Previous studies examining the delay to the onset of vasodilation have primarily focused on the onset of exercise, a setting complicated by the fact that the muscle pump and the vasodilator systems are both activated, making it difficult to attribute changes in blood flow to one or both. The goal here was to determine the delay to the onset of vasodilation after changes in work rate imposed by changes in treadmill grade (work intensity) during locomotion at a steady speed. The rationale was that constant speed would help ensure constant muscle pump activity (contraction frequency) such that vasodilator responses could be examined in isolation. Seven Sprague-Dawley rats underwent three trials each in which treadmill incline was suddenly (∼1 s) elevated from −10° to +10°. The delay to the onset of vasodilation averaged 5.0 ± 1.8 s, and this delay was not altered by inhibition of nitric oxide synthase. Similar or longer delays were seen during sinusoidal exercise. Thus there is a significant delay before the onset of vasodilation after an increase in work intensity (muscle force) during locomotory exercise at constant speed.
- muscle blood flow
- arterial pressure
- vascular conductance
the onset of locomotion and most forms of dynamic exercise are accompanied by a rapid increase in the blood flow to the muscles engaged in producing movement. The rise in blood flow is largely attributable to a rise in the vascular conductance calculated across muscle, which in turn is attributable to the muscle pump (4, 8, 17, 22) and to the inhibition of arteriolar smooth muscle after the production, release, diffusion, and transduction of vasodilator chemicals (6-8). The relative contribution of each of these two mechanisms is unclear. The delay to the onset of vasodilation is controversial and may depend on the mode of exercise. Some investigators report “immediate” vasodilation (no delay) (3), whereas others report delays of up to tens of seconds, depending on exercise mode and intensity (6). Previous studies examining the delay to the onset of vasodilation have focused on the onset of exercise. This setting is complicated by the fact that the muscle pump and the vasodilator systems are both activated, making it difficult to attribute changes in blood flow to one or both.
In the present study, we sought to test the metabolic vasodilation hypothesis as a mechanism for raising muscle blood flow at exercise onset by characterizing the dynamic properties of the vasodilatory responses to changes in work rate imposed by changes in treadmill grade during voluntary locomotion at constant speed. The rationale was that constant speed would help ensure constant muscle pump activity (contraction frequency) such that vasodilator responses could be examined in isolation. The following approaches were employed. The delay to the onset of muscle vasodilation after a sudden increase in treadmill grade (work intensity) during locomotion at a steady speed was examined in rats. This approach was repeated in rats after inhibition of nitric oxide synthase (NOS). We also examined responses when treadmill grade was altered continuously in a sinusoidal pattern in rats. Sinusoidal exercise was repeated in rats after inhibition of NOS to test whether this would alter the time course of vasodilation inasmuch as NOS inhibition has been shown to alter the time course of vasodilation at the onset of exercise (15).
All procedures met National Institutes of Health guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Iowa.
Twelve male Sprague-Dawley rats (250–450 g) were selected for their willingness to walk on a motor-driven treadmill (model 1010 Modular Treadmill, Columbus Instruments, Columbus, OH). The rats were familiarized with treadmill walking before the following aseptic surgical procedures were performed.
Rats were anesthetized with isoflurane. Each animal had an ultrasonic transit-time blood flow transducer (model 1.5RB, Transonic, Ithaca, NY) implanted on the terminal aorta through a midline abdominal incision. The probe cable was tunneled to an exit site on the back. The animal was given nalbuphine hydrochloride (1 mg/kg sc) for control of postoperative pain. The animal was allowed to recover until an acceptable blood flow signal was acquired (usually 2–3 days).
The flow transducer was connected to a flowmeter (model T106, Transonic). The animal was then placed on the treadmill. A pressure transducer (model PE10 EZ, Ohmeda, Madison, WI) connected to a length of water-filled tubing was mounted parallel to the walking surface of the treadmill. The pressure transducer was connected to a signal conditioner (model 6600, Gould Instrument Systems, Valley View, OH). Signals were displayed on a chart recorder (model MT95K2, Astro-Med, West Warwick, RI), digitized at 1 kHz, and written to a fixed disk of a microcomputer by using commercially available software (PONEMAH Physiology Platform, P3, Gould Instrument Systems).
Treadmill locomotion started at 10 m/min, and the animal walked at −10° for 2 min, +10° for 2 min, and then −10° for 2 min. As the animal continued to walk at 10 m/min, treadmill grade was cycled manually between +10° and −10° at 0.01 Hz for two cycles (180 s), 0.02 Hz for three cycles (150 s), 0.04 Hz for five cycles (120 s), 0.06 Hz for five cycles (90 s), 0.10 Hz for six cycles (60 s), 0.20 Hz for five cycles (25 s), and 0.5 Hz for five cycles (10 s). The foregoing trial served to familiarize the animal with the changes in treadmill incline, and the data were not included in the subsequent analysis. The animal then walked at 10 m/min −10° for 2 min, +10° for 2 min, and −10° for 2 min, after which the cyclic changes described above were imposed in reverse order (high to low frequency). The animal then walked at −10° for 2 min, +10° for 2 min, and −10° for 2 min, and the cyclic alterations described above were imposed in the original (low to high frequency) order. The animal then walked at 10 m/min −10° for 2 min, +10° for 2 min, and −10° for 2 min.
One to three days later, the animals were studied again. The animals performed the familiarization bout as described above. The animals were then injected withN ω-nitro-l-arginine methyl ester (l-NAME, 10 mg/kg ip; Sigma Chemical, St. Louis, MO). Approximately 10 min were allowed for hindlimb blood flow to decay to the new lower steady state. The animal then walked at 10 m/min −10° for 2 min, +10° for 2 min, and −10° for 2 min, after which the same cyclic changes were imposed in high- to low-frequency order. The animal then walked at −10° for 2 min, +10° for 2 min, and −10° for 2 min, and the same cyclic alterations in grade were imposed from the low to the high frequency. The animal then walked at 10 m/min −10° for 2 min, +10° for 2 min, and −10° for 2 min.
Stride frequency was measured in five rats to verify that alterations in grade did not alter stride frequency. While the animal walked at a constant speed of 7.5 m/min, treadmill incline was altered between −10° and +10° every 10 s for 90 s. This pattern was chosen because it shared characteristics of both the step-response and sinusoidal exercise protocols. At each foot strike, an observer activated a spike generator from which stride frequency was determined. The four complete cycles in each rat were ensemble averaged into one composite cycle, and the composite cycle from each rat was averaged together into the one cycle shown in Fig.1. It can be seen that stride frequency varied little and in no identifiable relationship to the changes in grade.
Data analysis was carried out by using 1-s averages of the data written to the fixed disk. For the control (no drug) condition, each of the three trials in which treadmill grade was increased in a step from −10° to +10° was analyzed individually. The baseline (average) blood flow and standard deviation of blood flow were calculated from the ten 1-s averages taken from the 10 s immediately preceding the onset of the sudden rise in grade. The delay to the onset of vasodilation was established by determining the time point after the onset of the sudden rise in grade at which a 1-s time-averaged blood flow exceeded a threshold level defined as two standard deviations above the baseline blood flow. The steady-state values of blood flow during locomotion at −10° and +10° were established by averaging blood flow over the final 15 s at each grade. The results from the multiple trials in a single rat were averaged together such that each animal contributed only once to the group mean data. Data collected after l-NAME administration were analyzed similarly.
For the sinusoidal alterations in treadmill grade, the lags in time from the peak in grade until the corresponding peak in blood flow, and from the trough in grade until the corresponding trough in blood flow, were identified by direct inspection, as were the values of the peak in flow and the trough in flow. The amplitude of the blood flow response was calculated as the difference between the peak in flow and the trough in flow. This was done for all frequencies except the two highest frequencies (0.20 and 0.50 Hz) for which there were no readily identifiable associations between treadmill grade and blood flow. The results from the multiple cycles at each frequency were averaged together for each of the two periods of exercise at each frequency. Data are presented as means ± SE.
The delays derived from the step increase in grade before and afterl-NAME treatment were compared by using a pairedt-test. For the remainder of the data, treatment effects were tested statistically by multiple linear regression by using a computer spreadsheet program. Dummy variables were used as independent variables to encode treatment effects and to account for interindividual variability among animals, analogous to a repeated-measures ANOVA (20). For amplitude, all 140 values (7 rats × 5 frequencies × 2 bouts/frequency × 2 for before and after l-NAME treatment) were entered into a single regression. For the independent variables, a dummy variable was used to encode for l-NAME treatment, and the actual quantitative values of frequency were entered into the regression in addition to the dummy variables used to account for the different animals. For delay, the analysis resulted in 280 values because the peak-to-peak and trough-to-trough delays were determined separately. All 280 of these values were entered into a single regression. For the independent variables, a dummy variable was used to encode forl-NAME treatment, a dummy variable was used to encode whether the delay was a peak-to-peak delay or a trough-to-trough delay, and the actual quantitative values of frequency were entered into the regression in addition to the dummy variables used to account for the different animals. On the basis of our results (see Fig. 7), it appeared that the difference between the peak-to-peak delays and the trough-to-trough delays was dependent on frequency. To test the hypothesis that the difference between the two delays was dependent on frequency, a final dummy variable, equal to the product of the dummy variable encoding peak-to-peak vs. trough-to-trough and frequency, was entered into the regression to test statistically for the apparent interaction of these two variables.
The responses of hindlimb blood flow to three trials of a step increase in treadmill grade during constant-speed locomotion in a single rat are shown in Fig. 2. The delay until the onset of vasodilation was 7 s for two of the trials and 8 s for the third trial. The absolute value of the blood flow averaged over the 10 s preceding the increase in treadmill grade was 32 ml/min in this rat.
Under control conditions, resting blood flow averaged 21.7 ± 8.9 ml/min and l-NAME treatment reduced blood flow to 10.4 ± 4.3 ml/min. The group mean responses of hindlimb blood flow to a step increase in treadmill grade during constant-speed locomotion before and after NOS inhibition are shown in Fig.3. NOS inhibition reduced baseline blood flow but did not alter the dynamics of the vasodilation in that the responses under the two conditions parallel one another. The average delay was 5.0 ± 1.8 s (range 2.0–7.3 s) and 7.0 ± 2.4 s (range 3.7–11.0 s) for the control andl-NAME conditions, respectively (P = 0.16).
Figure 4 shows hindlimb blood flow in response to sinusoidal changes in treadmill grade in a single rat. Figure 4 A shows that blood flow lags grade by ∼10 s when treadmill grade is altered at 0.01 Hz. Figure 4 B shows that blood flow lags grade by ∼7 s when treadmill grade is altered at 0.06 Hz. At this higher frequency, it can be seen that the blood flow responses are out of phase with the changes in work performed. Blood flow is highest when the animal is doing the least work and lowest when the animal is doing the greatest amount of work.
Figure 5 shows the average hindlimb blood flow from seven rats in response to sinusoidal changes in treadmill grade. Figure 5 A shows that blood flow lags grade by ∼10 s when treadmill grade is altered at 0.01 Hz. Figure5 B shows that blood flow lags grade by ∼6 s when treadmill grade is altered at 0.06 Hz. At this frequency, it can be seen that the blood flow responses are out of phase with the changes in work performed. Blood flow is highest when the animal is doing the least work and lowest when the animal is doing the greatest amount of work.
Figure 6 shows the influence of NOS inhibition on hindlimb blood flow responses to sinusoidal changes in treadmill grade in a single rat. Figure 6 A shows the lag in blood flow when treadmill grade is altered at 0.04 Hz when NOS function was intact. Figure 6 A also illustrates that the vasoconstriction after a decrease in grade proceeds more rapidly than does the vasodilation that follows an increase in grade. Figure6 B shows that NOS inhibition reduced the mean level of blood flow but had little effect on the dynamics of vasodilation.
The peak-to-peak (vasodilation) and trough-to-trough (withdrawal of vasodilation) delays across frequencies are shown in Fig.7. There was a statistically significant main effect of frequency (P < 0.001), indicating that the delays decreased with increasing frequency. There was a statistically significant main effect of increasing grade vs. decreasing grade (P < 0.01) on delay. In addition, there was a statistically significant interaction between frequency and grade (P < 0.05) signifying that as frequency decreased, the peak-to-peak delay increased more so than the trough-to-trough delay. l-NAME treatment had no effect on delay (P = 0.11).
The amplitudes of the changes in hindlimb blood flow to sinusoidal alterations in treadmill grade during constant-speed locomotion in rats are shown in Fig. 8. The amplitudes at the lowest frequency exceeded the difference in the steady-state values, and amplitude decreases with increasing frequency. NOS inhibition reduced amplitude. There were statistically significant main effects of frequency (P < 0.001) and ofl-NAME treatment (P < 0.001) on amplitude.
The increases in muscle blood flow and muscle vascular conductance achieved during dynamic exercise are tightly coupled to the amount of work performed by muscle, and metabolic vasodilation is an important regulatory mechanism by which muscle blood flow is coupled to local energy demands (14). The common view is that vasodilator substances within active muscle accumulate in a manner governed by the balance between the energy expended by the muscle and the blood flow through the muscle and that metabolic vasodilation constitutes the primary determinant of the vascular conductance achieved during locomotion. Because arterial pressure changes far less than does muscle blood flow in the transition from rest to locomotion (20 vs. >200%), the blood flow achieved is tightly coupled to vascular conductance.
The muscle pump exerts important influences on both the pressure-volume (capacitive) (11, 19) and the pressure-flow characteristics (17, 18) of the peripheral circulation. A number of investigators employing a broad mixture of different exercise conditions have concluded that the muscle pump can augment blood flow across muscle (5, 13, 15, 17, 18, 22). There is also evidence against the importance of the muscle pump (10). A common assumption is that contraction frequency constitutes a major determinant of muscle pump efficacy (5, 13, 15, 17, 22) just as cardiac frequency can constitute a major determinant of cardiac pump efficacy, and several studies have provided evidence in support of this idea (13, 15, 17). The importance of contraction force on the blood flow-raising function of the muscle pump appears far less important. For example, if increases in muscle force augment muscle pump function, then the effectiveness of muscle pumping would be expected to increase along with increasing treadmill grade (muscle force). However, treadmill grade exerts little influence on the initial rise in blood flow at locomotion onset, suggesting that muscle force production is not a strong determinant of muscle pump function (15). For these reasons, the approach in the present study of varying treadmill grade at constant-speed locomotion should induce little alteration in muscle pump function, and thus the changes observed should largely reflect the local action of vasodilator systems.
Our goal was to determine whether the metabolic vasodilator systems that match blood flow to metabolism in muscle during exercise have sufficient dynamic characteristics to account for the rise in blood flow observed at locomotion onset. The rise in blood flow at locomotion onset is essentially immediate even at extremely low treadmill speeds and grades. For example, within 1 s of the onset of locomotion, there is a detectable rise in blood flow in rats walking at 5 m/min and −10° incline (15) and in dogs walking at 3.2 km/h and 0% grade (17). In dogs, a doubling of treadmill speed from a low to a moderate level leads to a doubling of the immediate rise in hindlimb conductance in dogs walking on the flat (15,17). These investigators attributed this initial (2–3 s) rise in blood flow and calculated conductance to more effective muscle pumping at the higher contraction frequency. After a delay of ∼10 s during which time conductance was relatively unchanged, they observed conductance to undergo a second rise that they attributed to the action of vasodilator substances. They also found that vascular conductance rose more smoothly to its steady-state level in dogs walking uphill at a moderate speed, suggesting that vasodilation might begin much sooner at this workload (thereby encroaching on the short window of opportunity for gauging the effectiveness of the muscle pump). For these reasons, we purposely selected relatively low treadmill speeds for the present study. For the rats, we selected 10 m/min. Importantly, this speed is twofold greater than a speed (5 m/min) at which an immediate increase in flow is observed at locomotion onset, meaning that the overall level of metabolic activity in the present study was clearly sufficient to address our overall goal.
Moreover, the alterations in blood flow directed to the muscles actively engaged in producing the alterations in force output in the present study are much larger than indicated by the percent changes in terminal aortic flow. For example, terminal aortic flow rose from 22 ml/min at rest to 29 ml/min during locomotion at 10 m/min −10° incline in the present study, an apparent increase of only 32%. However, rat skeletal muscle blood flow rises from 0.3 ml · min−1 · g−1 at rest to 1 ml · min−1 · g−1across a wide range of treadmill speeds (15–45 m/min), giving an increase of 0.7 ml · min−1 · g−1(9). Thus the 7 ml/min increase in terminal aortic blood flow from rest to exercise observed in the present study can be attributed to only 10 g of active muscle. This 10 g of muscle is expected to have a resting blood flow of 3 ml/min, and during locomotion it would rise by 7 ml/min to 10 ml/min, i.e., over a threefold increase over resting levels. Similar calculations reveal that blood flow to active muscle rose by 100% from trough to peak during sinusoidal changes in treadmill incline at the low frequency studied. Thus we imposed robust alterations in vasodilator drive within the muscles actively engaged in producing the imposed changes in work rate. It is unknown whether the delay to the onset of vasodilation in response to similar changes in treadmill grade would be less, similar, or greater at higher exercise intensities. However, the observation that the time course of a change in oxygen uptake to a step change in exercise intensity is similar across a wide range of exercise intensities (1) suggests that it would be unaltered.
The delay to the onset of vasodilation after a step increase in treadmill grade in rats averaged 5 s. These estimates correspond closely to directly observed vasodilatory delays derived from both in situ (6) and isolated microvessels (24). Thus we confirm these findings and extend them to awake animals performing locomotion. The average responses shown in Fig. 3 suggest that vasodilation may begin somewhat earlier. However, this interpretation is complicated by the fact that relative early responses in one or two animals would bias the average upward even if there were no change at this time in the remaining animals.
Sinusoidal forcing is a common tool for perturbing a system to evaluate its dynamic characteristics. This approach has been successfully employed to evaluate respiratory (2) and thermoregulatory (25) exercise responses. To our knowledge, we are the first to employ this approach to evaluate muscle blood flow responses. We found that the delay of vasomotor responses to sinusoidal alterations in treadmill grade ranged from 3 to 12 s for frequencies ranging from 0.10 to 0.01 Hz. These findings indicate that the metabolic vasodilator systems that match blood flow to metabolism in muscle during exercise are relatively slow compared with the changes in blood flow observed at exercise onset (3, 4, 13, 15-17,21). Thus the changes in flow seen at exercise onset may be due to the muscle pump and/or to an entirely different class of vasodilator substances (14). The amplitude of the changes in blood flow at the lowest frequency exceeded the steady-state difference in blood flow observed between −10° and +10°. This finding may relate to the overshoot in muscle blood flow observed at the onset of exercise (17, 21). Amplitude decreased with increasing frequency. At a midrange frequency (∼0.06 Hz), the blood flow response was out of phase with the changes in grade such that the blood flow was highest when the animal was doing the least amount of work and vice versa. Thus, although blood flow rises and falls as grade is increased and decreased, there is a failure to “match” blood flow to the work being performed in a strict temporal sense. There was no readily discernable relationship between grade and flow at the highest frequencies studied (0.20 and 0.50 Hz), indicating that these frequencies were likely too fast for the dilator systems to respond.
Figure 6 illustrates the tendency for the dynamics of the response to increasing grade to differ from the responses to decreasing grade. Several of the phases of decreasing blood flow seen here proceed far more rapidly than the more slowly developing increases in blood flow. This observation was borne out across all animals, particularly at the lower frequencies studied (Fig. 7). This finding was not unexpected in light of the observations of Gorczyski et al. (6), who found that vascular recovery was relatively fast compared with the time it took for vasodilation to reach a steady state in response to muscle stimulation.
Inhibition of NOS is well documented to reduce resting and exercising blood flows (12, 16, 23), and this is what we found. NOS inhibition had little effect on the dynamics of vasodilation, suggesting that this system plays little or no role in determining these dynamics in rats under the conditions studied. NOS inhibition suppressed the difference between the peak-to-peak and the trough-to-trough delays in a manner that suggests that NO might prolong the vasodilation associated with an increase in grade. In dogs, it has been demonstrated that NOS inhibition slows the rise in blood flow at exercise onset (15). Whether this apparent difference stems from species differences or from a different role of nitric oxide at exercise onset compared with during ongoing exercise remains to be determined.
The delay to the onset of vasodilation after a step increase in treadmill grade averaged 5.0 s and was not altered by NOS inhibition in rats. During sinusoidal exercise, blood flow lagged changes in grade by 4 s at a high frequency to 10 s at a low frequency, and these lags were little altered by NOS inhibition. At an intermediate frequency, blood flow responses are out of phase with the changes in work performed; blood flow is highest when the animal is doing the least work and lowest when the animal is doing the greatest amount of work. Thus the metabolic vasodilator systems that are thought to match muscle blood flow to muscle metabolism appear to be too sluggish to account for the dynamics of blood flow adaptation seen at locomotion onset.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-46314.
Present address of T. M. Zidon: Dept. of Vet. Biomed. Sci., Univ. of Missouri, Columbia, MO 65211.
Address for reprint requests and other correspondence: D. Sheriff, 518 Field House, Dept. of Exercise Science, University of Iowa, Iowa City, IA 52242.
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