On the initiation of exercise, blood vessels in active skeletal muscle vasodilate, and the autonomic nervous system is engaged (i.e., sympathoexcitation). During exercise, blood flow to the active muscle increases to meet the oxygen and metabolic tissue needs. This is known as exercise-induced vasodilation (i.e., exercise hyperemia). In addition, a local neural-mediated reflex known as the exercise pressor reflex is activated to help redistribute blood flow to active muscle and to prevent blood pressure (BP) from falling (13). Besides a mechanical interaction between blood vessels, afferent nerve endings, and skeletal muscle (i.e., muscle pump) activating these blood flow regulators, it has been proposed that compounds from contracting skeletal muscle directly can affect blood flow by altering vascular tone and resistance (13,14). Currently, the role that muscle-derived compounds play in exercise hyperemia and in the exercise pressor reflex remains unclear.
Previous exercise studies have used infusion techniques to examine the influence of a range of metabolites on exercise-induced skeletal muscle vasodilation and the exercise pressor reflex. In these studies, specific metabolite(s) or antagonists are infused into the arterial supply of the limb while simultaneously measuring vascular responses (e.g., diameter, blood flow, or both), afferent nerve discharge, sympathetic engagement, BP, or a combination thereof (13,14). A major limitation with these studies is that the amount of the substances in question that actually reaches the interstitial environment is unknown. Moreover, it often is difficult to determine if these substances are initiating more of a systemic response through activation of sensory receptors or nerve endings in other parts of the body.
The technique of microdialysis overcomes some of these limitations. Microdialysis can measure potential muscle-derived compounds in the interstitial space that is in contact with blood vessels, nerve endings, and skeletal muscle cells at rest and during a variety of exercise paradigms.
This paper provides an overview of exercise-induced hyperemia, the exercise pressor reflex, and the technique of microdialysis. Microdialysis studies in which potential interstitial muscle-derived compounds involved in exercise-induced vasodilation and the exercise pressor reflex are examined.
EXERCISE FLOW REGULATION: VASODILATION AND EXERCISE PRESSOR REFLEX
Metabolic byproducts of muscle contractions can contribute to both vasodilation and engagement of the exercise pressor reflex that can evoke sympathoexcitation. As skeletal muscle contracts, muscle-derived compounds are produced intracellularly and are transported actively out of the cell and into the interstitial space. Compounds also may be produced in the interstitium and be released from endothelial cells during muscle contractions. Once in the interstitium, muscle-derived compounds can alter the degree of smooth vascular contraction and vascular tone through activation of membrane-bound metabolic receptors and nerve endings. The degree of vasodilation has been suggested to be proportional to the amount of metabolic factor(s) released from the contracting muscle or endothelium into the interstitial space (14).
During exercise, interstitial metabolites activate the exercise pressor reflex by stimulation of afferent pathways whose terminal ends are located in the interstitium of the exercising muscle. These thin myelinated and unmyelinated group III and IV muscle afferent fibers synapse in the dorsal horn of the spinal cord and project to neurons in the brainstem. Activation of this reflex results in elevation of heart rate (HR), BP, ventilation, and muscle sympathetic nerve activity (MSNA). The increase in these variables has been suggested to oppose the potent metabolic dilator system in exercising muscle. This muscle reflex is engaged when the muscle fatigues, when a mismatch occurs between blood supply and metabolic demand, or both (13).
USE OF MICRODIALYSIS TO EXAMINE THE INTERSTITIAL ENVIRONMENT
Microdialysis is an excellent method to measure a wide variety of metabolites and muscle-derived compounds directly in the interstitial space during exercise (10). The specific technique of microdialysis has been well described in a prior issue of Exercise and Sports Sciences Reviews (5). Our laboratory and others have used the linear probe microdialysis design with multiple microdialysis fibers inserted into the vastus lateralis or gastrocnemius muscle approximately 1 cm apart. To minimize any fiber breakage during strenuous exercise bouts, a 10-cm length of 5–0 suture is glued to the nondiffusible nylon tubing to give the microdialysis probe tensile strength (11). Operating on the principle of diffusion, a perfusate (i.e., extracellular physiological solution) is infused through the probe, where it will equilibrate with the fluid in the interstitium. The returning fluid, known as dialysate, is collected with small collection tubes or by metabolite sensitive electrodes that are connected to the outlet of the microdialysis tubing (Fig. 1).
Microdialysis has been used during steady-state rhythmic and static exercise paradigms with simultaneous measurement of blood flow, HR, BP, and MSNA. Because of the small volume of dialysate sample that is able to be collected, the number of metabolites that are able to be measured simultaneously is limited. An important limitation of this method is that the type of cell releasing the metabolite(s) cannot be determined with certainty. In addition, estimates of interstitial metabolite concentrations can be influenced by the limited time resolution and the fact that the concentration of the metabolite(s) may be different in different parts of the interstitium.
INTERSTITIAL METABOLITES AND EXERCISE-INDUCED VASODILATION
Microdialysis studies have investigated a range of interstitial muscle-derived compounds potentially involved with exercise-induced vasodilation. These include: adenosine, adenosine triphosphate (ATP), potassium (K+), phosphate, lactate, hydrogen (H+), nitric oxide (NO), bradykinin, and prostacyclin (PGE2). Blood flow was simultaneously measured during exercise in most of these studies.
Animal metabolite infusion studies have yielded conflicting data on the role of adenosine in exercise-induced vasodilation (14). Yet, human studies using microdialysis have shown a strong relationship between interstitial adenosine and increases in blood flow associated with incremental exercise. A fivefold increase in interstitial adenosine occurred from rest (220 ± 100 nmol·L−1) to 10 W with a gradual nonlinear rise as intensity was increased incrementally to a maximum of 50 W (∼2000 nmol·L−1 at max) during one-legged knee extension exercise in healthy young men (4). A strong correlation (r = 0.98) between interstitial adenosine and femoral blood flow (Doppler) was shown. Our laboratory has also examined changes in interstitial adenosine measured by microdialysis and femoral blood flow measured by Doppler during one-legged knee extension exercises at low to moderate intensities (30% and 60% Wmax) in healthy young men and women (10). Resting interstitial adenosine (332 ± 50 nmol·L−1) increased approximately two- to threefold during exercise and returned toward baseline values in recovery. This response was intensity dependent (Fig. 2). A significant correlation between interstitial adenosine and flow/conductance (r = 0.69) also was demonstrated. Thus, in our study, approximately 50% of the variability in limb flow can be explained by changes in interstitial adenosine. In another study, during incremental dynamic plantar flexion exercise in healthy young subjects (9 men and 1 woman), interstitial adenosine was shown to have an approximately twofold increase with increasing intensity (7). Thus, human microdialysis studies demonstrated that: (1) interstitial adenosine increases with exercise in an intensity dependent relationship, and (2) a temporal association exists between interstitial adenosine and limb blood flow during rhythmic leg exercise.
The primary source of the adenosine is unclear. Adenosine may come from contracting muscle or may be produced in the interstitium as a result of the availability of interstitial adenosine substrates (e.g., ATP, adenosine diphosphate [ADP], adenosine monophosphate [AMP]) and enzymes (4). However, adenosine may also be released during exercise by the endothelium and erythrocytes.
Microdialysis has also been used to measure interstitial ATP as well as other purine compounds, such as ADP and AMP, during progressive one-legged knee extension exercise as previously described (4). Interstitial ATP, ADP, and AMP, along with adenosine and blood flow, progressively increased during the exercise bouts (10–50 W;Fig. 3). Adenosine triphosphate concentration at rest was 0.13 ± 0.03 μmol·L−1 increased approximately 14- and 34-fold during exercise (10 and 50 W, respectively), which corresponded to an approximate 11- and 28-fold increase in blood flow. Thus, ATP seems to be associated with vasodilation.
Like adenosine, the primary source of ATP is unclear. Whether muscle contractions lead to an ATP exocytosis is unknown. In addition, during exercise, ATP has been demonstrated to be released by sympathetic nerve endings and erythrocytes.
Several studies have examined the influence of exercise on interstitial K+. Interstitial K+ was measured in six young males during one-legged knee extensor exercise (6). Interstitial K+ was shown to increase with increasing intensities (4.19 ± 0.09, 6.17 ± 0.19, 7.48 ± 1.18, 9.04 ± 0.74 mM at rest; 10, 30, and 50 W, respectively). Venous K+ also was measured and was shown to be lower in value than interstitial K+. Our laboratory has also examined changes in interstitial K+ and femoral blood flow during one-legged knee extension exercise as previously noted (10). Interstitial K+ was collected on-line with the use of microelectrodes. Interstitial K+ increased during exercise (3.91 ± 0.10 to 5.52 ± 0.53 meq·L−1; 60% Wmax) and returned toward baseline in recovery. Interstitial K+ increased in conjunction with an increase in workload. A correlation between MBV and K+ (r = .53) also was demonstrated. Thus, interstitial K+ seems to be associated with exercise hyperemia.
Our laboratory has examined changes in interstitial phosphate measured by microdialysis and femoral blood flow during one-legged knee extension exercise at low to moderate intensities as previously described (10). Interstitial phosphate increased during exercise (approximately 50%) and continued to increase in recovery. No correlation with blood flow and phosphate was demonstrated. Thus, it seems unlikely that this metabolite plays a role in exercise hyperemia.
Our laboratory measured interstitial lactate with microdialysis and femoral blood flow with Doppler during a one-legged knee extension paradigm (10). Interstitial lactate increased during exercise (1.19 ± 0.46 to 2.01 ± 0.72 nM; 60% Wmax) and continued to increase in recovery. No correlation between flow and lactate was demonstrated. Thus, like phosphate, it is unlikely that this interstitial metabolite plays a role in exercise hyperemia.
Studies examining the effect of exercise on interstitial pH (i.e., H+ ions) has yielded conflicting data. Our laboratory has examined changes in interstitial H+ measured by microdialysis collected on-line through flowthrough pH microelectrodes with femoral blood flow during one-legged knee extension exercise (10). Interstitial H+ decreased (98.70 ± 8.54 to 69.41 ± 5.87 nM; 60% Wmax) during the exercise bout, becoming alkalotic, but then returned toward baseline in recovery. An alkalization during muscle contractions has also been reported during static exercise paradigms (11). The alkalization (i.e., reduction of H+ ions) during exercise may represent a balancing of the relative concentrations of strong cations such as K+ and anions such as lactate and phosphate to preserve ionic neutrality within a given space. In another exercise study (15) using pH-sensitive fluorescent dye, interstitial pH fell (i.e., H+ ions increased), causing the interstitial environment to become more acidic during the one-legged knee extension exercise in an intensity-dose manner. In this report, bicarbonate was added to the perfusate, whereas bicarbonate was not added to perfusate in the former study. Thus, the role for H+ with exercise hyperemia is unclear.
Interstitial NO has been measured using NO-sensitive electrodes. These electrodes have been placed on the outlet of the microdialysis tubing during one-legged dynamic knee extension exercise (2). During a 30-W exercise bout, interstitial NO was shown to increase with exercise. However, NO synthase inhibition does not alter exercise blood flow, and NO synthase inhibition does not increase other interstitial metabolites (e.g., adenosine, K+, and prostacyclin) during exercise (3). These and other infusion studies (14) suggest that NO may not play a direct role in evoking or sustaining exercise hyperemia but may play a role in determining blood flow during rest and recovery.
Langberg et al. (7) have used microdialysis to measure interstitial bradykinin in the calf muscle during 10 min of incremental dynamic plantar flexion exercise (0.75 W, 2 W, 3.5 W, and 4.75 W). Interstitial bradykinin increased with exercise (23.1 ± 4.9 to 110.5 ± 37.9 nmol·L−1) and decreased in recovery; however, there were no clear intensity-dependent responses as was seen with adenosine. Although blood flow was not measured simultaneously, it was suggested by the authors that bradykinin may contribute to skeletal muscle vasodilation at low workloads.
Microdialysis has been used to examine the interstitial prostacyclin during exercise. During 30 min of one-legged knee extension at 30 W, interstitial prostacyclin increased from rest (1.17 ± 0.20 to 1.97 ± 0.30 ng·mL−1). However, prostacyclin continued to increase in recovery (2.76 ± 0.38 ng·mL−1) (3). The authors suggest that prostacyclin may have a role in exercise hyperemia; however, its role may be more important in postexercise hyperemia.
In summary, microdialysis studies during rhythmic exercise have demonstrated that interstitial adenosine, ATP, and K+ seem to be strongly associated with exercise hyperemia. At lower exercise intensities, interstitial bradykinin also may be involved. The role of interstitial H+ on blood flow remains unclear. Changes in interstitial phosphate, lactate, NO, and prostacyclin do not seem to play a role in exercise hyperemia; however, NO and prostacyclin may be more involved in postexercise hyperemia.
INTERSTITIAL METABOLITES AND EXERCISE PRESSOR REFLEX
Microdialysis studies have also investigated a range of muscle-derived compounds potentially involved with the exercise pressor reflex. These compounds include: adenosine, ATP, K+, phosphate, lactate, and H+. Static exercise usually was used to evoke the pressor response and the rise in muscle metabolites. As opposed to rhythmic exercise, static exercise evokes less vasodilation, and thus BP changes are more reflective of peripheral sympathetic constrictor tone. In these studies, the exercise paradigms need to be sufficiently vigorous to elevate muscle metabolites but not so vigorous that the probes no longer function, that systemic effects of the metabolites are seen, or both. Most of these studies used HR, BP, and MSNA as indices of sympathoexcitation (13).
Previous studies examining the role of adenosine have used bolus administrations of adenosine and demonstrated an increase in MSNA, BP, or both (13); however, it is difficult in these studies to exclude the influence of arterial chemoreceptor responses. In addition, because the endothelium provides a large barrier to some metabolites, a large quantity of the metabolite needs to be administered to cross into the interstitium. However, microdialysis studies allow direct examination of interstitial adenosine.
Interstitial adenosine has been studied in cats using an isolated hindlimb perfusion model coupled with microdialysis (12). The cat hindlimb was perfused with bovine red cells to which two different perfusions of adenosine (100 μM and 20 mM) were added. Despite large increases in interstitial adenosine (3- and 2500-fold for 100 μM and 20 mM adenosine infusions, respectively), there were no significant increases in HR or BP. The lack of a pressor response was not the result of technical concerns with the preparation, because boluses of potassium chloride and phosphate did evoke pressor responses. This suggests that adenosine does not stimulate group III and IV muscle afferents.
Our laboratory recently published two papers (8,9) that suggest that ATP may evoke the exercise pressor reflex. In the first, we found that arterial infusions of ATP evoked a rise in blood pressure that was mediated by Purinergic 2×3 receptors (9). We also observed that ATP sensitizes mechanically sensitive muscle afferents. In the second paper, microdialysis methods were used to demonstrate that interstitial ATP rises with twitch contractions (8) (Fig. 4). The increase in interstitial ATP was linked to the amount of tension generated. Additionally, this study suggested that the ATP was released from skeletal muscle. Furthermore, the findings of this report did not suggest that sympathetic or motor nerve sources, or both, were responsible for the rise in interstitial ATP with twitch contractions.
Changes in interstitial K+ as measured by microdialysis have been examined in humans. Our laboratory has measured interstitial K+ by flowthrough K+ microelectrodes during intermittent static quadriceps exercise (25% muscle voluntary contraction for 20 s by 5 s at relaxation) (11). Dialysate K+ increased with exercise (Δ.6 ± 0.1 meq·L−1) and rapidly returned toward baseline in recovery. This study and other studies (13) suggest that interstitial K+ may play a role in evoking the exercise pressor reflex. However, a recent report by Daley et al. (1) using venous K+ values as surrogate markers for interstitial K+ suggested that it does not evoke the muscle reflex.
We investigated the role of deprotonated phosphate (H2PO4−) as measured by 31P-nuclear magnetic resonance in evoking the pressor reflex during ischemic handgrip exercise in humans (13). The cellular concentration of H2PO4− was correlated to MSNA. We also directly measured interstitial phosphate with microdialysis during intermittent static quadriceps exercise as previously noted (11). Corrected dialysate phosphate increased with exercise (Δ0.18 ± 0.08 nM) and returned toward baseline in recovery. The increases in dialysate phosphate were associated with increases in MSNA, HR, and BP responses. Thus, it seems that interstitial phosphate may play a role in evoking the exercise pressor reflex.
We also measured interstitial lactate during static leg exercise as previously described (11). Resting interstitial lactate levels (1.1 ± 0.1 mM) increased to 1.6 ± 2 mM during exercise and further increased in recovery (2.0 ± 0.2 mM). Although during exercise interstitial lactate increased along with HR, BP, and MSNA, these latter variables returned to baseline in recovery, unlike interstitial lactate. Thus, our data suggest that interstitial lactate does not contribute to the direct stimulation of the exercise pressor reflex because of the different temporal pattern observed for the reflex responses.
Human studies have shown that during static exercise, pH becomes alkalotic and dialysate H+ levels decrease (69.4 ± 3.7 to 16.7 ± 3.8 nM) but return to baseline in recovery (11). Thus, based on the findings from our group, we do not believe that interstitial acidosis is necessary to evoke the exercise pressor reflex.
In summary, studies during exercise have demonstrated that interstitial ATP and phosphate may play a role in evoking the pressor reflex. We do not believe that interstitial adenosine, lactate, H+, K+, or a combination thereof play a prominent role in this process.
In this report, we examined the role that a number of substances may play in evoking vasodilation and in initiating the exercise pressor reflex. The technique of microdialysis has allowed a direct determination of the changes in the interstitial milieu during dynamic and static exercise paradigms, overcoming many of the limitations of metabolic infusion studies. During rhythmic exercise, interstitial adenosine, ATP, and K+ were associated with exercise hyperemia. Interstitial ATP and phosphate seem to play a role in evoking the exercise pressor reflex. It is clear that different interstitial muscle-derived compounds may be involved, depending on the exercise paradigm. Whether interstitial metabolites work synergistically or at different times during the exercise bout needs further investigation. Future studies also will require improved microdialysis methodologies to obtain better time resolution of changes in interstitial metabolites. Only then will it be possible to understand how skeletal muscle blood flow and autonomic control are regulated in health and disease.
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