INTRODUCTION

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THE MUSCLE REFLEX is a sympathetically mediated response to stimulation of group III and IV muscle afferents (4, 18). Activation of the reflex results in elevations in heart rate, blood pressure, and ventilation, as well as muscle sympathetic nerve activity (MSNA) (17, 18). This reflex is most often associated with exercise, where a number of different compounds associated with muscle contraction have been postulated to evoke this response. These include lactic acid (23, 29), phosphate (30), K⁺ (24, 25), and adenosine (5-7). Adenosine has attracted substantial attention, since it has also been identified to contribute to the regulation of peripheral vasodilation (2, 16). Thus its role in the regulation of blood flow makes it a favorable candidate for also activating the muscle reflex.

The thin-fiber muscle afferents responsible for activating the muscle reflex reside in the interstitial space of skeletal muscle (4, 18). Many of the previous reports identifying adenosine as a stimulator of these afferents have used intravascular injections of adenosine (6, 7). However, the half-life of adenosine in blood is very short (<1 s) (19), and it has been proposed that vascular endothelium functions as an impermeable metabolic barrier for adenosine (20). Therefore, without measuring actual interstitial adenosine concentrations, it is impossible to determine how much adenosine, if any, diffused into the interstitial space during intravascular adenosine administration studies. On the other hand, it is well known that adenosine is a potent stimulator of other arterial chemoreceptors (1), such as those that reside in the carotid body (33). Moreover, our laboratory recently demonstrated in humans that adenosine administered into the femoral artery elevates MSNA via stimulation of chemosensitive receptors other than muscle afferents (16). Therefore, strong evidence exists for the conclusion that exogenously administered adenosine does not activate the muscle reflex.

In contrast, some controversy still exists regarding whether endogenously produced adenosine may have the capacity to stimulate group III and IV muscle afferents. The rationale for this contention is that interstitial adenosine has been shown to be increased during exercise (10), when MSNA and the exercise pressor reflex are activated (27). However, a number of other substances such as lactic acid, phosphate, and K⁺, which have been postulated to contribute to the activation of the muscle reflex, are also elevated (14, 15). Therefore, it is impossible under these conditions to distinguish which putative stimulator of the muscle reflex is contributing to its activation.

To make conclusive observations regarding the potential effects of adenosine on the activation of the muscle reflex, it is necessary to measure actual interstitial adenosine concentrations along with simultaneous determinations of heart rate and blood pressure under conditions where just interstitial adenosine levels are elevated. Furthermore, it is equally important to elevate interstitial adenosine levels without the confounding influences of adenosine on central arterial chemoreceptors. One possible method for accomplishing these aims is to use an isolated perfused muscle preparation, where adenosine can be perfused into the muscle without the confounding influences of systemic spillover. Furthermore, microdialysis of the muscle will allow determination of interstitial adenosine levels in conjunction with simultaneous measurements of heart rate and blood pressure. Therefore, the purpose of the present study was to measure interstitial adenosine levels as well as heart rate and blood pressure responses during isolated perfusion of the cat triceps surae muscle group with control blood followed by blood containing 20 mM or 100 µM adenosine.

	METHODS

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Animal preparation. Experiments were conducted in six adult male cats (6.0 ± 0.2 kg) that were initially preanesthetized with ketamine (25 mg/kg im). The cats were then anesthetized with isoflurane (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 bicarbonate was infused and ventilatory rate was altered to maintain an arterial pH of 7.35-7.45, PCO₂ of 30-40 mmHg, and HCO₃ 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.

Microdialysis probes. The semipermeable fibers (Spectrum Laboratories, Laguna, CA) used to construct the microdialysis probes had a molecular weight cutoff of 13,000 (0.20 mm ID, 0.22 mm OD). Each end of a single fiber was inserted ~1 cm into a hollow polyamide tube (0.25 mm ID, 0.36 mm OD) and glued. The actual probe length (distance between the 2 polyamide tubes) was 4 cm.

Microdialysis probe insertion. Four microdialysis probes were inserted into the triceps surae muscle of each leg. The probes were inserted into the muscle via a 22-gauge 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 a Ringer solution. In an effort to minimize the possibility of draining the interstitial space (13), the perfusate contained 3.0 mM glucose and 0.5 mM lactate. The dialysate was collected in 250-µl microcentrifuge tubes and immediately sealed to prevent evaporation and stored at 80°C until analyzed.

Determination of probe recovery. To fully utilize the microdialysis technique, an in vivo estimate of the extraction fraction of the compound being measured in the interstitial space needs to be made, which is defined as "probe recovery." This determination is necessary to calculate actual interstitial concentrations and to document any possible changes in probe recovery during the course of the experiment. In the present study, the "internal reference" method introduced by Scheller and Kolb (26) was used. With this method, a small amount of radioactive tracer, in the form of the compound being investigated, was added to the microdialysis 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. (12), 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. Therefore, in the present study, a very small amount of [2-³H]adenosine (<0.2 µCi/ml) was added to the final microdialysis perfusion solution as the internal reference marker for the determination of probe recovery.

Blood perfusate. The blood perfusate was composed of washed bovine erythrocytes to give a final hematocrit of 39.1 ± 1.1%. The blood perfusate further contained filtered BSA fraction V (42.9 g/l of plasma) as well as ions to give a final concentration of K⁺ of 5.88 ± 0.03 mM, Na⁺ of 122 ± 3 mM, Ca²⁺ of 1.72 ± 0.04 mM, Cl of 115 ± 1 mM, and HCO₃ of 19.3 ± 0.2 mM. The blood perfusate was oxygenated before use, yielding a final composition of pH 7.36 ± 0.02, 36 ± 2 mmHg PCO₂, 446 ± 14 mmHg PO₂, 12.7 ± 0.4 g/dl Hb, and 103.0 ± 0.1% O₂ saturation. Four drums were each filled with 300 ml of blood, placed in a heated perfusion box (37°C), and continually rotated to ensure adequate mixing. Two of the drums were used for the perfusion of control blood (no additives), while the other two were used for the perfusion of blood containing 20 mM or 100 µM adenosine, respectively. To ensure minimal degradation of adenosine while in the blood, adenosine was added to the blood perfusate drums ~1 min before the perfusion of the triceps surae muscle group. The rationale for choosing these two adenosine concentrations was that interstitial adenosine concentrations have never been measured during exogenous adenosine administration; therefore, it was impossible to estimate the amount of adenosine that would diffuse from the blood into the interstitial space. Pilot studies revealed that hindlimb perfusion with 20 mM adenosine resulted in a supraphysiological increase in interstitial adenosine, while perfusion with 100 µM adenosine resulted in a more physiological increase in interstitial adenosine.

Experimental protocol. Ninety minutes after the insertion of the microdialysis probes, cannulation of the popliteal artery and vein of one leg was initiated. This length of time was allowed, as microdialysis probe insertion results in some cellular disruption, and it has been shown in adipose tissue that interstitial ATP levels are transiently elevated (3). However, it has also been noted that the elevation in ATP levels declined to basal levels after only 30 min. Despite this observation, a 90-min equilibration period was used before the experiment was initiated to ensure that the external environment surrounding the probes had returned to normal. The popliteal artery and vein were cannulated with 20- and 18-gauge Teflon catheters, respectively, and the arterial catheter was advanced so that the tip was ~0.5 cm from the flow probe. The vasculature was flushed with 3 ml of heparinized saline (10 IU/ml), and control blood perfusion was immediately initiated. The blood perfusion rate was adjusted until it matched that observed in the popliteal artery before cannulation (Table 1). The blood was perfused through the triceps surae muscle only once (single pass), and blood perfusion rate was confirmed by the timed collection of venous blood in a graduated cylinder.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the experimental protocol where the triceps surae muscle group was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 µM adenosine for 10 min.

Analysis. Heart rate and blood pressure were calculated during the last 15 s of each blood perfusion period. The collected dialysate samples were analyzed for adenosine using the method of Tullson et al. (32) and HPLC. 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 [2-³H]adenosine.

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 adenosine. The probe recoveries were then used to calculate the actual interstitial concentrations of adenosine as follows
where D_c and P_c represent the dialysate and perfusate concentrations of adenosine.

Statistics. Changes in heart rate, blood pressure, probe recovery, blood perfusion rate, and interstitial adenosine from control to adenosine blood perfusion were compared using a paired Student's t-test. Values are means ± SE, and significance was accepted at P < 0.05.

	RESULTS

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Stimulation of the muscle reflex. Bolus injections (1 ml) of 50 mM phosphate (NaH₂PO₄) and/or saturated KCl into the popliteal artery resulted in activation of the muscle reflex and substantial increases in blood pressure (Fig. 2). The delta increase (P < 0.05) in systolic and diastolic blood pressure after injection of 50 mM phosphate (NaH₂PO₄) was 31.3 ± 4.9 and 25.3 ± 4.6 mmHg, respectively. Similarly, the delta increase (P < 0.05) in systolic and diastolic blood pressure after injection of saturated KCl was 36.0 ± 7.7 and 29.8 ± 8.9 mmHg, respectively. This procedure was conducted before and during perfusion of the triceps surae muscle group with both 20 mM and 100 µM adenosine. Under these conditions, despite the presence of adenosine, a pressor reflex was still evoked by the bolus injections of phosphate and KCl. It should be noted that 1-ml bolus injections of saline were used as volume controls and did not result in any significant shifts in blood pressure or heart rate.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Real-time trace of 1-ml bolus injections of saline (control) as well as 50 mM phosphate (NaH2PO4) and saturated (sat) KCl into the popliteal artery of the cat. Phosphate (PO4) and KCl evoked stimulation of the muscle reflex, as shown by increases in blood pressure (BP). This procedure was conducted to ensure the viability of the preparation.

Systemic infusion of blood containing adenosine. The infusion of blood containing 20 mM adenosine into the systemic circulation of the cat via the right jugular vein resulted in an immediate and dramatic decrease in blood pressure (Fig. 3). On the other hand, the infusion of blood containing 100 µM adenosine into the systemic circulation via the right jugular vein resulted in a gradual increase in systolic blood pressure and, subsequently, mean arterial pressure (Fig. 3). The pressor effects evoked by the infusion of both adenosine blood solutions were maintained for as long as the adenosine blood solutions were infused.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Systemic infusion of blood containing 20 mM (A) and 100 µM (B) adenosine resulted in a dramatic decrease and gradual increase in blood pressure, respectively. Pressor effects evoked by infusion of both adenosine blood solutions were maintained for the duration of the infusion.

20 mM and 100 µM blood adenosine perfusions. Skeletal muscle interstitial adenosine concentrations were elevated (P < 0.05) ~2,500- and 3-fold after perfusion of the triceps surae muscle group with blood containing 20 mM and 100 µM adenosine, respectively (Fig. 4). Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure (Table 1, Figs. 5 and 6). Furthermore, review of the heart rate and blood pressure data revealed no significant differences in these variables before or after cannulation and perfusion with control blood.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Perfusion of the triceps surae muscle group with control blood and then with blood containing 20 mM (A) and 100 µM (B) adenosine resulted in ~2,500- and 3-fold elevations, respectively, in interstitial adenosine concentrations. *Significantly different from control, P < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Typical blood pressure traces comparing perfusion of control blood (A) with perfusion of blood containing 20 mM adenosine (B). No significant changes in heart rate or blood pressure were observed.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Typical blood pressure trace comparing perfusion of control blood (A) with perfusion of blood containing 100 µM adenosine (B). No significant changes in heart rate or blood pressure were observed.

	DISCUSSION

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This study represents the first time that skeletal muscle interstitial adenosine concentrations have been determined during exogenous adenosine administration in conjunction with simultaneous heart rate and blood pressure measurements. The major findings were that skeletal muscle interstitial adenosine levels were increased in response to intravascular adenosine perfusion. Furthermore, the greater the level of adenosine that was perfused through the triceps surae muscle group of the cat, the greater the increase in interstitial adenosine concentration. However, elevated muscle interstitial adenosine concentrations did not result in a corresponding increase in heart rate or blood pressure, suggesting that adenosine does not stimulate group III and IV muscle afferents.

In the present study a very unique experimental model was assembled to examine the possible role of adenosine in evoking the muscle reflex. These included decerebration, triceps surae muscle microdialysis, and vascular isolation and perfusion. It is evident that a substantial amount of surgery and instrumentation was necessary, and therefore it was also necessary to ensure that a viable muscle reflex was still intact before initiating the perfusion protocols. In the present study, 1-ml bolus injections of phosphate (NaH₂PO₄) and/or saturated KCl evoked the muscle reflex before and after adenosine blood perfusion. In addition, bolus injections of phosphate and/or KCl elicited a pressor response while 20 mM and 100 µM adenosine was being perfused into the triceps surae muscle group. These data suggest that, after surgery and experimental instrumentation, an intact and viable muscle reflex existed. Furthermore, the fact that a pressor response was evoked while interstitial adenosine levels were elevated strongly suggests that increased interstitial adenosine concentrations do not interfere with the normal function and activation of thin-fiber muscle afferents by other chemical stimuli.

One critical aspect of the model used in the present study was that all the vasculature not directly supplying or draining the triceps surae muscle group as well as surrounding muscles needed to be isolated and tied off. In addition, the femoral and iliac veins needed to be tied off to eliminate any possible drainage from the triceps surae muscle group arising from the collateral circulation. Subsequently, when the artificial blood solutions were perfused through the triceps surae muscle group, no spillover into the systemic circulation should occur. To confirm this, the 20 mM and 100 µM adenosine blood solutions were infused into the jugular vein of the cat after completion of the muscle perfusion protocols to document changes in heart rate and blood pressure associated with adenosine's interaction with arterial chemoreceptors. It was observed that the systemic infusion of 20 mM and 100 µM adenosine resulted in a decrease and an increase in blood pressure, respectively. In contrast, perfusion of the triceps surae muscle group with the two adenosine blood solutions resulted in no significant changes in heart rate or blood pressure. These findings support the contention that there was no spillover of the blood from the isolated perfused triceps surae muscle group into the systemic circulation, thereby eliminating the potential confounding effects of systemic adenosine interactions.

A number of key studies suggesting that adenosine stimulates the muscle reflex have been performed by Costa and Biaggioni (6, 7), who used intravascular injections of adenosine. In one set of studies, adenosine was injected into the brachial artery of humans, which resulted a doubling of MSNA. In a similar set of experiments, adenosine was injected distal to a pneumatic cuff inflated to 50 mmHg to arrest the venous circulation, and an increase in MSNA similar to that reported during static handgrip exercise was observed. Although these studies were informative, one of the major drawbacks was that the pneumatic cuff was only inflated to 50 mmHg and forearm blood flow was not monitored. Therefore, the authors could not conclusively exclude the possibility that the injected adenosine may have also stimulated arterial chemoreceptors. In an effort to overcome this limitation, MacLean et al. (16) injected adenosine into the femoral artery of humans distal to a blood pressure cuff inflated to 220 mmHg. When adenosine was injected under these conditions, no increase in MSNA was observed during the 40 s of cuff inflation. This duration of time was more than twice the onset latency period observed for adenosine injection alone. However, on cuff deflation, there was a very rapid increase in MSNA (onset latency of 9 s), which was similar in magnitude to that observed for adenosine injection alone. These data clearly demonstrate that adenosine injected into the femoral artery of humans does not stimulate thin-fiber muscle afferents, and it is most likely that arterial chemoreceptors mediated the sympathetic activation associated with exogenous adenosine administration.

Despite the strong in vivo human experimental evidence reported by MacLean et al. (16) and earlier work in animals by others (23, 31), a limitation to these studies is that interstitial adenosine concentrations have never been measured during exogenous adenosine administration. The terminal ends of group III and IV afferents reside in the interstitial space of skeletal muscle (4, 18). Therefore, without determining actual interstitial adenosine concentrations, it is impossible under these conditions to know how much adenosine, if any, diffuses into the interstitium. In fact, it has been suggested that the highly efficient nucleoside-uptake system of the endothelium provides an impermeable barrier for adenosine transport into the interstitium (20). In addition, the half-life of adenosine in blood is very short because of the rapid uptake of adenosine into red blood cells (19). In the present study, the increase in interstitial adenosine represents <3% of the total amount of adenosine perfused through the triceps surae muscle group. Furthermore, the concentrations of adenosine used in the present study were perfused for a total of 10 min, and thus the muscle was exposed to supraphysiological levels of adenosine. In contrast, no in vivo human study has used an intravascular injection of adenosine that approaches the levels used in the present study. These data illustrate the impermeable nature of the endothelium and the large dose of adenosine needed before a change in interstitial adenosine is observed. Therefore, in the previously described studies, it is most likely that a negligible amount of the administered adenosine ever reached the interstitial space.

In an effort to overcome the limitations imposed by the short half-life of adenosine, Rotto and Kaufman (23) conducted a series of experiments in which they injected 2-chloroadenosine into the femoral artery of cats and directly measured the activity of group III and IV muscle afferents (the receptive fields of which reside in the triceps surae muscle group). The advantage of this protocol is that 2-chloroadenosine is not taken up by red blood cells and, subsequently, has a much longer half-life. These researchers found that 2-chloroadenosine had only trivial effects of the discharge of group III (3 of 23 afferents) and group IV (4 of 23 afferents) muscle afferents, and their findings are in agreement with those in the present study. It has been speculated that the discrepancies between the findings in the cat and those reported by Costa and Biaggioni (7) in the human may be due to species differences. Although this is a possibility, the present findings, in conjunction with previous findings by Rotto and Kaufman as well our previous human observations (16), strongly suggest that exogenously administered adenosine does not stimulate thin-fiber muscle afferents in either species.

It has been proposed that endogenously produced adenosine may play a role in the activation of thin-fiber muscle afferents (7). The rationale for this hypothesis is that adenosine has been shown to be elevated in the interstitial space of exercising skeletal muscle (10) and that infusion of theophylline before handgrip exercise resulted in an attenuation of the MSNA response (7). However, theophylline is a broad, nonspecific adenosine receptor antagonist, and there is no evidence to suggest that it alters skeletal muscle interstitial adenosine concentrations or influences the activation of thin-fiber muscle afferents by other chemical stimuli. In fact, it may be that theophylline attenuates MSNA responses to exercise by interacting with arterial chemoreceptors known to be activated by adenosine (8, 9, 33). In addition, during exercise a number of other substances such as lactic acid, phosphate, and K⁺, which have been postulated to contribute to the activation of the muscle reflex, are also elevated (14, 15). Therefore, it is impossible under these conditions to determine which putative stimulator of the muscle reflex is contributing to its activation.

To overcome these limitations, the isolated perfused cat triceps surae muscle preparation was used. This preparation allows the manipulation of interstitial adenosine levels without altering the interstitial concentrations of other putative stimulators of the muscle reflex. It was observed that perfusion of the triceps surae muscle group with blood containing 20 mM and 100 µM adenosine significantly elevated the interstitial concentration of adenosine. However, there were no significant increases in heart rate or blood pressure. These data provide the first direct evidence that elevated interstitial adenosine concentrations do not stimulate the muscle reflex. In addition, these findings support our previous in vivo human experiments which showed that adenosine elevates MSNA via central, rather than peripheral, mechanisms (16).

Since interstitial adenosine levels have never been determined during exogenous adenosine infusion, it was difficult to estimate the amount of adenosine needed to result in a change in interstitial adenosine concentrations. Perfusion of the triceps surae muscle group with blood containing 20 mM adenosine resulted in a 2,500-fold increase in interstitial adenosine concentrations. Although this increase was unmistakable and clearly demonstrates that elevated interstitial adenosine levels do not activate the muscle reflex, the increase in interstitial adenosine was unphysiological in nature. Therefore, one of our goals was to find a concentration of adenosine to perfuse through the triceps surae muscle group that would result in a physiological increase in interstitial adenosine similar to that observed during exercise. In the present study, perfusion of the triceps surae muscle group with blood containing 100 µM adenosine resulted in a final interstitial adenosine concentration of 4.1 ± 1.2 µM. This magnitude of increase in interstitial adenosine is similar to that observed during maximal one-legged knee extension exercise (10), when one would expect the muscle reflex to be maximally activated. However, in the present study, there were no indications that this level of increase in interstitial adenosine resulted in the stimulation of thin-fiber muscle afferents. These data clearly demonstrate that supraphysiological as well as physiological increases in skeletal muscle interstitial adenosine concentrations do not activate the muscle reflex.

In summary, this study demonstrates that, during constant skeletal muscle adenosine perfusion conditions, <3% of the adenosine diffuses into the interstitial space. These data confirm the impermeable nature of the endothelium to adenosine transport and the subsequently large doses of adenosine that need to be presented to the muscle before increases in interstitial adenosine concentrations are observed. However, when interstitial adenosine levels were elevated, independent of any changes in other putative stimulators of the muscle reflex, no significant changes in heart rate or blood pressure were observed. These data demonstrate that elevated skeletal muscle interstitial adenosine concentrations do not active the muscle reflex. In addition, these findings support our previous in vivo human observations which demonstrated that adenosine elevates MSNA via stimulation of chemosensitive receptors other than muscle afferents (16).