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Am J Physiol Heart Circ Physiol 292: H2491-H2497, 2007. First published January 26, 2007; doi:10.1152/ajpheart.01396.2006
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Limitations to vasodilatory capacity and VO2 max in trained human skeletal muscle

Jeremy Barden,1 Lesley Lawrenson,1 Jennifer G. Poole,1 Jeannie Kim,1 D. Walter Wray,1 Damian M. Bailey,2 and Russell S. Richardson1

1Department of Medicine, University of California San Diego, La Jolla, California; and 2School of Applied Science, University of Glamorgan, Glamorgan, South Wales, United Kingdom

Submitted 20 December 2006 ; accepted in final form 24 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To further explore the limitations to maximal O2 consumption (VO2 max) in exercise-trained skeletal muscle, six cyclists performed graded knee-extensor exercise to maximum work rate (WRmax) in hypoxia (12% O2), hyperoxia (100% O2), and hyperoxia + femoral arterial infusion of adenosine (ADO) at 80% WRmax. Arterial and venous blood sampling and thermodilution blood flow measurements allowed the determination of muscle O2 delivery and O2 consumption. At WRmax, O2 delivery rose progressively from hypoxia (1.0 ± 0.04 l/min) to hyperoxia (1.20 ± 0.09 l/min) and hyperoxia + ADO (1.33 ± 0.05 l/min). Leg VO2 max varied with O2 availability (0.81 ± 0.05 and 0.97 ± 0.07 l/min in hypoxia and hyperoxia, respectively) but did not improve with ADO-mediated vasodilation (0.80 ± 0.09 l/min in hyperoxia + ADO). Although a vasodilatory reserve in the maximally working quadriceps muscle group may have been evidenced by increased leg vascular conductance after ADO infusion beyond that observed in hyperoxia (increased blood flow but no change in blood pressure), we recognize the possibility that the ADO infusion may have provoked vasodilation in nonexercising tissue of this limb. Together, these findings imply that maximally exercising skeletal muscle may maintain some vasodilatory capacity, but the lack of improvement in leg VO2 max with significantly increased O2 delivery (hyperoxia + ADO), with a degree of uncertainty as to the site of this dilation, suggests an ADO-induced mismatch between O2 consumption and blood flow in the exercising limb.

exercise; blood flow; vasodilatory reserve

THE ISOLATED QUADRICEPS MUSCLE mass employed in knee-extensor (KE) exercise is such that O2 delivery is not restrained by the finite limit of cardiac output (1, 3) or by the relatively small dimensions of the femoral artery (20). Pioneering studies during development of the KE exercise paradigm elevated the maximum recorded muscle blood flow in humans from the previously accepted value of 60–100 to 250–300 ml·min–1·100 g muscle–1 (1, 2). Subsequent studies suggested that even these large increases in absolute muscle blood flow may not be truly maximal (31); this was later confirmed in a study of highly trained athletes performing KE exercise during a rapid incremental protocol in which a maximum blood flow of 385 ml·min–1·100 g muscle–1 was achieved (28). However, despite these increases in maximum recorded muscle perfusion, it has been noted that neither muscle blood flow nor maximal O2 uptake (VO2 max) has revealed any sign of achieving a maximal value or plateau (28, 32).

Muscle blood flow is precisely regulated to match the metabolic requirements of skeletal muscle in humans (2). For example, under hypoxic conditions, the typical response in the peripheral circulation is an increase in vascular conductance, which elevates muscle blood flow, offsetting the reduction in arterial O2 content (CaO2) and maintaining O2 delivery (31). However, maximal work capacity in hypoxia is usually compromised, inasmuch as O2 availability, specifically the PO2 gradient from blood to muscle, is reduced and contributes to the early termination of graded exercise (22, 25). It has been suggested that this failure of maximum leg blood flow in hypoxia to exceed that attained in normoxia is an indication that blood flow is very close to maximal in both conditions (28). In support of the importance of O2 availability and a potential ceiling to muscle blood flow, KE exercise in 100% O2 (hyperoxia) facilitates a greater work rate (WR) and VO2 max, but maximal muscle blood flow does not surpass that achieved in hypoxia or normoxia (22).

The concentration of adenosine (ADO), an endogenous vasoactive metabolite, in the interstitium is closely associated with exercise intensity (9). Because the predominance of the enzyme responsible for ADO formation in skeletal muscle (5'-nucleotidase) is membrane bound, ADO is released from skeletal muscle into the interstitial space, where A1, A2A, and A2B receptors are localized to vascular smooth muscles and endothelial cells (9, 13). Therefore, it is not surprising that endogenous ADO has been implicated in the control of exercise-induced vasodilation, with the intra-arterial infusion of exogenous ADO at rest and during submaximal exercise resulting in muscle blood flows that are comparable to those during maximal exercise (19). In addition, intra-arterial ATP infusion in one leg during two-leg maximal cycling exercise has been shown to increase leg vascular conductance and decrease O2 extraction in the infused leg (5). However, exogenous ADO has not been superimposed on maximal KE exercise to examine the extent and importance of a skeletal muscle vasodilatory reserve under these conditions (19).

Therefore, the objectives of this study are as follows. First, we sought to determine whether there is a skeletal muscle vasodilatory reserve capacity during maximal KE exercise through pharmacological vasodilation with ADO during hyperoxia. The inclusion of hypoxia in these studies afforded an interesting comparison with the ADO effects, inasmuch as hypoxia has also been recognized to significantly increase blood flow at a given work intensity. Second, we aimed to further explore the limitations to VO2 max through maximal exercise bouts performed during progressive levels of varied O2 supply (hypoxia, hyperoxia, and hyperoxia + ADO). Specifically, it was hypothesized that, in exercise-trained skeletal muscle, 1) hypoxia would elevate skeletal muscle blood flow, but diminished O2 availability would limit VO2 max, and therefore blood flow would fail to surpass that attained in hyperoxia, 2) arterial ADO infusion at 80% of maximal WR (WRmax) in hyperoxia would increase vascular conductance and elevate blood flow beyond that in hyperoxia and hypoxia, revealing a skeletal muscle vasodilatory reserve, and 3) the progression from hypoxia to hyperoxia to hyperoxia + ADO would provoke a stepwise elevation in O2 delivery, leading to concomitant increases in WRmax and VO2 max.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Six bicycle-trained (>250 miles/wk) men volunteered to participate in the study. A complete health history and physical examination were completed, the protocol was approved, and informed consent was obtained according to the University of California Human Research Protection Program requirements. The physical characteristics of the subjects are presented in Table 1.


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  		Table 1. Subject characteristics

 
Preliminary tests. For characterization of their exercise capacity, the subjects performed an initial incremental cycle ergometry test that began with a 5-min warm-up at 100 W that was increased in 50-W increments to 300 W and then in 25-W increments every subsequent minute. Once they were familiar with the KE exercise paradigm, the subjects performed a graded maximal KE exercise test in hyperoxia (100% O2). This pretest provided values for hyperoxic WRmax that were used on the experimental day (see below).

Exercise model. The KE exercise paradigm has been described previously (1). Briefly, the subjects were seated on an adjustable chair, with a cycle ergometer placed behind them. Resistance to KE exercise was provided by friction on the flywheel, which was turned by the subject via a metal bar connected to the crank of the ergometer and a boot attached to the ankle of the subject. Sixty dynamic contractions of the KE muscles per minute were maintained at each WR. The range of motion was such that a contraction of the quadriceps femoris muscle caused the lower part of the leg to extend from ~90° to ~170° flexion. By design, the momentum of the flywheel returned the relaxed leg (subjects relaxed after flexion) to the starting position. Anecdotal subject reports, force traces, electromyography, and T2-weighted magnetic resonance imaging (21) support the conclusion that active contractions are limited to the quadriceps muscles during this exercise modality.

Experimental protocol. After catheter placement, three bouts of progressive KE exercise were performed: 1) hyperoxia (100% O2) and sham infusion (saline), 2) hypoxia (12% O2) and sham infusion (saline), and 3) hyperoxia + ADO, in which an intra-arterial infusion of ADO was begun after 80% of the hyperoxic WRmax (determined during pretesting). The three exercise bouts were performed in accordance with a randomized single-blind design, punctuated by a rest period of ~1 h to ensure that the previous exercise bouts did not affect performance in the subsequent bouts. For each exercise bout, WR was increased from an unweighted warm-up followed by 20, 40, 60, 80, 90, and 100% of maximum pretest (hyperoxic) values with additional 10% increments in WR to failure, if possible. Each WR was continued until the subjects were unable to maintain a cadence of 60 rpm for ≥120 s. This protocol ensured that WRmax was achieved. The sequence of events at each WR was as follows: 1) measurement of femoral venous blood flow, 2) collection of 3-ml blood samples for blood gas measurements (PO2, PCO2, pH, and arterial O2 saturation), 3) measurement of femoral venous blood flow (duplicate flows), 4) blood pressure measurement, and 5) challenge with the next WR.

Catheter placement and blood flow measurements. Subjects returned to the laboratory within 2 wk of preliminary studies; at this time, three catheters (radial artery, femoral artery, and femoral vein) and a thermocouple (femoral vein) were placed using sterile technique, as previously reported. Briefly, two 1.25-mm-OD catheters (model DSA 400L, Cook, Bloomington, IN) were introduced pericutaneously, one in the left femoral vein and one in the left femoral artery below the inguinal ligament, and advanced 8 cm distally and proximally, respectively. Placement of the femoral venous catheter was such that the infusion of cold saline facilitates thorough mixing of blood and infusate across the lumen of the vein. Subsequently, a 20-gauge, 3.2-cm-long arterial catheter was inserted pericutaneously into the radial artery of the nondominant hand for arterial blood sampling. A thin (0.64-mm-diameter) polyethylene thermocouple (model IT-18, Physitemp Instruments, Clifton, NJ), was advanced from approximately the same location as the venous catheter proximally 10 cm into the left femoral vein toward the heart.

Muscle blood flow during KE exercise was measured by the thermodilution technique, as previously described (24). Measured blood flow is accepted as a close approximation of quadriceps muscle blood flow, because blood is directed toward working muscle, and the quadriceps represents the sole working muscle group of the leg during KE exercise (21). Venous and infusate temperatures during the 15-s saline infusions were measured by two digital thermometers (model IT-18, Physitemp Instruments) interfaced to a personal computer (Biopac System, Santa Barbara, CA). Muscle blood flow data were collected after a metabolic steady state was achieved at each WR (2–4 min, depending on the exercise intensity) and were calculated using a heat balance equation (1). Muscle O2 delivery was calculated as follows: blood flow x CaO2. Net venous lactate outflow from the quadriceps was calculated as follows: blood flow x ([venous lactate] – [arterial lactate]), where [venous lactate] and [arterial lactate] represent venous and arterial lactate concentrations. Constant blood pressure measurements from within the femoral artery and femoral vein were monitored at heart level by a pressure transducer (Transpac IV, Abbot Laboratories). Mean arterial and venous blood pressures were calculated through the integration of each pressure curve, and vascular conductance was calculated as follows: blood flow ÷ mean arterial pressure.

Femoral arterial infusion of ADO. During preliminary laboratory visits, thigh length, circumference, and skinfold measurements were obtained and used to estimate the muscle mass of the quadriceps femoris of each subject (18). The thigh volume was then divided by the concentration (3 mg/ml) of the commercially available sterile ADO solution used in the study (Adenoscan, Fujisawa Healthcare). The quotient of this calculation (in ml/min) was equal to the rate at which the syringe pump (model PHD 2000, Harvard Apparatus) was set to achieve the target arterial infusion rate of 1 mg·min–1·l–1 of thigh volume. This rate was selected to minimize the systemic effects of ADO (14) and in recognition of the previous finding that, at least during submaximal exercise, greater concentrations result in a minimal increase in leg blood flow (19). Sham saline infusion rates were identical to the calculated ADO infusion rate for each subject.

Muscle O2 transport conductance and mean capillary PO2. Through numerical integration, the value for muscle O2 transport conductance (DO2), which is assumed to be constant across the capillary, that would yield the measured femoral venous PO2, given the measured arterial PO2, was calculated. The numerical average of all PO2 values computed, equally spaced in time (t), along the capillary from the arterial to the venous end, was considered to be mean capillary PO2 (Pco2) (34). The fundamental iterative process of determining PcFormula and DO2 is based on the following equation

Formula
where intracellular PO2 [PO2(i)] is assumed to be close to zero during relatively intense exercise (26).

Blood analyses. The 3-ml arterial and femoral venous blood samples withdrawn anaerobically at each WR were measured for their PO2, PcFormula, pH, O2 saturation, and Hb concentration. An IL 1306 blood-gas analyzer and an IL 482 CO-Oximeter (Instrumentation Laboratories, Lexington, MA) were used for all measurements.

Statistical analyses. Data were subjected to and passed normality tests before a one-factor repeated-measures ANOVA followed by post hoc Bonferroni-corrected paired-samples t-tests using a commercially available software package (Graphpad, Instat, San Diego, CA). The sample size for each group (n = 6) was initially found to be sufficient via preliminary power analyses and confirmed by the achievement of adequate power in statistical testing of the major variables (e.g., VO2, O2 delivery, and muscle blood flow). Variables were considered significantly different when P ≤ 0.05. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
WRmax and overview of data presentation. WRmax was significantly attenuated in hypoxia but was not significantly different between hyperoxia and hyperoxia + ADO (Table 2). However, four of six subjects did achieve a greater WRmax in the hyperoxia + ADO trial, but, as noted, this did not achieve statistical significance. To allow a clear understanding of the physiological responses to each condition, the data are presented across submaximal efforts to WRmax, expressed as a percentage of hyperoxic WRmax (Figs. 1 and 2). In an effort not to lose interesting data, we plotted values for the four subjects who attained a greater WRmax in the hyperoxia + ADO trial in isolation at the corresponding mean WRmax attained. Hematologic, metabolic, and vascular responses at WRmax are presented in Table 2.


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  		Table 2. Hematologic, metabolic, and vascular responses to maximal exercise

 

Figure 1
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  		Fig. 1. Relation between leg vascular conductance, quadriceps muscle blood flow, O2 delivery, and percent maximal hyperoxic work rate (WRmax). All values were recorded during incremental single-leg knee-extensor (KE) exercise. Four subjects exceeded 100% WRmax in hyperoxia + adenosine (Hyper + ADO) trial. Tendency for this single data point to be higher or lower than the preceding point (100% WRmax Hyper + ADO) is not purely the result of a varied sample size but, rather, reflects a trend in the data. *Significantly different from hyperoxia and hypoxia; {dagger}significantly different from hyperoxia and Hyper + ADO; #significantly different from hyperoxia (P < 0.05).

 

Figure 2
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  		Fig. 2. Relation between arterial-venous O2 difference, O2 consumption (VO2), venous lactate outflow, and percent WRmax. All values were recorded during incremental single-leg KE exercise. Four subjects exceeded 100% WRmax in hyperoxia + ADO trial. Tendency for this single data point to be higher or lower than the preceding point (100% WRmax Hyper + ADO) is not purely the result of a varied sample size but, rather, reflects a trend in the data. *Significantly different from hyperoxia and hypoxia; {dagger}significantly different from hyperoxia and Hyper + ADO; #significantly different from hyperoxia; §significantly different from hypoxia and Hyper + ADO (P < 0.05).

 
Blood pressure, vascular conductance, blood flow, and O2 delivery. Blood pressure rose across work rates in all conditions and was different only at maximal exercise; i.e., it was lower in the hypoxia trial, during which a lower WRmax was achieved (Table 2). Vascular conductance increased in all conditions as the WR was increased toward maximum. Before the infusion time point (≤80% of hyperoxic WRmax), leg vascular conductance in the hypoxia trial was higher at every WR than in the hyperoxia and hyperoxia + ADO trials (Fig. 1). Beyond the infusion time point (>80% of hyperoxic WRmax), leg vascular conductance in the hyperoxia + ADO trial was increased to a level similar to that in the hypoxia trial and higher than in the hyperoxia trial. At the highest WR in the hyperoxia + ADO trial (n = 4), conductance fell to a level similar to that in the hypoxia and hyperoxia trials.

Consistent with the increased leg vascular conductance before the infusion time point in hypoxia, blood flow was elevated in the hypoxia trial compared with the hyperoxia + ADO and hyperoxia trials. Beyond the infusion time point, blood flow in the hyperoxia + ADO trial increased above that in the hyperoxia and hypoxia trials. As with conductance, at the highest WR in the hyperoxia + ADO trial (n = 4), blood flow fell but remained significantly higher than that attained at WRmax in the hyperoxia and hypoxia trials (Table 2, Fig. 1).

Submaximal O2 delivery was equivalent in all exercise conditions, despite differences in the amount of O2 carried per unit of blood. As would be expected, O2 delivery increased significantly and in a fashion similar to blood flow beyond the infusion time point in the hyperoxia + ADO trial (Table 2, Fig. 1).

Arterial-venous O2 difference, VO2, and lactate outflow. Before the infusion time point, the arterial-venous O2 difference was reduced in the hypoxia trial compared with the hyperoxia and hyperoxia + ADO trials at a similar WR. The arterial-venous O2 difference in the hyperoxia + ADO trial plummeted shortly after the point of infusion and then returned to the level observed in hypoxia; however, the arterial-venous O2 difference remained significantly lower in the hyperoxia + ADO than in the hyperoxia trial (Table 2, Fig. 2).

Muscle VO2 before the infusion time point increased in a linear fashion as WR was increased toward maximum and was similar in all three exercise conditions. VO2 tended to reach a plateau in the hypoxia, but not the hyperoxia + ADO or hyperoxia, exercise bout. VO2 in the hyperoxia + ADO trial fell initially beyond the infusion time point, but this fall was followed by a rise in VO2 to levels that were similar to those in the hypoxia trial, although they were still lower than levels attained in the hyperoxia trial (Table 2, Fig. 2).

For the first two submaximal exercise levels, venous lactate outflow was similarly low in all three exercise conditions. However, from 60% of the hyperoxia WRmax, venous lactate outflow was significantly elevated in the hypoxia trial compared with the hyperoxia + ADO and hyperoxia trials. The similar relation between WR and venous lactate outflow in the hyperoxia + ADO and hyperoxia trials was not altered by commencement of the infusions. Venous lactate outflow in the hypoxia and hyperoxia + ADO trials (n = 4) was greater than that at WRmax in the hyperoxia trial (Table 2, Fig. 2).

PCO2 and DO2 at maximal exercise. At WRmax, with the theoretically appropriate anchoring of these data through zero as a starting point (if PcFormula = 0, so must VO2), there was a proportionate change in calculated PcFormula and VO2 max as a result of the transition between exercise in hypoxia and in hyperoxia. Despite an increased PcFormula in the hyperoxia + ADO trial, a proportionate increase in VO2 max was not attained. Rather, VO2 max in the hyperoxia + ADO trial dropped to levels similar to those in the hypoxia trial and significantly less than those in the hyperoxia trial (Table 2, Fig. 3).


Figure 3
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  		Fig. 3. Relation between maximal VO2 (VO2 max) and mean capillary PO2 (PcFormula) in hypoxia, hyperoxia, and hyperoxia + ADO (HyperA80%) at maximal KE exercise. Solid line represents muscle O2 diffusion conductance, the slope of which (including zero) has been proposed to, at least partially, dictate potential rise and fall in skeletal muscle VO2 max with varied O2 driving pressures in the capillary (capillary PO2) (24). Dotted line also represents muscle O2 diffusion conductance, but the slope is reduced, because the adenosine-induced increase in PcFormula failed to concomitantly increase VO2 max. §Significantly different from hypoxia and hyperoxia + ADO (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study combined different levels of inspired O2 with pharmacological vasodilation to explore the relation between O2 availability, O2 delivery, and VO2 in maximally exercising muscle. During maximal exercise, O2 delivery rose progressively from hypoxia (12% O2) to hyperoxia (100% O2) and hyperoxia with intra-arterial ADO (hyperoxia + ADO) infused at 80% of WRmax. Acute ADO infusion increased vascular conductance and exercising leg blood flow, implying a potential vasodilatory reserve in exercising skeletal muscle. However, skeletal muscle VO2 was diminished after ADO infusion, despite an increase in O2 supply, suggesting attainment of maximal metabolic capacity, a red cell transit time limitation, a disruption of local VO2-to-blood flow matching, or a combination of these events. Taken together, these findings support the concept of a vasodilatory reserve during intense exercise and exemplify the deleterious effect of disrupting the precise matching of metabolism and perfusion in the exercising limb.

Peripheral vascular conductance during exercise. The sympathetic response to whole body exercise is a global vasoconstriction of arterioles in all except essential organs (30). Sympathetic responsiveness, which is blunted within exercising muscle, is one mechanism by which blood flow is accurately distributed to the sites of muscular work (33, 35). Additionally, norepinephrine spillover in active muscle is proportional to the overall mass of muscle involved in the exercise (23, 31). However, even during single-leg KE exercise, where there is no imminent threat of approaching maximal cardiac output or need to optimally distribute blood flow, the vasculature of the exercising quadriceps muscle can be interpreted as remaining partially vasoconstricted (19). This experimental paradigm may then be superior to the whole leg-cycling approach previously employed by Calbet et al. (5) to explore the possibility of a vasodilatory reserve in exercising skeletal muscle of humans.

In agreement with other studies (22), the present data reveal a gradual rise in vascular conductance up to ~70% of quadriceps VO2 max, with higher vascular conductance in the hypoxia trial than in the hyperoxia and hyperoxia + ADO trials during submaximal exercise (Fig. 1). With the infusion of ADO, vascular conductance rose above that of the hyperoxia trial to match that of the hypoxia trial until WRmax. The increased vascular conductance in the hypoxia trial during submaximal exercise agrees with previous studies that have recognized a vasodilatory reserve capacity during submaximal KE exercise (5, 19, 32) but extends these observations to WRmax with the infusion of ADO (Fig. 1). With the disclaimer that the present investigation cannot unequivocally determine that the ADO-induced increase in muscle blood flow was confined to the exercising quadriceps muscle, these data may be interpreted as evidence of a vasodilator reserve capacity within the vasculature of an isolated muscle group at WRmax, implying that peak muscle blood flow is not limited by vascular structure during maximal KE exercise.

Data from the present study extend findings from recent work by Calbet et al. (5), where ATP was infused during cycle-ergometer exercise to determine the impact of ATP-induced vasodilation on VO2 and O2 extraction during near-maximal and maximal cycling exercise. They reported that ATP infused during 92–100% of maximal effort significantly increased leg vascular conductance and decreased O2 extraction, with no change in leg VO2 and small increases in leg blood flow. Although this study elegantly demonstrated the vasodilatory capacity of ATP, the two-leg-cycling exercise paradigm with infusions and measurements in only one leg somewhat limits comparison of these findings with the present data. In contrast, the present study employed the single-leg KE exercise paradigm, which is not hindered by a nontreated limb contributing to the exercise. Additionally, in the study of Calbet et al., the ATP infusion failed to increase leg blood flow at maximal exercise; therefore, maximal O2 delivery was unaltered, a fundamental tenet of a study designed to examine the effect of convective O2 transport on VO2 max. Thus the present combination of an isolated skeletal muscle model, alterations in inspired fraction of O2, and ADO infusion, which increased O2 delivery, has more definitively documented the impact of increased O2 delivery and O2 utilization in an exercising human limb. However, it must be emphasized that although the work of KE exercise is almost exclusively performed by the quadriceps muscle group in this limb (21), the present study can neither confirm nor deny that the likely mismatch that occurred in the face of increased O2 delivery was limited to shifts in intramuscle (quadriceps) or intermuscle (whole limb) perfusion. This likely shift of muscle blood flow from where it would be optimally directed is an important observation that speaks to the importance of balancing vasoconstriction and vasodilation in active and inactive muscles during exercise, a topic that has received considerable attention recently (33, 35).

Teleologically, it is possible that vascular conductance in the hyperoxia + ADO trial was increased to levels similar to those observed in the hypoxia trial (Fig. 1), not by chance but, rather, because A1 receptors potentially mediate hypoxia-induced vasodilation by an endothelial release of ADO (19). Indeed, the findings of a matched vascular conductance in the hypoxia and hyperoxia + ADO trials support this current mechanistic dilator theory. Incremental rates of ADO infusion during submaximal exercise have previously revealed a leveling off of the dilatory response with concentrations above that used in the present investigation (19). However, with only the single level of hypoxia and the single ADO infusion rate used here, it is not clear whether the maximum vasodilatory capacity has been reached or whether more severe levels of hypoxia or a larger dose of ADO could further increase vascular conductance.

O2 supply dependence in skeletal muscle. Conceptually, the reduction or elevation of O2 availability should have different effects on muscle metabolism, depending on the mitochondrial capacity of a muscle. This appears to be the case, inasmuch as the VO2 max of sedentary subjects during cycle exercise has been characterized as limited by O2, whereas the VO2 max of exercise-trained (cycling) subjects appears to be limited by O2 supply (6, 10, 22). In another model of small muscle mass (plantar flexion) exercise, phosphocreatine rephosphorylation time (an index of metabolic capacity) was shortened in hyperoxia only in exercise-trained subjects, whereas it was elongated in all healthy subjects in hypoxia (8). Furthermore, in exercise-trained subjects performing KE exercise, Richardson et al. (22) reported that muscle VO2 max could be increased in hyperoxia and attenuated in hypoxia (22). Interestingly, several KE exercise studies have failed to elevate VO2 max with hyperoxia, but these studies did not utilize 100% O2, nor was long-term exercise-trained skeletal muscle investigated (16, 17). The elevation of the O2 driving gradient with hyperoxia (100% O2) in chronically trained cyclists has yielded the highest increment in VO2 max and the highest human mass-specific VO2 max, apparently without reaching the metabolic limits of these subjects (22).

In the present study, O2 supply was raised beyond traditional hyperoxic values with concomitant ADO infusion, significantly elevating O2 delivery and the O2 driving gradient from blood to cell. Studies that have examined the relation between mean PcFormula and VO2 max under conditions of altered O2 availability support the concept of diffusion-limited VO2 max in exercise-trained human muscle during KE exercise (22). The present hypoxia-to-hyperoxia data support this concept, but the ADO infusion data during hyperoxia reveal that, despite an elevated PcFormula (compared with hyperoxia and hypoxia trials), the muscle failed to correspondingly increase VO2 max (Fig. 3). With this approach to understanding the determinants of VO2 max, there are two distinct interpretations of these findings. 1) Metabolic limitation, at which point an elevated O2 supply provides no further benefit, is attained. However, the present observation that VO2 max was actually attenuated (rather than reaching a plateau) implies that other factors may have been involved. 2) In the hyperoxia + ADO trial, a fall in O2 diffusional conductance, as a direct consequence of increased muscle blood flow and the resultant decrease in red blood cell transit time or the indiscriminate dilation of vascular beds that were less involved in the exercise, disturbed the local matching of perfusion and metabolism (5). Each of these possibilities, individually or in combination, could account for the high PcFormula but lack of an increase in VO2 max during the hyperoxia + ADO trial (Fig. 3).

Skeletal muscle blood flow and O2 availability. During submaximal exercise in hypoxia, the increase in active muscle blood flow and vascular conductance is supported by the present data and the majority of investigations in humans who have reduced CaO2 with anemia, hypoxia, or anemia + hypoxia (Fig. 1) (4, 10–12, 24, 29, 32). Somewhat surprisingly, this response to decreased O2 availability during exercise is tightly coupled with the fall in arterial oxyhemoglobin and seems largely independent of O2 dissolved in the blood (7, 29). However, at maximal exercise in this and other studies (11, 12, 22, 24), maximal blood flow did not increase to compensate for the reduced CaO2 that accompanied hypoxia, and thus maximal O2 delivery and muscle VO2 max were reduced. Evidently, as seen in the hyperoxia + ADO trial, a greater blood flow could have been achieved (Table 2, Fig. 1); yet, during the hypoxia trial, this elevated blood flow was not attained. Presumably, this is evidence of O2 supply limitation, with the added vasodilation of hypoxia having little impact on the diminished O2 driving gradient from blood to cell, which is drastically reduced in this condition (22). Therefore, these data again support the concept that when O2 availability is reduced, even to a highly perfused exercising muscle mass, factors that determine diffusional O2 transport from blood to muscle play a key role in determining VO2 max (Table 2, Fig. 3).

ADO, heart rate, and skeletal muscle blood flow. As anticipated, ADO infusion increased peripheral vascular conductance and blood flow and convective O2 delivery to the leg. However, despite the short half-life of ADO in vivo (15) and the direction of ADO toward the exercising leg, there was still a clear central effect, inasmuch as heart rate was significantly elevated by >20 beats/min at WRmax compared with preinfusion (Table 2). This suggests an increased cardiac output, which is supported by the observation that arterial ADO infusion had no effect on blood pressure and, therefore, was not predominantly due to baroreceptor unloading (Table 2). It is possible that the heart rate response was mediated, at least in part, by an ADO-sensitive pool of central afferent nerves previously demonstrated to be absent in the leg (14).

After the initial large increase in leg blood flow in the hyperoxia + ADO trial, at WRmax blood flow did not continue to increase in a linear fashion, although it remained significantly higher than WRmax during the hyperoxia and hypoxia trials (Fig. 1). This response was very consistent with the hyperbolic rise and fall in all four subjects with >100% WRmax. Although the exact mechanism responsible for the decline in peak blood flow observed in the hyperoxia + ADO trial is beyond the scope of the present study, several possibilities deserve consideration and possibly future investigation: elevation of norepinephrine spillover from sympathetic nerves potentially driven by the systemic effects of ADO on muscle sympathetic nerve activity (14), reduction in the effective concentration and, therefore, in the vasodilatory signal as a result of increased blood flow (35), and/or a resetting of ADO receptor saturation or sensitivity (19).

Venous lactate outflow. In each protocol, lactate outflow rose exponentially with increasing WR, presumably as a consequence of increasing catecholamines and the transition from slow- to fast-twitch muscle fibers with increasing exercise intensity (27). As previously recognized (27), this process was accelerated in the hypoxic trial, perhaps additionally driven by a reduced intracellular PO2 and culminating in a greater venous lactate outflow at WRmax than in the hyperoxia trial (Fig. 2). In the hyperoxia + ADO trial, the attenuated VO2 max and elevated venous lactate outflow at WRmax imply that anaerobic glycolysis may have contributed to the sustained exercise beyond the WRmax achieved in this trial (n = 4; Table 2). It was anticipated that the elevated O2 delivery afforded by the ADO-induced dilation would reduce lactate outflow, much as hyperoxia does compared with normoxia (22). Although the exact mechanism responsible for the promotion of this nonoxidative metabolism in the hyperoxia + ADO trial at WRmax is unknown, it is likely related to the impact of ADO on local blood flow distribution in relation to metabolism.

Experimental considerations. Although the underlying tenet of the KE exercise employed in the present study is that, under normal conditions, blood flow is directed to the principal leg muscles recruited [i.e., the quadriceps (21)], we recognize the possibility that the ADO infusion may have provoked vasodilation in nonexercising tissue of the infused leg, which may have yielded far more gross changes in blood flow-to-metabolism matching.

In summary, this study has revealed a leg vasodilatory reserve capacity during maximal isolated small muscle mass exercise, evidenced by an increased leg vascular conductance during hypoxia and local intra-arterial ADO infusion in the hyperoxia + ADO trial. Although the ADO infusion during the hyperoxia + ADO trial facilitated an increase in O2 supply over and above that achieved in the hyperoxia trial, this intervention failed to increase skeletal muscle VO2 max. Inasmuch as the ADO infusion actually attenuated muscle VO2 max to the level achieved in hypoxia, it is likely that alterations in other factors, such as red blood cell transit time and the matching of muscle blood flow to metabolism within the quadriceps and possibly within other areas of the leg, also played a role in this outcome.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded in part by National Heart, Lung, and Blood Institute Grant HL-17731, the Sam and Rose Stein Institute for Research on Aging, and Tobacco-Related Disease Research Program Grant 15RT-0100.


   ACKNOWLEDGMENTS
 
We thank the subjects who participated in the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. S. Richardson, Dept. of Medicine, 9500 Gilman Dr., Univ. of California San Diego, La Jolla, CA 92093-0623 (e-mail:rrichardson{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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