INTRODUCTION

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WHEN OXYGEN DELIVERY to active skeletal muscle is insufficient to meet metabolic demands, metabolite concentrations increase and stimulate metabolically sensitive group III and IV afferent nerve endings within the active muscle, eliciting a reflex increase in efferent sympathetic nerve activity and systemic arterial pressure (SAP) termed the muscle metaboreflex. Recently, Sheriff (20) investigated the length of time to the onset of the pressor response induced by total vascular occlusion of the terminal aorta in dogs performing treadmill exercise from mild to moderate workloads. Completely restricting blood flow to the active skeletal muscle of the hindlimbs evoked a substantial pressor response with a latency that was inversely related to workload and markedly shorter than the latency to increments in sympathetic nerve activity attributed to metaboreflex activation previously reported during static muscle contractions (7, 18, 19). However, only SAP was recorded in that study. Inasmuch as SAP is affected by changes in cardiac output (CO) and peripheral vasoconstriction, that study did not distinguish the mechanism of the pressor response or determine whether differential latencies exist among the efferent mechanisms of this reflex.

To our knowledge, the recent investigation by Sheriff (20) is the only study that has examined the latency of the metaboreflex during whole body dynamic exercise. However, several investigators have examined the mechanisms that mediate the metaboreflex-induced pressor response during dynamic exercise. In dogs, it is known that during mild dynamic exercise, graded partial reductions in active skeletal muscle perfusion induce substantial increases in both SAP and CO but cause only minor changes in total vascular conductance (TVC) (14, 21, 31). Relatively small changes in TVC indicate that little net peripheral vasoconstriction is occurring, although there is strong evidence that vasoconstriction does occur in nonischemic vascular beds such as the skeletal muscle of the forelimbs and kidneys (11, 12, 21). In contrast, several studies have shown that in situations in which CO does not or cannot increase, peripheral vasoconstriction can induce a pressor response of similar magnitude. In dogs performing mild dynamic exercise during constant heart rate (ventricular pacing) and ₁-adrenergic blockade, metaboreflex activation via graded partial reduction of hindlimb blood flow, as described above, evokes a pronounced increase in SAP that is entirely mediated via peripheral vasoconstriction, inasmuch as CO remained unchanged (21). Moreover, complete thigh occlusion in humans performing dynamic leg exercise causes a significant pressor response even though CO decreases slightly (1). In this study (1), humans did perform occlusive leg exercise; however, blood flow to the lower extremities was not measured before occlusion, and examining the latencies of the hemodynamic variables was not an objective. Thus the specific aims of the present study were to determine the mechanisms that mediate the pressor response during total vascular occlusion of active skeletal muscle and whether these efferent mechanisms demonstrate differential latencies to muscle metaboreflex activation.

Because Asmussen and Nielsen (1) showed that during occlusive leg exercise a significant pressor response occurs, whereas CO is actually reduced slightly, and because an additional study by Toska et al. (26) showed that the immediate reduction in CO that occurs during occlusive leg exercise is primarily the result of a vagally mediated bradycardia, compounded with the short latency of SAP during metaboreflex activation shown by Sheriff (20), we hypothesized that at least the initial pressor response to total vascular occlusion should be mediated via peripheral vasoconstriction, with increases in CO occurring at some time thereafter.

	METHODS

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All experiments were performed using seven conscious dogs of either gender (21-26 kg) selected for their willingness to run on a motor-driven treadmill. All procedures were reviewed and approved by the Institutional Animal Care Committee and conformed to National Institutes of Health guidelines.

Surgical preparation. Each animal was prepared in a series of surgical sessions with at least 1 wk between surgeries and between the last surgery and the first experiment. For all procedures anesthesia was induced with Pentothal Sodium and maintained with isoflurane. Cefazolin (500 mg iv) was administered both pre- and postoperatively, and then cephalexin (30 mg/kg by mouth, 2 times/day) was given to prevent postoperative infection. During recovery from surgery, buprenorphine (0.015 mg/kg iv) and acepromazine (0.1 mg/kg im) were administered for analgesia and sedation when necessary.

Experimental procedures. All experiments were performed after the animals had fully recovered from surgery and were active, afebrile, and of good appetite. The animal was brought to the laboratory and allowed to roam freely for 15-30 min. The animal was then directed to the treadmill, and the blood flow transducers were connected to the flowmeters (Transonic Systems). The FAP, SAP, and CVP catheters were connected to pressure transducers (Transpac IV, Abbott Laboratories). Heart rate (HR) was monitored via a cardiotachometer triggered by the CO signal. All data were sampled by a laboratory computer at 1,000 Hz, and mean values for each cardiac cycle were saved on a hard disk for subsequent analysis.

Data analysis. The vascular conductances through the forelimb or kidney were calculated as FLBF or RBF divided by (SAP  CVP), respectively. Changes in RVC are presented as a percentage of the change from the average control level. Although the trend and latency for RVC are very similar across all dogs included in this study, there was some variability in the absolute RBF values. In three dogs RBF ranged from 61.1 to 87.1 ml/min; however, in three other dogs RBF was substantially higher (108.6 to 161.8 ml/min). TVC was calculated as CO/(SAP  CVP). Nonischemic vascular conductance (NIVC) was calculated as (CO  TAQ)/(SAP  CVP). Subtracting TAQ allows isolation of the systemic responses without interference from the substantial mechanical decrease in TVC that results from occluder inflation.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Data from 1 experiment showing method of calculation of latencies for systemic arterial pressure (SAP), nonischemic vascular conductance (NIVC), cardiac output (CO), heart rate (HR), and renal vascular conductance (RVC, expressed as a percentage of control). For SAP, CO, HR, and RVC, the time period between occlusion and intersection of the short, stable period and linear regression is defined as the latency for that variable. Latency for NIVC was defined as the time period between occlusion and the highest point, just as constriction begins. Arrows correspond to onset of response for each variable. This experiment was performed at the highest exercise intensity (6.4 km/h, 10% grade); however, patterns of responses were very similar in all dogs and at both exercise intensities.

Statistical analysis. A two-way ANOVA for repeated measures was used to compare the latencies at the lower exercise intensity with the corresponding latencies at the higher exercise intensity as well as to compare each of the latencies within a workload. Individual means were compared using the test for simple effect. The software used for all statistical analysis was SYSTAT (version 5.02). An alpha level of P < 0.05 was used to determine statistical significance.

	RESULTS

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Figure 2 shows the responses to total vascular occlusion of the terminal aorta at the mild exercise intensity, 3.2 km/h at 0% grade. This figure contains averaged data from seven dogs (n = 6 for RVC, n = 5 for FLVC). The latencies for SAP, vascular conductance, and RVC were not statistically different from each other and occurred much earlier than the latencies for CO and HR.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   SAP, vascular conductance (VC), total vascular conductance (TVC), NIVC, CO, HR, RVC, forelimb vascular conductance (FLVC), and central venous pressure (CVP) vs. time in response to complete terminal aortic occlusion during the lower exercise intensity (3.2 km/h, 0% grade). Data are averaged from 7 animals (n = 7 for SAP, VC, CO, and HR; n = 6 for RVC; n = 5 for FLVC). Arrows correspond to onset of response ± SE for each variable. Vertical dashed line represents occluder inflation; horizontal lines from 0 to 80 s represent average control level 1 min before occlusion for each variable. Note that SAP begins to increase at same point that NIVC and RVC begin to decrease.

Figure 3 shows the responses to total vascular occlusion of the terminal aorta at the moderate exercise intensity, 6.4 km/h at 10% grade. Figure 3 is based on averaged data from seven dogs (n = 6 for RVC, n = 5 for FLVC). The response patterns of all variables were similar to those during the lower exercise intensity but were larger in magnitude and occurred more rapidly.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   SAP, VC, CO, HR, RVC, FLVC, and CVP vs. time in response to complete terminal aortic occlusion during the higher exercise intensity (6.4 km/h, 10% grade). Data are averaged from 7 animals (n = 7 for SAP, VC, CO, and HR; n = 6 for RVC; n = 5 for FLVC). Arrows correspond to onset of response ± SE for each variable. Vertical dashed line represents occluder inflation; horizontal lines from 0 to 60 s represent average control level 1 min before occlusion for each variable. Note that SAP begins to increase at same point that NIVC and RVC begin to decrease.

Figure 4 shows the latencies for SAP, NIVC, CO, HR, and RVC at both mild and moderate exercise intensities. Each latency at the lower exercise intensity was statistically different (P < 0.05) from the same latency at the higher exercise intensity. As stated earlier, latencies for FLVC and CVP were not calculated.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Latencies for SAP, NIVC, CO, HR, and RVC in response to complete terminal aortic occlusion during 3.2 km/h, 0% grade and 6.4 km/h, 10% grade. Note that as exercise intensity increases, latency for each variable decreases. * P < 0.05 vs. 3.2 km/h, 0% grade.

Table 1 shows the P values calculated when all latencies were compared within each exercise intensity. Note that the latencies for HR and CO were significantly longer than those for SAP, NIVC, and RVC, which were not different from each other.

	DISCUSSION

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There are two new findings in this study: 1) the efferent mechanisms mediating metaboreflex-induced pressor responses exhibited differential latencies, with vasoconstriction significantly preceding the metaboreflex-mediated rise in CO, and 2) the first phase of the metaboreflex pressor response seen during total vascular occlusion in dogs during mild and moderate exercise is mediated primarily via peripheral vasoconstriction.

Response patterns and latencies. The patterns of response for all variables at the lower exercise intensity were similar to the corresponding response patterns at the higher exercise intensity. In addition, all latencies were inversely related to exercise intensity. The arterial pressure responses and latencies were very similar to those described by Sheriff (20), who recently investigated the effect of total vascular occlusion of the terminal aorta in dogs performing dynamic exercise of mild to moderate exercise intensities. He found that there was an initial rise in SAP within the first 5 s, followed by a short stable period lasting several seconds. This short, stable period is likely the result of buffering by the arterial baroreflex due to the large decrease in vascular conductance caused by the occlusion (26). This was very well supported by our findings; however, in the study by Sheriff (20), only SAP was monitored. The present data indicate that the effect of a mechanical decrease in vascular conductance does likely evoke baroreflex responses. Specifically, a reflex vasodilation occurred, as indicated by an increased TVC, although RVC did decrease very rapidly, indicating vasoconstriction [likely caused by autoregulation (13)]. However, because the net total effect was vasodilation (increased TVC), other vascular beds must have dilated. In addition, HR and CO were rapidly decreased, likely caused by arterial baroreceptor-mediated parasympathetic activation, elicited by the initial rise in SAP as a result of the mechanical effects of total occlusion, as described by Toska et al. (26). After a short latency, TVC then decreased to control levels, indicating significant vasoconstriction, which was evidenced by the rapid rise in SAP. Before the vasoconstriction occurred, HR and CO reversed direction and began to increase back toward control levels. However, this response pattern is likely not caused by the metaboreflex and, importantly, is similar to that seen during rapid forms of baroreflex activation. For example, Strange et al. (25) observed that pulsatile negative pressure applied over the carotid sinus (neck suction) caused rapid reductions in HR that quickly returned toward control level. Using a similar technique but with static pressure, Eckberg (5) found an initial peak (within 2 s) increase in P-P interval, followed by a rapid decrease that reached a stable level that was higher than the control level. Stephenson and Donald (24) found that rapid increases in isolated carotid sinus pressure of conscious dogs resulted in significant rapid initial reductions in HR that rebounded to a steady-state level that was less than the control level. Furthermore, dogs are known to have a strong Bainbridge reflex (2, 3, 6, 27). Therefore, the immediate rise in CVP (stimulating the Bainbridge reflex) during both workloads coupled with the "rebound effect" from the baroreflex are possibilities that may explain the initial rise in HR and CO back toward control levels. Finally, we believe that the initial increases in HR were not mediated via the metaboreflex, inasmuch as the HR then decreased during mild exercise at 24 s. It seems highly unlikely that the metaboreflex would cause HR to increase, then decrease, and then increase again. Therefore, we believe that the metaboreflex-induced latencies for HR and CO occurred during the subsequent rises in both variables, which were significantly longer than the latency for TVC during both workloads. In addition, the latencies for TVC, RVC, and SAP were not different from each other, which indicates that vasoconstriction plays a major role in at least the initial phase of the pressor response. This pattern of metaboreflex activation is in stark contrast to what is observed in graded partial occlusions, in which the pressor response is primarily caused by increases in CO (14, 21, 31).

Potential influences of other cardiovascular reflexes. In the present study hindlimb occlusion caused significant increases in SAP and CVP; therefore, the arterial and cardiopulmonary baroreflexes are likely to be active throughout the entire duration of the occlusion, and previous studies have shown that both of these baroreflexes attenuate muscle metaboreflex pressor responses (4, 16, 22, 30). Interestingly, a recent study by Potts and Mitchell (17) showed that neural input from skeletal muscle afferents resets the threshold of the carotid baroreflex to a higher pressure, which indicates that the extent of arterial baroreceptor buffering of the muscle metaboreflex may wane as muscle afferents become progressively more active. We do not know whether the baroreflexes modulate the latency of the muscle metaboreflex. However, Sheriff et al. (22) showed that the baroreflex does not affect the threshold level of hindlimb perfusion required to activate the muscle metaboreflex during mild exercise; rather, the baroreflex acts to attenuate the magnitude of the pressor response. Similarly, Waldrop and Mitchell (30) demonstrated larger pressor responses to hindlimb ventral root stimulation in anesthetized baroreceptor-denervated cats than in baroreceptor-intact cats. Therefore, we speculate that the baroreflex likely acts to attenuate the rate of increase in SAP, CO, and peripheral vasoconstriction without affecting the latency of the responses. However, this has yet to be investigated.

Mechanisms of the pressor response. Blood pressure can rise through increases in CO and/or peripheral vasoconstriction. Previously, Asmussen and Nielsen (1) demonstrated that the interruption of leg blood flow during exercise on a bicycle ergometer resulted in a significant pressor response that was mediated via peripheral vasoconstriction, inasmuch as CO was reduced slightly. Toska et al. (26) showed that the immediate reduction in HR, and thus CO, that occurred just after the inflation of leg cuffs during leg exercise was eliminated by atropine, albeit a smaller, slower HR decrease still remained. Muscle metaboreflex activation during constant heart rate after ₁-adrenergic blockade results in large increases in arterial pressure primarily mediated via peripheral vasoconstriction, whereas before blockade and during the same type of exercise, similar but slightly larger pressor responses occurred mostly because of increases in CO (21). These studies, in concert with that of Sheriff (20), who showed that the latency of the metaboreflex is quite short (as fast as 5 s during moderate exercise), led us to hypothesize that at least the initial pressor response during metaboreflex activation should be mediated via peripheral vasoconstriction.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Observed pressor responses (SAPobs) as well as predicted individual effects of how peripheral vasoconstriction (SAPNIVC), CO (SAPCO), and mechanical effect of occluder (SAPmech) would affect SAP if other variables are held constant shown for mild (3.2 km/h, 0% grade; top) and moderate exercise (6.4 km/h, 10% grade; bottom). Vertical arrow represents onset of observed pressor response ± SE. Bottom panel only contains data through 60 s because dogs were unable to run with a normal gait any longer at that workload.

Importance of metaboreflex-mediated peripheral vasoconstriction. Peripheral vasoconstriction is an important mechanism utilized to mobilize blood centrally and maintain or increase CVP, which is crucial in helping to raise CO (18). Figure 3 shows that there is a significant vasoconstriction that occurred from 12 to 20 s. Interestingly, after 14 s, there was a continued rise in CVP throughout the duration of the occlusion that occurred despite an increase in CO. An increase in CO alone with no other compensatory mechanisms should cause CVP to fall because of the inverse relationship that exists between CO and CVP (23). Once beyond its latency there is by definition a significant elevation in CO. In the present study during both workloads, even after CO increased, CVP was either maintained (mild exercise) or increased (moderate exercise). A recent study by Sheriff et al. (21) showed that metaboreflex activation in dogs, via graded partial occlusion, is capable of eliciting substantial central blood volume mobilization. Therefore, the peripheral vascular adjustments mediated by the metaboreflex include those that act to maintain or increase ventricular filling pressure, which is important in the ability to increase CO despite increasing ventricular afterload.