The role played by purinergic 2Y receptors in evoking the muscle chemoreflex is not well defined. To shed light on this issue, we compared the pressor responses with popliteal arterial injection of UTP (1 mg/kg), a selective P2Y agonist, with those to popliteal arterial injection of ATP (1 mg/kg), a P2X and P2Y agonist, and to α,β-methylene ATP (50 μg/kg), a selective P2X1 and P2X3 agonist, in decerebrate unanesthetized cats. We found that injection of ATP and α,β-methylene ATP increased mean arterial pressure by 19 ± 2 and 15 ± 4 mmHg, whereas UTP had no affect on arterial pressure. In addition, the pressor responses to injection of ATP and α,β-methylene ATP were abolished by section of the sciatic nerve, demonstrating that they were reflex in origin. We conclude that P2Y receptors on thin fiber muscle afferents play no role in evoking the muscle chemoreflex.
- groups III and IV muscle afferents
- autonomic nervous system
- neural control of circulation
- phrenic nerve activity
the exercise pressor reflex is manifested by increases in arterial pressure, heart rate, and breathing and is evoked by the contraction-induced stimulation of groups III and IV afferent endings in skeletal muscle (11). Recently, purinergic 2 (P2) receptors, which are stimulated by ATP, have been shown to play a significant role in the generation of the exercise pressor reflex (4). In contrast, purinergic 1 (P1) receptors, which are stimulated by adenosine, have been shown to play no role in eliciting this reflex (4). P2 receptors have been divided into two subtypes. The first, P2X, is a cation channel and the second, P2Y, is G protein coupled. Much of the evidence that P2 receptors play a role in evoking the exercise pressor reflex has come from studies in which it was attenuated by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (4), an agent that blocks P2, but does not block P1 receptors (9). Although PPADS antagonizes most P2X receptors, it also antagonizes P2Y1 receptors (9). PPADS may also be a weak antagonist to P2Y2, P2Y4, and P2Y6 receptors (9). Overall, interpretation as to which P2 receptor was blocked by PPADS may not be apparent when the antagonist is used in vivo.
At this point in time, highly selective antagonists for P2X or for P2Y receptors do not exist. Consequently, one must use another approach to shed light on the role played by P2 receptor subtypes in evoking the exercise pressor reflex. We, therefore, chose to stimulate P2 receptors with selective agonists injected into the arterial supply of the hindlimb skeletal muscles to see if their injection increased arterial pressure, heart rate, and phrenic nerve discharge, an index of ventilation. These cardiovascular and phrenic effects comprise the muscle chemoreflex (14) and are very similar to those attributed to the exercise pressor reflex, which is evoked by static contraction of hindlimb muscles.
All procedures were reviewed and approved by the Institutional Care and Use Committees of the Pennsylvania State University College of Medicine. Adult male cats (n = 12, 3.4 ± 0.3 kg, range: 3.1–4.0 kg) were initially anesthetized with a mixture of 5% halothane and oxygen. The right jugular vein and common carotid artery were cannulated for the delivery of drugs and fluids as well as for the measurement of arterial blood pressure, respectively. The carotid arterial catheter, whose tip was located in the descending thoracic aorta was connected to a pressure transducer (model P23 XL, Statham) to monitor blood pressure. Heart rate was calculated beat-to-beat from the arterial pressure pulse by a Gould Biotach amplifier. The trachea was cannulated, and the lungs were ventilated mechanically (Harvard Apparatus). Arterial blood gases and pH were measured by a blood gas analyzer (model ABL-700, Radiometer), and Pco2 and arterial pH were maintained within normal range by either adjusting ventilation or intravenous administration of sodium bicarbonate (8.5%). A temperature probe was passed through the mouth to the stomach. Temperature was continuously monitored throughout each experiment and maintained at 37–38°C by a water-perfused heating pad.
The left common iliac artery and vein were isolated, and snares were placed around these vessels to trap the injectate in the leg (see below). The left triceps surae muscles and sciatic nerve were isolated; the latter so it could be sectioned if a pressor response to injection was found (see below). Before the start of the experiment, all visible branches of the left sciatic nerve were cut except for that supplying the triceps surae muscles. After the left popliteal artery was isolated for the injection of purines and capsaicin, the cat was placed in a Kopf stereotaxic and spinal unit. While the lungs were ventilated with the halothane-oxygen mixture, we decerebrated the cat at the midcollicular level. All neural tissue rostral to the plane of section was removed, and the cranial vault was filled with agar. Next, the halothane oxygen mixture was slowly withdrawn over a 30-min period until the cat's lungs were ventilated with room air. We used a decerebrate preparation instead of an anesthetized one because the former yields a larger exercise pressor reflex than does the latter (7). We reasoned that this would also be the case for the muscle chemoreflex.
The left phrenic nerve was exposed using a lateral cervical approach. The nerve was cut and its central end was draped over a bipolar hook electrode and covered with petroleum jelly to prevent desiccation. The electrode was attached in series with a high-impedance probe (model HIP 511, Grass) and then amplified (model P511, Grass). Phrenic nerve activity (PNA) was displayed on a storage oscilloscope (Hewlett-Packard) and made audible. The amplifier was filtered between 100 Hz and 3 kHz.
We examined the arterial pressure, heart rate, and ventilatory responses to injecting ATP (1,000 μg/kg), UTP (5, 20, 50, 200 and 1,000 μg/kg), α,β-methylene ATP (50 μg/kg), and capsaicin (2 μg) into the left popliteal artery of decerebrate, unanesthetized cats. The molecular weights of ATP, UTP, and α,β-methylene ATP are 551.1, 484.1, and 505.5, respectively. Five minutes prior to injections, we paralyzed the cat with rocuronium bromide (2–4 mg iv). The injection volumes ranged from 0.3 to 1.0 ml and required 5–10 s to complete. Each of the purines was dissolved in saline. The largest doses of ATP and UTP (i.e., 1,000 μg/kg) were injected in a concentration of 5 mg/ml. The pHs of the two solutions were 3.65 for ATP and 4.76 for UTP. Previously, we showed that injection of 1 ml of HCl with pHs as low as 2.46 into the arterial supply of the triceps surae muscles of cats had no effect on either mean arterial pressure, heart rate, or ventilation (13). Capsaicin was placed in solution as previously described (8). The doses of UTP were injected in ascending order, and the interval between injections was 20 min. Not every dose of UTP was given to every cat. The lowest three doses of UTP were selected because they are similar to those of α,β-methylene ATP shown previously to evoke reflex pressor responses in decerebrate unanesthetized cats (3). The higher two doses of UTP were selected because they are in the range of those of ATP shown previously to evoke the reflex pressor response in decerebrate unanesthetized cats (10). To minimize circulation of the injectate to other vascular beds, such as those in the lungs as well as the carotid and the aortic bodies, we occluded the left common iliac artery and vein as well as the popliteal artery and vein. We used UTP because it is a selective agonist for P2Y2 and P2Y4 receptors (19), which are the predominate P2Y receptors found on feline dorsal root ganglion cells (15). UTP does not stimulate P2X receptors (19). We used α,β-methylene ATP because it is a selective P2X1- and P2X3-receptor agonist (1), of which the latter is widely found on feline dorsal root ganglion cells (15). Moreover, α,β-methylene ATP has been demonstrated previously to evoke reflex pressor responses in unanesthetized decerebrate cats (3, 10). Finally, we used capsaicin because it is well established to evoke the muscle chemoreflex (2), and its use allowed us to demonstrate the reactivity of our preparation.
Baseline mean arterial blood pressure and heart rate were measured immediately before a maneuver, and peak mean arterial blood pressure and heart rate were measured within the 30-s period immediately following injection of each agent. Phrenic nerve activity was smoothed, rectified, and then integrated. Thirty second values were used to compare the differences between baseline and the response to each maneuver. Statistical comparisons were performed with two-way repeated-measures ANOVA. If significant main effects were found with an ANOVA, post hoc tests were performed with the Holm-Sidak method to determine significant differences between individual means. The criterion for statistical significance was P < 0.05. All values are expressed as means ± SE.
We found that popliteal arterial injection of ATP (1 mg/kg), α,β-methylene ATP (50 μg/kg), and capsaicin (2 μg) increased mean arterial pressure (P < 0.05) by 19 ± 2, 15 ± 4, and 31 ± 5 mmHg, respectively (n = 6). These increases were abolished by cutting the sciatic nerve (Fig. 1). In contrast, popliteal arterial injection of UTP (1 mg/kg) had no effect on arterial pressure in any of the six cats tested (Figs. 1 and 2). Injection of ATP and capsaicin significantly increased heart rate (P < 0.05), effects that were also abolished by cutting the sciatic nerve. Injection of either α,β-methylene ATP (50 μg/kg) or UTP (1 mg/kg) did not change heart rate. ATP (1 mg/kg), but not capsaicin, UTP (1 mg/kg), or α,β-methylene ATP (50 μg/kg), significantly increased phrenic nerve activity (P < 0.05); this increase was also abolished by cutting the sciatic nerve. Finally, UTP injected into the popliteal artery in doses of 5, 20, 50, and 200 μg/kg had no effect on arterial pressure, heart rate, or phrenic nerve activity. However, in each of three cats, UTP (1 mg/kg), injected through the carotid arterial catheter into the thoracic aorta, decreased mean arterial pressure (from 120 ± 13 to 70 ± 7 mmHg) and heart rate (from 137 ± 21 to 73 ± 29 beats/min). Phrenic nerve discharge was not measured in these three cats.
Although eight subtypes of P2Y receptors are expressed on dorsal root ganglion cells of cats, the P2Y2 and P2Y4 proteins predominate (15). In fact, P2Y2 and P2Y4 receptors are expressed to a greater extent in the dorsal root ganglia of cats than are P2X3 receptors (15), which when stimulated by α,β-methylene ATP in our experiments evoked reflex pressor responses. Nevertheless, the failure of UTP to evoke a muscle chemoreflex might be attributed to the fact that this purine is susceptible to breakdown by ectonucleotidases, whereas α,β-methylene ATP is resistant to breakdown by this extracellular enzyme. To control for this possibility we injected ATP into the popliteal artery in a dose identical to that of the dose of UTP injected. We found that popliteal arterial injection of ATP, which is also susceptible to extracellular breakdown by ectonucleotidases, evoked a muscle chemoreflex, whereas injection of UTP did not.
In our experiments, popliteal arterial injections of ATP increased MAP, HR, and phrenic nerve discharge, whereas popliteal arterial injections of α,β-methylene ATP only increased MAP. Nevertheless, the differences in heart rate and phrenic nerve discharge evoked by the two substances were small and may have been caused by different levels of thin fiber muscle afferent stimulation. Comparison of the reflex effects of the two substances is not possible because ATP and α,β-methylene ATP were injected in different doses, had different selectivities for P2X3 receptors, and were degraded differently by ectonucleotidases.
Our finding that UTP did not evoke the muscle chemoreflex causes us to question the role played by P2Y receptors in evoking the exercise pressor reflex. Specifically, if P2Y2 and P2Y4 receptors on groups III and IV muscle afferents are stimulated by ATP release during muscular contraction, the stimulation of these receptors by an exogenous agonist should evoke a reflex pressor response. Clearly this was not found when UTP was injected into the popliteal artery in our experiments. Any interpretation of our finding that UTP failed to evoke the muscle chemoreflex must be limited to the doses used in our experiments. Nevertheless, the doses used in our experiments were relatively large and their effects, if any, would be expected to be revealed in our unanesthetized preparation. Finally, our finding that UTP evoked large decreases in arterial pressure when injected into the thoracic aorta shows that this P2Y receptor agonist was biologically active in our preparation. The origin of the responses to UTP injection remains to be investigated.
Controversy exists over whether P2Y receptors stimulate sensory nerves directly or potentiate their responses to other stimuli. For example, stimulation of P2Y receptors with UTP has been reported to stimulate directly cutaneous afferents in mice (18). Likewise, stimulation of P2Y receptors with UTP increased intracellular calcium concentrations and released CGRP from isolated DRG neurons in rats (17). In a contrasting study, UTP had no direct effect on either CGRP or substance P release from isolated rat DRG cells, but was found to potentiate the capsaicin-induced release of these neuropeptides (6). Similarly, UTP did not increase CGRP release from an in vitro preparation of the dura mater, but potentiated the proton-induced release of this neuropeptide (20). Our findings are the first to examine the effects of UTP in a non-rodent species and clearly indicate that the UTP did not evoke the muscle chemoreflex.
Immunohistochemical techniques have revealed that there are marked differences between the distribution of P2 receptor subtypes on the dorsal root ganglion cells of cats and rats. For example, the P2Y1 receptor is rarely found on cells in the feline DRG, whereas it is found frequently on cells in the rat DRG (15, 16). In addition, ∼33% of the dorsal root ganglion cells in the cat stain positive for P2X3 receptors, whereas ∼90% of the dorsal root ganglion cells in the rat stain positive for this receptor (15, 16). We note with interest that in both cats and rats, DRG cells staining positive for P2X3 almost never stain positive for neurofilament protein 300, a marker of myelinated axons (15, 16). This immunocytochemical finding fits well with the electrophysiological findings that P2 agonists stimulated only group IV and a few group III muscle afferents conducting impulses under 4 m/s (5, 12).
In summary, we found that UTP, in the doses injected, did not evoke the muscle chemoreflex in decerebrated unanesthetized cats. In contrast, ATP, α,β-methylene ATP, and capsaicin were found to evoke this reflex, demonstrating that our preparation was fully capable of generating this chemoreflex. Our findings support the hypothesis that ATP release by contracting skeletal muscle stimulates P2X receptors on groups III and IV muscle afferents to evoke part of the metabolic component of the exercise pressor reflex. Our findings do not support the hypothesis that P2Y receptors on these afferents are stimulated by ATP release to evoke this reflex. Nevertheless, our findings do not exclude the possibility that P2Y receptors on groups III and IV muscle afferents are sensitized by ATP release, thereby potentiating the exercise pressor reflex.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-30710.
We thank Jennifer Probst for her technical assistance.
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