Blocking the Transient Receptor Potential Vanilloid-1 Does Not

Blocking the transient receptor potential vanilloid-1 does not reduce the exercise pressor reflex in healthy rats Guillaume Ducrocq, Juan Estrada, Joyce Kim, Marc Kaufman

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Guillaume Ducrocq, Juan Estrada, Joyce Kim, Marc Kaufman. Blocking the transient receptor potential vanilloid-1 does not reduce the exercise pressor reflex in healthy rats. AJP - Regulatory, Integrative and Comparative Physiology, American Physiological Society, 2019, 317 (4), pp.576 - 587. 10.1152/ajpregu.00174.2019. hal-02969591

HAL Id: hal-02969591 https://hal.archives-ouvertes.fr/hal-02969591 Submitted on 16 Oct 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Am J Physiol Regul Integr Comp Physiol 317: R576–R587, 2019. First published July 31, 2019; doi:10.1152/ajpregu.00174.2019.

RESEARCH ARTICLE Neural Control

Blocking the transient receptor potential vanilloid-1 does not reduce the exercise pressor reﬂex in healthy rats

X Guillaume P. Ducrocq, Juan A. Estrada, X Joyce S. Kim, and Marc P. Kaufman Heart and Vascular Institute, Pennsylvania State University College of Medicine, Hershey, Pennsylvania Submitted 12 June 2019; accepted in ﬁnal form 26 July 2019

Ducrocq GP, Estrada JA, Kim JS, Kaufman MP. Blocking the 28). Group III and IV muscle afferents synapse in laminae I, transient receptor potential vanilloid-1 does not reduce the exercise II, and V of the dorsal horn of the spinal cord (5, 26) and pressor reflex in healthy rats. Am J Physiol Regul Integr Comp Physiol then project to brainstem to exert their sympathoexcitatory 317: R576–R587, 2019. First published July 31, 2019; doi:10.1152/ effects (17). ajpregu.00174.2019.—Controversy exists regarding the role played Recently, studies have identified the receptors that activate by transient receptor potential vanilloid-1 (TRPV1) in evoking the exercise pressor reflex. Here, we determine the role played by TRPV1 the group III–IV afferent fibers, whose contraction-induced in evoking this reflex while assessing possible confounding factors stimulation evokes the exercise pressor reflex. So far, there is arising from TRPV1 antagonists or from the vehicle in which they good agreement that purinergic 2X receptors (15, 42), acid- were dissolved. The exercise pressor reflex was evoked in decere- sensing ion channels (42), and transient receptor potential brated, anesthetized Sprague-Dawley rats by electrical stimulation of (TRP) ankyrin-1 (23) play important roles in evoking the the tibial nerve to contract the triceps surae muscles statically. This reflex. In contrast, conflicting results exist regarding the role procedure was repeated before and after injection of the TRPV1 played by TRP vanilloid-1 (TRPV1) in evoking the exercise blockers: capsazepine (100 ␮g/100 ␮L), ruthenium red (100 ␮g/100 ␮ ␮ ␮ pressor reflex (22, 29, 38, 45). TRPV1 is expressed in rat dorsal L), or iodoresiniferatoxin (IRTX; 1 g/100 L). We found that root ganglion cells innervating the hindlimb muscles (29); it is capsazepine decreased the exercise pressor reflex when the drug was Ͻ Ͼ dissolved in DMSO (Ϫ10 Ϯ 9 mmHg; P ϭ 0.015; n ϭ 7). However, activated by low pH ( 5.5) and heat ( 43°C) and by vanilloid similar reduction was found when DMSO alone was injected (Ϫ8 Ϯ 5 compounds, such as capsaicin (4, 31). The hypothesis that mmHg; P ϭ 0.023; n ϭ 5). Capsazepine, dissolved in ethanol (2 Ϯ 6 TRPV1 plays a role in evoking the exercise pressor reflex may mmHg; P ϭ 0.49; n ϭ 7), ruthenium red (Ϫ4 Ϯ 12 mmHg; P ϭ 0.41; have arisen from findings that the injection of capsaicin into the n ϭ 7), or IRTX (4 Ϯ 18 mmHg; P ϭ 0.56; n ϭ 7), did not arterial supply of hindlimb skeletal muscle evoked a strong significantly decrease the exercise pressor reflex. In addition, we reflex pressor response (19, 47), an effect that, in turn, was found that capsazepine and ruthenium red had “off-target” effects. blunted by injection of TRPV1 antagonists (22, 38, 45). Two Capsazepine decreased the pressor response evoked by intra-arterial findings, however, cast doubt on this hypothesis, namely, that injection of bradykinin (500 ng/kg; Ϫ12 Ϯ 13 mmHg; P ϭ 0.028; n ϭ ␣ ␤ ␮ Ϫ Ϯ ϭ muscle interstitial pH does not decrease below 6.9 (43) and that 9) and - -methylene ATP (10 g/kg; 7 8 mmHg; P 0.019; during exhaustive exercise muscle temperature, does not in- n ϭ 10), whereas ruthenium red decreased the ability of the muscle to produce and sustain force (Ϫ99 Ϯ 83 g; P ϭ 0.020; n ϭ 7). Our data crease above 39°C (12). Furthermore, skeletal muscle does not therefore suggest that TRPV1 does not play a role in evoking the produce vanilloid substances (8). exercise pressor reflex. Additionally, given their strong off-target The lack of consensus regarding the role played by TRPV1 effects, capsazepine and ruthenium red should not be used for study- in evoking the exercise pressor reflex might have been caused ing the role played by TRPV1 in evoking the exercise pressor reflex. by differences in animal species (i.e., cats vs. rats), animal health, TRPV1 antagonists, and/or the solvents used to dissolve blood pressure; exercise; rats; transient receptor potential vanilloid-1 the antagonists. For example, the findings showing that capsazepine or iodoresiniferatoxin (IRTX) decreased the pressor response to static contraction have not been accompanied by INTRODUCTION the proper vehicle controls (29, 38), raising the possibility that the reduced pressor reflex found following capsazepine or The exercise pressor reflex, in combination with sympatho- IRTX injections was caused by the vehicle alone. In addition, lysis, functions to increase arterial blood flow to contracting the effect of TRPV1 antagonists might have been misinter- muscles (1, 32, 36). The afferent arm of the reflex is composed preted because of the drugs acting on other receptors. For of group III and IV fibers (25) whose endings are found in the example, capsazepine, in addition to inhibiting TRPV1 effec- interstitial space of skeletal muscle, small vessels, or connec- tively, has been found to inhibit voltage-gated calcium chan- tive tissue (40, 46a). For the most part, the endings of group III nels in vitro (10). Likewise, ruthenium red, acting as a nonse- afferents respond to mechanical distortion of their receptive lective TRP antagonist (2), also inhibits TRP ankyrin-1 (18), fields (20, 28, 33), whereas the endings of group IV afferents receptors that, in turn, have been shown to play a role in respond to byproducts of muscle contraction (20, 21, 27, evoking the exercise pressor reflex (23). In addition, ruthenium red blocks ryanodine receptors (48), an effect that might have reduced the ability of the muscle to produce force by reducing Address for reprint requests and other correspondence: G. P. Ducrocq, Heart 2ϩ and Vascular Institute, Pennsylvania State Univ. College of Medicine, 500 the sarcoplasmic release of Ca . It is therefore possible that University Dr., C2810, Hershey, PA 17033 (e-mail: [email protected]). the injection of TRPV1 antagonists might have reduced the

R576 0363-6119/19 Copyright © 2019 the American Physiological Society http://www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS R577 exercise pressor reflex by mechanisms other than just TRPV1 Animal Characteristics, Wellness, and Sample Size inhibition. Experiments were conducted at constant room air temperature Therefore, the main purpose of the present study was to (21°C) on 91 male Sprague-Dawley rats (Charles River), weighing determine the role played by TRPV1 in evoking the exercise 300–500 g. pressor reflex in healthy rats. To block TRPV1, we injected Rats were housed within the central animal facility of the Penn- capsazepine, ruthenium red, or IRTX into the arterial supply of sylvania State University College of Medicine, with access to food the triceps surae muscles. To determine if the vehicles in which and water ad libitum, and were exposed to a 50:50 light/dark cycle. these TRPV1 antagonists were dissolved affected the exercise All attempts were made to minimize animal discomfort and pain. pressor reflex, we tested the effect of dissolving capsazepine in two different solvents, namely, DMSO and ethanol. Finally, to Surgical Procedures determine possible confounding factors of TRPV1 antagonism, At the beginning of every surgery, the animal was anesthetized by we measured the effect of capsazepine on the pressor response ␣ ␤ inhalation of 4% of isoflurane with oxygen. We started the surgical evoked by bradykinin, - -methylene ATP, and diprotonated procedure only when the corneal reflex stopped and when pinching phosphate, each of which is a metabolic byproduct of contrac- the hindpaw did not produce a withdrawal reflex. tion but does not directly activate TRPV1. We also determined With the rat in supine position, the neck area was opened to expose the effect of ruthenium red on the ability of the muscle to the trachea, which was cannulated with a 2-cm long 14G Teflon tube. produce and sustain force. We tested the following hypotheses Lungs were mechanically ventilated (model 683; Harvard Apparatus, that: 1) capsazepine dissolved in DMSO decreased the exercise Holliston, MA), and the amount of isoflurane was reduced to 2%. The pressor reflex, but this decrease was due to the DMSO alone; left and right common carotids were isolated from the vagus nerve and 2) capsazepine dissolved in ethanol decreased the exercise cannulated using RenaPulse High Fidelity Pressure Tubing (RPT040; Braintree Scientific, Braintree, MA) to record arterial blood pressure pressor reflex, but this decrease was confounded by the fact (P23XL; Gould-Statham Instruments, Los Angeles, CA) and to draw that capsazepine decreased the pressor responses to bradykinin, arterial blood samples, respectively. Similarly, the right jugular vein ␣-␤-methylene ATP, and diprotonated phosphate; 3) ruthe- was cannulated (RPT040) to inject drugs into the systemic circulation. nium red decreased the exercise pressor reflex, but this de- An incision was made on the ventral part of the left thigh to expose crease was confounded by the fact that the ability of the muscle the inguinal fat pad. Once the inguinal fat pad has been reflected to produce force was reduced; and 4) IRTX had no effect on upward to expose the junction of the femoral, superficial epigastric, the exercise pressor reflex. muscular branch, and saphenous arteries, the superficial epigastric artery was cannulated with the tip of the tubing (SUBL-140; Braintree Scientific) positioned just before the anastomosis with the femoral METHODS artery. This cannulation permits injection of solutions directly into the Ethical Approval arterial circulation of the hindlimb. A snare (2.0 silk suture) was placed around the femoral artery and vein, which when tightened, The Institutional Care and Use Committee of the Pennsylvania trapped the solution into the hindlimb circulation. Finally, the mus- State University College of Medicine approved all of the procedures. cular branch and saphenous arteries were ligated (4.0 or 5.0 silk The authors understand and conformed to the ethical guidelines of the suture) to increase the probability that any injected solution entered journal for animal use in research. the circulation of the triceps surae muscles.

Table 1. Peak pressor and cardioaccelerator responses to intra-arterial injection of chemicals, static contraction, or stretch of triceps surae muscles

Stimulus (Index) Baseline Peak 95% CI P Value Cohen’s d Capsaicin (n ϭ 35) MAP 132 Ϯ 24 176 Ϯ 29 [29 59] P Ͻ 0.001 d ϭ 1.67 HR 455 Ϯ 64 461 Ϯ 65 [3 6] P Ͻ 0.001 d ϭ 0.92 Bradykinin (n ϭ 15) MAP 116 Ϯ 31 138 Ϯ 32 [6 38] P Ͻ 0.001 d ϭ 1.37 HR 419 Ϯ 54 421 Ϯ 54 [1 4] P Ͻ 0.001 d ϭ 0.59 ␣-␤-Methylene ATP (n ϭ 10) MAP 127 Ϯ 39 150 Ϯ 38 [7 40] P Ͻ 0.001 d ϭ 2.16 HR 461 Ϯ 57 464 Ϯ 57 [1 6] P Ͻ 0.001 d ϭ 0.58 Diprotonated phosphate (n ϭ 10) MAP 127 Ϯ 25 166 Ϯ 37 [11 66] P Ͻ 0.001 d ϭ 1.68 HR 473 Ϯ 55 479 Ϯ 52 [2 10] P Ͻ 0.001 d ϭ 1.32 Static contraction (n ϭ 34) MAP 105 Ϯ 25 130 Ϯ 31 [17 35] P Ͻ 0.001 d ϭ 1.53 HR 386 Ϯ 42 396 Ϯ 40 [7 14] P Ͻ 0.001 d ϭ 0.94 BPI 2,999 Ϯ 757 3,328 Ϯ 856 [216 442] P Ͻ 0.001 d ϭ 1.11 Passive stretch (n ϭ 33) MAP 120 Ϯ 23 158 Ϯ 30 [37 51] P Ͻ 0.001 d ϭ 2.04 HR 394 Ϯ 41 401 Ϯ 39 [4 9] P Ͻ 0.001 d ϭ 0.76 BPI 3,474 Ϯ 699 3,989 Ϯ 738 [425 605] P Ͻ 0.001 d ϭ 1.46 Results are presented as means Ϯ SD; n ϭ sample size. Ninety-ﬁve percent conﬁdence interval (CI) is presented as the lower and upper boundary of the interval containing the true value of the effect of stimulus on the corresponding index. BPI, blood pressure index (in millimeters of mercury times s); HR, heart rate (in beats/minute); MAP, mean arterial blood pressure (in millimeters of mercury).

Capsazepine (in 10% ethanol ; n = 9) With the use of a blunted spatula, we decerebrated the rat by sectioning Ͻ1 mm rostral to the superior colliculus (9). The isoﬂurane P = 0.013; d = 0.96 P = 0.15; d = 0.50 100 25 was then discontinued, and the lungs were ventilated with room air. ) -1 Blood arterial PO2 (100–150 mmHg), PCO2 (35–40 mmHg), pH 80 20 Ϫ (7.35–7.45), and [HCO3 ] (22–26 mM) was regularly monitored and 60 15 kept within physiological range. Body temperature was maintained 40 10 around 37°C using a heating lamp. At the end of the experiment, the decerebrated rats were killed by intravenous injection of 3 mL of a

peak MAP (mmHg) 20 5

peak HR (beats.min supersaturated KCl solution into the systemic circulation. 0 0 Before After Capsazepine Before After Capsazepine Experimental Procedures Capsazepine (in 0.5% DMSO ; n = 7) Contraction of the triceps surae muscles. Baseline tension of the P = 0.038; d = 0.78 P = 0.088; d = 0.70 triceps surae muscles was set at 100 g. The motor threshold was then

70 ) 8 -1 determined by progressively increasing the current of a single pulse 60 6 (0.01 ms) applied to the tibial nerve until a muscle twitch was 50 observed. The stimulator output was then set at a current intensity that 40 4 evoked a twitch tension equal to 90% of the maximal twitch tension 30 (~1.60 times motor threshold). Given that ruthenium red inhibits 20 2 ryanodine channels in the muscle ﬁbers (48), we anticipated that peak peak MAP (mmHg) 10 peak HR (beats.min tension of the static contraction would be reduced after injecting the 0 0 Before After Capsazepine Before After Capsazepine IRTX (n = 7) Bradykinin (n = 9) P < 0.001; d = 1.91 P = 0.11; d = 0.53

60 ) 8 -1 P = 0.028; d = 0.84 P = 0.28; d = 0.34 50 40 8 6 ) -1 40 30 6 30 4 20 20 4 2 peak MAP (mmHg) 10 peak HR (beats.min

peak MAP (mmHg) 10 2 0 0 Before After IRTX Before After IRTX peak HR (beats.min 0 0 Before After Capsazepine Before After Capsazepine Ruthenium Red (n = 6) (in 10% ethanol) (in 10% ethanol)

P = 0.001; d = 1.89 P = 0.20; d = 0.85 α-β-Methylene ATP (n = 10) 6

70 ) -1 60 5 P = 0.019; d = 1.02 P = 0.85; d = 0.07 40 ) 12 50 4 -1 40 10 3 30 30 8 2 20 20 6

peak MAP (mmHg) 10 1 peak HR (beats.min 4 0 0

peak MAP (mmHg) 10

Before After Ruthenium Red Before After Ruthenium Red peak HR (beats.min 2 0 0 Fig. 1. Capsazepine, iodoresiniferatoxin (IRTX), and ruthenium red reduced Before After Capsazepine Before After Capsazepine the pressor response to intra-arterial injection of capsaicin. Individual (closed (in 10% ethanol) (in 10% ethanol) circles) or group (shaded bars) data for mean arterial blood pressure (MAP) or heart rate (HR) are presented as the peak difference (⌬) measured before vs. Diprotonated Phosphate (n = 10) after injecting capsaicin (0.5 ␮g/mL) into the superﬁcial epigastric artery. Transient receptor potential vanilloid-1 was inhibited by injecting 0.1 mL P = 0.66; d = 0.12 P = 0.038; d = 0.61 ␮ ␮ ␮ ␮ 80 12 capsazepine (100 g/100 L), ruthenium red (100 g/100 L), or IRTX (1 ) -1 ␮g/100 ␮L) into the superﬁcial epigastric artery. 10 60 8 For the contracting or stretching experiments, an incision was made 40 6 on the skin located at mid-distance between the two great trochanters 4

peak MAP (mmHg) 20 2

with the rat in prone position. Two other incisions were made along peak HR (beats.min each side of the pelvis to hold the ilium bones with a metal clamp. For 0 0 the contracting experiments, the popliteal fossa was opened to expose Before After Capsazepine Before After Capsazepine (in 10% ethanol) (in 10% ethanol) and isolate the tibial nerve, which was hooked with bipolar stainless- steel electrodes. The femur was attached to a small metal clamp to Fig. 2. Capsazepine decreased the pressor responses to intra-arterial injection prevent knee movements during the contracting or stretch procedure. of bradykinin and ␣-␤-methylene ATP. Individual (closed circles) or group Each paw was secured to the experimental table, and the head of the (shaded bars) data for mean arterial blood pressure (MAP) and heart rate (HR) are presented as the peak difference (⌬) measured before and after injecting rat was secured using a customized stereotaxic unit. To record the bradykinin (500 ng/kg), ␣-␤-methylene ATP (10 ␮g/kg), or diprotonated force produced by the triceps surae muscles, the calcaneus bone was phosphate (86 mM; pH ϭ 6.0) into the superﬁcial epigastric artery. Transient severed, and its attached Achilles tendon was connected to a force receptor potential vanilloid-1 was inhibited by injecting 0.1 mL capsazepine transducer (FT03; Grass Instrument, Quincy, MA) and a rack and (100 ␮g/100 ␮L), dissolved in 10% ethanol and 10% Tween 80, into the pinion. superﬁcial epigastric artery.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS R579 drug. Therefore, the stimulation current intensity was set to evoke a artery using the superficial epigastric artery catheter. The snare was twitch equal to 50% of the maximal twitch tension (~1.25 times motor released 2 min after injecting capsazepine or 10 min after injecting threshold). This procedure allowed us to increase the current intensity ruthenium red or IRTX. After a washout period of 5 min, the once the drug had been injected to match the peak twitch tension. stretching procedure was repeated. During static contraction, the tibial nerve was stimulated for 30 s Chemical injection. We first determined the effect of capsazepine, at 40 Hz (0.01 ms pulse duration) to increase arterial blood ruthenium red, and IRTX on the pressor responses to the TRPV1 agonist pressure reflexively (39). After a recovery period of at least 10 capsaicin, injected into the superficial epigastric artery. We also deter- min, the contraction procedure was repeated to verify that the mined the effect of capsazepine on the pressor responses to “non- pressor response was reproducible. After 10 min of recovery, the TRPV1” agonists (i.e., bradykinin, ␣-␤-methylene ATP, and diproto- snare around the femoral artery and vein was tightened, and nated phosphate) injected into the femoral artery using the superficial capsazepine (100 ␮g/100 ␮L; 0.1 mL), ruthenium red (100 ␮g/100 epigastric artery catheter. A non-TRPV1 agonist was defined as a drug ␮L; 0.1 mL), or IRTX (1 ␮g/100 ␮L; 0.1 mL) was injected into the that does not activate TRPV1 (i.e., pH Ͻ 5.5, heat Ͼ 43°C, or vanilloids). femoral artery using the superficial epigastric artery catheter. The Bradykinin, ATP, and diprotonated phosphate were chosen because they snare was released 2 min after injection of capsazepine or 10 min after are produced by the contracting muscles and are known to evoke a reflex injection of ruthenium red or IRTX (38). Following a washout period pressor response when injected into the arterial supply of skeletal muscle of 5 min, the stimulation current intensity was adjusted, if necessary, (13, 14, 24, 41). During these experiments, the rat was paralyzed with to evoke the same twitch tension as that evoked before injecting the intravenous injection of pancuronium bromide (1 mg/mL; 0.2 mL). drug. Before the injection, the snare placed around the femoral artery and vein To control for the effect of ruthenium red on the ability of the was tightened to trap the solution into the hindlimb circulation. Then, muscle to produce force, a supramaximal twitch was evoked just capsaicin (0.5 ␮g/mL; 0.1 mL), bradykinin (500 ng/kg; 0.2 mL) (35), before injecting the drug and just before contracting the triceps surae ␣-␤-methylene ATP (10 ␮g/kg; 0.2 mL) (14), and diprotonated phos- muscles after the TRPV1 blocker injection. Given that the supramaxi- phate (86 mM; pH 6.0; 0.2 mL) (13, 23) were injected into the femoral mal twitch elicits a complete spatial recruitment of the muscle fibers, artery using the superficial epigastric artery catheter to increase blood a decrease of the peak tension demonstrated that the ability of the pressure reflexively. The snare was released 2 min after injecting the muscle fiber pool to produce force has been reduced. drug. After 15 min of recovery (30 min for the bradykinin trials), To control that the tibial nerve stimulation did not electrically capsazepine (100 ␮g/100 ␮L; 0.1 mL), ruthenium red (100 ␮g/100 ␮L; activate the axons of the group III and IV afferents, which evoke the 0.1 mL), or IRTX (1 ␮g/100 ␮L; 0.1 mL) was injected into the femoral exercise pressor reflex, the rat was paralyzed by intravenous injection artery using the superficial epigastric artery catheter. The snare was of pancuronium bromide (1 mg/mL; 0.2 mL), and the tibial nerve was released 2 min after injecting capsazepine or 10 min after injecting stimulated for 30 s at 40 Hz, with the highest stimulation parameters ruthenium red or IRTX. After a washout period of 5 min, capsaicin, used to evoke contraction. If an increase in blood pressure was bradykinin, ␣-␤-methylene ATP, or diprotonated phosphate was rein- observed, then the data were excluded from the data set. jected. Because bradykinin can be considered as a secondary TRPV1 Stretch of the triceps surae muscles. The pressor response evoked agonist (37, 44), the bradykinin experiment was reproduced using IRTX by stretching the triceps surae muscles was compared before and after to verify that if capsazepine blocks the response to bradykinin, then the injecting the TRPV1 antagonists, capsazepine, ruthenium red, and effect was mediated through TRPV1 inhibition. IRTX. To stretch the triceps surae muscles, the tension was passively Control for an effect of the drug vehicle. If one of the TRPV1 raised using a rack and pinion until reaching a fixed tension corre- antagonists decreased the pressor response to one of the stimuli sponding to ~750–850 g from a baseline tension of 100 g (⌬650–750 studied, then we attempted to replicate the decrease in another group g). The tension was released 30 s later. After 10 min of recovery, the of rats for which we injected the vehicle in which the drug was snare around the femoral artery and vein was tightened, and capsaz- dissolved. This experiment was conducted to test the possibility that epine (100 ␮g/100 ␮L; 0.1 mL), ruthenium red (100 ␮g/100 ␮L; 0.1 the decrease in any pressor response was caused by the drug and not mL), or IRTX (1 ␮g/100 ␮L; 0.1 mL) was injected into the femoral caused by the vehicle in which it was dissolved.

Table 2. Effect of vehicle controls for TRPV1 antagonists on the peak pressor and cardioaccelerator responses to injecting chemicals into superﬁcial epigastric artery

10% Ethanol/10% Tween 80 0.5% DMSO 100% Saline 0.1% Ethanol

Chemicals Before After Before After Before After Before After Capsaicin MAP 36 Ϯ 11 42 Ϯ 25 63 Ϯ 29 70 Ϯ 26 35 Ϯ 838Ϯ 10 34 Ϯ 20 43 Ϯ 25 HR 10 Ϯ 913Ϯ 11 4 Ϯ 24Ϯ 22Ϯ 33Ϯ 23Ϯ 12Ϯ 1 n 6444 Bradykinin MAP 23 Ϯ 829Ϯ 14 Not tested Not tested 21 Ϯ 12 19 Ϯ 8 HR 2 Ϯ 13Ϯ 21Ϯ 12Ϯ 2 n 5 3 ␣-␤-Methylene ATP MAP 22 Ϯ 12 21 Ϯ 9 Not tested Not tested Not tested HR 3 Ϯ 12Ϯ 1 n 9 Diprotonated phosphate MAP 33 Ϯ 24 37 Ϯ 26 Not tested Not tested Not tested HR 4 Ϯ 44Ϯ 2 n 5 Results are presented as means Ϯ SD increase in mean arterial blood pressure (MAP, in mmHg) or heart rate (HR, in beats/min), evoked by each chemical; n ϭ sample size. Note that no signiﬁcant difference in the pressor or cardioaccelerator response was found for any of the vehicles that were injected into the superﬁcial epigastric artery. TRPV1, transient receptor potential vanilloid-1.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. R580 TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS Blue dye control. To determine that injections into the femoral Capsazepine (MedChemExpress) was dissolved in ethanol or DMSO. artery accessed the arterial circulation of the triceps surae muscles, we For the ethanol solution, 10 mg capsazepine was diluted into 1 mL injected 0.2–0.3 mL Evans Blue dye into the femoral artery using the ethanol. Tween 80 (1 mL) was added, and the solution was shaken superficial epigastric artery catheter after tightening the snare placed several times to avoid visible flakes. The final volume was obtained by around the femoral artery and vein. We considered that the solution the addition of 8 mL saline. For the DMSO solution, 10 mg capsazepine spread into the muscle when the belly of the triceps surae muscles was was diluted in 50 ␮L DMSO, which was the smallest volume of solvent stained blue. If the color of the muscles did not change, then we that could be used without producing visible flakes or precipitate. Five excluded the data from the study. microliters of that solution was withdrawn and mixed with a drop of Drug preparation. The stock solution for each of the drugs used in Tween 80 and 0.995 mL saline to obtain a 0.5% DMSO solution. the present experiments was stored in 1 mL aliquots at Ϫ20°C. On the IRTX (1 mg; Tocris) was diluted in 0.1 mL ethanol with a drop of day of the experiment, we diluted the stock solution to the needed Tween 80. Saline (0.9 mL) was added, and the solution was properly concentration with 0.9% saline solution. Bradykinin (Sigma-Aldrich), shaken to avoid the appearance of visible flakes. That solution (0.1 ␣-␤-methylene ATP (Tocris), and ruthenium red (Tocris) were dis- mL) was mixed with 9.9 mL saline to bring the concentration of the solved in saline. Diprotonated phosphate was made by dissolving 153 stock solution down to 100 ␮g/mL. mg (43 mM) Na2HPO4 and 129 mg (43 mM) NaH2PO4 into 25 mL of 10 mM HEPES. The pH of the solution was decreased to 6.0 by Data Analysis adding HCl and if necessary, adjusted on a weekly basis. Capsaicin (10 mg; Sigma-Aldrich) was dissolved in 0.1 mL ethanol Tension and arterial blood pressure signals were amplified (Gould with a drop of Tween 80. Then, 9.9 mL saline was added. The solution Universal and Pressure Processors; Gould-Statham Instruments, Los was stirred and gently heated to avoid visible flakes. Saline (40 mL) Angeles, CA), displayed, and recorded at 1 kHz using an analog-to- was finally added until obtaining a clear solution. digital converter (Micro1401 MKII; Cambridge Electronic Design,

A Before capsazepine After capsazepine (in 0.5% DMSO) (in 0.5% DMSO) 1000 (g)

Tension 0 200 BP

(mmHg) 100 ) -1 420 HR 380

(beats.min 0 1020304050607080900 102030405060708090 Time (s) Time (s) B P = 0.015; d = 0.88 P = 0.003; d = 1.05 P = 0.13; d = 0.60

50 700 ) 50 -1 600 40 40 500 30 400 30 20 300 20 200 10 BPI (mmHg.s) 10

peak MAP (mmHg) 100 peak HR (beats.min 0 0 0 Before After Capsazepine Before After Capsazepine Before After Capsazepine 106 ± 25 111 ± 26 369 ± 35 348 ± 59

P = 0.80; d = 0.18 P = 0.18; d = 0.53 1.2 30 1.0 25 0.8 20 0.6 15

TTI (kg.s) 10 Tension (kg) Tension 0.4 0.2 5 0 0 Before After Capsazepine Before After Capsazepine 83 ± 13 88 ± 16 Fig. 3. Effect of capsazepine dissolved in DMSO on the exercise pressor reﬂex. A: representative sample. B: individual (closed circles) and group (gray bars; n ϭ 7) data for mean arterial pressure (MAP), blood pressure (BP) index (BPI), heart rate (HR), peak tension, and tension-time index (TTI) evoked by static contraction of the triceps surae muscles before and after injecting 0.1 mL capsazepine (100 ␮g/100 ␮L) dissolved in 0.5% DMSO into the superﬁcial epigastric artery. The numbers (Ϯ) below the MAP, HR, and tension graphs represent the baseline values (in grams for tension). ⌬, difference.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS R581 Cambridge, UK) and its associated commercially available software Statistical Analysis (Spike2; 7.20; RRID:SCR_000903; Cambridge Electronic Design). Ϯ Heart rate was determined beat by beat from the pulsatile wave of the Data in RESULTS are presented as the means SD difference blood pressure signal and expressed as beats per minute. To determine between baseline and post-TRPV1 antagonist. With the use of a the effect of the TRPV1 antagonists on the pressor response to Kolmogorov-Smirnoff test, we verified that our samples respected a chemical injections, we calculated the difference between the peak normal distribution. Then, paired Student’s t tests were used to mean arterial pressure and its corresponding baseline value, measured determine the following: 1) the pre- to poststimulus (i.e., contraction, before and after injection of the antagonist. For ␣-␤-methylene ATP stretch, capsaicin, etc.) change in peak pressor response and peak injections, specifically, the pressor response depicted a double-peak heart rate or 2) the pre- to postinjection effect of TRPV1 blockers on response. As the second peak was most likely caused by recirculation the peak pressor response and peak heart rate evoked by the different of the drug, we analyzed the peak pressor response recorded during stimuli. For contraction or stretch experiments, these analyses were the first peak only. To determine the effect of TRPV1 antagonists on also conducted on the blood pressure index, peak tension, and the pressor response to 30 s of static contraction or a passive stretch, tension-time index. The level of significance was set at P Ͻ 0.05. we calculated the peak pressor response (as described above) and the The effect size was calculated using Cohen’s d (3). A Cohen’s d change in the blood pressure index. The blood pressure index was index for effect size was considered small, medium, or large when calculated by first integrating the area under the curve during the 30-s d was close to 0.2, 0.5, or 0.8, respectively (3). When individual contraction or stretch period and then subtracting from this value the data are not presented, effect size was also calculated using 95% area under the curve measured 1 s before the contraction or stretch and confidence intervals (⌬CI) (6). ⌬CI is presented as the lower and multiplied by 30. With the use of a similar method, we calculated the upper limit of the interval that should, if this experiment is change in peak tension produced by the contraction or stretch, as well repeated, contain 95% of the time of the true value of the treated as the tension-time index (i.e., the equivalent of the blood pressure effect (6). Statistical analyses were conducted using Statistica 8.0 index for tension). (RRID:SCR_014213; StatSoft, Tulsa, OK).

A Before 0.5% DMSO After 0.5% DMSO

500 (g)

Tension 0 140

BP 80

(mmHg) 20 ) -1 400 380

HR 360 0 102030405060708090 0 1020304050607080 90 (beats.min Time (s) Time (s) B P = 0.023; d = 1.10 P = 0.045; d = 1.60 P d 50 ) 25 = 0.15; = 0.90

700 -1 40 600 20 500 30 400 15 20 300 10 200 10 BPI (mmHg.s) 5 peak MAP (mmHg) 100 peak HR (beats.min 0 0 0 Before After DMSO Before After DMSO Before After DMSO 80 ± 9 98 ± 8 415 ± 24 406 ± 22

P = 0.36; d = 0.36 P = 0.80; d = 0.02 1.0 25

0.8 20

0.6 15

0.4 10 TTI (kg.s) Tension (kg) Tension 0.2 5

0 0 Before After DMSO Before After DMSO 79 ± 3 86 ± 13 Fig. 4. Effect of DMSO on the exercise pressor reﬂex. A: representative sample. B: individual (closed circles) and group (shaded bars; n ϭ 5) data for mean arterial pressure (MAP), blood pressure (BP) index (BPI), heart rate (HR), peak tension, and tension-time index (TTI) evoked by static contraction of the triceps surae muscles before and after injecting 0.5% DMSO (0.1 mL) into the superﬁcial epigastric artery. The numbers (Ϯ) below the MAP, HR, and tension graphs represent the baseline values. ⌬, difference.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. R582 TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS RESULTS blood pressure response to ␣-␤-methylene ATP or diprotonated phosphate was not tested. The vehicle in which capsaz- Pressor Response Evoked by Chemical Injections epine (10% ethanol, 10% Tween 80, 80% saline), ruthenium Injections of capsaicin, bradykinin, ␣-␤-methylene ATP, red (saline), or IRTX (0.01% ethanol) was dissolved had no and diprotonated phosphate into the superficial epigastric ar- effect on the blood pressure or the cardioaccelerator responses tery significantly increased arterial blood pressure and heart to the different drugs tested (P Ͼ 0.12; Table 2). rate (Table 1). Capsazepine, ruthenium red, and IRTX signif- Pressor Responses Evoked by Contraction icantly attenuated the pressor response to capsaicin injection, showing that these antagonists effectively blocked TRPV1 Static contractions of the triceps surae muscles significantly (Fig. 1). However, capsazepine also decreased the pressor elevated mean arterial blood pressure, blood pressure index, responses evoked by injections of bradykinin and ␣-␤-meth- and heart rate (Table 1). The blocking of TRPV1 with capsaz- ylene ATP, showing that the effect of capsazepine was not epine dissolved in 0.5% DMSO significantly reduced the peak limited to TRPV1 antagonism (Fig. 2). The effect of capsaz- pressor response and the blood pressure index evoked by static epine on bradykinin was not replicated using IRTX (12 Ϯ 20 contraction of the triceps surae muscles (Fig. 3). However, the mmHg; ⌬CI ϭ [Ϫ9 34]; P ϭ 0.20; d ϭ 0.15; n ϭ 6), suggest- injection of a capsazepine-free solution, containing only 0.5% ing that the inhibition of the bradykinin pressor response by DMSO to control for an effect of the vehicle, produced a capsazepine was not secondary to TRPV1 antagonism. Cap- similar reduction of the peak pressor response and blood sazepine had no effect on the peak pressor response evoked by pressure index evoked by static contraction, suggesting that the injection of diprotonated phosphate. The effect of IRTX on the effect of capsazepine was, at least partly, due to the vehicle in

A Before capsazepine After capsazepine (in 10% ethanol / 10% Tween 80 / 80% saline) (in 10% ethanol / 10% Tween 80 / 80% saline) 2000

(g) 0 Tension

100 BP 60 (mmHg) )

-1 400

360 HR

(beats.min 0 1020304050607080900 102030405060708090 Time (s) Time (s) B P = 0.58; d = 0.18 P = 0.15; d = 0.50 P = 0.49; d = 0.18 25 350 ) 20 -1 20 300 250 15 15 200 10 10 150 100 5 5 BPI (mmHg.s)

peak MAP (mmHg) 50 0 0 peak HR (beats.min 0 Before After Capsazepine Before After Capsazepine Before After Capsazepine 102 ± 24 101 ± 26 365 ± 39 355 ± 31

P d P d 2.0 = 0.72; = 0.09 35 = 0.17; = 0.35 30 1.5 25 20 1.0 15 TTI (kg.s) Tension (kg) Tension 0.5 10 5 0 0 Before After Capsazepine Before After Capsazepine 90 ± 10 90 ± 12 Fig. 5. Effect of capsazepine dissolved in ethanol on the exercise pressor reﬂex. A: representative sample. B: individual (closed circles) and group (shaded bars; n ϭ 7) data for mean arterial pressure (MAP), blood pressure (BP) index (BPI), heat rate (HR), peak tension, and tension-time index (TTI) evoked by static contraction of the triceps surae muscles before and after injecting 0.1 mL capsazepine (100 ␮g/100 ␮L) dissolved in 10% ethanol and 10% Tween 80 into the superﬁcial epigastric artery. The numbers (Ϯ) below the MAP, HR, and tension graphs represent the baseline values. ⌬, difference.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS R583 which capsazepine was dissolved (Fig. 4). In contrast, capsaz- evoked by passive stretch, whereas the peak pressor response epine dissolved into 10% ethanol and 10% Tween 80, IRTX, or remained unchanged (Table 3). Similar to the static contraction ruthenium red did not have a significant effect on the peak data, vehicle (i.e., 0.5% DMSO) injection decreased the blood pressor response or the blood pressure index evoked by static pressure index evoked by passive stretch without significantly contraction (Figs. 5, 6, and 7). Except for ruthenium red, changing the peak pressor response (Table 3). injection of TRPV1 antagonists or 0.5% DMSO solution did not significantly change peak tension or the tension-time index. DISCUSSION Ruthenium red significantly decreased the tension-time index evoked by a 30-s static contraction and decreased the peak The primary goal of our experiments was to shed light on the force produced by a supramaximal twitch (Ϫ99 Ϯ 83 g; controversy over the role played by TRPV1 in evoking the ⌬CI ϭ [Ϫ176 Ϫ 22]; P ϭ 0.020; d ϭ 0.99; n ϭ 7), showing exercise pressor reflex arising from static contraction of the triceps that the drug impaired the ability of the muscle to produce and surae muscles. We found that three structurally different TRPV1 sustain force. antagonists, namely, capsazepine, IRTX, and ruthenium red, when dissolved in either ethanol or saline, had no effect on Pressor Response Evoked by Passive Stretch the exercise pressor reflex, despite the fact that each of these Passive stretch of the triceps surae muscles significantly antagonists significantly attenuated the reflex pressor re- elevated blood pressure, blood pressure index, and heart rate sponses to capsaicin injection into the arterial supply of the (Table 1). Except for capsazepine dissolved in 0.5% DMSO, hindlimb muscles. Although we found that capsazepine, TRPV1 inhibition did not significantly change the peak pressor dissolved in DMSO, did attenuate the exercise pressor response, blood pressure index, or peak heart rate evoked by reflex, we replicated this finding when we injected DMSO in passive stretch (Table 3). Capsazepine dissolved in 0.5% the same concentration and volume as that used to place DMSO significantly decreased the blood pressure index capsazepine into solution. Our results thus showed that

A Before IRTX After IRTX 1500

(g) 500 Tension 140

BP 100 (mmHg)

) 60 -1 300

HR 260 0 1020304050607080900 102030405060708090

(beats.min Time (s) Time (s) B P d P d P d 60 = 0.56; = 0.22 600 = 0.57; = 0.27 ) 14 = 0.61; = 0.17 -1 50 500 12 40 400 10 8 30 300 6 20 200 4 BPI (mmHg.s)

peak MAP (mmHg) 10 100 2 peak HR (beats.min 0 0 0 Before After IRTX Before After IRTX Before After IRTX 119 ± 17 118 ± 22 357 ± 70 372 ± 50

P d 1.5 = 0.72; = 0.32 30 P = 0.17; d = 0.42 25 1.0 20 15

0.5 TTI (kg.s) 10 Tension (kg) Tension 5 0 0 Before After IRTX Before After IRTX 84 ± 14 88 ± 19 Fig. 6. Effect of iodoresiniferatoxin (IRTX) on the exercise pressor reﬂex. A: representative sample. B: individual (closed circles) and group (gray bars; n ϭ 7) data for mean arterial pressure (MAP), blood pressure (BP) index (BPI), heat rate (HR), peak tension, and tension-time index (TTI) evoked by static contraction of the triceps surae muscles before and after injecting 0.1 mL IRTX (1 ␮g/100 ␮L) into the superﬁcial epigastric artery. The numbers (Ϯ) below the MAP, HR, and tension graphs represent the baseline values. ⌬, difference.

A Before Ruthenium Red After Ruthenium Red

(g) 500

Tension 0 150 BP 50 (mmHg) ) -1 450 HR 350

(beats.min 0 1020304050607080900 102030405060708090 Time (s) Time (s) B P = 0.41; d = 0.31 50 P = 0.77; d = 0.09 ) 40 P = 0.61; d = 0.14

600 -1 40 500 30 30 400 300 20 20 200 10 10 BPI (mmHg.s) peak MAP (mmHg) 100 peak HR (beats.min 0 0 0 Before After Ruthenium Red Before After Ruthenium Red Before After Ruthenium Red 101 ± 21 104 ± 22 387 ± 32 377 ± 33

P = 0.013; d = 0.98 0.8 P = 0.21; d = 0.51 25 20 0.6 15 0.4

TTI (kg.s) 10 Tension (kg) Tension 0.2 5

0 0 Before After Ruthenium Red Before After Ruthenium Red 88 ± 7 85 ± 13 Fig. 7. Effect of ruthenium red on the exercise pressor reﬂex. A: representative sample. B: individual (closed circles) and group (shaded bars; n ϭ 7) data for mean arterial pressure (MAP), blood pressure (BP) index (BPI), heat rate (HR), peak tension, and tension-time index (TTI) evoked by static contraction of the triceps surae muscles before and after injecting 0.1 mL ruthenium red (100 ␮g/100 ␮L) into the superﬁcial epigastric artery. The numbers (Ϯ) below the MAP, HR, and tension graphs represent the baseline values. ⌬, difference.

TRPV1 played no role in the pressor response evoked by most likely caused by the drug acting as a ryanodine channel static contraction in healthy rats. inhibitor (48) that, in turn, decreased the ability of the muscle Our finding that blockade of TRPV1 played no role in fibers to release Ca2ϩ. With the knowledge beforehand that evoking the exercise pressor reflex agreed with studies in cats ruthenium red would decrease the peak force of contractions, or rats with chronic femoral artery ligation, showing that IRTX we evoked a submaximal contraction before injecting the drug. had no effect on this reflex (22, 45). This finding, however, Most of the time, this procedure allowed us, after injecting contrasts with that of Smith et al. (38) and Mizuno et al. (29), ruthenium red, to increase the current intensity to match the who found that injection of capsazepine, IRTX, and ruthenium peak tension of the initial contraction. Despite this precaution, red into the arterial supply of the hindlimb muscles of decer- we were still not able to match the tension-time index, sug- ebrated rats with freely perfused hindlimb muscles attenuated gesting that in addition to ruthenium red decreasing the ability the exercise pressor reflex. In part, this contrast might be of the muscle to produce force, it decreased the ability of the explained by the solvents used to place capsazepine and IRTX muscle to sustain force. Smith et al. (38) did not report the into solution. This possibility, however, is difficult to assess tension-time index. Consequently, our data reveal the possibil- because the solvent used to dissolve these antagonists was not ity that the decrease in blood pressure, resulting from the described by Smith et al. (38) and Mizuno et al. (29). injection of ruthenium red in their study, was the result of a Ruthenium red was the third TRPV1 antagonist used in our decrease in the tension-time index. experiments. As ruthenium red was dissolved in saline, our Studies using analgesic balms containing capsaicin, topi- result, showing that the exercise pressor reflex was not atten- cally applied to the skin overlying limb muscles, have been uated following ruthenium red injection, cannot be explained reported to attenuate the exercise pressor reflex in both animals by the solvent in which the drug was dissolved. Alternatively, (16, 30) and humans (7, 46). At first glance, these studies might we found that ruthenium red markedly decreased the ability of be viewed as complementary to the findings of Smith et al. (38) the triceps surae muscles to produce force, an effect that was and Mizuno et al. (29). Likewise, some investigators may

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00174.2019 • www.ajpregu.org Downloaded from journals.physiology.org/journal/ajpregu (089.158.232.103) on May 6, 2020. TRPV1 AND THE EXERCISE PRESSOR REFLEX IN RATS R585 Table 3. Effect of TRPV1 antagonists on the peak pressor and cardioaccelerator responses to stretch of triceps surae muscles

TRPV1 Antagonist (Index) Before Injection After Injection 95% CI P Value Cohen’s d Capsazepine (10% ethanol/10% Tween 80; n ϭ 8) MAP 27 Ϯ 13 25 Ϯ 16 [Ϫ95] P ϭ 0.29 d ϭ 0.18 HR 9 Ϯ 97Ϯ 6[Ϫ62] P ϭ 0.25 d ϭ 0.40 BPI 417 Ϯ 247 328 Ϯ 208 [Ϫ367 189] P ϭ 0.47 d ϭ 0.28 Capsazepine (0.5% DMSO; n ϭ 9) MAP 38 Ϯ 12 41 Ϯ 15 [Ϫ49] P ϭ 0.40 d ϭ 0.30 HR 9 Ϯ 88Ϯ 8[Ϫ22] P ϭ 0.93 d ϭ 0.24 BPI 589 Ϯ 378 397 Ϯ 350 [Ϫ905 Ϫ273] P ϭ 0.003 d ϭ 0.95 Ruthenium red (n ϭ 4) MAP 47 Ϯ 844Ϯ 19 [Ϫ36 29] P ϭ 0.74 d ϭ 0.17 HR 4 Ϯ 14Ϯ 2[Ϫ55] P ϭ 0.22 d ϭ 0.19 BPI 540 Ϯ 174 468 Ϯ 176 [Ϫ274 130] P ϭ 0.22 d ϭ 0.42 IRTX (n ϭ 8) MAP 39 Ϯ 15 37 Ϯ 17 [Ϫ97] P ϭ 0.48 d ϭ 0.18 HR 5 Ϯ 44Ϯ 3[Ϫ31] P ϭ 0.23 d ϭ 0.39 BPI 555 Ϯ 220 549 Ϯ 233 [Ϫ113 103] P ϭ 0.47 d ϭ 0.50 Results are presented as means Ϯ SD increase in mean arterial blood pressure (MAP, in mmHg), heart rate (HR, beats/min), or blood pressure index (BPI; measured as mmHg·s), evoked by passive stretch; n ϭ sample size. Ninety-five percent confidence interval (CI) is presented as the lower and upper boundary of the interval containing the true value of the effect of the transient receptor potential vanilloid-1 (TRPV1) antagonist. IRTX, iodoresiniferatoxin. interpret this finding as evidence that TRPV1 plays a role in ity exists that a compensatory mechanism could have hidden evoking the exercise pressor reflex. However, it is important to the antagonism of TRPV1 in our experiments. For example, realize that capsaicin is an explosive stimulus to group IV Stone et al. (42) showed in healthy rats that the exercise pressor muscle afferents (19) and that its prolonged application may reflex was attenuated only if a combination of three antagonists prevent these thin fiber afferents from evoking the reflex was administered; in contrast, the injection of each antagonist because they have been depleted of a neurotransmitter. In separately had no effect. Stone et al. (42) concluded that the addition, this interpretation does not address the issue of a exercise pressor reflex was determined by redundant mecha- vehicle-induced effect, which is just as applicable in these nisms that compensated one for the other. Nevertheless, given experiments as it was in the experiments described above in the unphysiological thresholds required for the activation of the which TRPV1 antagonists were reported to attenuate the ex- TRPV1 (i.e., pH Ͻ 5.5, heat Ͼ 43°C) and given the absence of ercise pressor reflex. an attenuation of the exercise pressor reflex following injection The secondary goal of our experiments was to determine of capsazepine, IRTX, or ruthenium red, we think it is unlikely the selectivity of capsazepine for TRPV1. We found that that static contraction activated TRPV1 in decerebrated rats capsazepine, dissolved in ethanol and Tween 80, attenuated with freely perfused hindlimb muscles. the pressor reflex evoked by injection of bradykinin and ␣-␤-methylene ATP into the arterial supply of the hindlimb Perspectives and Significance muscles. This finding was surprising because there is no evidence that either bradykinin or ␣-␤-methylene ATP di- Our results suggest the following: 1) TRPV1 does not rectly stimulates TRPV1. Instead, one can argue that bra- play a role in evoking the exercise pressor reflex in healthy dykinin and ␣-␤-methylene ATP stimulate TRPV1 by an rats; 2) capsazepine and ruthenium red should not be used to indirect action (34, 37, 44). For example, activation of study the role played by TRPV1 in evoking the exercise bradykinin 2 receptors has been found to release a lipoxy- pressor because of their “off-target” effects; and 3) DMSO, genase byproduct that, in turn, activated TRPV1 (37). If this in a concentration of 0.5% or greater, should be avoided as were the case in our experiments, then IRTX should have a vehicle to dissolve TRPV1 antagonists to study the exer- also decreased the pressor response to bradykinin injection. cise pressor reflex. It did not, which suggests that the effect of capsazepine on the pressor response to bradykinin injection was not the GRANTS result of TRPV1 antagonism. We speculate that the decrease Funding for this study was provided by the National Institute of Arthritis in the pressor responses to bradykinin and ␣-␤-methylene and Muskuloskeletal and Skin Diseases (Grant R01-AR-059397) and National ATP injections was the result of an off-target action of Heart, Lung, and Blood Institute (Grant P01-HL-134609). capsazepine, of which the blocking of voltage-gated calcium channels is one (10). This off-target action might neverthe- DISCLOSURES less also be expected to decrease the pressor response to No conflicts of interest, financial or otherwise, are declared by the authors. diprotonated phosphate injection, but it did not, and we can offer no explanation as to why this is the case. AUTHOR CONTRIBUTIONS G.P.D. and M.P.K. conceived and designed research; G.P.D. performed Study Limitation experiments; G.P.D. analyzed data; G.P.D., J.A.E., J.S.K., and M.P.K. inter- preted results of experiments; G.P.D. prepared figures; G.P.D. drafted manu- Although our findings strongly suggest that TRPV1 does not script; G.P.D., J.A.E., J.S.K., and M.P.K. edited and revised manuscript; play a role in evoking the exercise pressor reflex, the possibil- G.P.D., J.A.E., J.S.K., and M.P.K. approved final version of manuscript.

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