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1Department of Medicine, Division of Cardiology, Pennsylvania State University College of Medicine, Hershey; and 2Lebanon Veterans Affairs Medical Center, Lebanon, Pennsylvania
Submitted 6 June 2005 ; accepted in final form 29 September 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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hydrogen ion; thin-fiber afferent nerve; muscle reflex
Lactic acid appears to contribute very little to the pressor response evoked by active skeletal muscle, because it is in the form of the lactate ion. Thus the search for the "source" of H+ ion has shifted to the diprotonated form of phosphate (H2PO4–). Static muscle contraction elevates interstitial phosphate concentrations (21). Compared with the monoprotonated form of phosphate (HPO42–), the diprotonated form (H2PO4–) evokes a large increase in blood pressure when injected into the arterial supply of the cat hindlimb (29). Thus H2PO4– has been suggested as a stimulant of muscle afferents. However, the receptors and mechanisms that activate muscle afferents through which H2PO4– mediates the pressor responses remain unknown.
The phosphate data mentioned above were obtained in human and cat studies. The muscle pressor reflex is similarly expressed in rats (30). Thus, in this report, we investigated the role played by H2PO4– in evoking a muscle-based pressor response in rats.
Purinergic P2X receptors, vanilloid type 1 receptor (VR1), and acid-sensing ion channels (ASIC) appear on unmyelinated and thinly myelinated afferent nerve fibers (1, 2, 20). Previous studies by our laboratory and others have shown that these receptors may play an important role in modulating the discharge of muscle afferent nerves, and thus their engagement may contribute to the magnitude of the pressor response seen with muscle contraction (7, 9, 17–19). 
,
-Methylene ATP and capsaicin increase discharge of group III and IV hindlimb muscle afferent fibers via stimulation of P2X and VR1 receptors, respectively (8, 12), and H+ stimulates ASIC (but not VR1) and evokes a pressor response (17).
In this report, we examine the role played by P2X, VR1, and ASIC in mediating the pressor response observed when H2PO4– (pH 6.0) is injected into the rat hindlimb. We hypothesized that H2PO4– stimulates VR1 and ASIC but not P2X. Our data support the hypothesis. Moreover, simultaneous VR1 and ASIC blockade with capsazepine and amiloride led to a reduction in the observed pressor response with H2PO4–, but the combined attenuation was less than the algebraic sum seen when the blockers were given alone. Finally, resiniferatoxin (RTX) pretreatment (a maneuver that destroys sensory fibers with VR1 receptors) markedly reduced but did not eliminate the pressor response seen with H2PO4–. A preliminary report of these findings has been reported (15).
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METHODS |
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General Methods
Animal surgical preparation. The rats were anesthetized by inhalation of an isoflurane-O2 mixture (2–5% isoflurane in 100% O2). An endotracheal tube was inserted and attached to a ventilator (model AWS, Hallowell EMC). Polyethylene (PE-50) catheters were inserted into an external jugular vein and the common carotid artery for drug administration and measurements of arterial blood pressure, respectively. A continuous infusion of physiological saline (0.1 ml/h) into the jugular vein was established by using a syringe pump (Medical Industries). This maintained fluid balance and basal blood pressure. The femoral arteries and arterial collaterals were carefully isolated in both hindlimbs. An incision was made in the femoral artery. A PE-10 catheter was inserted into the femoral artery for injections into the arterial blood supply of the hindlimb muscles of each leg, as previously described (17, 18). The skin covering the triceps surae muscle and femoral region was surgically separated from the muscle below. The femoral and sciatic nerves of both legs were isolated carefully so that they could be sectioned at the end of the study. The animals were artificially ventilated, and respiratory parameters were monitored by connecting a pneumotachograph (Fleisch) to a respiratory gas monitor (Datex-Ohmeda, Madison, WI) and maintained within normal ranges, as previously described (17, 18). Body temperature was continuously monitored with a rectal thermometer (series 400, Yellow Springs Instruments) and maintained at 37.5–38.5°C by a heating pad and external heat lamps.
Decerebration.
A detailed procedure has been described in our laboratory's previous report (17). A transverse section was made anterior to the superior colliculus and extending ventrally to the mamillary bodies. This approach afforded the opportunity to examine the effect of muscle afferent-mediated pressor responses without considering the confounding effects of anesthesia. Once the decerebration was complete, anesthesia was removed from the inhaled mixture. A recovery period of 
40 min after decerebration was employed to allow sufficient time for elimination of the effects of anesthesia gas from the preparation.
Measurements of cardiovascular activities. Arterial blood pressure was measured by connecting the carotid arterial catheter to a pressure transducer (model P23ID, Statham). Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 s. HR was determined from the arterial pressure pulse. All measured variables were continuously recorded on an eight-channel chart recorder (model TA 4000, Gould, Valley View, OH) and stored on an iMac computer that used the PowerLab system (ADInstruments, Castle Hill, Australia).
Drugs and Injected Solutions
As previously described (29), H2PO4– and HPO42– were made by mixing equimolar concentrations of NaH2PO4 and Na2HPO
and were buffered in 10 mM of HEPES (Aldrich) dissolved in 0.9% sodium chloride, respectively. The pH of H2PO4– and HPO42– was adjusted on the day when an experiment was performed.
Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate acid (PPADS), capsazepine, amiloride, and RTX were purchased from Sigma. PPADS was dissolved in saline before it was used. Capsazepine was dissolved in 20% Cremophor in distilled water to make a stock solution of 10 mg/ml. Amiloride was prepared in saline to make a stock solution of 1 mM. RTX was dissolved in 10% Tween 80 and 10% alcohol in normal saline. The appropriate concentrations described below in this report were adjusted by dissolving those stock solutions in saline when they were used.
The injection volume was 0.1–0.2 ml, and the duration of injection was 30 s. At least 20 min were allowed between successive injections, unless otherwise specified.
Experimental Protocol
The responses induced by 15, 50, and 86 mM of H2PO4– were examined. On the basis of the data, 86 mM of H2PO4– were chosen in the following studies. The same concentration of HPO42– (pH 7.5) was also injected as a control. In this report, vehicle control experiments were performed for each compound, including saline, HEPES, and Cremophor.
Study series 1: The effect of PPADS on the pressor response induced by H2PO4–. A previous report demonstrated that H2PO4– injected into the blood supply of the cat hindlimb evoked a pressor response (29). The purpose of this protocol was to examine whether this pressor response was attenuated by blocking P2X receptors on thin-fiber muscle afferents in the rat hindlimb (n = 12). After a postsurgical recovery period, H2PO4– (86 mM, pH 6.0) was injected. Next, PPADS (0.5, 1, and 2 mM) was injected into the arterial blood supply of the hindlimb muscles 5 min before injections of H2PO4–. Finally, recovery responses for H2PO4– were performed.
Study series 2: The effect of capsazepine on the pressor response induced by H2PO4–. H2PO4– was injected, and the pressor response was determined. Then, to examine if VR1 blockade attenuated the phosphate response, capsazepine (0.25, 0, 5, and 1 mg/kg) was injected 5 min before a repeated injection of H2PO4–. A recovery experiment was also examined. This study was performed in 22 decerebrate rats.
Study series 3: The effect of amiloride on the pressor response induced by H2PO4–. After the control condition, amiloride (6 µg/kg) was injected into the femoral artery 5 min before H2PO4– to examine whether ASIC blockade attenuated the H2PO4–-induced pressor response (n = 14). It is noted that the dose of amiloride used significantly blunted the pressor response induced by lactic acid in rats (17). Thus we believe that it is appropriate to choose this dose to examine if blockade of ASIC can blunt the pressor response induced by acid phosphate in this report.
Study series 4: The effect of combined pretreatment with capsazepine and amiloride on the H2PO4–-induced pressor response. As in previous parts of this study, H2PO4– was injected, and the pressor response was determined. Capsazepine and amiloride were injected 5 min before H2PO4–. It is noted that the antagonists were given in a random fashion. Then capsazepine and amiloride were injected 5 min before repeated injections of H2PO4–. There was a 2-min interval between intra-arterial injection of capsazepine and amiloride. Following each injection of H2PO4–, the same volume of saline was injected to wash out the injectate, and a period of at least 30 min was allowed before administration of the next drug. In addition, H2PO4– was injected after those series of injections to obtain recovery data. Finally, the reflex pressor response induced by arterial injection of H2PO4– was also examined after section of the sciatic nerves. This study was performed in 16 decerebrate rats.
Study series 5: Arterial injections of H2PO4– in rats after treatment with RTX to destroy afferent nerves containing VR1. The rats were injected with RTX (400 µg/kg intraperitoneal) to produce a prolonged desensitization of VR1 4–5 days after RTX injection (26). The control rats were injected with the vehicle for RTX. The animals were instrumented as previously described. A recovery period was allowed after surgery. H2PO4– was injected into the arterial supply of the hindlimb muscles of 11 decerebrate rats.
Based on prior work, a period of 5 min was chosen so that an antagonist had sufficient time to be delivered to the tissue before being eliminated. For example, a half-life for antagonists such as these (in plasma and smooth muscle tissue) is 
10–20 min (25, 31). Thus we believe that a 5-min time period between injections of antagonist and phosphate was appropriate.
Experimental Data Analysis
All measured variables were continuously recorded on an eight-channel chart recorder. These variables were also sampled by an iMac computer that was equipped with a PowerLab data-acquisition system. Computer-acquired data were used in post hoc analyses. Control values were determined by analysis of 30 s of the data immediately before arterial injection. The peak response of each variable was determined by the peak change from the control value.
Experimental data (MAP and HR) were analyzed statistically by using a one-way repeated-measures ANOVA. As appropriate, Tukey's post hoc analyses were utilized to determine differences between groups. Values are means ± SE. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed by using SPSS for Windows (version 11.5, SPSS, Chicago, IL).
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RESULTS |
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Baseline values for MAP and HR and their response to H2PO4– injections are presented in Table 1. H2PO4– (15, 50, and 86 mM) injected into the femoral artery significantly increased blood pressure by 1.3 ± 0.8, 9.3 ± 1.4, and 25.12 ± 6.23 mmHg in decerebrated rats, respectively. HR was not significantly affected by this injection. The same concentration of HPO42– injected into the artery did not alter blood pressure (n = 5). The MAP was 106.4 ± 6.2 mmHg before and 108.2 ± 8.6 mmHg after injection. In addition, vehicle for phosphate, HEPES, did not significantly alter blood pressure (n = 4). The MAP was 102.2 ± 4.5 mmHg before and 105.3 ± 7.2 mmHg after injection.
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Effects of Simultaneous VR1 and ASIC Blockade on H2PO4–-Induced Reflex Pressor Response
H2PO4– increased MAP by 32.5 ± 5.6 mmHg, whereas H2PO4– increased MAP by only 4.5 ± 2.2 mmHg after VR1 and ASIC blockade. Capsazepine and amiloride reduced the pressor response to H2PO4– by 87%. Of note, the blood pressure attenuation when both drugs were given was greater than the attenuation seen when the VR1 and ASIC blockers were given alone (87 vs. 60 and 52%, respectively, P < 0.05). These results are shown in Fig. 3.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Thin-fiber muscle afferents are stimulated by a variety of metabolic by-products of muscular contraction. Lactic acid, H+, phosphate, and ATP may all play a role in stimulating these afferent fibers (6–8, 19, 27–29). In this report, we examined the role phosphate plays in stimulating muscle nerves, causing blood pressure to rise. In a prior study, NMR methods were employed to measure muscle concentrations of H2PO4– and H+ during handgrip in humans (21). The results of these experiments suggested that H2PO4– but not H+ correlated with muscle sympathetic nerve activity during exercise. In this same paper, cat experiments showed that H2PO4– injected into the arterial blood supply of the hindlimb muscles evoked a larger increase in blood pressure than HPO42– (29). Additionally, the magnitude of the pressor response to phosphate seemed to parallel the H2PO4–/HPO42– dissociation curve. Recently, Hoheisel et al. (11) demonstrated that intramuscular injections of an acidic phosphate solution excited single-fiber group IV muscle afferents. The receptors mediating the effect of H2PO4– were not determined (11).
Potential Receptor Mechanism Mediating the H2PO4– Effect
The major findings in this report are that a pressor response elicited when H2PO4– was injected into the arterial blood supply of the muscles was significantly attenuated after VR1 and ASIC receptors were blocked with capsazepine and amiloride. By contrast, P2X blockade with PPADS did not attenuate phosphate-induced responses. The same concentration of PPADS attenuated the pressor response evoked by arterial injection of ,-methylene ATP into the hindlimb muscle (19). If acid phosphate had stimulated P2X receptor, PPADS should have blunted the phosphate-induced response.
P2X receptors are present on thin-fiber afferent nerves (2). There is accumulating evidence that ATP-mediated stimulation of P2X receptors plays an important role in evoking muscle reflex response and mediating the autonomic adjustments to exercise (7–9, 16, 18, 19). Published studies have demonstrated that skeletal muscle contraction leads to an increase in the ATP in the muscle interstitial space (10, 16, 24), which reflexly evokes a pressor response via stimulation of ATP-sensitive P2X receptor (but not P2Y receptor) on sensory afferents in the hindlimb muscle. The findings of this report would seem to exclude the possibility that part of the pressor response seen with P2X stimulation is due to a direct effect of inorganic phosphate.
VR1 appear preferentially on metabolite-sensitive thin-fiber sensory neurons (20). These receptors are located on afferents in a variety of tissues and mediate the effect of the vanilloid compound capsaicin. When thin-fiber muscle afferent nerves are stimulated by capsaicin, a muscle pressor reflex is initiated (17, 18). For example, when capsaicin is injected into the arterial supply of the dog hindlimb, blood pressure rises by 20%, and this effect is abolished by sectioning afferent nerves from the hindlimb musculature (4). Capsaicin stimulates 71% of group IV and 26% of group III dog hindlimb muscle afferent fibers (12). Of note, a recent report has suggested that intramuscular H2PO4– stimulated 56% of group IV muscle afferents that were capsaicin sensitive (11).
Interestingly, in the present report, VR1 and ASIC blockade reduced the H2PO4– response by 60 and 52%, respectively. Combined VR1 and ASIC blockade caused nearly complete blockade (87% reduction). It is important to note that prior work suggests that the effect of lactic acid and H+ is mediated via stimulation of ASIC (17), but not via engagement of skeletal muscle VR1 sensory afferents (Table 2).
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It is more difficult to understand the role played by the VR1 receptor. The VR1 receptor does not appear to play a role in the lactic acid response (Table 2). Thus the effect of H2PO4– on VR1 is not likely to be mediated by the delivery of H+ to the receptor, because, if this were the case, the lactic acid effect (as well as the H2PO4–) should have been blocked by the VR1 blocker capsazepine (17). Furthermore, it is unlikely that inorganic phosphate per se is mediating the observed response. If this were the case, then the monoprotonated form of phosphate should have evoked a pressor response. Thus we would speculate that activation of the VR1 by phosphate requires the simultaneous engagement of some linked receptor by the hydrogen ion. Perhaps phosphate engaged the VR1 as H+ activated a secondary linked membrane receptor such as the ASIC. Clearly, further work will be necessary to better understand this intriguing issue.
Limitations
Amiloride is a nonspecific ASIC blocker. ASIC subunits are expressed on thin-fiber afferent nerves (14). Unfortunately, aside from Psalmotoxin 1, which only blocks ASIC 1a (5), no specific pharmacological blockers of the family ASIC are presently available (13). Thus, until a group of ASIC-specific blockers becomes available, one must consider the possibility that the effects seen with amiloride could be due to blockade of another member of the epithelial Na channel/degenerin family of ion channels (13).
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GRANTS |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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,-Methylene ATP elicits a reflex pressor response arising from muscle in decerebrate cats. J Appl Physiol 93: 834–841, 2002.This article has been cited by other articles:
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  A. R. Light, R. W. Hughen, J. Zhang, J. Rainier, Z. Liu, and J. Lee Dorsal Root Ganglion Neurons Innervating Skeletal Muscle Respond to Physiological Combinations of Protons, ATP, and Lactate Mediated by ASIC, P2X, and TRPV1 J Neurophysiol, September 1, 2008; 100(3): 1184 - 1201. [Abstract] [Full Text] [PDF]
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 J. Xing, L. Sinoway, and J. Li Differential responses of sensory neurones innervating glycolytic and oxidative muscle to protons and capsaicin J. Physiol., July 1, 2008; 586(13): 3245 - 3252. [Abstract] [Full Text] [PDF]
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Z. Gao, S. Koba, L. Sinoway, and J. Li 20-HETE increases renal sympathetic nerve activity via activation of chemically and mechanically sensitive muscle afferents J. Physiol., May 15, 2008; 586(10): 2581 - 2591. [Abstract] [Full Text] [PDF]
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 R. C. Drew, M. P. D. Bell, and M. J. White Modulation of spontaneous baroreflex control of heart rate and indexes of vagal tone by passive calf muscle stretch during graded metaboreflex activation in humans J Appl Physiol, March 1, 2008; 104(3): 716 - 723. [Abstract] [Full Text] [PDF]
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S. G. Hayes, A. E. Kindig, and M. P. Kaufman Blockade of acid sensing ion channels attenuates the exercise pressor reflex in cats J. Physiol., June 15, 2007; 581(3): 1271 - 1282. [Abstract] [Full Text] [PDF]
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Z. Gao, J. D. Li, L. I. Sinoway, and J. Li Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response J Appl Physiol, June 1, 2007; 102(6): 2288 - 2293. [Abstract] [Full Text] [PDF]
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 Z. Gao, V. Kehoe, J. Xing, L. Sinoway, and J. Li Temperature modulates P2X receptor-mediated cardiovascular responses to muscle afferent activation Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1255 - H1261. [Abstract] [Full Text] [PDF]
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