Vasodilatation, oxygen delivery and oxygen consumption in rat hindlimb during systemic hypoxia: roles of nitric oxide

Abstract

1. We have investigated the relationship between O₂ delivery (D_O2) and O₂ consumption (V_O2) in hindlimb muscle of anaesthetised rats during progressive systemic hypoxia. Since muscle vasodilatation that occurs during hypoxia is nitric oxide (NO) dependent, we examined the effects of the NO synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME).

2. In control rats (n = 8), femoral vascular conductance (FVC) increased at each level of hypoxia. Hindlimb D_O2 decreased with the severity of hypoxia, but muscle V_O2 was maintained until the critical D_O2 value (D_O2,crit) was reached at 0.64 ± 0.06 ml O₂ min⁻¹ kg⁻¹; below this V_O2 declined linearly with D_O2. This is a novel finding for the rat but is comparable to the biphasic relationship seen in the dog.

3. In another group of rats (n = 6), l-NAME caused hindlimb vasoconstriction and attenuated the hypoxia-evoked increases in FVC. D_O2 was so low after l-NAME administration that V_O2 was dependent on D_O2 at all levels of hypoxia.

4. In a further group (n = 8), femoral blood flow and D_O2 were restored after l-NAME by infusion of the NO donor sodium nitroprusside (20 μg kg⁻¹ min⁻¹. Thereafter, hypoxia-evoked increases in FVC were fully restored. Nevertheless, D_O2,crit was increased relative to control (0.96 ± 0.07 ml O₂ min⁻¹ kg⁻¹, P < 0.01).

5. As NOS inhibition limited the ability of muscle to maintain V_O2 during hypoxia, we propose that hypoxia-induced dilatation of terminal arterioles, which improves tissue O₂ distribution, is mediated by NO. However, since the hypoxia-evoked increase in FVC was blocked by l-NAME but restored by the NO donor, we propose that the dilatation of proximal arterioles is dependent on tonic levels of NO, rather than mediated by NO.

Systemic hypoxia is characterised by reduced arterial oxygen content and results in vasodilatation in limb muscle (Marshall & Metcalfe, 1988; Marshall, 1995). In the rat this vasodilatation is, at least in part, dependent on nitric oxide (NO), as it can be severely attenuated by the NO synthase (NOS) inhibitor l-NAME (Skinner & Marshall, 1996; Bryan & Marshall, 1999a). However, the physiological consequence of the NO-dependent vasodilatation has not been fully investigated.

In larger mammals, such as the dog, hindlimb muscle has the ability to maintain oxygen consumption (V_O2) at a constant rate in the face of reduced oxygen delivery (D_O2) until a critical D_O2 value (D_O2,crit) is reached, below which V_O2 declines linearly with D_O2 (Duran & Renkin, 1974; Granger et al. 1976; Cain & Chapler, 1979; Samsel & Schumacker, 1988; Curtis et al. 1995). Thus, two phases are seen in the relationship between V_O2 and D_O2; a delivery-independent phase and a delivery-dependent phase. It has been proposed that the mechanism responsible for the delivery-independent phase resides within the terminal arterioles: they dilate when D_O2 is reduced, allowing a more homogeneous distribution of the available O₂. At D_O2,crit the terminal arterioles have dilated maximally, and can play no further role in the regulation of V_O2 (Granger et al. 1976)_. In agreement with this hypothesis, when D_O2 is normal, the distribution of oxygen to different parts of dog muscle is heterogeneous, as detected by multiple measurements of tissue P_O2 and when D_O2 decreases during systemic hypoxia, the distribution of tissue P_O2 values becomes more homogeneous (Harrison et al. 1990). If this hypothesis is correct, any disruption of this microcirculatory response to reduced D_O2 would be expected to alter D_O2,crit (Curtis et al. 1995).

In small mammals, such as the rat, it is not clear whether muscle V_O2 is regulated in this manner. Indeed, it has been suggested from experiments in which D_O2 was altered by changing blood flow or perfusion rate, that V_O2 is always delivery dependent in the skeletal muscle of smaller mammals, even under resting conditions (Honig et al. 1971; Grubb & Folk, 1978). However, our recent studies on the rat have demonstrated that during moderate systemic hypoxia hindlimb D_O2 is compromised, but V_O2 remains constant (Marshall & Davis, 1999). Further, our direct observations, using intravital microscopy on the spinotrapezius muscle of the rat, have shown that terminal arterioles undergo a heterogeneous response to hypoxia, such that some dilate and others constrict, implying a redistribution of blood flow within the muscle (Mian & Marshall, 1991a). This redistribution of flow could act to divert blood from well-perfused areas of muscle to those that are more hypoxic (Marshall, 1995), raising the possibility that the mechanisms that regulate hindlimb V_O2 in the rat are similar to those in the dog.

Thus, a primary aim of the present study was to test this hypothesis over a wider range of D_O2 values. We have investigated the effects of graded systemic hypoxia upon V_O2 within the hindlimb of the anaesthetised rat. We have shown a biphasic relationship between hindlimb V_O2 and D_O2, which allowed the calculation of D_O2,crit within the rat hindlimb for the first time. Moreover, since we already know that the vasodilator response of rat hindlimb to systemic hypoxia is NO dependent (Skinner & Marshall, 1996; Bryan & Marshall, 1999a), further experiments were performed to evaluate the role of NO in determining D_O2,crit.

Some of the results of the present paper have already been reported in brief (Edmunds & Marshall, 2000).

METHODS

Experiments were performed on male Wistar rats (200-250 g) in which anaesthesia was induced with an oxygen-halothane mixture (3.5 % halothane) and maintained with Saffan (Schering-Plough Animal Health, Welwyn Garden City, UK) delivered at 7-12 mg kg⁻¹ h⁻¹, i.v., during surgery and at 4-8 mg kg⁻¹ h⁻¹, i.v., during the experimental period (Bryan & Marshall, 1999a). The surgery required to record physiological variables was similar to that described previously (Bryan & Marshall, 1999a; Marshall & Davis, 1999). Briefly, the trachea was cannulated so that inspired O₂ could be altered by changing the mixture of N₂ and O₂ delivered across the side-arm of the cannula. Arterial blood pressure (ABP) was recorded from the left brachial artery and femoral blood flow (FBF) was recorded from the right femoral artery via a transonic flow probe (0.7 V) connected to a T106 flow meter (Transonic Systems Inc., Ithaca, NY, USA). Femoral vascular conductance (FVC) was computed on-line as FBF divided by ABP. A cannula was inserted into the ventral tail artery to allow infusion of the NO donor sodium nitroprusside (SNP, see below). Samples of arterial blood were taken from a cannula in the right femoral artery, while samples of venous blood from the right hindlimb were taken from a cannula placed in the left femoral vein, and advanced so that the tip lay at the bifurcation of the inferior vena cava. The position of this cannula was confirmed post mortem. Arterial and venous blood samples (65 μl) were analysed for oxygen content using a co-oximeter (IL-682 CO-Oximeter, Instrumentation Laboratory, Lexington, MA, USA). This, with the measurement of FBF, allowed calculation of hindlimb D_O2 and V_O2. Because the muscles perfused by the iliac/femoral artery cannot be accurately removed for weighing (see Marshall & Davis, 1999), values of D_O2 and V_O2 are expressed per unit bodyweight. At the end of each experiment animals were killed by anaesthetic overdose followed by cervical dislocation.

Protocols

In control rats (Group 1, n = 8), cardiovascular variables were measured continuously while the animal spontaneously breathed 21 % O₂. After a 25 min period of stabilisation, the inspirate was changed, for 5 min periods, to a range of different hypoxic mixtures: 14, 12, 10, 9, 8, 7, 6 and sometimes 5 % O₂ in N₂. The order of the hypoxic mixtures was randomised and at least 15 min breathing 21 % O₂ was allowed between successive periods of hypoxia. During the fifth minute of each hypoxic challenge, blood samples were taken from the femoral vein and artery cannulae, so that hindlimb D_O2 and V_O2 could be calculated at each level of hypoxia. For each rat, D_O2 was plotted against V_O2 and regression lines were fitted to the delivery-dependent and delivery-independent portions of the D_O2-V_O2 curve using the dual-line least-squares method described by Samsel & Schumacker (1988). The intercept of these two regression lines indicated D_O2,crit (Fig. 1).

Figure 1. The relationship between D_O2 and V_O2 within the hindlimb of one animal in the control group during graded systemic hypoxia.

The filled circles represent the delivery-dependent phase of this relationship and the filled squares show the delivery-independent phase. The two distinct phases were separated using the dual-line least-squares method described by Samsel & Schumacker (1988). The intercept of these two lines defines D_O2,crit. The data shown are representative of the eight different experiments within this group.

In Group 2 rats (n = 6), after the initial stabilisation period (see above), l-NAME was given (10 mg kg⁻¹, i.v.) and 20 min later the protocol described above was performed. Administration of l-NAME resulted in a substantial vasoconstriction within the hindlimb, such that FBF and D_O2 were severely reduced during air breathing (see Results). Therefore, in a further two groups of animals (Groups 3 and 4, n = 8 for each), SNP (10 or 20 μg kg⁻¹ min⁻¹, i.a.) was continuously infused, after administration of l-NAME, so as to restore FVC or FBF, respectively, during air breathing, to levels pertaining to those before l-NAME by restoring a basal level of NO to the vasculature. The SNP infusion was commenced 20 min after l-NAME and the protocol described above was then performed.

An additional series of experiments was conducted (Group 5, n = 6), in which FBF was restored after l-NAME by infusion of the prostacyclin analogue iloprost (1 μg kg⁻¹ min⁻¹, i.a.). Following this, the protocol described above was repeated again, except that an additional level of hypoxia (16 % inspired O₂) was included to allow the accurate determination of D_O2,crit. The lowest level of hypoxia, 5 % inspired O₂, was not used in this protocol.

All variables were recorded on an Apple Power Mac computer (4400/160) using Maclab 8/s (AD Instuments, Hastings, West Sussex, UK). l-NAME and SNP were supplied by Sigma with saline as the vehicle. Iloprost was purchased from Schering Health Care Limited (West Sussex, UK), with saline as the vehicle.

Statistical analysis of data

All data are expressed as means ±s.e.m. Changes in FVC were computed as the integrated FVC, in conductance units (CU), for the 5 min period during the hypoxic stimulus minus the integrated baseline FVC taken for 5 min before hypoxia. Effects of hypoxia on mean arterial pressure (MAP) and FBF within groups were analysed using Student's paired t test. MAP and FVC were compared at each level of hypoxia between different groups of animals by using one-way ANOVA for multiple comparisons and Tukey's post hoc test for differences at particular levels of hypoxia. D_O2,crit values for Groups 1 and 3 were compared using Student's unpaired t test. P < 0.05 was considered significant.

RESULTS

Control animals (Group 1)

Arterial P_O2 values obtained by similar levels of systemic hypoxia have previously been noted (Marshall & Metcalfe, 1988). The cardiovascular changes evoked by episodes of acute systemic hypoxic were comparable to those we have described before (Marshall & Metcalfe, 1988; Skinner & Marshall, 1996; Bryan & Marshall, 1999a). Briefly, hypoxia caused a decrease in MAP and an increase in FVC, the change being significant at each level of hypoxia: P < 0.01 in each case for MAP (Fig. 2), and P < 0.05 in each case for FVC (Fig. 3), indicating vasodilatation within the hindlimb. The increase in FVC in the face of decreased MAP acted to maintain FBF at near-control values at all levels of hypoxia (Table 1), except when breathing 6 % O₂ when FBF decreased (FBF at 21 vs. 6 % O₂: 1.8 ± 0.2 vs. 1.0 ± 0.2 ml min⁻¹, P < 0.05).

Figure 2. Changes in MAP evoked by graded systemic hypoxia.

Each column represents the mean ±s.e.m. of the change in MAP, measured from the baseline immediately preceding the hypoxic challenge, recorded over 5 min periods of hypoxia. The percentage O₂ in the inspirate is indicated above the columns. Control animals (Group 1, ▪), after l-NAME, 10 mg kg⁻¹, i.v. (Group 2, □), after l-NAME with SNP, 10 μg min⁻¹ kg⁻¹, i.a. (Group 3,) and after l-NAME with SNP, 20 μg min⁻¹ kg⁻¹, i.a. (Group 4,). *P < 0.05 and **P < 0.01, when compared with Group 1; †P < 0.05 and ††P < 0.01, Group 3 or 4 vs. Group 2.

Figure 3. Changes in FVC evoked by graded systemic hypoxia.

Each column represents the mean ±s.e.m. of changes in FVC (in conductance units, CU), measured from the baseline immediately preceding the hypoxic challenge, recorded over 5 min periods of hypoxia, the percentage O₂ in the inspirate being indicated below the columns. Control animals (Group 1, ▪), after l-NAME, 10 mg kg⁻¹, i.v. (Group 2, □), after l-NAME with SNP, 10 μg min⁻¹ kg⁻¹, i.a. (Group 3,) and after l-NAME with SNP, 20 μg min⁻¹ kg⁻¹, i.a. (Group 4,). *P < 0.05 and **P < 0.01, when compared with Group 1; †P < 0.05 and ††P < 0.01, when compared with Group 2.

Table 1.

Mean arterial blood pressure (MAP) and femoral blood flow (FBF) during air breathing and at the fifth minute of each hypoxic challenge for control, L-NAME (10 mg kg⁻¹) and L-NAME (10 mg kg^-1) with SNP (20 μg kg⁻¹ min⁻¹) groups

	Control Group 1	L-NAME Group 2	L-NAME + SNP Group 4
MAP (mmHg)
21% O2	119 ± 2	149 ± 1***	86 ± 5***†††
14% O2	77 ± 4	137 ± 4***	72 ± 5†††
12% O2	71 ± 4	122 ± 4***	65 ± 3†††
10% O2	67 ± 3	115 ± 3***	62 ± 4†††
9% O2	59 ± 4	101 ± 7***	58 ± 4†††
8% O2	62 ± 2	88 ± 6***	57 ± 3†††
7% O2	50 ± 4	86 ± 7**	55 ± 3††
6% O2	60 ± 4	78 ± 6	68 ± 7
FBF (min min−1)
21% O2	1.8 ± 0.2	0.9 ± 0.1*	1.6 ± 0.2†
14% O2	1.9 ± 0.1	0.8 ± 0.1**	1.9 ± 0.2††
12% O2	1.7 ± 0.2	1.0 ± 0.1*	2.2 ± 0.2††
10% O2	2.0 ± 0.2	0.9 ± 0.1**	2.2 ± 0.2††
9% O2	2.0 ± 0.2	1.0 ± 0.1**	1.9 ± 0.2†
8% O2	1.7 ± 0.2	0.8 ± 0.1*	2.2 ± 0.2†††
7% O2	1.8 ± 0.2	0.9 ± 0.2*	2.4 ± 0.3†††
6% O2	1.0 ± 0.2	0.8 ± 0.2	1.9 ± 0.2*

Values are means ± S.E.M.

^*P < 0.05

^**P < 0.01

^***P < 0.001 vs. Control

^†P < 0.05,

^††P < 0.01

^†††P < 0.001, SNP with L-NAME vs. L-NAME alone.

The relationship between D_O2 and V_O2 in Group 1 is shown in Fig. 4A. The protocol we used achieved a wide range of hindlimb D_O2 values. It can be seen that V_O2 was maintained during moderate decreases in D_O2, i.e. V_O2 was delivery independent. Only when D_O2 was more severely reduced did V_O2 decline linearly with D_O2, and thus became delivery dependent. Calculation of D_O2,crit from each individual animal gave a value of 0.64 ± 0.06 ml O₂ min⁻¹ kg⁻¹, which corresponds with the inflection of the group data shown in Fig. 4A.

Figure 4. Effects of graded systemic hypoxia on D_O2 and V_O2 of the rat hindlimb.

Each value represents the mean ±s.e.m. for V_O2 and D_O2 during air breathing (highest D_O2) or after 5 min periods of hypoxia (D_O2 decreasing progressively with the severity of each hypoxic challenge). A shows data for Group 1 (control animals, ▪), B shows data for Group 2 (l-NAME, 10 mg kg⁻¹, i.v., ▴), C shows data for Group 4 (SNP infusion at 20 μg kg⁻¹ min⁻¹, i.a., after l-NAME, •) and D shows data for Group 5 (iloprost infusion at 1 μg kg⁻¹ min⁻¹, i.a., after l-NAME, ♦).

l-NAME (Group 2)

The NOS inhibitor l-NAME caused an increase in MAP (before vs. after l-NAME: 115 ± 6 vs. 149 ± 1 mmHg, P < 0.01). This was accompanied by a decrease in FBF (before vs. after l-NAME: 1.4 ± 0.2 vs. 0.9 ± 0.1 ml min⁻¹, P < 0.05) and in FVC (before vs. after l-NAME: 0.0123 ± 0.0018 vs. 0.0060 ± 0.0005 CU, P < 0.01), indicating vasoconstriction within the hindlimb. In Group 2, the falls in MAP were significantly different from those recorded in Group 1, reflecting the fact that the falls in MAP evoked by moderate hypoxia (≤ 9 % inspired O₂) were smaller than those seen in Group 1 (Fig. 2). However, during more severe hypoxic challenges (≤ 8 % inspired O₂) the decreases in MAP were similar in Group 1 and Group 2 (Fig. 2). The increases in FVC evoked during each hypoxic challenge were severely attenuated in Group 2 when compared to those in Group 1, except during 6 % O₂ (Fig. 3). As a consequence of the decrease in baseline FBF induced by l-NAME (Table 1), D_O2 during air breathing was considerably reduced, relative to that of Group 1 (0.62 ± 0.07 vs. 1.20 ± 0.10 ml O₂ min⁻¹ kg⁻¹, respectively, P < 0.01; see Fig. 4B). Under these conditions, V_O2 decreased linearly with D_O2 over the complete range of hypoxic challenges (Fig. 4B). Thus, there was no delivery-independent portion of the relationship between V_O2 and D_O2, and D_O2,crit could not be calculated.

Restoration of FVC and FBF after l-NAME (Groups 3, 4 and 5)

In Group 3, infusion of SNP at the lower rate of 10 μg min⁻¹ kg⁻¹, after l-NAME, restored FVC during air breathing to values pertaining to those before l-NAME (before l-NAME vs. after l-NAME with SNP: 0.0151 ± 0.0008 vs. 0.0160 ± 0.0015 ml min⁻¹ mmHg⁻¹). However, this infusion of SNP caused MAP to fall to below that seen during air breathing prior to l-NAME (before l-NAME vs. after l-NAME with SNP: 116 ± 6 vs. 91 ± 5 mmHg, P < 0.01), and this, in turn, meant that FBF was slightly decreased relative to that recorded before l-NAME (1.4 ± 0.1 vs. 1.8 ± 0.1 ml min⁻¹, respectively, P = 0.07). As a consequence, D_O2, when breathing air, was also slightly decreased relative to control animals (0.97 ± 0.08 vs. 1.20 ± 0.10 ml O₂ min⁻¹ kg⁻¹, respectively), and although this difference did not achieve statistical significance (P = 0.1), D_O2,crit could not be calculated during progressive systemic hypoxia for the same reasons as described for Group 2 (data not shown). Interestingly, this infusion rate of SNP completely restored the FVC response to the range of hypoxic challenges, despite NOS inhibition (Fig. 3).

In Group 4, FBF was restored in the presence of l-NAME to values similar to those seen prior to l-NAME, by infusion of SNP at the higher rate of 20 μg min⁻¹ kg⁻¹ (FBF before l-NAME vs. after l-NAME and SNP: 1.7 ± 0.1 vs. 1.6 ± 0.2 ml min⁻¹, Table 1). As with the lower infusion rate of SNP, MAP during air breathing fell to below the value seen before l-NAME (86 ± 5 vs. 114 ± 2 mmHg, respectively, P < 0.01, Table 1). Further, FVC was slightly increased relative to FVC measured before l-NAME (0.0220 ± 0.0039 vs. 0.0144 ± 0.0018 CU, respectively, P = 0.07). As intended, this higher infusion rate of SNP after l-NAME did restore hindlimb D_O2 during air breathing to values similar to those seen in control animals (Fig. 4A vs. C). And V_O2 during air breathing in Group 4 was similar to that in Group 1 (Fig. 4A vs. C). Further, from Fig. 4C it can be seen that when D_O2 was reduced by systemic hypoxia, two phases occurred in the relationship between D_O2 and V_O2, a delivery-dependent phase and a delivery-independent phase. However, the D_O2,crit in Group 4 was calculated to be 0.96 ± 0.07 ml O₂ min⁻¹ kg⁻¹, which was significantly greater (P < 0.01) than that in Group 1 (0.64 ± 0.06 ml O₂ min⁻¹ kg⁻¹). This is particularly remarkable since the increases in FVC evoked by the hypoxic challenges in Group 4 were at least as great as those evoked in Group 1 (Fig. 3). In fact the increases in FVC evoked by more severe hypoxic challenges (≤ 8 % inspired O₂) were greater in Group 4 than in Group 1.

In Group 5, infusion of iloprost (1 μg kg⁻¹ min⁻¹) after l-NAME restored FBF to values similar to those observed before l-NAME (FBF before l-NAME vs. after l-NAME and iloprost: 1.9 ± 0.1 vs. 1.8 ± 0.2 ml min⁻¹). The changes in the FVC integral during the hypoxic challenges were not significantly different from those seen in the presence of l-NAME alone (Group 2), the smallest change being seen when animals breathed 14 % O₂ (0.04 ± 0.1 CU) and the largest when animals breathed 7 % O₂ (1.1 ± 0.5 CU). Importantly, the hypoxia-evoked increases in FVC in Group 5 were smaller than those observed in the control animals (Group 1; P < 0.001). Although one animal in this group failed to maintain V_O2 as D_O2 decreased, calculation of D_O2,crit for the other animals within this group gave a value of 0.94 ± 0.05 ml O₂ min⁻¹ kg⁻¹, Fig. 4D). This was shown to be significantly higher (P < 0.01) than the D_O2,crit calculated in control (Group 1) animals, but not significantly different from that calculated in Group 4. Interestingly, V_O2 measured during air breathing in Group 5 was significantly greater that V_O2 calculated during air breathing in Group 1 (V_O2 in Group 1 vs. Group 5: 0.46 ± 0.03 vs. 0.60 ± 0.06 ml O₂ min⁻¹ kg⁻¹, P < 0.05, Fig. 4).

DISCUSSION

The major findings of this study can be summarised as follows: when D_O2 to rat hindlimb was decreased by graded levels of systemic hypoxia, hindlimb V_O2 remained constant until a critical level of D_O2, below which V_O2 declined linearly with D_O2. This biphasic relationship between D_O2 and V_O2 is comparable to that previously reported for larger animals and allowed calculation of D_O2,crit in the rat hindlimb for the first time. Blockade of NO synthesis with l-NAME caused tonic vasoconstriction, a reduction in FBF during air breathing, and a severe attenuation of the hindlimb vasodilatation observed during hypoxia. Concomitantly, D_O2 was so severely reduced that D_O2,crit could not be calculated as V_O2 was linearly dependent on D_O2 over the full range from air breathing to severe hypoxia. However, infusion of SNP in the presence of l-NAME restored FBF and consequently D_O2 when air breathing to values observed in the absence of l-NAME. Under these conditions, the hindlimb vasodilatation evoked by hypoxia was completely restored despite continued NOS blockade, and was even potentiated at more severe levels of hypoxia. Concomitantly, the biphasic relationship between V_O2 and D_O2 was restored, but D_O2,crit was significantly greater than that calculated in the absence of l-NAME.

Regulation of hindlimb V_O2 in response to reduced D_O2

The results presented herein confirm and extend previous observations from our laboratory showing that the rat hindlimb can maintain its V_O2 in the face of a decrease in D_O2 during moderate, acute systemic hypoxia (Marshall & Davis, 1999). It seems probable that the mechanisms responsible for this ‘autoregulation’ of V_O2 in the rat are similar to the mechanisms proposed for this phenomenon in the dog. Namely, as D_O2 decreases, the arterioles, and particularly the terminal arterioles, dilate, improving the distribution of O₂ through the capillary network and allowing more efficient utilisation of the O₂ that is present (Granger et al. 1976). This hypothesis is especially attractive when considered in conjunction with microvascular responses directly observed in the spinotrapezius muscle of the rat (Mian & Marshall, 1991a): the terminal arterioles show a heterogeneous mixture of constrictor and dilator responses to systemic hypoxia, suggesting a redistribution of blood flow within skeletal muscle (Marshall, 1995).

Our findings therefore contrast with those of earlier studies that addressed the relationship between D_O2 and V_O2 in skeletal muscles of smaller mammals such as the rat (Honig et al. 1971; Grubb & Folk, 1978). In these studies, D_O2 was changed by increasing flow either to perfused hindlimb muscle (Grubb & Folk, 1978) or to the gracilis muscle (Honig et al. 1971). When D_O2 increased, V_O2 also increased, suggesting that in rat skeletal muscle at rest, V_O2 is limited by the amount of O₂ available to it. Our studies clearly show this is not the case: rat hindlimb muscle can maintain V_O2 at a constant rate, even when D_O2 is decreased. The reason for the discrepancy between the present and previous studies performed on the rat is unclear. However, the surgical intervention used in the previous studies was substantial, whereas the surgery performed in our studies was kept to a minimum (see Marshall & Davis, 1999). Microvasculature damage caused by surgical trauma could reduce gross muscle blood flow and therefore D_O2, or alter the ability of the microvessels to regulate V_O2. In accord with the present findings, it may be noted that whole-body V_O2, which would be expected to strongly reflect skeletal muscle V_O2, was maintained in the rat during hypoxia- and hyperoxia-induced changes in total O₂ transport: total V_O2 only decreased when total O₂ transport was reduced below a critical level (Adams et al. 1982).

The effects of l-NAME

We have previously shown that during air breathing, l-NAME causes vasoconstriction within the rat hindlimb, which results in a decrease in FBF despite the concomitant increase in MAP (Skinner & Marshall, 1996; Bryan & Marshall, 1999a). The present study showed that this fall in FBF was so severe that D_O2 decreased to below the D_O2,crit value recorded in the absence of l-NAME. Thus V_O2 fell with D_O2 over the full range of hypoxic challenges. This new finding suggests that in vivo experiments on the rat in which l-NAME is given systemically should be interpreted with caution for, even during air breathing, muscle could be relatively hypoxic as V_O2 may already be dependent on D_O2.

In dog hindlimb, inhibition of NO synthesis with l-NAME was also shown to increase V_O2 (King et al. 1994; Vallet et al. 1994), probably by removing the inhibitory influence of basal NO synthesis on mitochondrial respiration (Shen et al. 1995). This was not seen in our experiments. However, any such effect would have been masked if V_O2 had already been compromised by the limitation of blood flow caused by l-NAME. Indeed, when FBF was restored after NOS inhibition by infusion of the prostacyclin analogue iloprost, hindlimb V_O2, during air breathing, was significantly elevated relative to V_O2 in control animals.

In the present study l-NAME severely attenuated the increase in FVC evoked by progressive systemic hypoxia. This finding is consistent with our previous reports (Skinner & Marshall, 1996; Bryan & Marshall, 1999a), but it contrasts with the conclusion drawn by Vallet et al (1994) that the dilatation evoked in the dog hindlimb by systemic hypoxia is not sensitive to l-NAME. However, Vallet et al. (1994) used one level of severe hypoxia in which arterial P_O2 was reduced to 28 mmHg. This value is somewhat lower than arterial P_O2 measured in rats breathing 6 % O₂ (34 ± 2 mmHg; Mian & Marshall, 1995) and as can be seen from Fig. 3, l-NAME alone had no effect on the increase in FVC evoked in the rat by 6 % O₂. Thus, the results of Vallet et al (1994) do not exclude the possibility that hindlimb dilatations evoked in the dog by less severe hypoxic challenges are NO dependent. Indeed, moderate hypoxia does cause a NO-dependent vasodilatation in the dog diaphragm, whereas the dilatation induced by severe hypoxia is resistant to NOS inhibition (Ward, 1996). Our own findings that the increase in FVC evoked when control animals breathed 6 % O₂ was smaller than that evoked by less severe hypoxic challenges (e.g. change in FVC during 7 % O₂vs. 6 % O₂: 3.78 ± 0.64 vs. 1.79 ± 0.71 CU, P < 0.05), and that the increase in FVC evoked by 6 % O₂ was not NO dependent, could reflect the O₂ dependency of the NOS enzyme (see Stuehr, 1999, for review). Thus, during a severe hypoxic challenge (6 % O₂), arteriolar O₂ tension may be reduced to levels that limit NOS activity, so that NO plays a less important role in the dilatation. Under these conditions, the muscle vasodilatation may be more dependent on other substances such as adrenaline (Mian & Marshall, 1991b) and prostacyclin (Abbas & Marshall, 2000).

The effects of SNP infusion after l-NAME

Haemodynamic effects

We successfully used infusions of SNP in the presence of l-NAME to restore either FVC or FBF during air breathing by adding a tonic level of NO back to the hindlimb under conditions in which NO synthesis could not occur. With the lower infusion rate of SNP (10 μg kg⁻¹ min⁻¹), which restored FVC, there was a disproportionate decrease in MAP during air breathing, to below the level recorded before l-NAME. For this reason FBF and therefore D_O2 were not fully restored: the higher infusion rate of SNP (20 μg kg⁻¹ min⁻¹) was required to do this. The reason for the disproportionate decrease in MAP is unclear, but there are several potential explanations. Firstly, the vascular resistance of the other major tissues that contribute to total peripheral resistance and thereby ABP may have a smaller tonic release of NO than skeletal muscle, and therefore require a lower infusion rate of SNP after administration of l-NAME to restore tone. Secondly, different vascular beds may be more sensitive than skeletal muscle to a given concentration of NO and therefore SNP. Finally, tissues that have a higher blood flow per gram tissue weight would receive more SNP during infusion, and so may show a disproportionately greater dilatation.

Both of the infusion rates of SNP when given after l-NAME restored or even potentiated the increases in FVC evoked by hypoxia, an effect that was not observed in Group 5 animals when FBF was restored by infusion of iloprost. These hypoxia-induced changes in FVC are likely to be largely attributable to dilatation of the more proximal arterioles within skeletal muscle (Mian & Marshall 1991a) as these sections are responsible for the majority of the resistance within this vascular bed (Froneck & Zweifach, 1975). Thus, a reasonable interpretation of our results is that the hypoxia-induced increase in FVC that is blocked by l-NAME is due to dilatation of the proximal arterioles, and that it is dependent on NO, rather than mediated by an increased release of NO, as it can be restored during NOS inhibition providing a basal level of NO is present. It should be noted that this proposal differs from another potential explanation: hypoxia-evoked muscle vasodilatation is mediated by the release of NO (Pohl & Busse, 1989; Skinner & Marshall, 1996; Blizter et al. 1996; Bryan & Marshall, 1999a). The interpretation of other studies in which basal levels of NO were not restored after NOS inhibition should be viewed with similar caution.

Vasodilatation that requires the presence of NO, but not the increased release of NO, is a novel finding with respect to the muscle vasodilatation during systemic hypoxia, but similar observations have been made on the cerebral vascular response to hypercapnia: the NO donors SIN-1 and SNAP reversed the attenuation of the hypercapnic cerebovasodilatation produced by NOS inhibitors (Iadecola & Zhang, 1994; Iadecola et al. 1994). It has been known for some time that vasodilators that increase cGMP and cAMP can act in a synergistic manner (DeWit et al. 1994), probably through cGMP-mediated inhibition of a cAMP phosphodiesterase leading to decreased catabolism of cAMP (Delpy et al. 1996). Thus, tonic production of NO could be required for the full response of the proximal arterioles to cAMP-dependent vasodilators that are known to be released during hypoxia, for example prostaglandins, circulating catecholamines and possibly adenosine (Mian & Marshall, 1991b; Bryan & Marshall, 1999b; Abbas & Marshall, 2000).

This proposal is consistent with the observation that during more severe levels of hypoxia (≤ 8 % oxygen), the evoked increases in FVC after l-NAME and during SNP infusion were greater than in control animals (Group 4 vs. Group 1, Fig. 3). For if, when O₂ tension falls during severe systemic hypoxia, the activity of NOS is limited (Stuehr, 1999; see above), the synergistic influence of NO and cGMP may also be lost. Since there is no reason to suppose that the concentration of NO liberated from SNP is affected by low O₂ tension, relatively more NO would have been present in Group 4 than in Group 1 during severe systemic hypoxia. This, in turn, would facilitate vasodilatation.

Effects on D_O2 and V_O2

As indicated in the Results, D_O2,crit could not be calculated in Group 3 (in which SNP was infused at the lower rate after l-NAME), even though D_O2 during air breathing (0.97 ± 0.08 ml O₂ min⁻¹ kg⁻¹) was higher than D_O2,crit calculated in control animals (0.64 ± 0.06 ml O₂ min⁻¹ kg⁻¹). This suggests, indirectly, that D_O2,crit was increased by NOS inhibition. When SNP was infused at the higher rate (Group 4), D_O2 during air breathing was restored. Additionally V_O2 during air breathing was similar to that of the control animals, and since tonically released NO is known to decrease mitochondrial respiration, this provides further evidence that this infusion of SNP replaced basal levels of NO. In Group 4 the biphasic relationship between D_O2 and V_O2 was restored, allowing calculation of D_O2,crit. But this was shown to be significantly higher than in control animals. This was confirmed when FBF was restored after NOS inhibition by iloprost. This provides strong evidence that the defence of V_O2 during moderate decreases in D_O2 is mediated by an increased release of NO. Considering the proposal that the dilatation of the terminal arterioles is important in maintaining V_O2 when D_O2 is compromised (Granger et al. 1976), and our own evidence that the more terminal arterioles act to improve muscle O₂ distribution through a heterogeneous mixture of dilator and constrictor responses (Mian & Marshall, 1991a), we propose that their dilatation is, at least in part, mediated by NO release.

In some contrast with the present findings, double blockade of NOS and cyclo-oxygenase enzymes did not alter D_O2,crit calculated for dog hindlimb during progressive ischaemia (Curtis et al. 1995). This raises the possibility that different substances are responsible for determining D_O2,crit when D_O2 is reduced by ischaemia than when D_O2 is reduced by hypoxia. Certainly D_O2,crit for dog hindlimb was calculated to be 5.4 ml min⁻¹ (kg muscle weight)⁻¹ during ischaemia (Curtis et al. 1995), but as high as 9.8 ml min⁻¹ (kg muscle weight)⁻¹ when using combined data from anaemic hypoxia and systemic hypoxia (Cain, 1977). Interestingly, if we assume hindlimb muscle weight of the rats used in the present study to be 13-15 g (see Marshall & Davis, 1999), then D_O2,crit can be estimated as 11.0 ml min⁻¹ (kg muscle weight)⁻¹ for rats during progressive systemic hypoxia, a value that correlates well with that reported by Cain et al. (1977) for systemic hypoxia/anaemia.

In conclusion, the present study demonstrates that progressive systemic hypoxia evokes vasodilatation in rat hindlimb and shows for the first time that O₂ consumption of rat hindlimb remains constant until O₂ delivery is reduced by about 50 %. We propose that the increase in gross muscle vascular conductance is attributable to dilatation of the more proximal arterioles that is NO dependent, but not necessarily NO mediated, whereas the maintenance of V_O2 is attributable to dilatation of the terminal arterioles that is mediated by increased release of NO.