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J Appl Physiol 85: 1583-1592, 1998;
8750-7587/98 $5.00
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Vol. 85, Issue 4, 1583-1592, October 1998

SPECIAL COMMUNICATION
Effects of hindlimb contraction on pressor and muscle interstitial metabolite responses in the cat

Dave A. MacLean1,2, Kathryn F. LaNoue2, Kristen S. Gray1,3, and Lawrence I. Sinoway1,3

1 Section of Cardiology and 2 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey 17033; and 3 Lebanon Veterans Affairs Medical Center, Lebanon, Pennsylvania 17042

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We used the microdialysis technique to measure the interstitial concentration of several putative metabolic stimulants of the exercise pressor reflex during 3- and 5-Hz twitch contractions in the decerebrate cat. The peak increases in heart rate and mean arterial pressure during contraction were 20 ± 5 beats/min and 21 ± 8 mmHg and 27 ± 9 beats/min and 37 ± 12 mmHg for the 3- and 5-Hz stimulation protocols, respectively. All variables returned to baseline after 10 min of recovery. Interstitial lactate rose (P < 0.05) by 0.41 ± 0.15 and 0.56 ± 0.16 mM for the 3- and 5-Hz stimulation protocols, respectively, and were not statistically different from one another. Interstitial lactate levels remained above (P < 0.05) baseline during recovery in the 5-Hz group. Dialysate phosphate concentrations (corrected for shifts in probe recovery) rose with stimulation (P < 0.05) by 0.19 ± 0.08 and 0.11 ± 0.03 mM for the 3- and 5-Hz protocols. There were no differences between groups. The resting dialysate K+ concentrations for the 3- and 5-Hz conditions were 4.0 ± 0.1 and 3.9 ± 0.1 meq/l, respectively. During stimulation the dialysate K+ concentrations rose steadily for both conditions, and the increase from rest to stimulation (P < 0.05) was 0.57 ± 0.19 and 0.81 ± 0.06 meq/l for the 3- and 5-Hz conditions, respectively, with no differences between groups. Resting dialysate pH was 6.915 ± 0.055 and 6.981 ± 0.032 and rose to 7.013 (P < 0.05) and 7.053 (P < 0.05) for the 3- and 5-Hz conditions, respectively, and then became acidotic (6.905, P < 0.05) during recovery (5 Hz only). This study represents the first time simultaneous measurements of multiple skeletal muscle interstitial metabolites and pressor responses to twitch contractions have been made in the cat. These data suggest that interstitial K+ and phosphate, but not lactate and H+, may contribute to the stimulation of thin fiber muscle afferents during contraction.

microdialysis; potassium; pH; phosphate; lactate; decerebration

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

MUSCULAR CONTRACTION results in the elevation of heart rate, blood pressure, and ventilation, and this response is collectively known as the exercise pressor reflex (20, 21). This reflex plays a key role in the response of the cardiovascular system to exercise. It has been demonstrated that the afferent arm of this reflex is composed of group III and IV afferents, which reside in the interstitial space of the exercising muscle (3, 20, 21). Activation of these thin fiber afferents can result from mechanical deformation of their receptive fields (13, 16) and from stimulation by metabolites produced by the exercising muscle (14, 24, 30, 32, 38). More specifically, excitation of chemosensitive nerve endings has been observed in response to lactic acid (24, 30, 32), phosphate (32), K+ (27, 28, 38), and H+ (26, 36).

Although these previous studies have provided valuable information and guidance regarding the possible stimulators of thin fiber muscle afferents, several drawbacks are apparent. Foremost, actual interstitial concentrations have not been quantified in contracting hindlimb muscle. The direct determination of interstitial concentrations is important for a number of reasons. First, the group III and IV muscle afferents are located in the interstitium, and even the changes that occur in response to normal exercise are unknown. Second, by determining interstitial concentrations in conjunction with cardiovascular responses during exercise, it will be possible to examine the magnitude of change in the putative regulators and how closely changes in each of these regulators follow cardiovascular changes. The use of microdialysis and the findings from this study will also provide valuable information for use in the design of future studies. For example, by measuring interstitial metabolite concentrations in response to arterial infusions, it will be possible to determine whether metabolite concentration changes are evoked in the interstitium by the substances in question. This is particularly important, since many previous studies have used large, relatively unphysiological doses to examine the effects of such compounds as lactate and phosphate (24, 32). Subsequently, these findings can be compared with those obtained during normal muscle contraction (as in this study) to see whether the same magnitude of change and cardiovascular responses are observed.

The microdialysis technique was first described by Delgado et al. (5) and is based on the principle of simple diffusion through a semipermeable membrane. Briefly, a microdialysis probe is inserted into the target tissue and perfused with a physiological solution. As the perfusate passes through the probe, compounds in the perfusate as well as in the interstitial space can diffuse into and out of the probe. The dialysate is then collected and analyzed, and by making an in vivo calibration of the exchange fraction (probe recovery) across the probe membrane, actual interstitial concentrations can be calculated. Therefore, the aims of the present study were to 1) determine the magnitude of the pressor response associated with 3- and 5-Hz twitch stimulation, 2) simultaneously measure and quantify the interstitial concentration of several of the putative stimulators of the exercise pressor reflex, and 3) compare the changes in interstitial metabolites with the cardiovascular responses associated with exercise.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animal preparation. Experiments were conducted in 10 adult cats (4.4 ± 0.3 kg) of both genders (3 for control and 7 for experimental studies). The cats were preanesthetized with ketamine (25 mg/kg im). The cats were then anesthetized with isoflurane gas (Forane) once the trachea was cannulated and attached to a ventilator (model 683, Harvard). The ventilator was set with a tidal volume of 20 ml/stroke and a rate of 20-30 strokes/min. Blood gases were frequently checked throughout the experiment, and HCO-3 was infused and the ventilatory rate was altered to maintain an arterial pH of 7.35-7.45, a PCO2 of 30-40 Torr, and an HCO-3 concentration of 20-25 mmol/l. The right jugular vein and carotid artery were cannulated for the systemic infusion of drugs and the continuous measurement of blood pressure, respectively. Body temperature was maintained at 37-38°C by the use of heating pads and lamps.

Before the decerebration procedure, the left carotid artery was tied off and the cats were injected with dexamethasone (4 mg iv). This steroid helps prevent decerebration-induced brain stem edema (22). The cat's head was then fixed in a Kopf stereotaxic instrument, and the decerebration was performed as anesthesia was continued. The majority of the temporal and parietal plates was removed, and the dura was incised and reflected laterally. The two cortical hemispheres were removed by making a transverse incision just anterior to the superior colliculus and extending it ventrally to the mammillary bodies. The brain rostral to the incision was removed, and bleeding was controlled with cotton gauze (previously soaked in boiling saline), absorbable hemostat, and gentle application of manual pressure. The calvarium was then packed with moist gauze, and the skin covering the cranium was clamped shut. Once the decerebration was complete, the anesthetic agent was removed from the inhalation mixture (100% O2). It should be noted that decerebration is an important component of this work, inasmuch as it allows the examination of autonomic reflex responses without the confounding effects of anesthesia (11). In these experiments no decerebrate rigidity was observed, inasmuch as the red nucleus remained intact (23).

The sciatic nerve was carefully dissected out using a posterior approach, and all branches not directly innervating the triceps surae muscle were severed. The popliteal fossa was freed of all fat and nonessential tissue, and mineral oil was poured in to fill the fossa. The sciatic nerve was then placed on an isolated stimulating electrode. The Achilles tendon was cut, tied, and attached to a tension transducer (model FT10, Grass Instrument, Quincy, MA). The cat was placed in a Kopf spinal unit, and the hips were stabilized via bilateral hip spikes and the lower leg via ties around the ankle and knee. The skin over the triceps surae muscles on both hindlimbs was dissected away and prepared for microdialysis probe insertion. Before microdialysis probe insertion, motor threshold was determined by gradually increasing the voltage to the sciatic nerve until a muscle contraction was observed.

Microdialysis probes. The fibers used to construct the microdialysis probes were obtained from an artificial dialysis kidney (GFE18) that had a molecular weight cutoff of 3,000 (0.20 mm ID, 0.22 mm OD). Each end of a single fiber was inserted ~1 cm into a hollow nylon tube (0.50 mm ID, 0.63 mm OD) and glued. The actual probe length (distance between the 2 nylon tubes) was 4 cm. 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 a 3-cm piece was glued to the nylon tubing on one side of the probe and a 3-cm piece was glued to the nylon tubing on the other side. Thus the suture was glued only to the nylon tubing but spanned the distance of the probe. This modification allowed the microdialysis probes to function very well during muscle contraction paradigms and is illustrated in Fig. 1. It was further determined in vitro that the addition of the suture did not effect the diffusion of any of the measured parameters across the microdialysis membrane (data not shown).


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  		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 microdialysis probes to function during muscle contraction without fracturing.

Four microdialysis probes were inserted into the triceps surae muscle of each leg. The probes were inserted into the muscle via a 14-gauge (Venflon, IV) cannula in the direction parallel to muscle fiber orientation. After insertion the microdialysis probes were attached to a perfusion pump (model 102, CMA) and perfused at a rate of 5 µl/min with Ringer solution. In an effort to minimize the possibility of draining the interstitial space (18), the perfusate contained 3.0 mM glucose and 0.5 mM lactate and had a pH of 7.301 ± 0.062. The collection 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). 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 dialysate was collected in 100-µl microcentrifuge tubes, immediately sealed to prevent evaporation, and stored at -80°C until analysis.

Determination of probe recovery. To fully utilize the microdialysis technique, we estimated the in vivo extraction fraction of the compounds being measured in the interstitial space (probe recovery). By determining probe recovery, we were able to calculate actual interstitial concentrations and document any changes in probe recovery associated with muscle contraction. In the present study the "internal reference method" introduced by Scheller and Kolb (29) 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 in the study of Kurosawa et al. (17), where the simultaneous measurement of tracer loss and compound recovery proved to be 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 experimental conditions where probe recovery may change during the paradigm (such as during exercise). 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.

Experimental procedures. Resting data were collected 60 min after the insertion of the microdialysis probes. This length of time was allowed, inasmuch as microdialysis probe insertion results in some cellular disruption, and it has been shown in adipose tissue that interstitial ATP levels are transiently elevated (2). 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. Resting blood pressure, heart rate, K+, and pH values were recorded while dialysate was collected for biochemical analysis. The sciatic nerve was then stimulated, in random order, at 3 or 5 Hz (0.1-ms duration, 2 and 3 times motor threshold) for 5 min. Heart rate, blood pressure, K+, and pH were recorded throughout the 5-min stimulation period as well as during the 10-min recovery period. Dialysate was also collected throughout the stimulation and recovery periods. The same protocol was conducted on the contralateral leg following the procedures outlined above. In most preparations, several different stimulation protocols could be conducted on each triceps surae muscle group.

Control experiments. In three separate cats we performed additional experiments to ensure that the stimulation parameters used to evoke muscle contraction did not also directly stimulate group III afferents and thereby evoke a pressor response independent of muscle contraction. In these cats the surgical procedures outlined above were followed, except microdialysis probes were not inserted into the triceps surae muscle groups. The sciatic nerve was then stimulated at one, two, and three times motor threshold (5 Hz) for 60 s, with 15-min recovery periods between stimulation trials. This time period was selected, as peak pressor responses were normally observed during this time frame. Furthermore, a number of different trials were conducted in each leg, and thus a shorter time period was selected to prevent muscle fatigue from repeated stimulation. Heart rate and blood pressure were recorded at rest and during stimulation and recovery. The cats were then given an intravenous injection of vecuronium (0.1 mg/kg), and 15 min later the stimulation protocols were repeated and all variables were recorded. This drug completely paralyzes the animal, and thus electrical stimulation of the sciatic nerve does not result in any muscle tension development. The effects of vecuronium were allowed to dissipate (120 min), the nerve was restimulated, and tension development and pressor responses were recorded.

Analysis. Heart rate, blood pressure, and developed triceps surae muscle tension were continuously recorded (model TA4000 recorder, Gould, Valley View, OH), and the onset latency for an increase in blood pressure was determined for each stimulation protocol. The peak increase in blood pressure, heart rate, and tension during stimulation was also determined. Meanwhile, K+ and pH values were manually recorded during the experiment, and the peak increase in these variables during stimulation was determined; the data are presented as such. The collected dialysate samples were analyzed for lactate and phosphate using luminometric procedures in combination with an NADH-dependent luciferase (39). This analytic 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 lactate and phosphate analysis was conducted on a single 5-µl sample. 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 activity of L-[U-14C]lactate.

Calculations. Probe recovery based on the internal reference method was calculated as follows
recovery = (P<SUB>dpm</SUB> − D<SUB>dpm</SUB>)/P<SUB>dpm</SUB>
where Pdpm and Ddpm 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
interstitial = [(D<SUB>c</SUB> − P<SUB>c</SUB>)/recovery + P<SUB>c</SUB>]
where Dc and Pc represent, respectively, the dialysate and perfusate concentrations of lactate.

There is no reliable internal reference marker available for phosphate; however, the molecular weights of phosphate and lactate are approximately the same, and both are anions released by the muscle during contraction. Therefore, we used the probe recoveries for lactate to correct the changes in dialysate phosphate concentrations that resulted from contraction-related changes in probe recovery. Subsequently, all dialysate phosphate concentrations were corrected for changes in probe recovery from rest to stimulation and from rest to recovery.

Not every stimulation protocol was conducted on each muscle group. Furthermore, although four microdialysis probes were initially inserted into the triceps surae muscle of each leg, as the experiments progressed several probes ceased to function (because of breakage or collapse 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 steadily decreased during the experiment or was very low from the onset. These observations indicate that factors aside from muscle contraction (e.g., lymphocytes) were interfering with 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 a definite criterion for acceptance. In the present study, strict criteria for acceptance were set before the experiments as follows: 1) a microdialysis probe must perfuse continuously throughout 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 was not met, 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 individual cats, legs (triceps surae groups), and microdialysis probes used in generating the data varies between protocols. Therefore, the sample size for each measured variable is summarized in Table 1.

                              
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  		Table 1.   Sample size for each measured variable and stimulation frequency

Statistics. Changes in tension were analyzed using a two-way ANOVA testing for changes over time as well as comparisons between the two stimulation intensities (3 vs. 5 Hz). 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 heart rate, mean arterial pressure, probe recovery, and interstitial metabolites (lactate, phosphate, corrected phosphate, K+, pH, and H+) were analyzed using a two-way ANOVA comparing changes over time (rest vs. stimulation, rest vs. recovery, and stimulation vs. recovery) and comparing the two stimulation intensities (3 vs. 5 Hz). 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. Values are means ± SE, and significance was accepted at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Control experiments. Substantial pressor responses (heart rate and blood pressure) were observed during electrical stimulation of the sciatic nerve at two and three times motor threshold in all cats. Meanwhile, after the administration of vecuronium, a noticeable increase in blood pressure was observed in ~50% of the cats at three times motor threshold. In contrast, during muscular paralysis no noticeable increase in blood pressure was observed during stimulation at two times motor threshold. These data suggest that the direct stimulation of the sciatic nerve at three times motor threshold (0.1-ms duration) may result in an increase in blood pressure independent of muscular contraction. Therefore, to eliminate the possible confounding effects of this finding on data presentation and interpretation, only heart rate and blood pressure data from animals stimulated at two times motor threshold are presented.

Tension, heart rate, and blood pressure. At the initiation of stimulation, substantial tension development was observed for 3- and 5-Hz twitch protocols (Fig. 2). The maximum amount of tension developed was 1.4 ± 0.2 and 2.0 ± 0.2 kg for the 3- and 5-Hz stimulation protocols (P < 0.05), respectively. Furthermore, mean peak tension developed by 30 s for both stimulation frequencies, then declined steadily over the remainder of the 5-min stimulation period. At the end of the contraction period, tension had decreased ~43 and 60% compared with the peak tension developed for the 3- and 5-Hz conditions, respectively.


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  		Fig. 2.   Tension development during 3- and 5-Hz twitch contractions. Stimulation resulted in substantial tension development for both contraction protocols. * Significant difference from rest; dagger  significant difference from 3-Hz twitch protocol (P < 0.05).

Resting heart rate and mean arterial pressure were 192 ± 17 beats/min and 120 ± 8 mmHg and 174 ± 10 beats/min and 128 ± 10 mmHg for the 3- and 5-Hz conditions, respectively. During muscle contraction, heart rate was consistently increased throughout the stimulation period for both stimulation frequencies, with the peak increase in heart rate being 20 ± 5 and 27 ± 9 beats/min for the 3- and 5-Hz protocols, respectively (Fig. 3). Similarly, mean arterial pressure was consistently increased above rest for all cats studied for both stimulation intensities, with the peak increase being 21 ± 8 and 37 ± 12 mmHg for the 3- and 5-Hz stimulation protocols, respectively (Fig. 3). However, because of the statistical correction needed to compensate for multiple comparisons, the increase in heart rate and mean arterial pressure was significant only for the 5-Hz group. The onset latency for an unquestionable increase in mean arterial pressure was 4.0 ± 1.9 and 4.1 ± 2.1 s for the 3- and 5-Hz conditions, respectively, and no difference between stimulation frequencies was noted. All cardiovascular variables had returned to baseline after 10 min of recovery.


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  		Fig. 3.   Heart rate and mean arterial pressure (MAP) during rest, stimulation, and recovery for 3- and 5-Hz twitch contractions. Increase in heart rate from rest to stimulation was 20 ± 5 and 27 ± 9 beats/min for 3- and 5-Hz conditions, respectively. Increase in MAP from rest to stimulation was 21 ± 8 and 37 ± 12 mmHg for 3- and 5-Hz conditions, respectively. * Significant difference from rest (P < 0.05).

Interstitial concentrations. Microdialysis probe recovery for lactate was 41.0 ± 3.7, 43.3 ± 3.6, and 37.8 ± 3.7% during rest, stimulation, and recovery, respectively, for the 3-Hz stimulation condition. There were no significant differences between rest and stimulation or between rest and recovery; however, a significant difference was found between stimulation and recovery (P < 0.05). During the 5-Hz contraction, microdialysis probe recovery for lactate was 36.9 ± 1.4, 38.4 ± 1.6, and 33.6 ± 1.5% during rest, stimulation, and recovery, respectively. Although the shifts in probe recovery were small, a significant difference was observed between stimulation and recovery (P < 0.05). The interstitial lactate concentrations were modestly, but significantly, elevated from rest to stimulation for the 3- and 5-Hz conditions (Fig. 4). After the 3-Hz stimulation protocol, the interstitial lactate levels returned to baseline after 10 min of recovery. In contrast, the interstitial lactate concentrations remained elevated (P < 0.05) during recovery after the 5-Hz stimulation procedure. The change in interstitial lactate from rest to stimulation was 0.41 ± 0.15 and 0.56 ± 0.16 mM for the 3- and 5-Hz stimulation protocols, respectively, and was not different between groups. In contrast, the change in interstitial lactate from rest to recovery was 0.20 ± 0.13 and 0.52 ± 0.12 mM for the 3- and 5-Hz conditions, respectively, and was different (P < 0.05) between groups.


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  		Fig. 4.   Interstitial lactate concentrations as well as dialysate and corrected dialysate phosphate levels during rest, stimulation, and recovery for 3- and 5-Hz twitch contractions. 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.

The dialysate phosphate concentrations were significantly increased from rest to stimulation for the 3- and 5-Hz trials (Fig. 4). However, the dialysate phosphate levels had returned to baseline after 10 min of recovery, and no significant differences were observed between the 3- and 5-Hz stimulation protocols at any time point. As stated previously, the dialysate phosphate concentrations were corrected for the very slight changes in probe recovery observed for lactate during the experiment. This was accomplished by adjusting all the stimulation and recovery dialysate phosphate values to the probe recovery values obtained at rest. With this correction, it was observed that the dialysate phosphate values were significantly increased from rest to stimulation for both stimulation frequencies (Fig. 4). Dialysate phosphate concentrations increased 0.19 ± 0.08 and 0.11 ± 0.03 mM from rest to stimulation for the 3- and 5-Hz stimulation protocols, respectively, and were not different between groups.

The resting dialysate K+ concentrations were 4.0 ± 0.1 and 3.9 ± 0.1 meq/l for the 3- and 5-Hz twitch protocols, respectively. During stimulation the dialysate K+ concentrations steadily rose and peaked after 4-5 min of contraction at 4.5 ± 0.1 (P < 0.05) and 4.8 ± 0.2 (P < 0.05) meq/l for the 3- and 5-Hz conditions, respectively (Fig. 5). Meanwhile, during the 10-min recovery period the dialysate K+ concentrations continually decreased to baseline levels. There were no significant differences between stimulation frequencies in any of the dialysate K+ values.


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  		Fig. 5.   Dialysate K+ and H+ concentrations and pH at rest and during stimulation and recovery for 3- and 5-Hz twitch contractions. Stimulation resulted in a peak increase in dialysate K+ concentration and pH and a decrease in H+ concentration. However, during recovery, dialysate pH decreased and H+ concentration increased for the 5-Hz twitch condition. There were no differences between the stimulation conditions. * Significant difference from rest (P < 0.05).

The resting dialysate pH values were 6.915 ± 0.055 and 6.981 ± 0.032 for the 3- and 5-Hz stimulation intensities, respectively. During stimulation the dialysate pH values continually increased from rest for both stimulation protocols and peaked at 7.024 ± 0.058 (P < 0.05) and 7.072 ± 0.034 (P < 0.05) for the 3- and 5-Hz conditions, respectively (Fig. 5). During recovery, dialysate pH steadily decreased for both stimulation protocols and plateaued at 6.871 ± 0.051 and 6.899 ± 0.038 for the 3- and 5-Hz conditions, respectively. There were no differences between stimulation intensities in any of the pH determinations; however, pH was significantly lower during recovery than during rest for the 5-Hz group. The pH values were converted to H+ concentrations, and these data are presented in Fig. 5. Both stimulation protocols demonstrated a decrease in H+ concentration during muscle contraction (P < 0.05) followed by an increase in H+ concentration toward baseline during recovery. For the 5-Hz condition, H+ increased above baseline (P < 0.05) by the end of the 10-min recovery period.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The purpose of the present study was to use the microdialysis technique to measure the interstitial concentrations of several of the putative stimulators of the metabosensitive arm of the exercise pressor reflex during 3- and 5-Hz twitch contractions in the decerebrate cat. The major findings were that both stimulation frequencies resulted in consistent increases in heart rate and mean arterial pressure, which were found to be significant for the 5-Hz condition. Furthermore, the interstitial concentrations of lactate, phosphate, and K+, as well as pH, were elevated above resting values during the contraction period. Meanwhile, during recovery, all variables fell to baseline except lactate, which remained significantly elevated, and pH, which decreased significantly below baseline, for the 5-Hz condition. Microdialysis has been used during exercise to examine changes in interstitial fluid of skin (4), brain (9), and spinal cord (8). However, this study represents the first time that interstitial concentrations have been made in contracting cat hindlimb muscle in conjunction with the measurement of cardiovascular responses.

In most previous studies examining pressor responses, a short-duration static (tetanic) stimulation protocol was used to produce muscle contraction. However, in the present study a 3- and a 5-Hz twitch stimulation protocol was used for a duration of 5 min. It has been demonstrated that the pressor response evoked by a static contraction is greater than that evoked by a twitch contraction in the same animal (12), and this is one of the primary reasons for its consistent use. The rationale for selecting a contraction protocol different from that traditionally used was the need to customize the stimulation protocol to answer the experimental questions posed. For example, to determine interstitial concentrations, a certain dialysate volume was necessary; therefore, in the present study, >= 5 min were needed to collect sufficient volume for analysis. Similarly, a stimulation protocol was needed that would result in sufficient tension development to evoke a substantial pressor response (3) without completely fatiguing the muscle. Therefore, because a static stimulation protocol would result in significant muscle fatigue long before 5 min, a twitch protocol was selected. In the present study, both stimulation frequencies resulted in substantial tension development with only a moderate loss in tension after 5 min (43 and 60% for the 3- and 5-Hz conditions, respectively). Similarly, muscle contraction at 3 and 5 Hz resulted in consistent pressor responses for all cats studied, as reflected by elevations in heart rate and blood pressure. These data suggest that our choice of stimulation frequencies and duration was very successful in evoking the cardiovascular changes desired.

The exercise pressor reflex plays an important role in the cardiovascular response to muscle contraction. Several investigators have studied the effects of twitch contractions on pressor responses. Different effects (pressor vs. depressor) were manifested depending on the continual use of anesthetic, even after decerebration (12, 15). However, consistent pressor responses have been observed for unanesthetized, decerebrate cats in response to twitch contractions (12). In the present study, 5-Hz stimulation resulted in a 27 ± 9 beat/min increase in heart rate as well as a 37 ± 12 mmHg increase in blood pressure. In a study by Iwamoto and co-workers (12) using unanesthetized decerebrate cats, 5-Hz twitch stimulation at three times motor threshold (0.1-ms duration) produced a 9.6 ± 1.9 beat/min and a 13.0 ± 2 mmHg increase in heart rate and blood pressure, respectively. This increase was significant, but the magnitude of increase is approximately threefold smaller than that observed in our study. Although our data confirm these earlier findings, a number of possibilities explain the differences between the magnitude of responses observed. In the study by Iwamoto et al., stimulation duration was only 30 s and the mode of excitation was via ventral root stimulation. In contrast, our stimulation duration was 5 min, and muscle contraction was achieved by direct stimulation of the sciatic nerve. In the present study the peak increase in heart rate and blood pressure frequently occurred >30 s after the beginning of contractions. We believe that these findings, in conjunction with the different mode of excitation, most likely explain the differences between the two studies in the magnitude of the pressor responses.

It has been demonstrated that the afferent arm of the exercise pressor reflex is composed of group III and IV afferents, which reside in the interstitial space of the exercising muscle (3, 20, 21). Activation of these thin fiber afferents can result from the mechanical deformation of their receptive fields (13, 16) and from stimulation by metabolites produced by the exercising muscle (14, 24, 26, 30, 32, 36, 38). Specifically, sympathoexcitation has been observed in response to lactic acid (24, 30, 32), phosphate (32), and K+ (27, 28, 38) as well as in response to muscle acidification (26, 36). The approach to examining the effects of these compounds has included nuclear magnetic resonance studies of forearm exercise followed by postexercise muscle ischemia in humans (1, 31, 32), muscle contractions and pharmacological blockers in humans and cats (6, 25, 26), and direct injection of these substances into the arterial supply of muscle (24, 28, 32). Although these studies have provided valuable information regarding the potential effects of these compounds, a major drawback is that the interstitial concentrations have not been measured simultaneously. In the present report we used the microdialysis method (19) to simultaneously measure the interstitial concentrations of multiple metabolites during bouts of muscle contraction.

One of the first identified and most investigated compounds that has been suggested to be a muscle afferent stimulant is lactate. However, substantial controversy exists regarding the role of lactic acid during muscle contraction. In a series of human studies from this laboratory (6, 33), glycogen depletion and dichloroacetate (an attenuator of lactate production) infusion resulted in an attenuation of muscle metaboreceptor-mediated responses and muscle sympathetic nerve activity, respectively. Similarly, intravenous dichloroacetate infusion in cats reduced the response of muscle afferents to contraction by 31% compared with control (30). Meanwhile, in animal studies in which lactic acid was infused into the arterial supply of muscle, cardiovascular and group III and IV afferent discharge responses were increased in response to the infusions (24, 30, 32). However, there are several drawbacks to these injection studies. 1) The dose of the infused lactic acid is often supraphysiological in nature. 2) There is no way to determine how much, if any, of the injected lactic acid actually reaches the interstitial space. 3) Without knowing the normal interstitial concentration of the compound during muscle contraction, it is impossible to know whether the injected metabolite is capable of evoking muscle afferent stimulation at a concentration normally seen during exercise. 4) Without the ability to measure interstitial concentrations, it is difficult to speculate on the possible effects of lactic acid on the dissociation of other compounds.

Aside from the concerns discussed above, a number of additional issues limit enthusiasm for the concept that lactate is an important, direct muscle afferent stimulant. Prior work has demonstrated that the ability of lactate to increase muscle afferent discharge and evoke a pressor reflex is associated with the pH of the infused lactate. For example, when lactate was injected at pH values within the physiological range, it was ineffective in evoking a pressor response (24, 32). On the basis of these observations, it has been suggested that the different effects of lactate and lactic acid may be due to the ability of lactic acid to acidify the interstitial space and/or some other metabolic by-product of contraction (32). Furthermore, recent work by Vissing et al. (37) in individuals with McArdle's disease (myophosphorylase deficiency) showed that muscle sympathetic nerve activity, heart rate, and blood pressure responses to forearm static exercise were not reduced, despite the fact that lactate, as well as H+, in these subjects was significantly impaired.

In the present study the interstitial lactate concentration was increased 0.41 ± 0.15 and 0.56 ± 0.16 mM during contraction in the 3- and 5-Hz stimulation protocols, respectively. This was only a modest increase in interstitial lactate (because of the low stimulation frequencies) and thus most likely had little effect on the dissociation of other compounds. Furthermore, in the present study, muscle interstitial lactate levels remained elevated above rest during recovery for the 5-Hz condition, yet the pressor responses returned to baseline during this period. Therefore, these observations provide further evidence for the lack of a role for interstitial lactate in directly stimulating the exercise pressor reflex in cats.

Another substance that has been suggested to be a potent muscle afferent stimulant is phosphate (32). It has been demonstrated that femoral arterial injection of the mono (HPO2-4)- and diprotonated (H2PO-4) forms of phosphate stimulates a pressor response; however, the magnitude of increase in the pressor response was substantially greater for H2PO-4 than for HPO2-4 (32). During muscle contraction, intramuscular phosphocreatine is broken down to provide immediate energy, and since the dissociation constant for the conversion of HPO2-4 to H2PO-4 is ~6.8, the H2PO-4 concentration will increase relatively dramatically as the muscle acidifies and Pi is generated. If this phosphate is rapidly transported into the interstitial space, then phosphate would be a logical choice for stimulating muscle afferents and mediating local vasodilation (10).

In the present study, 3- and 5-Hz twitch stimulation resulted in a significant increase in interstitial phosphate compared with rest. Even when the changes in probe recovery due to muscular contraction were taken into account, the increase in interstitial phosphate still existed. These data suggest that, even at low stimulation frequencies, interstitial phosphate is elevated and could be contributing to the exercise-induced pressor response. Additionally, in contrast to lactate, phosphate rose during exercise and fell during recovery, which more clearly follows the heart rate and blood pressure responses to the twitch contraction paradigms. These data further suggest that if phosphate was evoking a pressor response, only a modest change in interstitial phosphate was needed. As mentioned earlier, H2PO-4 results in a greater pressor response than HPO2-4 (32), and the ratio of H2PO-4 to HPO2-4 is a function of pH. In the present study the shifts in pH were modest and thus most likely would not have had a substantial influence on this ratio.

It is well known that there are a release and a net loss of K+ from muscle during continuous exercise (34). As a result, it has been suggested that an accumulation of K+ in the interstitial space may lead to muscle afferent stimulation (7, 27, 28, 38). In a number of studies the intra-arterial injection of KCl resulted in reflex elevations in heart rate and blood pressure as well as stimulation of group III and IV muscle afferents (27, 28). However, an interesting observation from these studies was that the pressor reflex was relatively short (~20-25 s), whereas the increase in K+ concentration lasted several minutes. The authors suggested that muscle afferents adapt quickly to increases in K+ concentrations, which would explain their short-lived pressor response. They further concluded that, on the basis of their observations, K+ is most likely not responsible for the maintenance of the pressor reflex during static contractions.

In the present study, interstitial K+ concentrations were generally observed to gradually increase over the 5-min stimulation period for both contraction protocols. This is in contrast to the studies by Rybicki and co-workers (27, 28), in which interstitial K+ levels were rapidly increased by intra-arterial injection and then remained elevated for several minutes. It is probable that under normal dynamic exercise conditions there is a gradual accumulation of K+ in the interstitium, which may contribute to the stimulation of thin fiber muscle afferents and the exercise pressor reflex. Therefore, this gradual accumulation of K+ over time may have helped counteract the adaptive effects observed by Rybicki et al. when muscle afferents were exposed to constantly elevated K+ levels.

In a further study by Rybicki et al. (27), gracilis muscle of five dogs was stimulated at 40 Hz for 60 s and interstitial K+ was estimated by an ion-sensitive electrode placed on the surface of the muscle. The tetanic contraction resulted in a 4.3 ± 0.3 mM increase in interstitial K+. In the present study, 3- and 5-Hz twitch contraction for 5 min resulted in a 0.6 ± 0.2 and 0.8 ± 0.1 meq/l increase in interstitial K+, respectively. The difference in the magnitude of increase in interstitial K+ between the two studies is most likely the result of several factors: species differences, the intensity and duration of the stimulation protocol, and the method utilized to determine interstitial K+ (i.e., surface electrode vs. microdialysis). Regardless, both studies demonstrated an increase in extracellular K+ during muscle contraction, and further studies are needed using the microdialysis technique in conjunction with more intense stimulation protocols to compare results between studies.

An interesting finding in this study was that, at rest, interstitial pH was similar to intramuscular pH compared with blood pH, and muscle contraction at both stimulation frequencies resulted in an alkalosis, rather than an expected acidosis. It has been proposed that the H+ concentration in a physiological solution can be described by the equilibrium between three systems (termed the "Stewart" approach): 1) strong ions, 2) weak acids, and 3) carbon dioxide (35). In other words, within a given space, pH is a dependent variable. It has further been suggested that the H+ concentration in the interstitial space (an ultrafiltrate of plasma) is only influenced by changes in strong ion difference. In the present study, during stimulation the increase in interstitial K+ was greater than that of lactate (both stimulation frequencies), and therefore, in an effort to maintain electrical neutrality, H+ was decreased. During recovery from 3-Hz stimulation, all values returned to baseline. In contrast, during recovery from 5-Hz stimulation, interstitial lactate remained elevated while the other cations returned to baseline. As a result, interstitial H+ was elevated to maintain electrical neutrality. These data suggest that during 3- and 5-Hz twitch contractions, H+ does not play a role in stimulating the exercise pressor response. Although the concentration changes noted in the interstitial space are consistent and support the Stewart hypothesis, further studies are needed in which all the variables that influence strong ion difference are measured, to completely understand the mechanism of change in interstitial H+ during muscular contraction.

Finally, caution must be used when interpreting these data. In this report we were unable to dissociate between the effects due to stimulation of mechanically sensitive afferents and those due to metabolites. It may be possible that change in interstitial lactate acted primarily to sensitize mechanoreceptors (30). Therefore, it is clear that further studies are needed to differentiate between these variables (mechano vs. metabo) and more effectively examine the relationship between muscle contraction, interstitial metabolite concentrations, and the engagement of the exercise pressor reflex.

In summary, the interstitial concentration of lactate, phosphate, and K+ and interstitial pH were significantly elevated during muscle contraction, whereas interstitial lactate remained elevated and interstitial pH decreased during recovery. Our data suggest that interstitial lactate and H+ are not directly responsible for stimulation of the exercise pressor reflex during 3- and 5-Hz twitch contractions. However, phosphate and K+ did follow the changes in heart rate and mean arterial pressure during exercise and recovery. Despite this finding, the present study does not allow the authors to speculate on the relative importance of these two substances in stimulating muscle afferents. Additionally, it is possible that under contraction conditions there is an interaction between metabolites, and thus more than one may contribute to the stimulation of muscle afferents. Moreover, it is conceivable that one compound may contribute more to the stimulation of the reflex early in exercise and another may contribute to and/or help maintain elevated cardiovascular responses later in exercise.

The use of intramuscular microdialysis allowed us to determine the actual interstitial concentrations of several metabolically and potentially neurologically important compounds. This will allow us to conduct subsequent experiments in which these substances are infused at physiological concentrations while cardiovascular and interstitial responses are simultaneously measured. These subsequent experiments will provide a more appropriate assessment of the effects that various compounds may have on the stimulation of the muscle metaboreflex.

    ACKNOWLEDGEMENTS

The authors thank Dr. Ralph Lydic for assistance with the decerebration technique, Joanne Kirchmer for excellent technical support, Allen Kunselman for statistical expertise, and Jennie Stoner for excellent secretarial skills.

    FOOTNOTES

This work was supported by a Department of Veterans Affairs Merit Review Award (L. I. Sinoway), National Institute on Aging Grant R01 AG-12227 (L. I. Sinoway), and National Institutes of Health-sponsored General Clinical Research Center with Division of Research Resources Grant M01 RR-10732.

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

Address for reprint requests: D. A. MacLean, Section of Cardiology, MC H047, The Pennsylvania State University, The Milton S. Hershey Medical Center, PO Box 850, Hershey, PA 17033.

Received 4 February 1998; accepted in final form 9 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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