralevic & Burnstock, 1998.)
Ventilation. We hypothesized that DPCPX would increase baseline VT in 1–7CH rats by removing a tonic effect of adenosine on central A1 receptors (see Schmidt et al. 1995). In fact, DPCPX had no effect on baseline RF and VT in N rats, or in 1–7CH rats. Yet, in a previous study, the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT) which can cross the blood–brain barrier, like DPCPX (see Lohse et al. 1988; Bisserbe et al. 1992), had no effect on baseline ventilation in N rats, but did increase baseline VT in 3–4 week CH rats, when the resting hyperventilation is mainly due to an increase in VT (Thomas & Marshall, 1997). Taken together, these results are consistent with evidence that further adaptation occurs in the ventilatory response to chronic hypoxia between 7 days and 3–4 weeks (Olson & Dempsey, 1978) and suggest this is associated with an increase in the tonic depressive influence adenosine has on ventilation by acting on central A1 receptors.
In our previous studies on N and 3–4 week CH rats, the increase in VT evoked by acute hypoxia (breathing 8% O2) waned during the stimulus and this effect was attenuated by 8-PT, consistent with removal of the inhibitory effect of stimulating central A1 receptors (Neylon & Marshall, 1991; Schmidt et al. 1995; Thomas & Marshall, 1997). In the present study, the increase in VT recorded at the 5th minute of hypoxia was not altered by DPCPX in N or 1–7CH rats. It may be that a longer period of acute hypoxia would have demonstrated such an effect, for in N rats, VT waned further between the 5th and 10th minute of acute hypoxia and the effect of 8-PT was more pronounced at the 10th minute (Thomas & Marshall, 1994). However, an alternative possibility is that the inhibitory effect of central A1 receptors on VT was counteracted by a stimulatory effect on ventilation, particularly in the 1–7CH rats.
Consistent with this idea, infusion of adenosine evoked an increase in RF in N rats and 1–7CH rats that was significantly attenuated by DPCPX in 1 and 3CH rats, though not in N or 7CH rats. This suggests a mechanism by which A1 receptor stimulation can increase RF, at least in 1 and 3CH rats. Since the stimulatory action of adenosine on peripheral chemoreceptors was attributed to A2 receptors rather than A1 receptors (Monteiro & Ribeiro, 1987; Kobayashi et al. 2000), and adenosine reduces VT and RF by acting on A1 receptors within the central nervous system (Wessberg et al. 1985; Schmidt et al. 1995), it seems we have uncovered a novel stimulatory influence of adenosine on RF via A1 receptors that is easier to demonstrate in 1–3CH rats. This action of A1 receptors could be in the carotid body, although the carotid bodies of N rats do not express A1 receptors (Kobayashi et al. 2000). However, given that DPCPX can pass the blood–brain barrier (see above), it could be within the central nervous system. It may be that in early chronic hypoxia, when endogenous levels of adenosine are high, this stimulatory effect of A1 receptors on ventilation is physiologically important in limiting the inhibitory effect of central A1 receptor stimulation. Accordingly, in 1 and 3CH rats, DPCPX tended to attenuate the increase in RF evoked by acute hypoxia (see Fig. 3) although this did not reach statistical significance.
Effects of 8-SPT
If it is assumed that DPCPX blocked A1 receptors, then subsequent administration of 8-SPT had the potential to block A2A, A2B and A3 receptors peripherally, but not within the central nervous system, for 8-SPT does not cross the blood–brain barrier (Evoniuk et al. 1987). In accord with this, 8-SPT abolished the increase in FVC that persisted in response to adenosine infusion after DPCPX in N rats and in 1, 3 and 7CH rats. Given that A2A receptor stimulation accounts for 50% of the increase in FVC evoked by adenosine infusion in N rats (Bryan & Marshall, 1999a), it is reasonable to propose that A2A receptors similarly contributed to the adenosine-evoked increase in FVC in 1, 3 and 7CH rats; we cannot deduce whether A2B or A3 receptors also played a part.
8-SPT also attenuated the fall in ABP that persisted in response to adenosine infusion after DPCPX in N and 1–7CH rats, but in each group a small fall of 20–30 mmHg persisted. Thus, the combination of the two antagonists, DPCPX and 8-SPT, did not achieve complete adenosine receptor blockade. A possible explanation is that adenosine infused systemically produces part of its effect on ABP by crossing the blood–brain barrier and causing cerebral vasodilatation by acting on A2A receptors on the vascular smooth muscle (Coney & Marshall, 1998): these receptors would not be accessible to 8-SPT. In agreement with this proposition, we previously reported that 8-SPT does not produce such effective attenuation of the fall in ABP and cerebral vasodilatation induced by systemic hypoxia as 8-phenyl theophylline (8-PT), which is similarly non-selective between adenosine receptor subtypes, but does cross the blood–brain barrier (see Thomas & Marshall, 1994; Thomas et al. 1994). The disparity between the effects of 8-SPT and 8-PT cannot be explained by differences in their potencies: 8-SPT is a more effective competitive antagonist than 8-PT (Ramagopal et al. 1988) and we gave 8-SPT at a 2-fold higher dose than 8-PT (20 versus 10 mg kg–1).
It must also be acknowledged that adenosine still evoked an increase in RF in N rats after DPCPX and subsequent 8-SPT, although any remaining increase in RF in the 1, 3 and 7CH rats no longer reached statistical significance. The most likely explanation for this finding is that the A2 receptors of the carotid body that presumably mediate the RF response to adenosine after DPCPX (Monteiro & Ribeiro, 1987; Kobayashi et al. 2000) were not fully blocked by 8-SPT.
Despite these limitations, our finding that 8-SPT given after DPCPX had no further effect on baseline FVC, and did not alter the increase in FVC that was evoked by acute hypoxia in N rats or in 1, 3 or 7CH rats, strongly suggests that tonically released adenosine does not act on A2A receptors, or indeed on A2B or A3 receptors, to contribute to the tonic muscle vasodilatation, or to the muscle vasodilator response to acute hypoxia in N rats (see Bryan & Marshall, 1999a), or in 1, 3 or 7CH rats. Thus, even though we now have evidence that adenosine is tonically released in skeletal muscle in 1 and 3CH rats, and that additional adenosine is released by acute hypoxia (see above), it seems the concentration of adenosine reached locally is not high enough to produce muscle vasodilatation via A2A receptors (see Bryan & Marshall, 1999a), or via A2B and A3 receptors. As indicated in the Introduction, the A2A, A2B and A3 receptors have affinities for adenosine that are orders of magnitude lower than A1 receptors (Ralevic & Burnstock, 1998).
On the other hand, the finding that acute hypoxia no longer evoked an increase in RF after 8-SPT in 1, 3 or 7CH rats indicates that additional adenosine released into plasma or the carotid body was sufficient to increase RF by stimulating the carotid body A2 receptors (Monteiro & Ribeiro, 1987; Kobayashi et al. 2000). The increase in RF in response to acute hypoxia that persisted after 8-SPT in N rats is consistent with incomplete blockade of the A2 receptors (see above).
Effects of aminoguanidine
In a previous study on N rats, 8-SPT reduced the increase in FVC evoked by acute hypoxia by 50% and attenuated the accompanying decrease in ABP, but subsequent administration of L-NAME decreased baseline FVC, increased baseline ABP and virtually abolished the increase in FVC and fall in ABP evoked by acute hypoxia (Skinner & Marshall, 1996). Thus, in N rats, NO has a substantial tonic dilator influence and a substantial component of the muscle vasodilator response to acute hypoxia is dependent on NO that is synthesized by eNOS (see Ray et al. 2002; Ray & Marshall, 2005), but is not mediated by adenosine. It was therefore reasonable in the present study to test whether the tonic vasodilator influence on muscle and ABP that persisted in 1 and 3CH rats after DPCPX and 8-SPT was dependent on NO synthesized by iNOS or by eNOS.
In fact, the highly selective iNOS inhibitor aminoguanidine had no effect on baseline FVC or ABP or on the changes induced in these variables by acute hypoxia in N, 1 or 3CH rats, even when given at 70 mg kg–1, which is 4 times the dose that completely blocks iNOS in sepsis in the rat (Scott & McCormack, 1999). Moreover, aminoguanidine had no effect on the muscle vasodilatation evoked by infusion of the NO-dependent dilator ACh (see Gardiner et al. 1989; Joly et al. 1994). Thus, we can conclude that NO generated by iNOS does not contribute to the tonic muscle vasodilatation of the 1 and 3CH rats, nor does it moderate the muscle vasodilatation evoked by ACh. Indeed, the finding that L-NAME given after aminoguanidine increased baseline ABP, reduced FVC and abolished the dilator response to ACh strongly suggests that NO generated by eNOS is responsible for the tonic dilator influence in 1–3CH rats. The present findings are therefore entirely consistent with our proposal (see Walsh & Marshall, 2006) that the accentuated tonic dilator influence of NO in the 1 and 3CH rats can be attributed to the stimulatory influence on eNOS of tonically released adenosine, other dilator substances released by the state of tissue hypoxia, and the increased shear stress that results from a gradually increasing Hct.
To summarize, the present study on N rats and 1–7CH rats has shown that in the first few days of hypoxia, tonically released adenosine makes a substantial contribution to the reduced baseline level of ABP and increased FVC by stimulating A1 receptors, but not A2A or other adenosine receptors. Concomitantly, adenosine reduces baseline HR in 1CH rats only and has no significant effect on baseline ventilation in 1, 3 or 7CH rats. We propose that the accentuated tonic release of NO that we previously reported in 1 and 3CH rats (Walsh & Marshall, 2006) is partly driven by locally released adenosine acting on endothelial A1 receptors. On the other hand, we conclude that NO synthesized by iNOS makes no contribution to the tonic vasodilatation. By the 7th day of hypoxia, when the tonic dilator influence of NO has waned (see Walsh & Marshall, 2006), we show that adenosine no longer exerts a tonic dilator influence on ABP or muscle vasculature. We conclude this arises because by this time the increase in Hct and angiogenesis has increased muscle O2 supply and removed the stimulus for adenosine release. Interestingly, the growth factor that is strongly implicated in angiogenesis under hypoxic conditions is vascular endothelial growth factor (VEGF) whose expression is increased by hypoxia, adenosine and NO and whose actions may be mediated by NO (Schweiki et al. 1992; Wu et al. 1996; Fischer et al. 1997). It is therefore a reasonable proposition that adenosine and NO play a dual role in early chronic hypoxia, inducing muscle vasodilatation that helps maintain O2 delivery and triggering the angiogenesis that helps to compensate for the tissue hypoxia (see Deveci et al. 2001).
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P < 0.05: 5th minute of adenosine infusion in CH versus N rats. *P < 0.05: baseline value before versus after DPCPX. •
P < 0.05: value recorded in 5th minute of adenosine infusion before versus after DPCPX. 
P < 0.05: value recorded in 5th minute of adenosine infusion after DPCPX versus after 8-SPT.
