INTRODUCTION

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SEVERAL REPORTS PROVIDE EVIDENCE that nitric oxide (NO) contributes to the active hyperemia in contracting skeletal muscle (6, 8, 21, 27). Specifically, muscle blood flow was substantially reduced when NO synthase (NOS) inhibition was induced before high-intensity muscle contractions in cat hindlimb (16), before moderate-intensity work in conscious dogs (27) and rats (8), and in humans when NOS inhibition was induced during moderate rhythmic handgrip exercise (6). Furthermore, NOS activity was increased in gastrocnemius muscles of rats during exhaustive treadmill running (25). The outcomes of these studies have led to the conclusion that NO plays a functional role in active hyperemia. This interpretation, however, must be viewed with caution, as reciprocal increases in endothelin (ET)-1 with NOS inhibition have been reported (1, 4, 24). Furthermore, the increase in vascular resistance observed with NOS inhibition is attenuated or abolished with ET_A- or ET_A+B-receptor blockade (14, 19, 22, 24). It is possible that ET-1-mediated vasoconstriction may account for some or all of the observed reduction in muscle blood flow during NOS inhibition in contracting skeletal muscle. Therefore, the first objective of the present experiments was to test the hypothesis that the decrease in blood flow during NOS inhibition is, in part, the result of an increase in vasoconstrictor tone due to the reciprocal increase in ET-1 rather than solely due to the loss of NO-mediated vasodilation.

The metabolic consequence(s) of the decrease in muscle blood flow with NOS inhibition during muscle contractions is not fully appreciated. A confounding factor is the level of O₂ demand during muscle contractions and NOS inhibition. At low-intensity contractions, a reduction in blood flow with NOS inhibition could be offset by an increase in O₂ extraction to maintain the O₂ uptake (O₂). For example, when blood flow was reduced with NOS inhibition in exercising dogs, O₂ extraction increased to prevent an O₂ limitation at submaximal work intensities (20). At higher work intensities, O₂ extraction may be near or at maximal levels with little reserve left to respond to a reduction in blood flow. This may have been the case when O₂ decreased with NOS inhibition in the in vivo autoperfused dog diaphragm contracting at 3 Hz (30). A compensatory increase in O₂ extraction occurred, yet it was insufficient to prevent a reduced O₂-to-tension development (TD) ratio (O₂/TD), indicating an O₂ limitation (30). In light of these findings and the paucity of studies in this area, our second objective was to test the hypothesis that an O₂ limitation would occur in high-intensity but not low-intensity contractions when O₂ delivery was reduced during NOS inhibition. We believe that, during low-intensity contractions, an ample O₂ extraction reserve is available to prevent an O₂ limitation with the reduction in blood flow with NOS inhibition. During high-intensity contractions, however, both the blood flow and O₂ extraction are already at or near maximal levels; therefore, any reduction in blood flow with NOS inhibition and the absence of an O₂ extraction reserve would lead to a significant O₂ limitation.

	METHODS

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The animals used in this study were provided by the Queen's University Animal Care facility and were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Mongrel dogs (n = 24) with an average weight of 25.2 ± 1.0 kg were anesthetized with pentobarbital sodium (32 mg/kg iv); additional anesthetic was given as needed. The animals were intubated and ventilated to maintain arterial PCO₂ between 30 and 35 Torr.

The venous outflow from the left gastrocnemius muscle was isolated as previously described (13, 28). The muscle weight averaged 106 ± 5 g. Briefly, an incision was made through the skin from midthigh to ankle along the left hindlimb, and overlying muscles were doubly ligated and cut to expose the gastrocnemius muscle. All venous drainage into the popliteal vein was ligated, except for that from the gastrocnemius muscle. The tendon was cut close to its insertion, placed in a metal clamp, and attached to a force transducer. The sciatic nerve was sectioned, and the distal end was placed in a stimulating electrode. Optimal muscle length was set by determining the length at which the greatest peak tension was obtained during supramaximal stimulation. A two-channel catheter, with an electromagnetic flow probe (Narcomatic) in one channel, was placed in the femoral vein draining the muscle. The second channel allowed for in vivo zeroing of the flow probe without interruption of venous drainage from the muscle. Venous flow from the muscle was returned to the animal via a venous reservoir attached to a catheter in the right femoral vein. The left femoral artery was ligated and catheterized with a two-channel catheter: one line allowed for autoperfusion of the muscle with blood from the right femoral artery, whereas the other contained an in-line pump. Muscle perfusion pressure was measured using a pressure transducer attached to a t connection in the arterial line just before the catheter insertion in the left femoral artery. Once the initial muscle perfusion pressure was determined, the muscle was pump perfused at constant perfusion pressure for the duration of the experiment. In this manner, any changes in muscle vascular resistance would be reflected by the magnitude of changes in muscle blood flow. A catheter was placed in the left brachial artery for measurement of arterial blood pressure and withdrawal of arterial blood samples.

Once surgical preparations were complete, a 30-min period was allowed for stabilization of cardiovascular and metabolic parameters. To test the first hypothesis that the decrease in blood flow during NOS inhibition is, in part, the result of an increase in vasoconstrictor tone due to the reciprocal increase in ET-1, NOS inhibition was induced during low-intensity contractions with and without ET_A-receptor blockade. Specifically, in one group of eight animals [2 twitches (tw)/s], resting measures of blood flow and perfusion pressure were made, and arterial and muscle venous blood samples were taken. The muscle was then stimulated (4-6 V, 0.4-ms duration) to contract isometrically at 2 tw/s; an intensity equivalent to that of 50% high-intensity O₂ for this type of muscle contraction (13). Once steady state was achieved (~5-8 min), measurements were repeated, and then N-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg iv) was administered. Measurements were obtained at 2, 5, 10, and 15 min after administration of L-NAME. Our laboratory has previously established that this concentration of L-NAME produces effective NOS inhibition for at least 1 h (12). Furthermore, elevations in plasma ET-1 levels have been noted by 15 min of NOS inhibition (24). In a second group of eight animals (BQ-123 + 2 tw/s), resting muscle measurements were made, and the specific ET_A-receptor antagonist BQ-123 [cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu); 0.1 ml · kg muscle¹ · min¹ ia] was infused into the muscle circulation. This concentration of BQ-123 effectively reverses the changes in muscle vascular resistance and blood flow during ET-1 administration in the resting canine gastrocnemius muscle preparation (15). Another set of resting measures was obtained at 30 min of BQ-123 infusion, and then the same contraction protocol as described for the 2 tw/s group was followed. To test our second hypothesis that an O₂ limitation will occur in high-intensity but not low-intensity contractions when O₂ delivery is reduced during NOS inhibition, the muscle was stimulated to contract isometrically at 4 tw/s in a third group of 8 animals (4 tw/s). The same protocol as that for the 2 tw/s group was followed. The 4 tw/s contraction rate represents a high, if not maximal, level of twitch contractile rate in this muscle preparation (13).

All blood samples were analyzed for PO₂, PCO₂, and pH using a Radiometer BMS-MKII blood-gas analyzer, and these data were later corrected to the temperature of the dog at the time of sampling. In addition, all blood samples were analyzed for O₂ concentration using an Instrumentation Laboratories CO-oximeter IL-482. The resulting values were corrected for O₂ in solution using the factor 0.003 ml O₂ · dl¹ · Torr PO₂¹. Muscle O₂ was calculated using the Fick principle. O₂ extraction ratio was calculated as the arteriovenous O₂ difference across the muscle divided by the arterial O₂ concentration. TD was calculated as the peak twitch height minus the resting tension and is reported in grams of developed force per gram wet muscle weight. O₂/TD was determined and represents that amount of O₂ used per gram per twitch of muscle contraction and can be used as an indicator of inequity between O₂ demand (TD) and O₂ supply (O₂). A reduction in this ratio indicates that more force production is achieved with a lesser amount of O₂, presumably through the use of anaerobic sources, which would be an indication of an O₂ limitation during muscle contractions.

All values are reported as means ± SE. Differences for a given measured variable over time were determined using a single repeated-measures ANOVA. The Bonferroni analysis (29) was used for the post hoc multiple comparisons of differences between means. Therefore, the initial -level was set at 0.10 for the ANOVA to set significance of the Bonferroni analysis at P < 0.025. Differences between groups were determined by using unpaired t-tests, and differences in resting muscle values before and after treatment with BQ-123 were determined by using a paired t-test. Significance for the t-test analyses was accepted at P < 0.05.

	RESULTS

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Determination of the contribution of ET-1 to the decrease in muscle blood flow during NOS inhibition. The values for muscle blood flow, O₂ extraction ratio, and O₂/TD are shown in Fig. 1, A, B, and C, respectively. Muscle blood flow averaged 455 ± 23 ml · kg¹ · min¹ during steady-state contractions at 2 tw/s. By 2 min after L-NAME administration, muscle blood flow decreased 17% to 378 ± 17 ml · kg¹ · min¹ (P < 0.05) and remained at that level for the duration of the muscle contractions. The values for muscle blood flow in the BQ-123 + 2 tw/s group were not different than those obtained in the 2 tw/s group at any time during the protocol. Muscle O₂ extraction ratio averaged 0.71 ± 0.02 during steady-state contractions at 2 tw/s and increased further to 0.78 ± 0.01 post-L-NAME (P < 0.05). A similar response was observed in the BQ-123 + 2 tw/s group; the values for O₂ extraction ratio were not different from those observed in the 2 tw/s group except at 2 min post-L-NAME. The values for the O₂/TD averaged 5.8 ± 0.4 and 5.4 ± 0.3 in the 2 tw/s and BQ-123 + 2 tw/s groups, respectively. No significant change in the O₂/TD was observed in either group after administration of L-NAME except at 2 min post-L-NAME in the 2 tw/s group. The values between the groups were not different.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Mean ± SE values for muscle blood flow (A), O2 extraction ratio (B), and O2 uptake-to-tension development (TD) ratio (C) during low-intensity isometric twitch contractions [2 twitches (tw)/s], before and after nitric oxide synthase inhibition, with and without endothelin A-receptor blockade [cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ-123)]. , 2 tw/s (n = 8); , BQ-123 + 2 tw/s (n = 8). L-NAME, N-nitro-L-arginine methyl ester. * Significant difference from respective control value within each group at P < 0.025 (initial   no. of paired comparisons).  Significant difference between groups at P < 0.05.

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Comparison of the vascular and metabolic alterations during low-intensity and high-intensity contractions with NOS inhibition. The values for muscle blood flow, O₂ extraction ratio, and O₂/TD for the 2 tw/s and 4 tw/s groups are depicted in Fig. 2, A, B, and C, respectively. Blood flow decreased significantly in both groups after NOS inhibition; however, the extent of the reduction was only 10% in the 4 tw/s group compared with 19% in the 2 tw/s group (P < 0.05). In the 2 tw/s group, the O₂ extraction ratio increased (P < 0.05) after NOS inhibition, but no such response occurred in the 4 tw/s group. The O₂/TD decreased transiently at 2 min post-NOS inhibition in the 2 tw/s group (P < 0.05). In the 4 tw/s group, however, the O₂/TD was significantly reduced at 5 and 15 min post-NOS inhibition, indicating a sustained O₂ limitation in this group.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Mean ± SE values for muscle blood flow (A), O2 extraction ratio (B), and O2 uptake-to-TD ratio (C) during low-intensity (2 tw/s) and high-intensity (4 tw/s) isometric twitch contractions, before and after NOS inhibition. , 2 tw/s (n = 8); , 4 tw/s (n = 8). * Significant difference from respective control value within each group at P < 0.025 (initial   no. of paired comparisons).

	DISCUSSION

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The primary findings of the present study were that 1) ET_A-receptor blockade had no effect on the reduction in blood flow observed during NOS inhibition with low-intensity muscle contractions, and 2) O₂ extraction increased to compensate for the reduction in blood flow after NOS inhibition during low-intensity (2 tw/s) contractions, but no such compensatory response in O₂ extraction occurred during high-intensity (4 tw/s) contractions. The O₂/TD decreased with NOS inhibition during high-intensity but not low-intensity contractions, suggesting that the failure of O₂ extraction to increase at 4 tw/s resulted in an O₂ limitation. Secondary findings were that ET_A-receptor blockade 1) had no effect on resting muscle vascular tone, and 2) resulted in reduced force generation during low-intensity contractions.

Our finding that muscle blood flow decreased after NOS inhibition during low-intensity muscle contractions is in agreement with observations of others (6, 8, 21, 27). The magnitude of the change in blood flow was less in the present study (19%) compared with that of Shen et al. (27), who reported an increase in muscle resistance of 56% in dogs running at moderate intensity on a treadmill. Furthermore, Hirai et al. (8) demonstrated a 30% decrease in blood flow to primarily red muscles in rats running at moderate intensity on a treadmill, whereas Poucher (21) reported a 40% decrease in vascular conductance during high-intensity contractions of the gastrocnemius muscle in anesthetized cats. In humans, a 30% decrease in forearm blood flow occurred during rhythmic hand gripping at 15% of high-intensity voluntary contractions (6). Factors that may contribute to the variability observed between the above studies include differences in muscle type, animal model, and/or mode of exercise.

Substantial evidence exists for a reciprocal increase in ET-1 production with NOS inhibition (1, 4, 19, 22, 24). Our laboratory recently observed a similar response in the gastrocnemius muscle preparation such that the increase in skeletal muscle vascular resistance after NOS inhibition with L-NAME (20 mg/kg iv) was reversed with ET_A-receptor blockade (BQ-123, 0.1 ml · kg¹ · min¹ ia) (14). Given these findings, we hypothesized that the decrease in muscle blood flow observed during NOS inhibition and muscle contractions was not solely a function of the removal of NO-mediated dilation but was, in part, the result of the added vasoconstrictor tone associated with increased ET-1 levels. Our findings did not support our hypothesis. To our knowledge, this is the only study to determine that ET_A-receptor blockade had no effect on the observed reduction in blood flow in contracting muscle during NOS inhibition. This is in direct contrast to that observed under resting conditions in skeletal muscle.

A second major finding of the present study was that the decrease in muscle blood flow with NOS inhibition during high-intensity contractions (4 tw/s) may have resulted in an O₂ limitation due to the lack of an O₂ extraction reserve; this was not the case with low-intensity contractions (2 tw/s). These data confirmed our initial hypothesis that, as contraction intensity was increased and maximal O₂ extraction was reached, any decreases in blood flow with NOS inhibition would result in an O₂ limitation. A few studies have measured all of these related components, namely blood flow, O₂, O₂ extraction, and muscle force production. Specifically, in the in vivo isolated, autoperfused contracting dog diaphragm preparation (3 Hz), blood flow and O₂ decreased and O₂ extraction increased with no change in TD after NOS inhibition (30). We calculated the O₂/TD using the data presented by Ward and Hussain (30) and found a significant decrease, indicating that an O₂ limitation was present. In another study, during graded treadmill exercise in conscious dogs that were pretreated with L-NAME, blood flow decreased and O₂ extraction increased to maintain O₂ at mild and moderate work rates, but no such changes were observed at high-intensity work rates (20). At low-intensity work, Ward and Hussain demonstrated an O₂ limitation in dog diaphragm with NOS inhibition, whereas both O'Leary et al. (20) and our present data clearly support an O₂ extraction compensation for the reduction in blood flow to prevent an O₂ limitation at this lower work intensity.

In contrast to our observations, two studies by other investigators using the in situ canine gastrocnemius muscle preparation found that NOS inhibition had no effect on O₂ or TD during high-intensity isometric twitch contractions (4 tw/s) (3) or during isometric tetanic contractions (2). These findings appear to be in contrast to our observations with regard to the possibility of an O₂ limitation during NOS inhibition at high work intensity. However, the methods and protocols in these two studies may provide an explanation for our contrasting findings. Barclay and Woodley (3) employed an isometric 4 tw/s contraction protocol; however, the values for O₂, blood flow, and TD were reported at 15 min of contractions (before NOS inhibition with N-nitro-L-arginine) and at 60 min of contractions (45 min post-N-nitro-L-arginine). At 4 tw/s, a substantial amount of muscle fatigue occurs over time. At 15 min of contractions, the values for blood flow, O₂, and TD would be expected to already be lower than those that we measured between 5 and 8 min of contractions. More importantly, 45 min elapsed after NOS inhibition, which would allow sufficient time for other factors (i.e., metabolic vasodilation) to compensate for the initial fall in blood flow with NOS inhibition. We specifically designed our protocol to measure the vascular and metabolic consequences within a short period of time after NOS inhibition (~15 min), given the fact that metabolic autoregulation might obscure these changes over time. Barclay and Woodley (3) also employed a second protocol in which NOS inhibition was induced before 4 tw/s contractions and measures were obtained at 5 min of contractions. Again, the investigators found no effect of NOS inhibition on muscle blood flow, O₂, or TD. We designed the present experiments to prevent the possibility that, with NOS inhibition before muscle contractions, other dilatory mechanisms are recruited to meet the metabolic need of the tissue, which would obscure a role of NOS dilation; this possibility was acknowledged by Barclay and Woodley and was also shown in previous experiments in our laboratory (13). In the study by Ameredes and Provenzano (2), tetanic contractions were used and NOS inhibition was induced with a lesser used L-arginine analog, L-argininosuccinic acid; these represent vastly different experimental conditions compared with our study. The increased metabolic demand of tetanic contractions compared with twitch contractions may be sufficient to overcome any increase in vascular resistance with NOS inhibition. Given the above explanations, we believe that this is the first study to demonstrate that NO contributes to active hyperemia during both low- and high-intensity twitch contractions and that an O₂ limitation may have occurred during high-intensity contractions as indicated by a reduction in O₂/TD.

It is important to consider whether or not a decrease in O₂/TD is truly representative of an O₂ limitation at 4 tw/s during NOS inhibition. Three factors require consideration. First, additional empirical evidence, such as muscle lactate production data, may have helped to substantiate the existence of an O₂ limitation; however, we did not obtain these measures. Another potential indicator of an O₂ limitation may be found in the muscle venous pH values. If an O₂ limitation were present, an increased acid flux from the muscle might be expected as a consequence of increase lactate production and/or other metabolites and should be manifested by a reduction in muscle venous pH values during the period of O₂ limitation compared with control conditions. No such decrease in muscle venous pH was observed in the 4 tw/s group during NOS inhibition, which does not support our conclusion of an O₂ limitation.

Second, the lack of a further increase in O₂ extraction during the modest ischemia induced by NOS inhibition at 4 tw/s needs to be addressed. In studies in which blood flow was mechanically reduced by 50%, O₂ extraction increased 41% at both 3 Hz (0.39-0.55) and 5 Hz (0.44-0.61) (9) and from 0.57 to 0.62 (a 9% increase) during 4-Hz contractions in the canine gastrocnemius muscle preparation (7). The peak values for O₂ extraction in the above studies were much less than those observed in our study. It has been our experience that O₂ extraction increases to fairly high levels, even during mild contraction intensities (10, 11, 13). Further increases in O₂ demand were met by modest increases in O₂ extraction and large increases in blood flow (10, 11, 13). We believe this to be further evidence that, at 4 tw/s, O₂ extraction was near maximal, and, when flow was reduced by even a modest amount (10%), the muscle was unable to increase O₂ extraction further, and O₂/TD fell. However, Gorman et al. (7) demonstrated that O₂/TD did not decrease during 4 tw/s contractions in the same muscle preparation; however, the authors acknowledged that outlying data were obtained in one of the six dogs studied, leading to a large standard deviation and limited interpretation of the O₂/TD findings. Also, in other types of O₂ supply limitation, i.e., severe hypoxic hypoxia, when O₂/TD was reduced during 4 tw/s, O₂ extraction increased from 0.75 to 0.85 (10). Unlike severe hypoxic hypoxia, the 10% reduction in blood flow in the present study may not have been a sufficient stimulus to increase O₂ extraction further during NOS inhibition and high-intensity contractions.

A third factor that may cause difficulty in the interpretation of the O₂/TD data is that NO has direct negative effects on both mitochondrial respiration and TD (17). During NOS inhibition, it is possible that, for a given workload, the removal of this negative influence may result in increases in both O₂ and TD. The relative effect of NO on each of these parameters in unknown under these conditions; however, to reduce the O₂/TD, TD would have had to increase further than O₂. While we are unable to assess the contribution of the direct actions of NOS inhibition on muscle respiration and force development, it is an important physiological phenomenon, which must be taken into consideration in the interpretation of our O₂/TD data.

In summary, our conclusion that an O₂ extraction limitation was responsible for the reduction in O₂/TD with NOS inhibition during high-intensity twitch contractions is one interpretation of our findings. However, in lieu of the above discussion, it is also possible that the decreased O₂/TD may be a unique consequence of the ischemia associated with NOS inhibition in which muscle contraction efficiency is enhanced.

Another finding of the present study was that resting skeletal muscle vascular resistance was not altered after ET_A-receptor blockade. This is in contrast to previous data from our laboratory in which muscle vascular resistance was significantly reduced (~25%) after ET_A-receptor blockade (14). One explanation for this contrariety may be differences in vessel shear stress under constant perfusion pressure conditions, which were used in the present study, compared with constant flow conditions in our laboratory's previous study. ET receptors are upregulated by shear stress (23), and plasma ET-1 levels are elevated at low levels of shear stress (16). Under constant-flow conditions, basal ET-1 levels may be greater, and, therefore, ET_A-receptor blockade would reduce basal vascular resistance, whereas no change would be expected under constant perfusion pressure conditions.

Finally, it is of interest that TD and the corresponding O₂ values were lower in the BQ-123 + 2 tw/s group compared with the 2 tw/s group before and after NOS inhibition. This suggests that the ability of the muscle to develop force was reduced with blockade of ET_A receptors, thereby supporting the concept that ET-1 enhances muscle contractility. ET-1 exerts an inotropic effect in rat papillary muscle (18), rat ventricular muscle (26), and rat left atrium, right atrium, and right ventricle (5). In the present study, we can only speculate that the decreased TD in the presence of ET_A-receptor blockade was due to the loss of ET-1 inotropic effects during contractions, because we did not specifically measure ET-1 levels nor did we perform any specific contractile mechanism investigations. Our findings and those in cardiac muscle support the conclusion that ET-1 may also exert an inotropic action in skeletal muscle, a finding that requires further investigation.

In summary, we found that NO-mediated dilation contributed ~19 and 10%, respectively, to the blood flow responses during low- and high-intensity muscle contractions in the canine gastrocnemius muscle. Our findings did not support our initial hypothesis that the reduction in flow during muscle contractions in the presence of NOS inhibition was in part due to the reciprocal increase in ET-1. Accordingly, we concluded that NO-mediated dilation contributed to the active hyperemic response during both low-intensity and high-intensity muscle contractions. A major finding of the present study was that, during high-intensity contractions, the decrease in blood flow with NOS inhibition was associated with a decrease in O₂/TD and the lack of a further increase in O₂ extraction. These findings may be interpreted as an O₂ limitation due to the lack of a compensatory increase in O₂ extraction. However, an equally probable interpretation is that the decrease in O₂/TD may reflect an enhanced efficiency of muscle contractions, which is a unique consequence of the ischemia associated with NOS inhibition.