Role of opioid receptors in modulation of P2X receptor-mediated cardiac sympathoexcitatory reflex response

Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents’ activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,β-methylene ATP (α,β-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,β-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,β-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.


Results
Dose responses. Intrapericardial application of increasing doses of α,β-meATP, a selective P2X receptor agonist every 20 min evoked graded excitatory cardiovascular responses ( Fig. 1A and supplemental Fig. 1A), while HR was unchanged in this group of barodenervated and vagotomized rats (n = 9). In contrast, intrapericardial application of ADP, a selective P2Y receptor agonist, did not alter mean arterial pressure (MAP) (Fig. 1B and supplemental Fig. 1B, P > 0.05) and heart rate (HR) in separate baro-vagal denervated rats (n = 9). Application of the vehicle (PBS) also did not alter MAP and HR. The baseline of MAP and HR before application of α,β-meATP were 91 ± 8 mmHg and 384 ± 14 beats/min. CSR responses to activation of P2X receptors before and after procaine. Representative tracings of blood pressure in top panels 1-3 in Fig. 2A display the changes of arterial pressure in response to intrapericardial α,β-meATP (125 nmol) before and after administration of procaine into pericardium of a baro-vagal denervated rat. Administration of α,β-meATP increased arterial blood pressure with MAP elevation by 27 mmHg (Fig. 2A1), which was eliminated by intrapericardial procaine (Fig. 2A2). The MAP response to application of α,β-meATP recovered to the pre-procaine level 40 min after third response (Fig. 2A3,C). The MAP responses to repeated intrapericardial applications of α,β-meATP (125 nmol) were consistent before and after intrapericardial application of vehicle in seven baro-vagal denervated rats ( Fig. 2B and supplemental Fig. 2A). Application of α,β-meATP slightly decreased HR from 386 ± 18 to 382 ± 16 bpm (P > 0.05) in these rats. In contrast, blockade of cardiac neuronal transmission with intrapericardial procaine eliminated almost the MAP responses to application ofα,β-meATP ( Fig. 2C and supplemental Fig. 2B) in eight other rats. The baselines of HR and MAP prior to each response were in a similar range ( Table 1). Application of neither vehicle nor procaine itself changed MAP and HR ( Table 1).
Baseline MAP (89 ± 5 mmHg) and HR (381 ± 11 beats/min) were similar in both group of rats, one with intact vagal and sympathetic afferent nerves and the other with baro-vagal denervation (Table 1). In the vehicle treated group (n = 7) repeat intrapericardial application of α,β-meATP evoked consistent increases in MAP ( Fig. 5A and supplemental Fig. 5A). The magnitude of MAP response to α,β-meATP was similar between vagus-intact and vagotomized rats. In the opioid receptor antagonist treated group (n = 8), intrapericardial naloxone enhanced the α,β-meATP-evoked MAP response, which recovered 40 min after washout of naloxone ( Fig. 5B and supplemental Fig. 5B). Intrapericardial α,β-meATP stimulation also increased but slightly HR by 13 ± 6 beats/min from baseline of 378 ± 12 beats/min in this group, a response that was unaltered by naloxone application into pericardium (13 ± 6 vs. 16 ± 7 beats/min, after vs. before naloxone, P > 0.05). Intrapericardial vehicle and naloxone did not alter the baseline of HR and MAP.

Discussion
To our knowledge, this study is the first to assess whether stimulation of P2 receptors in the heart evokes excitatory cardiovascular reflex responses and if these reflex responses are modulated by peripheral opioids. It is known that the cardiac sympathoexcitatory reflex responses can deteriorate ischemic events leading to increase in morbidity and mortality in patients with ischemic heart disease 3,4,39,40 . In the present study, we observed that activation of P2X receptors with intrapericardial application of α,β-meATP, a mimetic of ATP and selective P2X receptor agonist provoked CSR reflex responses in a dose-dependent manner, while activation of P2Y receptor with ADP didn't alter the CSR responses. The excitatory responses were eliminated after blockade of P2X receptors with their selective antagonist A-317491. Local blockade of cardiac afferent neurotransmission with intrapericardial application of the local anesthetic procaine also abolished the α,β-meATP induced hypertensive responses. In addition, blockade of opioid receptors with intrapericardial naloxone enhanced the P2X receptor-mediated reflexes in both vagus-intact and vagotomized animals. Taken together, these data indicate that activation of cardiac P2X receptors, but not P2Y, is capable of provoking cardiac sympathoexcitatory reflex responses through stimulation of cardiac sympathetic afferents. Endogenously produced opioids suppress the P2X-mediated CSR response through activation of peripheral opioid receptors.
The heart receives sympathetic and vagal efferent and afferent innervation, as well as intrinsic cardiac nerve supply. It is well known that stimulation of cardiac vagal afferents with ischemia, chemical, or electrical stimuli leads to vasodepressor and bradycardia responses, while activation of cardiac sympathetic afferents evokes vasopressor and tachycardia responses 3,5,13,41 . Myocardial ischemia increases extracellular ATP concentration 8,9 and P2 receptors are located on parasympathetic sensory neurons like nodose ganglia and vagal afferent endings localized in lung and myocardium 11,[42][43][44] . Studies reported that administration of ATP into coronary artery induces a cardiac vagal depressor reflex 2,11 . Hence, it is accepted that the increased ATP during myocardial ischemia mainly triggers cardiac vagal vasodepressor reflex by stimulating P2X 2/3 receptors located on vagal sensory nerve terminals in the heart 2,10,11 . However, evidence also suggests that P2 receptors are located on spinal sensory neurons in the dorsal root ganglia and potentially on afferent terminals in the heart 8,9,42,45,46 , but the importance of extracellular ATP in provoking cardiac vasopressor and tachycardia responses remains unknown. The present study for the first time has provided evidence to demonstrate that intrapericardial application of α,β-meATP, an ATP analog, evokes pressor reflex responses in both vagotomized and vagus-intact rats (Figs 2 and 5). The α,β-meATP-mediated vasopressor reflex can be eliminated by blockade of cardiac afferent neurotransmission with intrapericardial application of local anesthetic drug procaine, suggesting that intrapericardial application of α,β-meATP highly likely stimulates cardiac sympathetic sensory nerve endings that is located more superficial and nearer to the epicardial surface of the heart 47,48 . This is consistent with our previous observation that epicardial application of ATP and α,β-meATP excites ischemically sensitive cardiac sympathetic afferents 12 .  www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ Action of extracellular ATP is mediated by the P2 receptors including ionotropic P2X and metabotropic P2Y families 18 . P2X receptor activation causes ion flux through the ligand-gated ion channels in cell membrane, while activation of the P2Y receptor essentially causes an intracellular-reaction cascade through a G-protein coupled mechanism. In the nervous system, both the P2X and P2Y receptor subtypes are presented in the DRG 23,24,45,46 and these DRG P2 receptors could be transported to the axonal nerve ending in the heart similar to the transport of DRG vanilloid receptors 49 . In the present study, we have observed that excitation of P2Y receptor with ADP fails to elicit vasopressor response although our earlier studies have shown that ADP excites cardiac sympathetic afferent 12 . The following factors are potentially responsible for this discrepancy. First, multiple P2Y receptor subtypes including P2Y1,2,4,6 are expressed in rat heart and coronary arteries and the P2Y receptor mediates inhibition of the heart 10,50 . ADP can induce relaxation of coronary small arteries through activation of P2Y receptors 51,52 . Second, rapid breakdown of ADP to adenosine, which in turn leads to direct negative inotropic and chronotropic effect through action on P1 purinoceptors located in the heart 53 . Last and most importantly, the cardiac sympathetic afferent response to ADP occurs at the sensory nerve fiber site. However, to evoke cardiac-sympathoexcitatory reflex responses it is necessary to excite the entire CSR reflex arc neural pathway that includes sensory neuron and fiber, integrative center(s) in the brain and spinal cord, sympathetic efferent nerve and effector organ in the cardiovascular system. It is possible that the ADP-induced afferent activation is insufficient to activate the entire neural pathway involved in the CSR reflex arc.
In contrast, the present study has documented that activation of P2X receptors with α,β-meATP evokes CSR responses, and this is consistent with our previous data that stimulation of P2X receptor activates cardiac sympathetic afferents 12 . Moreover, the α,β-meATP induced vasopressor response is eliminated by selective blockade of P2X 2/3 and P2X 3 receptors with A-317491. Previous studies have documented that among the members of the P2X receptor family, the heteromeric P2X 2/3 as well as homomeric P2X 1 , P2X 2 and P2X 3 receptors are sensitive to α,β-meATP 54 . By using A-317491, a potent and selective P2X 2/3 and P2X 3 receptor antagonist 55 , our data suggest www.nature.com/scientificreports www.nature.com/scientificreports/ that the P2X 2/3 and P2X 3 receptors located on cardiac sympathetic sensory neurons, but not P2Y receptors, are involved in the ATP-evoked CSR responses.
In addition to the P2X receptor-mediated CSR responses, the P2X receptor-mediated direct action as well as the P2Y and P1 receptor-induced effects in the heart also may contribute to the interplay between purinergic and adrenergic signaling in regulation of heart. In this respect, studies indicate that mRNA and protein of all P2X subtypes are expressed on cardiac myocytes 56 . ATP can produce positive chronotropic and inotropic effects on the heart and induce contractile responses of coronary arteries through direct activation of P2X receptors 57 . On the other hand, multiple P2Y receptor subtypes including P2Y1,2,4,6 are expressed in rat heart and coronary arteries 10,50 . Activation of P2Y receptors directly inhibits heart and adenosine-P1 receptors can induce negative inotropic and chronotropic effect and anti-β adrenergic actions in the heart 53,58 . Studies also have shown that ATP and its rapid breakdown products such as ADP and adenosine evoke endothelium-dependent or independent vasodilation in isolated human coronary arteries and other arteries through activation of P2Y or P1 receptors 51,52 . Additionally, ATP and noradrenaline as co-transmitters are released by sympathetic efferent nerves 10,52 . The released ATP inhibits the release of noradrenaline in the heart through its action on P2Y receptors located on the sympathetic terminals 59,60 , while noradrenaline can suppress the release of ATP from sympathetic nerves 59 . Hence, a physiological interplay between purinergic and adrenergic signaling in the heart warrants further studies.
Previously, multiple studies have shown that opioid receptor including µ-, δ-, and κ-receptors are located on small-, medium-and large-diameter sensory neurons in the DRG, nodose and trigeminal ganglia of animals and humans 32,61,62 . Fields and his colleagues 32 have shown multiple subtypes of opioid receptors located on primary afferent fiber terminals. The three subtypes of opioid receptors belong to the superfamily of G protein-coupled receptor (GPCR). Investigators have documented that opioids induce variable somatic and visceral sensory neural responses. In this regard, stimulation of opioid receptors on pelvic and gastric vagal sensory nerves suppresses visceral pain 63,64 . Others reported that opioid peptides excite rat mesenteric afferents and mouse DRG neurons, which can be eliminated by blockade of opioid receptors 65,66 . We found that endogenous opioids modulate the responses of cardiac sympathetic afferents to exogenous ATP and myocardial ischemia 38 , suggesting that endogenous opioids likely suppress the P2X receptor activation-evoked CSR responses by inhibiting the excitability of ischemically sensitive cardiac spinal afferents through stimulation of opioid receptors located on the cardiac spinal afferent terminals. Our speculation was consistent with the findings of other investigators. First, He and his colleagues 67 have shown that µ-opioid receptors are expressed and co-localized with TRPV1 receptors on the cardiac sensory nerve terminals. Second, Chizhmakov and his colleagues 68 reported that leu-enkephalin and morphine inhibit ATP-evoked excitation of somatic C-fiber sensory nerves through a GPCR-dependent mechanism, an effect that can be reversed by naloxone. Our speculation also is supported by our own findings showing that specific blockade of opioid receptors with naloxone enhanced the α,β-meATP-evoked vasopressor response in both vagus-intact and vagotomized rats. Moreover, stimulation of opioid receptors with intrapericardial application of DAMGO (400 nmol), an opioid receptor agonist, reduced the P2X receptor-mediated CSR responses by 48% in three vagus-intact rats in our pilot study (unpublished data). Additionally it is interesting to note that in rats with both vagal and sympathetic cardiac nerves intact, intrapericardial naloxone similarly exaggerated the α,β-meATP evoked hypertensive responses, although opioid receptors also are located on vagal afferents and nodose ganglion neurons 63,64,68 . Together, the present data suggest that blockade of opioid receptors by intrapericardial naloxone mainly enhances the α,β-meATP induced excitatory cardiac-cardiovascular reflex responses in rats. physiological and clinical implications. Clinical observations illustrate that angina pectoris can be accompanied by either vasopressor and tachycardia or vasodepressor and bradycardia 69-71 since myocardial ischemia through release of many mediators strongly stimulates both vagal and sympathetic cardiac sensory nerve fibers [11][12][13]41,72 . Excitation of myocardial sympathetic afferents (i.e., cardiac spinal afferent) leads to the excitatory cardiovascular reflexes including hypertension and tachycardia 1,3,5,13 , while stimulation of cardiac vagal afferents causes vasodepressor and bradycardia 2,41 . Anatomic studies have shown that P2X receptors are located on both cardiac vagal and sympathetic sensory nerves 2, [10][11][12]45,46 . Cardiac vagal afferent fibers are located nearer to the endocardial layer 48 , while sympathetic afferent fibers mainly are located on more superficial and nearer to the epicardial surface of the heart 47,48 . In addition, studies have shown that ATP breakdown occurs very rapidly and its half-life is about 0.2 s when perfused in the circulation 73,74 . Therefore, clinicians likely observe hypotension and bradycardia responses when patients suffer from subendocardial ischemia because of regional ischemia increasing ATP locally, which in turn stimulates cardiac vagal afferents. This is supported by previous studies conducted by Xu and his colleagues 2 . In contrast, when transmural ischemia occurs in patients, hypertension and tachycardia responses are observed as the locally increased ATP largely activates cardiac sympathetic afferents located closer to the epicardial surface of the heart, which is supported by both the present results and our earlier study 12 .
In summary, the novel evidence generated from the present study demonstrate that activation of P2X 2/3 and P2X 3 receptors, but not P2Y receptors, evokes cardiac hypertension response through stimulation of cardiac sympathetic sensory nerve fibers, the response that can be reduced by endogenous opioids through excitation of opioid receptor mechanisms. Since myocardial ischemia leads to release of both ATP and opioids into the extracellular space in the heart 8,9,75 , the interactions between opioids and ATP highly likely contribute to the net cardiovascular responses during myocardial ischemia. These new findings extend our knowledge of ischemic mediators like extracellular ATP produced during myocardial ischemia in stimulating cardiac sensory neurons-cardiovascular reflex responses, while endogenous opioids in suppressing the CSR responses through activation of opioid receptors located on cardiac spinal afferent terminals 67  www.nature.com/scientificreports www.nature.com/scientificreports/ reflexes through activating endogenous opioid pathways 28,30 , suggesting a possibility that acupuncture could attenuate the CSR reflex response, which needs further exploration. The role of opioid receptor subtypes on the P2X receptor-mediated CSR responses also requires further exploration.

Methods
Surgical preparation. All experimental preparations and protocols were reviewed and approved by the Animal Care and Use Committee at the University of California, Irvine. The investigation conformed to the American Physiological Society's "Guiding Principles in the Care and Use of Animals. " Adult Sprague-Dawley (SD) male rats (350-550 g) were anaesthetized initially with ketamine (100 mg/kg, im) followed by α-chloralose (50-60 mg/kg, iv). Additional doses of α-chloralose (25-30 mg/kg, iv) were given as necessary to maintain an adequate level of anesthesia assessed by observing the absence of a conjunctival reflex. A femoral vein was cannulated to administer drugs and fluids. Systemic arterial blood pressure was monitored by a pressure transducer attached to a carotid artery cannula. The trachea was intubated and respiration was maintained artificially (model 661, Harvard ventilator, Ealing, South Natick, MA, USA). Rats were ventilated with room air supplemented with 100% O 2 through the respirator. Arterial blood gases and pH were measured with a blood gas analyzer (ABL 5, Radiometer America, Inc., West Lake, OH) and were maintained within physiological limits (PO 2 > 100 mmHg, PCO 2 30-40 mmHg, pH 7.35-7.45) by adjusting the respiratory rate, tidal volume or by administering NaHCO 3 (1 M, iv). Body temperature was monitored by a rectal thermistor and maintained at 36-38 °C with a circulating water-heating pad and heat lamp.

Sinoaortic denervation and cervical vagotomy.
To eliminate the influence of vagal cardiac afferents that could mask the CSR responses to stimulation of sympathetic afferents and minimize the BP "buffering" action of arterial baroreceptors, bilateral cervical vagotomy and sinoaortic denervation with sectioning of carotid sinus nerves were conducted in rats used in first four protocols (see in following protocols), as described previously 3,76 . Vagotomy was not performed in rats used in the last protocol (described in protocols section) for examining the CSR responses in vagus-intact animals. The barodenervation was verified by noting the absence of the normal decrease of heart rate (HR) in response to ~40-mmHg increase in arterial BP induced by administration of phenylephrine (10 µg/kg, iv) intrapericardial catheter insertion. To administer chemicals to the heart, a catheter was placed in the pericardial sac as previously described 17,77 . In brief, a high midline thoracotomy (the collarbone and the first two ribs) was conducted to expose the thymus and heart. A polyethylene-50 (PE) tubing with 6-8 small holes in the distal end was inserted into the pericardial space over the left ventricle through a small incision made on the thymus gland (on the midline aspect of the thymus). The catheter was then sealed into the thymus and pericardium by suturing together the two thymus lobes and the surrounding muscle tissue with a silk suture to prevent any leaks from pericardium. Various chemical solutions that can stimulate or inhibit cardiac afferent nerve endings described in the following protocols were injected through the PE-50 catheter into the pericardial space. At end of experiment, we injected 80 µl of 2% Chicago Sky blue into the pericardial space in each rat, and leakage of dye from the pericardium was assessed visually at autopsy. Leakage occurred in ~4% of all rats and the animals with leakage from the pericardium were excluded from this study.
Each drug was dissolved in phosphate buffer solution (PBS, pH 7.35) to a stock concentration. The pH of working solution of each drug was adjusted with 1 M of NaHCO 3 [8.4% (wt/vol)] to a final value of 7.35. Procaine, A-317491 and α,β-meATP were purchased from Sigma-Aldrich (St. Louis, MO). Naloxone was purchased from Tocris (Minneapolis, MN). The stock solution was prepared weekly and stored in a −20 °C freezer and the working solution was prepared daily. experimental protocols. Dose responses of reflexes to α,β-meATP. In this protocol we examined CSR responses following activation of P2X receptors with intrapericardial application of α,β-meATP and activation of P2Y receptors with intrapericardial ADP. After completion of the surgical preparation including denervation, a minimum of 45 minutes was allowed for stabilization of arterial pressure. The CSR responses including arterial blood pressure and heart rate (HR) to application of α,β-meATP were recorded with injection of 40 µl of various doses of α,β-meATP or PBS (vehicle) into the pericardial space in nine barodenervated and vagotomized rats. The vehicle (i.e., PBS) and α,β-meATP were applied randomly. A dose-response curve was generated with five doses of α,β-meATP (31,63,125,250, and 500 nmol). α,β-meATP is a selective P2X 1 , P2X 2/3 , and P2X 3 receptor agonist and mimetic of ATP that is produced during myocardial ischemia and participates in activation of cardiac spinal afferents 12 . In separate group (n = 9), the CSR responses to randomly intrapericardial ADP with four doses (500, 1000, 2000, 4000 nmol) were recorded. ADP is a selective P2Y receptor agonist. The heart was washed with intrapericardial injection of 100 µl of warm saline (35 °C) three times to wash out the drug after each application. To prevent tachyphylaxis, recovery periods of at least 20 min were provided between consecutive stimuli. α,β-meATP + procaine. The influence of blockade of cardiac nerve transmission with procaine on the CSR responses to cardiac P2X receptor activation in bilateral barodenervated and vagotomized animals was examined in this protocol. After stabilization, α,β-meATP (125 nmol, 40 µl) was injected into the pericardial space to evoke repetitive reflex increases in BP and HR. Warm saline (100 µl) was applied intrapericardially three times to wash (2019) 9:17224 | https://doi.org/10.1038/s41598-019-53754-6 www.nature.com/scientificreports www.nature.com/scientificreports/ out α,β-meATP after each application of the P2X receptor agonist. To prevent tachyphylaxis, recovery periods of at least 20 min were provided between consecutive stimuli. In this protocol, intrapericardial α,β-meATP was applied 4 times over a period of at least 100 min. After the first two consecutive application of α,β-meATP, 80 µl of 2% procaine were injected into the pericardial sack of eight rats. Next, third intrapericardial α,β-meATP was conducted 5 min after procaine, which was 20 min after the second dose of α,β-meATP. Previous studies have demonstrated that this dose of procaine eliminates cardiac reflex responses by blocking cardiac afferent neurotransmission 3,78 since cardiac spinal afferent nerve endings are located mainly in the epicardial layers of the myocardium 48 . 40 minutes later, a fourth application of α,β-meATP was performed to observe a recovery of BP/ HR responses to α,β-meATP. To evaluate the reproducibility of cardiovascular reflex responses to α,β-meATP, seven additional rats were studied used as time control group. Each animal in this group was treated identical with exception that an intrapericardial application of vehicle (PBS, 80 µl) was used in place of procaine. This time control group also served as control for the following two protocols.
α,β-meATP + A-317491. To evaluate the influence of blockade of P2X receptors on CSR responses to α,β-meATP, we recorded BP and HR responses following repeated application of α,β-meATP before and after intrapericardial application of A-317491, a selective P2X 2/3 and P2X 3 receptors antagonist in bilateral barodenervated and vagotomized animals (n = 7). After stabilization, in a similar fashion as above mentioned procaine protocol, 80 µl of A-317491 (800 nmol) was injected into pericardium after first two consecutive applications of α,β-meATP. The third intrapericardial α,β-meATP was conducted 5 min after A-317491, which was 20 min after the second application of α,β-meATP. Previous studies have demonstrated that A-317491 at this dose reduces α,β-meATP-induced pressor response by selective antagonism of P2X 2/3 and P2X 3 receptors 55 . Following each application of α,β-meATP, the heart was washed three times with 100 µl of warm saline. 40 minutes later, a fourth application of α,β-meATP was performed to observe a recovery of BP/HR responses to α,β-meATP.
α,β-meATP + naloxone. In eight animals, we examined the influence of blockade of opioid receptors with naloxone, a specific opioid receptor antagonist, on the cardiovascular responses (BP and HR) to stimulation of P2X receptors with α,β-meATP. Following each application of α,β-meATP, the heart was washed three times with 100 µl of warm saline. In a similar fashion as the A-317491 protocol, after the first two consecutive intrapericardial α,β-meATP, 80 µl of naloxone (8 µmol) was injected into pericardium and third intrapericardial α,β-meATP was performed 5 min after naloxone and 20 min after the second application of α,β-meATP. We have demonstrated that this dose of naloxone enhances cardiac afferent activity in response to ischemia by blockade of opioid receptors 38 . Next, α,β-meATP was reapplied into pericardium 40 minutes after the third application of α,β-meATP, to allow for a recovery of the reflex responses comparable to the control level.
α,β-meATP + naloxone in vagus-intact rats. To determine if intrapericardial α,β-meATP induce cardiac vagal reflex responses including vasodepressor and bradycardia that may affect the P2X receptor activation-mediated CSR responses and if naloxone modulate the α,β-meATP-evoked responses, we recorded BP and HR responses to repeated intrapericardial application of α,β-meATP in two groups of rats without bilateral vagotomy. In an identical manner as protocol 4, vehicle in first group (n = 7) and naloxone (8 µmol) in second group (n = 8) of rats were applied into pericardial space after the first two intrapericardial α,β-meATP. Data analysis. Arterial blood pressure and HR were recorded with a Spike 2 data-acquisition system (CED micro 1401 mkII) and stored on a computer hard drive (Dell). Mean arterial pressure (MAP) is expressed in mmHg and HR is expressed in beats per minute. Data are expressed as means ± SEM. The Shapiro-Wilk test was used to determine if the data were distributed normally. Normally distributed data in all protocols were compared with either a Student's paired t-test for paired data or a one way repeated-measures ANOVA followed by the Holm-Sidak's post hoc test. All statistical calculations were performed with SigmaStat software (Jandel scientific Software, San Rafael, CA). Values were considered to be significantly different when P < 0.05.

Data availability
The data sets generated and analyzed during the current study are available from the corresponding author on reasonable request.