INTRODUCTION

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THE EXERCISE PRESSOR reflex is generally defined as an elevation in heart rate, blood pressure, and ventilation in response to muscular activity (1, 26, 28). In addition, this reflex is thought to be responsible for the well-documented increase in muscle sympathetic nerve activity (MSNA) observed during handgrip exercise (9, 25, 27, 28, 39, 43, 46). The exercise pressor reflex is believed to be a feedback mechanism whereby mechanical and metabolic aspects of exercise result in the stimulation of autonomic responses and an increase in MSNA and subsequently vasomotor tone to nonexercising muscle (16, 26). It has been demonstrated that the afferent arm of this reflex is composed of thinly myelinated and unmyelinated groups III and IV muscle afferents, respectively (16). Furthermore, it has been demonstrated that groups III and IV muscle afferents respond to mechanical deformation of their receptive fields (18, 20) and to stimulation by metabolites produced by exercising muscle (19, 31, 38, 40, 48).

A number of previous studies have attempted to identify which metabolite(s) released from exercising skeletal muscle are responsible for stimulating these chemosensitive afferents. To date, the predominant substances identified as putative stimulators of the exercise pressor reflex include lactic acid (31, 38, 40), phosphate (40), K⁺ (33, 34, 48), and H⁺ (32, 43) as well as adenosine (7), prostaglandins (17), and bradykinin (6). It should be noted that the muscle afferents responsible for the stimulation of the exercise pressor reflex reside in the interstitial space (16). Therefore, any by-product(s) of muscle contraction would exert its effect in this compartment, and, to most accurately determine the possible effects that these putative stimulators may have on groups III and IV afferents, interstitial concentrations need to be determined. However, to date, no studies have attempted to measure or quantitate several of the putative stimulators of the exercise pressor reflex simultaneously in the interstitial space during exercise in humans.

Recent advancements in the microdialysis technique now allow the measurement of a wide variety of compounds in the interstitial space. The microdialysis technique was first described by Delgado et al. (8) and is based on the principle of simple diffusion through a semipermeable membrane. Briefly, a microdialysis probe is inserted in the muscle and perfused with a physiological solution. As the solution passes through the probe, compounds in the interstitial space and in the perfusate can diffuse into and out of the probe, respectively. The dialysate is then collected and analyzed, and, by making an in vivo calibration of the exchange fraction (termed "probe recovery") of the compound(s) being investigated across the probe membrane, actual interstitial concentrations can be calculated. Therefore, the purpose of the present study was to determine interstitial pH, K⁺, lactate, and phosphate concentrations during intermittent static quadriceps exercise in humans while simultaneously measuring MSNA, heart rate, and blood pressure. We hypothesize that static quadriceps exercise will result in an elevation in MSNA in association with an elevation in interstitial K⁺, lactate, and phosphate.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. The experimental protocol was approved by the Institutional Review Board of the Milton S. Hershey Medical Center. Each subject had the purposes and risks of the study explained to him, and written informed consent was obtained. Seven, healthy male subjects with a mean age of 32 yr (range 26-40 yr) participated in this study. The subjects were normotensive and were not taking any medication, and all refrained from the ingestion of caffeine-containing beverages for 24 h before the study.

Heart rate and blood pressure. Heart rate was monitored by standard electrocardiographic methods, and systemic blood pressure was measured continuously using the volume clamp method (Finapres; Ohmeda, Madison, WI). Resting blood pressure was determined using an automated sphygmomanometer (Dinamap; Critikon, Tampa, FL).

MSNA. The microneurographic technique provides direct recordings of sympathetic nerve traffic directed to blood vessels in skeletal muscle causing an increase in vascular tone and vasoconstriction (12, 36, 37, 43). The details of this method, as used in our laboratory, have previously been described (3, 29, 40). Briefly, multiunit recordings of postganglionic MSNA were obtained by using a tungsten electrode placed in a muscle fascicle within the peroneal nerve. The electrode has a 200-µm shaft that tapers to a 1- to 5-µm tip. A reference electrode was placed in the subcutaneous tissue over the fibular head and 1-3 cm from the active electrode. The neural signal was amplified 1,000 times by a preamplifier and 50-90 times by an amplifier. The resultant signal was fed through a bandpass filter (700 and 2,000 Hz), and the signal was rectified and integrated to obtain a mean voltage neurogram.

Microdialysis probes. The fibers used to construct the microdialysis probes were obtained from an artificial dialysis kidney (GFE18) that had a molecular mass cutoff of 3,000 Da. Each end of a single fiber was inserted ~1 cm in a hollow nylon tube (inner diameter = 0.50 mm, outer diameter = 0.63 mm) and glued. The actual probe length (distance between the 2 nylon tubes) was 4 cm (inner diameter = 0.20 mm, outer diameter = 0.22 mm). To provide tensile strength to the microdialysis probe so that it could withstand the forces generated by muscle contraction, a 10-cm piece of 5-0 suture (Ethicon) was glued to the nylon tubing. The suture was attached so that 3 cm was glued to the nylon tubing on one side of the probe and 3 cm was glued to the nylon tubing on the other. Thus the suture was only glued to the nylon tubing but spanned the distance of the probe. This modification is illustrated in Fig. 1 and allows the microdialysis probes to function very well during muscle contractions ranging in intensity from mild to maximum (23, 24).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Schematic representation of a microdialysis probe. A section of 5-0 suture was glued to nondiffusible nylon tubing to give the microdialysis probe tensile strength. This modification allowed the microdialysis probes to function during intermittent static quadriceps exercise without fracturing.

Microdialysis probe insertion. Three to five microdialysis probes (depending on muscle size and orientation) were inserted in the vastus lateralis muscle of the subject's left leg. The skin and subcutaneous tissue at the probe's entrance and exit sites was first anesthetized with a local injection (0.5-1.0 ml) of lidocaine plus epinephrine (20 mg/ml + 12.5 µg/ml). The probes were inserted in the muscle via a 14-gauge (Venflon; IV) cannula in a direction parallel to muscle fiber orientation (i.e., 45° moving proximally and laterally). The distance between the entrance and exit sites of the probes was ~9 cm, and the distance between each probe was ~2-3 cm. After insertion, the microdialysis probes were attached to a perfusion pump (CMA model 102) and perfused at a rate of 5 µl/min with a Ringer solution (K⁺ = 4.0 meq/l, pH = 7.3, phosphate = 0 mM). In an effort to minimize the possibility of draining the interstitial space (5, 22), the perfusate contained 3.0 mM glucose and 0.5 mM lactate. The outflow tube of one microdialysis probe was attached to a flow-through K⁺ microelectrode, whereas another probe was attached to a flow-through pH microelectrode (Microelectrodes, Londonderry, NH). There was a 6-min delay between the passage of the perfusate through the microdialysis probe and detection by the microelectrodes. However, this time delay was taken into consideration when calculating the data, and thus all data are presented in real time. These microelectrodes were connected to an Orion pH meter with a separate channel for ionic determinations, which allowed the manual recording of K⁺ and pH. The probes were perfused, and the subjects rested supine for 60 min before the experiment was initiated.

Determination of probe recovery. To fully utilize the microdialysis technique, an estimate of the in vivo extraction fraction of the compound being measured from the interstitial space needs to be made (defined as probe recovery). This determination is necessary to calculate actual interstitial concentrations and to document any changes in probe recovery associated with muscle contraction, since previous studies have shown that probe recovery can vary dramatically in response to varying workloads (11, 24, 30). In the present study, the "internal reference" method introduced by Scheller and Kolb (35) was used. With this method, a small amount of radioactive tracer, in the form of the compound being investigated, was added to the perfusate. It has been suggested that the relative loss of the isotope from the perfusate into the interstitial space represents probe recovery for that compound. This was confirmed in vitro by Kurosawa et al. (21) who proved that simultaneous measurement of tracer loss and compound recovery were similar. The major advantage of this method is that probe recovery can be determined for each collected sample, allowing the continual monitoring of probe recovery over time. Furthermore, this method is well suited for experiments in which steady-state conditions change, such as during exercise, or any other situation in which probe recovery may be affected. Therefore, in the present study, a very small amount of L-[U-¹⁴C]lactate (<0.2 µCi/ml) was added to the final perfusion solution as the internal reference marker for the determination of probe recovery.

Intermittent static quadriceps muscle contraction. To perform intermittent static muscle contractions, a specially designed quadriceps exercise device was used (Fig. 2). The subjects were placed supine on a padded table, and the hip and knee were flexed at angles of 45 and 90°, respectively, using an adjustable, padded triangular wooden support. A universal flat load cell (Strainsert, West Conshohocken, PA) was mounted directly beneath the subject's left ankle and was attached with nylon strapping. The load cell was calibrated before each study using 22.7- and 45.5-kg weights. Before the insertion of the microdialysis probes, each subject performed three maximum voluntary contractions (MVC) using the quadriceps exercise device.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the exercise apparatus used by the subjects to perform intermittent static quadriceps exercise. Microdialysis of the vastus lateralis muscle was performed on the left leg while simultaneous measurements of muscle sympathetic nerve activity (MSNA) were obtained from the right leg.

Experimental procedures. Sixty minutes after the insertion of the microdialysis probes, resting data collection was conducted. This length of time was allowed as it has been shown that, after microdialysis probe insertion, some cellular disruption occurs that transiently elevates the interstitial concentrations of several metabolites, including ATP (4). However, it has also been noted that the elevation in ATP declined to basal levels after only 30 min. Despite this observation, a 60-min equilibration period is used before the experiment is initiated to ensure that the external environment surrounding the probe has returned to normal. The subjects then statically contracted their quadriceps muscle at 25% of their largest predetermined MVC for 20 s followed by 5 s of relaxation. Exercise intensity was kept constant by visual feedback to the subjects from a meter measuring the force of the contraction. A total of 12 contractions (5 min) were performed, and the cue to contract/relax was relayed to the subjects via a continuously looping tape recording. The rationale for selecting this protocol was that an exercise intensity was needed that was high enough to elicit an increase in MSNA and an exercise duration that was long enough to provide the volume of dialysate necessary to analyze all the compounds under investigation. The present exercise paradigm satisfied both of these criteria without fatiguing the subjects before the end of the 5-min exercise period (for most subjects, 5 min was close to the maximum duration they could maintain at this exercise intensity). During exercise, blood pressure, heart rate, and MSNA were continuously recorded, whereas K⁺ and pH values were manually recorded every minute. Dialysate was collected over the entire contraction/relaxation period (5 min). The exercise bout was followed by a 5-min recovery period in which all of the above-mentioned variables were collected as previously described.

Analyses. Heart rate and blood pressure were determined for each minute during the experiment while the peak increase in MSNA during contraction was also determined. Meanwhile, pH and K⁺ concentrations were recorded every minute during the experiment by manually recording the digital readout from the pH meter. The collected dialysate samples were analyzed for lactate and phosphate using luminometric procedures in combination with an NADH-dependent luciferase (49). This analytical method is 100-1,000 times more sensitive than traditional fluorometric assays and thus can be conducted on smaller sample sizes, which is critical for quantitative microdialysis. Thus both lactate and phosphate analyses were conducted on 5 µl of dialysate. Furthermore, 5 µl of dialysate were pipetted into a 5-ml scintillation vial, and 3 ml of scintillation fluid were added for the determination of the specific activity of L-[U-¹⁴C]lactate.

It should be noted that several in vitro and in vivo experiments were conducted in an effort to validate the use of the on-line pH electrode to determine dialysate pH. In the first set of experiments, microdialysis probes identical to the ones used in the present study were placed in an in vitro perfusion system in which the solution surrounding the probe could be rapidly changed. The probes were perfused at a rate of 5 µl/min (the same as that used in the present study), and the time course of change in pH and the percent recovery of H⁺ were determined when the external pH was rapidly changed from 7.0 to 6.0. It was observed that, when the external pH was altered, a rapid change in pH was detected, and 75% of this change occurred in ~20 s; stable readings occurred after 50 s. It was further determined that the in vitro recovery for H⁺ was ~95%. In the second set of experiments, interstitial pH and probe recovery were determined using the traditional no net flux method of probe calibration (22), and these values were compared with those obtained by the on-line electrode method employed in the present study. Briefly, five different pH solutions were perfused through the probes in random order for 60 min, and dialysate pH was determined at the end of each of the 60-min perfusion periods. With this method, probe recovery was determined as the slope of the regression line of the perfusate versus the dialysate minus the perfusate concentration (22). The actual interstitial concentration was then calculated as the x-intercept (i.e., point of no net flux). This analysis was performed using an animal model (6 probes in the triceps surae muscle of 3 cats), and it was determined that the interstitial pH was 6.9 ± 0.1 and probe recovery was 70 ± 5%. These above experiments are presented to illustrate the accuracy and reliability of the on-line pH microelectrodes used in the present study.

Calculations. Probe recovery based on the internal reference method was calculated as follows
where P_dpm and D_dpm represent the disintegrations per minute in the perfusate and dialysate, respectively, for lactate. The probe recoveries were then used to calculate the actual interstitial concentrations of lactate as follows
where D_c and P_c represent the dialysate and perfusate concentrations of lactate.

To date, there is not a feasible internal reference marker available for phosphate; however, the molecular weights of phosphate and lactate are approximately the same, and they are both anions released by muscle during contraction (41). Therefore, it is possible to use the probe recoveries for lactate to correct the changes in dialysate phosphate concentrations that are a result of shifts in probe recovery. Subsequently, all dialysate phosphate concentrations were corrected for changes in probe recovery from rest to contraction and from rest to recovery.

It should be noted that each subject did not have the same number of microdialysis probes inserted in his muscle (due to muscle size and orientation), and, as the experiment progressed, several probes ceased to function (due to breakage or collapsing of the nylon tubing due to shearing forces of the fascia during muscle contraction). Similarly, it was observed that the probe recovery for a number of microdialysis probes either steadily decreased during the experiment or were very low from the onset. These observations indicate that some other factors, such as the infiltration of lymphocytes, etc., were affecting proper probe function. These types of conditions exist for all microdialysis experiments, and the critical factor is identifying them and not "blindly" accepting the data from a microdialysis probe without setting definite criteria for acceptance. In the present study, strict criteria for acceptance were set before the experiments. The criteria were as follows: 1) a microdialysis probe must perfuse continuously throughout all of the experimental protocols, 2) flow through the microdialysis probe must be maintained at 5 µl/min, 3) the dialysate must be clear and uncontaminated, and 4) probe recovery must not be <20%. If any one of these criteria were not met then the data from that microdialysis probe were omitted, not only for that protocol but for the whole experiment. As a result of these above-discussed factors, the number of microdialysis probes used in generating the data varies between subjects. Therefore, the sample size for each measured variable is summarized in Table 1.

Statistics. The changes in heart rate, blood pressure, K⁺, pH, and H⁺ over time (i.e., rest vs. each minute during contraction and recovery) were analyzed using a one-way repeated-measures ANOVA, and, if significant differences were indicated, a Bonferroni post hoc test (Bonferroni correction factor for multiple comparisons) was used to determine where the significant differences occurred. Similarly, changes in MSNA, probe recovery, and interstitial metabolites (lactate, phosphate, and corrected phosphate) were analyzed using a one-way repeated-measures ANOVA comparing changes from rest to contraction and rest to recovery. If significant differences were indicated, a Bonferroni post hoc test was used to determine where the significant differences occurred. All values are expressed as means ± SE, and significance was accepted at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Heart rate, blood pressure, and MSNA. At the initiation of muscle contraction, heart rate and blood pressure were increased above (P < 0.05) resting levels and remained elevated throughout the contraction period (Fig. 3). The peak increases in heart rate and blood pressure were 13 ± 3 beats/min and 20 ± 2 mmHg, respectively, and occurred after 5 min of exercise for both variables. After the last muscle contraction, both heart rate and blood pressure quickly decreased and were not significantly different from resting values after 1 and 2 min of recovery, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Heart rate (A) and mean arterial pressure (B) during rest, exercise, and recovery in humans. The peak increase (P < 0.05) in heart rate and mean arterial pressure from rest to exercise was 13 ± 3 beats/min (bpm) and 20 ± 2 mmHg, respectively, and occurred after 5 min of exercise for both variables. * Significant difference from rest (P < 0.05).

In the present study, it was only possible to obtain peroneal nerve recordings on five of the seven subjects. At rest, MSNA expressed as burst per minute or total amplitude (arbitrary units/min) was 11.8 ± 2.9 and 77.4 ± 18.6, respectively. The peak increase in MSNA from rest to exercise was 31.4 ± 4.8 burst/min (P < 0.05) and 262.6 ± 57.8 arbitrary units/min (P < 0.05). A representative neurogram from one subject is presented in Fig. 4. It should be noted that the MSNA responses from the subjects in this study (n = 5) were included with those of three other subjects in which only MSNA (no microdialysis) was measured during muscle contraction, and the results are presented in the work by Herr et al. (13).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Representative neurogram from one subject during intermittent static quadriceps exercise in humans. The peak increase in MSNA frequency and total amplitude from rest to exercise was 31.4 ± 4.8 burst/min (P < 0.05) and 262.6 ± 57.8 arbitrary units/min (P < 0.05), respectively.

Interstitial metabolites. Microdialysis probe recovery for lactate was 37 ± 3.0, 36.4 ± 2.7, and 30.2 ± 2.7% during rest, exercise, and recovery, respectively. There were no significant differences in probe recovery between rest and exercise; however, a significant difference was found to exist between rest and recovery. Interstitial lactate concentrations were modestly, but significantly, increased from rest to exercise, and, during recovery, interstitial lactate levels were increased even further (P < 0.05). The change in interstitial lactate from rest to exercise and from rest to recovery was 0.5 ± 0.1 and 0.8 ± 0.2 mM (P < 0.05), respectively (Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Interstitial lactate (A), dialysate phosphate (B), and corrected dialysate phosphate (C) concentrations during rest, exercise, and recovery in humans. Interstitial lactate was increased during exercise and continued to increase during recovery, whereas both dialysate and corrected dialysate phosphate were increased during exercise but returned to baseline during recovery. Dialysate phosphate values were corrected for the very small shifts in probe recovery that occurred during the experiment as determined by [14C]lactate (see Calculations). * Significant difference from rest (P < 0.05).

The dialysate phosphate concentrations were significantly increased from rest to exercise but had returned to baseline after 5 min of recovery, with the change in interstitial phosphate from rest to exercise being 0.15 ± 0.04 mM (Fig. 5). As stated previously, the dialysate phosphate concentrations were corrected for the changes in probe recovery observed for lactate during the experiment. This was accomplished by adjusting all of the exercise and recovery dialysate phosphate values to the probe recovery values obtained at rest. With this correction, it was observed that the dialysate phosphate concentrations were significantly increased from rest to exercise (change being 0.18 ± 0.08 mM) but had returned to baseline values after 5 min of recovery.

The resting dialysate K⁺ concentration was 4.2 ± 0.1 meq/l, and, during exercise, the dialysate K⁺ concentration rapidly increased and was already significantly different from rest after 1 min of contraction. As exercise continued, dialysate K⁺ levels steadily rose and peaked (P < 0.05) after 5 min of muscle contraction, with the mean increase in K⁺ being 0.6 ± 0.1 meq/l (Fig. 6). After the conclusion of exercise, the dialysate K⁺ concentration(s) rapidly decreased and were not significantly different from resting values after 3 min of recovery. The resting dialysate pH value was 7.162 ± 0.023, and, during static quadriceps exercise, dialysate pH steadily rose and was significantly different from rest after 1 min and remained elevated (P < 0.05) throughout the exercise period (Fig. 6). The mean peak increase in dialysate pH occurred after 5 min of muscle contraction, rapidly decreased during recovery, and was not different from resting pH after 1 min of recovery. The dialysate pH values were converted to H⁺ concentrations, and these data are presented in Fig. 6. The resting dialysate H⁺ concentration was 69.4 ± 3.7 nM, and, during exercise, the dialysate H⁺ concentration continually decreased (P < 0.05), with a mean decline of 16.7 ± 3.8 nM occurring after 5 min of exercise (P < 0.05). After the cessation of muscle contraction, H⁺ concentrations increased and were not significantly different from resting values after 1 min of recovery.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Dialysate K+ (A), pH (B), and H+ (C) concentrations during rest, exercise, and recovery in humans. At the onset of exercise, dialysate K+ and pH concentrations steadily rose, whereas dialysate H+ levels continuously decreased. The peak increase (P < 0.05) in dialysate K+ was 0.6 ± 0.1 meq/l, and the mean decline (P < 0.05) in dialysate H+ was 16.7 ± 3.8 nM, both occurring after 5 min of exercise. * Significant difference from rest (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study represents the first time that interstitial pH, K⁺, lactate, and phosphate have been measured during intermittent static exercise in humans. The exercise paradigm was performed by extending the leg at the knee against a fixed load, resulting in a muscle contraction isolated predominantly to the quadriceps muscle mass (2). This type of exercise was selected, since the vastus lateralis muscle is sufficiently large enough to accommodate a number of different microdialysis probes and the exercise is isolated to a single muscle group. This approach allows a more accurate and complete assessment of interstitial metabolite responses to muscle contraction in humans. The major findings of this study were that interstitial K⁺, pH, phosphate, and lactate were increased (P < 0.05) during quadriceps exercise in conjunction with an elevation in MSNA, heart rate, and blood pressure. During recovery, all variables returned to baseline values except interstitial lactate, which remained elevated (P < 0.05) throughout the recovery period.

It is well known that when groups III and IV muscle afferents, which reside in the interstitium of skeletal muscle, are stimulated, they evoke a pressor reflex. These fibers respond to both chemical and/or mechanical stimulation (14, 18), and prior work by Hayward et al. (10) has demonstrated that the mechanically responsive group III fibers increase their discharge after fatiguing bouts of muscle contraction. Based on these data, the authors speculated that some metabolic by-product(s) of contraction may be responsible for sensitizing these thin-fiber muscle afferents.

A number of models and approaches have been used to determine the chemical substance(s) that can stimulate or sensitize interstitial afferents and contribute to the pressor reflex. Prior animal studies have relied on the following two general approaches to this problem: 1) examination of the muscle reflex or afferent responses to localized chemical infusions and 2) measurement of responses during muscle contraction as venous or whole muscle metabolites are measured (38). Meanwhile, previous human work has relied on five approaches. These include 1) measurements of venous metabolites during contraction as reflex responses are measured, 2) the use of nuclear magnetic resonance to determine the intracellular high-energy phosphate concentrations and pH during exercise as reflexes are followed, 3) the use of pharmacological and metabolic interventions to selectively alter metabolite concentrations as reflexes are followed, 4) the examination of reflex responses in subjects with inborn errors of metabolism, and 5) the measurement of reflex and metabolic responses during periods of postexercise circulatory arrest. It should be noted that a very high percentage of these prior human studies have also employed handgrip exercise.

These prior animal and human experiments have suggested that lactate, phosphate, K⁺, and/or H⁺ may stimulate muscle afferents, and therefore their release may contribute to the magnitude of the exercise pressor reflex (31-34, 38, 40, 43). However, as mentioned earlier, the afferent nerve endings are not located on the luminal side of small intramuscular vessels or intracellularly, but rather reside in the interstitium. Therefore, without interstitial measurements of these various substances, it is impossible to determine the physiological and relative importance of these various substances in contributing to the regulation of the exercise pressor reflex.

In the present study, MSNA, heart rate, and blood pressure were observed to increase in response to quadriceps exercise, indicating the stimulation of thin fiber muscle afferents and engagement of the exercise pressor reflex. One of the most investigated substances suggested to be important in evoking the exercise pressor reflex is lactic acid. These studies have included the infusion and injection of lactic acid into the arterial supply of cats (31, 38, 40) and the use of dichloroacetate to reduce muscle lactate responses to exercise (9, 38). More recently, researchers have examined MSNA responses to exercise in humans with McArdle's disease (myophosphorylase deficiency) and compared these with normal subjects (45). These studies have provided valuable information, and, despite some conflicting early findings, it is now generally accepted that lactate "per se" has little effect on stimulating the exercise pressor reflex directly; however, it is possible that it may play a role in sensitizing muscle afferents during contraction. In the present study, interstitial lactate levels increased 0.5 ± 0.1 and 0.8 ± 0.2 mM from rest to exercise and from rest to recovery, respectively. It was interesting that interstitial lactate increased further during recovery, a behavior previously observed for the cat triceps surae muscle after stimulation at 5 Hz (23). This observation that lactate concentrations rose during contraction and remained elevated during recovery is different from the temporal pattern observed for the reflex responses. These data further support the contention that it is unlikely that lactate, in the absence of muscle contraction, is responsible for evoking the muscle reflex response. However, as mentioned above, these data do not exclude the possibility that lactate may play a role in sensitizing mechanically sensitive afferents to the effects of muscle contraction (38).

An interesting finding in this study was that intermittent static quadriceps exercise resulted in an interstitial alkalosis compared with an acidosis that one would have predicted from prior nuclear magnetic resonance and venous effluent measurements (37, 43). However, the work of Stewart (42) has suggested that the pH of any given compartment is a dependent variable based on the concentration of the following three main groups of independent variables: 1) strong ions, 2) weak acids, and 3) carbon dioxide (42). It has been suggested that, in the interstitial space (an ultrafiltrate of plasma), the predominant determinant of H⁺ is the strong ion difference. Therefore, under these circumstances, if the rise in K⁺ is sufficiently large relative to that of lactate, then an alkalosis would be predicted. In the present study, the increase in K⁺ was ~0.1 mM greater than that observed for lactate. Subsequently, a drop in H⁺, or an increase in pH, would be necessary to maintain electrical neutrality. It should be noted that, although the changes in the interstitial space are consistent with the Stewart (42) approach to ionic homeostasis, further studies are needed in which all of the variables that influence strong ion difference are measured. These studies would allow us to further explore the mechanisms responsible for the regulation of interstitial H⁺ concentrations during exercise.

It has been suggested that diprotonated phosphate may play a role in evoking the exercise pressor reflex (40). Specifically, it has been demonstrated that femoral arterial injections of both monoprotonated (HPO²₄) and diprotonated (H₂PO₄) phosphate evoked a pressor response that was reflex in nature (40). However, the magnitude of this reflex was substantially greater for the H₂PO₄ compared with the HPO²₄ form. In the present study, we observed that the interstitial concentration of phosphate rose during contraction and returned to baseline during the recovery period. Furthermore, even when the changes in probe recovery due to muscular contraction were taken into account, a significant increase in interstitial phosphate during exercise was observed. This pattern more closely matches that of the pressor response and is what one would expect if this substance was directly stimulating muscle afferents.

It is well established that H₂PO₄ is a more potent stimulator of thin fiber muscle afferents than HPO²₄ (40), and the dissociation constant for the conversion of HPO²₄ to H₂PO₄ is ~6.8. In our experiments, we observed an alkalosis during contraction that would tend to reduce the H₂PO₄-to-HPO²₄ ratio. However, the magnitude of the alkalosis was quite modest and most likely did not have any major influence on this ratio. It should be noted that these data strongly suggest that, if phosphate was responsible for either evoking or contributing to the muscle reflex, then only a modest change in interstitial phosphate was necessary. Last, the authors realize that further studies using 1) isolated hindlimb perfusions with varying phosphate concentrations with simultaneous interstitial phosphate measurements and 2) improved methodologies for the measurement of phosphate such that determinations can be made more frequently than every 5 min are needed to both qualitatively and quantitatively determine the role that phosphate may play in evoking the exercise pressor reflex.

During exercise, K⁺ is released from working muscle, and its concentration is believed to rise in the muscle interstitium (15, 47). A number of investigators have therefore suggested that interstitial K⁺ may play a role in stimulating groups III and IV muscle afferents and evoking the exercise pressor reflex (33, 34, 44, 48). Prior animal work in which intra-arterial injections of KCl were administered in dogs suggests that the time pattern of change in K⁺ concentrations does not closely follow the time course of change in the pressor reflex (33, 34). These studies showed that K⁺ levels remained elevated for several minutes after administration, whereas heart rate, blood pressure, and groups III and IV muscle afferent discharge returned to baseline after a much shorter period of time (<30 s). These findings suggested to the authors that muscle afferents adapt to the increases in K⁺ concentrations relatively quickly, and subsequently K⁺ may be directly involved in the initiation, but not the sustaining, of the muscle reflex.

In the present study, dialysate K⁺ concentrations gradually increase over the 5-min exercise period, with the peak increase in K⁺ being 0.6 ± 0.1 meq/l and the largest increase occurring during the first minute. During recovery, dialysate K⁺ concentrations decreased and were not significantly different from resting values after 3 min of recovery. However, it should be noted that heart rate and blood pressure returned to baseline values after only 1 min of recovery. The authors believe that the rapid increase during the first minute of exercise followed by the gradual accumulation of K⁺ over the last 4 min of contraction may have helped to counteract the adaptive effects observed by Rybicki et al. (33, 34) in which muscle afferents were exposed to rapid and constantly elevated K⁺ levels. Furthermore, we believe that the pattern of change in the autonomic variables measured in this study and those for K⁺ are sufficiently similar that we cannot exclude a role for K⁺ in either directly stimulating chemically sensitive afferents or in sensitizing mechanoreceptors. Another factor that cannot be overlooked is the possibility that more than one of the putative stimulators of the exercise pressor reflex may be working concurrently during muscle contraction. Similarly, it is quite possible that one metabolite contributes more to the reflex early in the exercise bout while another contributes more toward the end. For example, it may be that K⁺ is responsible for primarily initiating the pressor reflex and, as exercise continues and phosphate begins to accumulate, then this metabolite contributes to sustaining the reflex. This latter point may help to explain the disassociation between elevated dialysate K⁺ levels during recovery and the return of heart rate and blood pressure to baseline.

In summary, this study represents the first time that interstitial concentrations of a number of putative stimulators of thin fiber muscle afferents have been simultaneously measured in human subjects. In addition, this study represents the first report in which time course changes in dialysate pH and K⁺ have been documented during intermittent static quadriceps exercise in humans. Our findings suggest that lactate and H⁺ are not likely to be directly involved in stimulating the exercise pressor reflex under the exercise conditions present in this study. In contrast, the time course change in K⁺ and the mean change in phosphate did follow the pattern of changes observed for MSNA, heart rate, and blood pressure, suggesting a role for these substances in stimulating the muscle reflex. However, because the reflex involves both mechanical and chemical components and because in this experiment we did not modulate interstitial concentrations, we cannot make definitive cause and effect statements regarding the relative importance of these substances in stimulating muscle afferents. Despite these limitations, the authors believe that this approach is an important first step in determining the relative role that these substances play in evoking the exercise pressor reflex in humans.