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The Journal of Physiology logoLink to The Journal of Physiology
. 2003 Jun 26;551(Pt 2):515–523. doi: 10.1113/jphysiol.2003.048207

Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats

Matthew R Zahner *,†, De-Pei Li *, Shao-Rui Chen *, Hui-Lin Pan *,†
PMCID: PMC2343227  PMID: 12829722

Abstract

Myocardial ischaemia causes the release of metabolites such as bradykinin, which stimulates cardiac sensory receptors to evoke a sympathoexcitatory reflex. However, the molecular identity of the afferent neurons and fibres mediating this reflex response is not clear. In this study, we tested the hypothesis that the cardiogenic sympathoexcitatory reflex is mediated by capsaicin-sensitive afferent fibres. Enhanced immunofluorescence labelling revealed that vanilloid receptor 1 (VR1)-containing afferent nerve fibres were present on the epicardial surface of the rat heart. Resiniferatoxin (RTX), a potent analogue of capsaicin, was used to deplete capsaicin-sensitive afferent fibres in rats. Depletion of these fibres was confirmed by a substantial reduction of VR1 immunoreactivity in the epicardium and dorsal root ganglia. The thermal sensitivity was also diminished in RTX-treated rats. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anaesthetized rats during epicardial application of bradykinin or capsaicin. In vehicle-treated rats, epicardial bradykinin (10 μg ml−1) or capsaicin (10 μg ml−1) application produced a significant increase in RSNA and arterial blood pressure. The RSNA and blood pressure responses caused by bradykinin and capsaicin were completely abolished in RTX-treated rats. Furthermore, epicardial application of iodo-RTX, a highly specific antagonist of VR1 receptors, blocked capsaicin- but not bradykinin-induced sympathoexcitatory responses. Thus, these data provide important histological and functional evidence that the heart is innervated by VR1-expressing afferent nerves and these afferent nerves are essential for the cardiogenic sympathoexcitatory reflex during myocardial ischaemia.

Myocardial ischaemia activates cardiac sympathetic afferent nerve endings and elicits chest pain, which is often associated with an autonomic reflex characterized by an increase in blood pressure and sympathetic nerve activity (White, 1957; Webb et al. 1972; Baker et al. 1980; Li & Pan, 2000). The sensory signals triggering the sympathoexcitatory reflex are conducted through cardiac sympathetic afferents, primarily thinly myelinated Aδ-fibres and unmyelinated C-fibres, that project to the dorsal horn of the thoracic spinal cord (Blair et al. 1982; Kuo et al. 1984). Myocardial ischaemia causes the production and release of several metabolites including bradykinin (Pan et al. 2000). It has been well documented that bradykinin activates cardiac nociceptors and produces a sympathoexcitatory response through kinin B2 receptors (Baker et al. 1980; Veelken et al. 1996; Tjen-A-Looi et al. 1998; Li & Pan, 2000; Pan & Chen, 2002). Although the heart is innervated by both myelinated and unmyelinated afferent nerves, the phenotypes of the sensory neurons and afferent fibres that lead to chest pain and cardiac sympathetic reflexes during ischaemia are not fully known.

The vanilloid receptor (VR1; also known as TRPV1 channel) is a non-specific cation channel activated by capsaicin, noxious heat and protons (Caterina et al. 1997). Studies using VR1 knockout mice have shown that activation of the VR1 receptor is essential for sensing thermal pain (Caterina et al. 2000), although its role in sensing ischaemic visceral pain is not clear. Since VR1 is mainly located on small-sized dorsal root ganglia cells and nociceptors, it is considered to be a molecular sensor for noxious stimuli. It has been shown that epicardial application of capsaicin produces haemodynamic and sympathoexcitatory responses similar to those seen during myocardial ischaemia and epicardial bradykinin application (Staszewska-Woolley et al. 1986; Staszewska-Woolley & Woolley, 1989). However, there is no histological evidence documenting the presence of VR1-containing afferent nerve endings on the heart. Systemic administration of capsaicin to neonatal rats permanently depletes nociceptive primary afferents (Jancso et al. 1977; Scadding, 1980). Resiniferatoxin (RTX), on the other hand, is an ultra-potent analogue of capsaicin that binds VR1 receptors. RTX causes degeneration of capsaicin-sensitive afferent neurons in adult rats upon systemic administration (Szallasi & Blumberg, 1989; Khan et al. 2002; Pan et al. 2003). In this study, using RTX as a pharmacological tool, we tested the hypothesis that capsaicin-sensitive cardiac afferents mediate the cardiogenic sympathetic reflex.

METHODS

Experiments were conducted on male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) weighing between 300 and 350 g. The procedures and protocols were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine. To deplete capsaicin-sensitive primary afferents, rats were treated with a single intraperitoneal injection of RTX (200 μg kg−1, Sigma-RBI, St Louis, MO, USA) under halothane anaesthesia (Pan et al. 2003). A separate group of rats was treated with vehicle (10 % Tween-80 and 10 % alcohol dissolved in normal saline) as controls.

Behavioural assessment of thermal nociception

To verify the depletion of capsaicin-sensitive afferents by RTX, the nociceptive threshold of the hindpaw to a noxious thermal stimulus was tested in all rats 4–6 days after treatment. Rats were placed on the glass surface of a thermal plantar testing apparatus (model 336; IITC Inc./Life Science Instruments, Woodland Hills, CA, USA) and allowed to acclimatize for 30 min before testing. The temperature of the glass surface was maintained constant at 30 °C. A mobile heat source located under the glass was focused onto the hindpaw of the rats. A digital timer recorded the paw withdrawal latency. The withdrawal latencies of the left and right paws were averaged and the mean was used to indicate the thermal sensitivity (Chen & Pan, 2002).

Surgical preparations and renal nerve recordings

Rats were initially anaesthetized using 2 % halothane in O2. An adequate depth of anaesthesia was confirmed by the absence of a withdrawal response to a noxious stimulus (tail pinch). The trachea was cannulated, and rats were mechanically ventilated using a rodent ventilator (model 683, Harvard Apparatus, South Natick, MA, USA). Expired CO2 concentration was monitored with a CO2 analyser (Capstar 100, IITC/Life Science Instruments) and maintained at 4–5 % throughout the experiment. The left carotid artery was cannulated, and the arterial blood pressure was measured with a pressure transducer (PT300, Grass Instruments, Quincy, MA, USA). The left jugular vein was cannulated for intravenous injection of drugs. Halothane exposure was discontinued after α-chloralose (50–60 mg kg−1, I.V.) and sodium phenobarbital (20 mg kg−1, I.V.) were administered. Supplemental doses of sodium pentobarbital were given to maintain an adequate depth of anaesthesia. During renal nerve recordings, rats were treated with the neuromuscular blocker pancuronium bromide (1 mg kg−1, I.V.). The adequacy of anaesthesia during neuromuscular blockade was judged by the stability of the arterial blood pressure. A limited lateral thoracotomy was performed to expose the heart. The pericardium was left in place until just prior to the epicardial application of bradykinin. Body temperature was maintained at 37–38 °C with a heating lamp.

For renal sympathetic nerve recordings, the left kidney was exposed through a left flank incision via a retroperitoneal approach (Li et al. 2001). A small branch of the renal nerve was isolated and carefully dissected from the renal vasculature and surrounding tissue with the aid of an operating microscope. The renal nerve was cut distally to ensure that afferent activity was not recorded. The renal nerve was then bathed in mineral oil and placed on a stainless steel recording electrode. The nerve signal was amplified (× 20 000–30 000) and bandpass filtered (100–3000 Hz) by an alternating current amplifier (model P511, Grass Instruments). Renal sympathetic nerve activity (RSNA) was then monitored through an audio amplifier (model AM10, Grass Instruments). Renal nerve activity and arterial pressure were recorded using a Powerlab data acquisition system (model 4SP, Mountain View, CA, USA), displayed and stored on a Pentium computer. Heart rate was counted by triggering from the blood pressure pulse. Renal nerve activity was also fed into a second Pentium computer through an analog-to-digital interface for subsequent off-line analysis. Discharge frequency was recorded and quantified using a software window discriminator (DataWave Technologies, Longmont, CO, USA). Respective noise levels were subtracted from the nerve recording using DataWave software, as described previously (Li et al. 2001). At the end of the experiments, rats were killed by an intravenous injection of an overdose of sodium pentobarbital.

Experimental design

(1) Immunofluorescence labelling of VR1 receptors

To determine the location of VR1-expressing afferent terminals on the heart and examine the effect of RTX on the afferent neurons and nerves, immunofluorescence labelling experiments were conducted in the thoracic dorsal root ganglia and myocardium obtained from three RTX-treated and three vehicle-treated rats.

Dorsal root ganglia

Under deep anaesthesia with sodium pentobarbital (60 mg kg−1, I.P.), rats were intracardially perfused with 200 ml of ice-cold normal saline containing 1000 U heparin followed by 500 ml of 4 % freshly depolymerized paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) and then 200 ml of 10 % sucrose in 0.1 M PBS (pH 7.4). The thoracic dorsal root ganglia were removed quickly and postfixed for 2 h in the same fixative solution, and cryoprotected in 30 % sucrose in PBS for 48 h at 4°C. The sections were cut to a thickness of 35 μm and collected free-floating in 0.1 M PBS. For conventional immunofluorescence labelling of VR1 receptors in the dorsal root ganglia (Pan et al. 2003), the sections were rinsed in 0.1 M PBS and blocked in 4 % normal goat serum in PBS for 1 h. Then, sections were incubated with the primary antibody (rabbit anti-VR1 N-terminus, dilution 1:1000; Neuromics, Minneapolis, MN, USA) diluted in PBS containing 2 % normal goat serum, 0.3 % Triton X-100 and 0.05 % Tween-20, for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed in 0.1 M PBS and incubated with goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (dilution 5 μg ml−1; Molecular Probes). The sections were rinsed in 0.1 M PBS for 40 min, and mounted on slides, dried and coverslipped.

Myocardium

For immunofluorescence labelling of VR1 receptors in the myocardium, the VR1 immunoreactivity was enhanced with TSA (tyramide signal amplification). The rat heart was removed under deep anaesthesia with sodium pentobarbital (60 mg kg−1, I.P.) and fixed with 4 % paraformaldehyde in 0.1 M PBS for 3 days at 4°C, then cryoprotected in 30 % sucrose in PBS for 48 h at 4°C. The myocardium was cut to a thickness of 50 μm transmurally and collected free-floating in 0.1 M PBS. Sections were rinsed and incubated in 3 % H2O2 with 10 % methanol to quench endogenous peroxidase, and blocked in TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5 % blocking reagent) buffer. Then, sections were incubated with the primary antibody (rabbit anti-VR1 N-terminus, dilution 1:1000; Neuromics) for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:100; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. Finally, the sections were incubated with fluorescein isothiocyanate (FITC) conjugated to tyramide (TSA direct kit, PerkinElmer Life Sciences, Boston, MA, USA) according to the manufacturer's recommendation. The sections were rinsed for 30 min, and mounted on slides, dried and coverslipped. Sections were examined on a confocal scanning microscope (Zeiss, Germany) and areas of interest were photodocumented.

(2) Role of capsaicin-sensitive afferents in cardiac sympathetic reflex

Blood pressure and nerve activity responses to epicardial bradykinin and capsaicin were studied in 10 vehicle-treated and nine RTX-treated rats. After a high quality renal nerve recording was obtained, a 30 min stabilization period was allowed. Then, baseline RSNA and blood pressure were recorded for 3 min. Bradykinin (10 μg ml−1; Sigma) was dissolved in normal saline and applied to the anterior surface (≈1 cm2) of the left ventricle with a cotton applicator, as described previously (Li & Pan, 2000; Li et al. 2001). Following bradykinin application, the RSNA and blood pressure were recorded continuously for another 3 min. The blood pressure and RSNA responses to bradykinin were examined twice, separated by ≈15 min. Following each bradykinin application, the heart was washed using cotton applicators soaked in normal saline, and the blood pressure and RSNA were allowed to return to baseline levels. Upon completion of bradykinin application, capsaicin (10 μg ml−1; Sigma Chemical) was applied topically to the anterior of the left ventricle using the same protocol as described above. Because capsaicin depletes neurotransmitters from afferent nerves, it was always applied after bradykinin application.

To ensure that the sympathetic vasomotor tone was still intact in RTX-treated rats, an intracerebroventricular (I.C.V.) injection of a GABAA receptor antagonist, bicuculline (10 μg in 10 μl saline), was given. In five RTX-treated rats, a guide cannula was implanted in the lateral ventricle (coordinates from bregma: 1.4 mm lateral, 1.0 mm caudal, 3.8 mm ventral). After completion of the capsaicin application, bicuculline was injected over a 45 s period. Blood pressure and RSNA were continuously recorded for ≈15 min following bicuculline injection. To verify the correct placement of the guide cannula, 1 % Chicago Sky Blue dye (Sigma) was injected into the lateral ventricle upon completion the experimental protocols.

(3) Role of VR1 receptors in the bradykinin-induced cardiac sympathetic reflex

To determine whether VR1 receptors mediate the bradykinin-induced RSNA and blood pressure responses, we measured the sympathetic reflex response to epicardial application of bradykinin before and after blockade of VR1 receptors with a highly specific VR1 antagonist, iodo-RTX (Wahl et al. 2001). To block the regional VR1 receptors, 200 μm iodo-RTX (22.5 μg dissolved in 10 μl DMSO and 140 μl 2-hydroxypropyl-β-cyclodextran) was topically applied to the epicardial surface in the region where bradykinin and capsaicin were applied in seven normal rats. The iodo-RTX concentration was determined in a pilot study, and it was the minimal concentration required to completely block the blood pressure response to epicardial capsaicin (10 μg ml−1). In these experiments, baseline blood pressure and RSNA were recorded for 3 min followed by epicardial vehicle application (10 μl DMSO in 140 μl 2-hydroxypropyl-β-cyclodextran). Two minutes after vehicle application, 10 μg ml−1 bradykinin was applied to the same location on the epicardium as described above. After a recovery period of approximated 15 min to allow the blood pressure and RSNA return to the baseline, iodo-RTX (200 μm, ≈33 μl) was applied topically to the anterior of the left ventricle. When a stable baseline was established, the blood pressure and RSNA responses to epicardial bradykinin (10 μg ml−1) application were examined. Finally, to determine whether iodo-RTX blocked the cardiovascular reflex response to capsaicin, we measured the blood pressure and RSNA responses to epicardial application of 10 μg ml−1 capsaicin in the presence of iodo-RTX.

Data analysis

Values are presented as means ± s.e.m. Baseline blood pressure and RSNA were averaged during the 3 min control period. Maximum blood pressure and RSNA were measured approximately 20 s following bradykinin or capsaicin application. The RSNA is presented as the percentage change from baseline activity due to the variability in baseline RSNA in each animal. One-way ANOVA with Tukey's post hoc analysis was used to compare the difference between group means. P < 0.05 was considered statistically significant.

RESULTS

A total of 32 rats were used in this study. To confirm that RTX depletes capsaicin-sensitive afferent neurons and fibres, paw withdrawal latency in response to the noxious heat stimulus was measured before and 4–6 days after RTX or vehicle injection (Fig. 1). Baseline withdrawal latency prior to vehicle or RTX treatment did not differ significantly between groups. In the vehicle group (n = 18), the paw withdrawal latency was not significantly altered by vehicle injection (8.24 ± 0.24 vs. 8.33 ± 0.47 s). However, in RTX-treated rats (n = 14), the paw withdrawal latency was significantly increased from 7.14 ± 0.59 to 24.99 ± 1.60 s (P < 0.05).

Figure 1.

Figure 1

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Mean paw withdrawal latency in response to a radiant heat stimulus before and 5 days after RTX (200 μg kg−1, n = 14) or vehicle (n = 18) treatment. Data are presented as means ± s.e.m. *P < 0.05 compared with baseline values.

VR1 immunofluorescence labelling and the effect of RTX

Immunofluorescence labelling of VR1 receptors was conducted on the thoracic dorsal root ganglia and myocardium from vehicle- and RTX-treated rats. The omission of the primary antibody resulted in negative labelling in the dorsal root ganglia and myocardium. In vehicle-treated rats, VR1 immunoreactivity was present in both the myocardium (Fig. 2A) and dorsal root ganglia (Fig. 2C). Immunocytochemical labelling of VR1 receptors revealed an intricate network of thin and tortuous fibres in the myocardium primarily near the epicardial surface. There was dense VR1-positive labelling on the epicardial surface of both ventricles, although some sparse labelling was also seen in the deeper myocardium. There was no VR1 immunoreactivity in the endocardium and the atrial wall (data not shown). In all the thoracic dorsal root ganglia examined, VR1-positive neurons were mainly small- and medium-sized cells. In RTX-treated rats, VR1 immunoreactivity was nearly absent in the epicardium (Fig. 2B). Also, there was a substantial reduction of VR1-positive neurons in dorsal root ganglia (Fig. 2D).

Figure 2.

Figure 2

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Confocal images showing immunofluorescence labelling of VR1 receptors in the left ventricle of the heart (scale bars: 15 μm in A and B) and thoracic dorsal root ganglia (DRG, scale bars: 30 μm in C and D) in a vehicle-treated (Control) and an RTX-treated rat. Note the dense VR1 immunofluorescence on the epicardial surface of the heart in the control rat.

Cardiac-sympathetic reflex in vehicle- and RTX-treated rats

Representative RSNA and blood pressure responses to epicardial application of bradykinin (10 μg ml−1) and capsaicin (10 μg ml−1) in a vehicle-treated rat are shown in Fig. 3. Both bradykinin and capsaicin induced an immediate and profound increase in RSNA and blood pressure. The maximal response occurred approximately 15–20 s after drug application and gradually returned to baseline within 10 min. In 10 vehicle-treated rats, epicardial bradykinin (10 μg ml−1) significantly increased RSNA 169.9 ± 23.3 % from baseline and mean arterial pressure from 76.9 ± 1.7 to 105.9 ± 3.4 mmHg (P < 0.05, Fig. 4A and B). However, heart rate was not significantly increased from baseline (324.3 ± 12.3 to 334.7 ± 13.7 beats min−1). Repeat bradykinin application induced a response of similar magnitude in blood pressure and RSNA. Also, in vehicle-treated rats, epicardial capsaicin (10 μg ml−1) application produced a 96.5 ± 27.9 % increase in RSNA from baseline and increased mean arterial pressure from 77.1 ± 2.9 to 99.9 ± 3.3 mmHg (P < 0.05, Fig. 4A and B). Heart rate was not significantly increased from baseline (312.3 ± 9.8 to 317.3 ± 9.8 beats min−1) with epicardial application of capsaicin in vehicle-treated rats.

Figure 3.

Figure 3

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Original tracings from a vehicle-treated rat showing RSNA and blood pressure responses to epicardial application of bradykinin (BK, 10 μg ml−1) and capsaicin (CAP, 10 μg ml−1). Bradykinin or capsaicin was applied at the time points indicated by the arrows.

Figure 4.

Figure 4

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Group data showing the RSNA (A) and mean arterial pressure (MAP, B) responses to epicardial application of bradykinin (10 μg ml−1, n = 10) and capsaicin (10 μg ml−1, n = 9). RSNA responses evoked by bradykinin and capsaicin were measured as the percentage change from baseline RSNA prior to bradykinin application. Data are presented as means ± s.e.m. *P < 0.05 compared with respective controls.

Representative RSNA and blood pressure responses to epicardial application of bradykinin (10 μg ml−1) and capsaicin (10 μg ml−1) in a RTX-treated rat are shown in Fig. 5. In nine RTX-treated rats studied, neither bradykinin (10 μg ml−1) nor capsaicin (10 μg ml−1) increased RSNA or blood pressure (Fig. 6). To confirm that the sympathetic outflow was still intact in RTX-treated rats, a GABAA receptor antagonist, bicuculline (10 μg in 10 μl saline, I.C.V.), was injected at the end of the experiments. In all five RTX-treated rats tested, bicuculline caused a profound increase in both RSNA and mean arterial pressure within the first minute of administration (Fig. 5).

Figure 5.

Figure 5

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Original tracings showing lack of RSNA and blood pressure responses to epicardial application of bradykinin (BK, 10 μg ml−1) and capsaicin (CAP, 10 μg ml−1) in an RTX-treated rat. Intracerebroventricular bicuculline (BIC, 10 μg in 10 μl vehicle) injection increased blood pressure and RSNA. Bradykinin or capsaicin was applied at the time points indicated by the arrows.

Figure 6.

Figure 6

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Group data showing RSNA (A) and mean arterial pressure (MAP, B) responses to epicardial application of bradykinin (10 μg ml−1, n = 9) and capsaicin (10 μg ml−1, n = 9). RSNA responses evoked by bradykinin and capsaicin were measured as the percentage change from baseline RSNA prior to bradykinin application. Data are presented as means ± s.e.m.

Role of VR1 receptors in the bradykinin-induced cardiogenic sympathetic reflex

To determine the role of VR1 receptors in the bradykinin-elicited cardiac sympathetic response, we applied the VR1 receptor antagonist iodo-RTX (200 μm) to the epicardial surface of the left ventricle prior to bradykinin (10 μg ml−1) or capsaicin (10 μg ml−1) in seven normal rats. Representative tracings of the effect of iodo-RTX on bradykinin- and capsaicin-elicited RSNA and blood pressure responses are shown in Fig. 7. In the presence of vehicle alone, bradykinin (10 μg ml−1) elicited an immediate and profound increase in RSNA and blood pressure. The initial iodo-RTX application alone produced a small increase in RSNA and blood pressure that gradually subsided within 10 min. Because this iodo-RTX-elicited response occurred only with the initial application, we re-applied iodo-RTX before application of bradykinin or capsaicin. In all seven rats tested, iodo-RTX had no significant effect on bradykinin-elicited RSNA or blood pressure responses compared to the initial response to bradykinin (10 μg ml−1) in the presence of vehicle (Fig. 8). However, the capsaicin-elicited blood pressure and RSNA responses were completely abolished by iodo-RTX in all seven rats tested (Fig. 7 and Fig. 8). The mean arterial pressures before and after capsaicin application were 78.1 ± 2.8 and 81.0 ± 2.7 mmHg, respectively.

Figure 7.

Figure 7

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Original tracings showing the responses of RSNA and arterial blood pressure to epicardial application of vehicle, capsaicin (10 μg ml−1) and bradykinin (10 μg ml−1) in the presence of iodo-RTX (200 μm) in a normal rat. Bradykinin or capsaicin was applied at the time points indicated by the arrows.

Figure 8.

Figure 8

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Group data showing RSNA (A) and mean arterial pressure (MAP, B) responses to epicardial bradykinin (10 μg ml−1) plus vehicle or iodo-RTX (200 μm), and capsaicin (10 μg ml−1) plus iodo-RTX in 7 normal control rats. The RSNA following bradykinin or capsaicin application is presented as the percentage change from baseline RSNA. Data are presented as means ± s.e.m. *P < 0.05 compared with respective controls.

DISCUSSION

This is the first study to provide histological evidence that the heart is innervated by VR1-expressing afferent nerves. Also, we found that the cardiogenic sympathoexcitatory reflex response elicited by epicardial bradykinin was completely eliminated in RTX-treated rats. Thus, these data provide strong evidence that capsaicin-sensitive afferents are essential for the cardiac sympathetic response. Furthermore, this reflex response is independent of VR1 receptors because iodo-RTX did not significantly attenuate the reflex response to epicardial application of bradykinin in normal rats. This finding suggests that while capsaicin-sensitive afferents are important for the cardiac sympathetic reflex response, the bradykinin-elicited reflex response is not dependent upon VR1 receptors at the nerve endings of cardiac sympathetic afferents. These new findings are important for our understanding of the sensory mechanisms of cardiac pain and the phenotype of afferent neurons involved in the sympathetic reflex responses to myocardial ischaemia.

Capsaicin, the pungent ingredient in chilli peppers, activates VR1 receptors on small-diameter sensory fibres involved in nociception (Szallasi & Blumberg, 1999; Caterina et al. 2000). VR1 receptors are produced in a subpopulation of dorsal root ganglia cells and expressed throughout the entire length of the afferent nerve (Szallasi, 1995; Caterina et al. 1997; Ma, 2002). Double immunohistochemical labelling studies have shown that in addition to most unmyelinated C-fibres, VR1 receptors are also expressed in about 30 % of thinly myelinated afferent fibres (Ma, 2002). In visceral tissues, VR1-expressing sensory nerve endings have been identified recently in the rat urinary bladder (Avelino et al. 2002). Using the enhanced immunocytochemical labelling technique, we demonstrated for the first time that VR1-containing afferent nerves are widely distributed on the epicardial surface of the ventricle. Also, RTX treatment eliminated VR1-expressing afferent fibres on the epicardium. Although it has been reported that capsaicin can induce the cardiac sympathetic reflex (Staszewska-Woolley et al. 1986; Staszewska-Woolley & Woolley, 1991), to our knowledge there is no histological evidence documenting the presence of VR1-containing afferent nerve endings on the heart. While the conventional fluorescence immunocytochemical labelling was adequate in identifying VR1 receptors in dorsal root ganglia neurons, we were unable to detect VR1 immunoreactivity with this method in the myocardium, possibly due to the weak VR1 antigen present on the heart. Therefore, we used the TSA enhancing technique (Wang et al. 1999; Buki et al. 2000) to amplify the immunofluorescence signal. The principle of this enhanced labelling is that horseradish peroxidase-conjugated streptavadin bound to the biotinylated secondary antibody intensifies the fluorescence signal by catalysing the production of reactive fluorescent tyramide particles. The fluorescent tyramide particles bind to tyrosine moieties in close proximity to the horseradish peroxidase. The specificity of binding is attributed to the extremely short half-life of the tyramide radicals, thus only tyramide in close proximity to the horseradish peroxidase will bind tyrosine (Wang et al. 1999). With this increased sensitivity of the immunolabelling technique, we were able to demonstrate the presence of VR1 immunoreactivity predominantly on the epicardium. This histological finding is consistent with an earlier physiological study showing that topical phenol application to the epicardium abolishes excitatory cardiac sympathetic reflexes triggered by chemical stimulation of cardiac afferents in dogs (Barber et al. 1984). Also, we have shown in previous electrophysiological studies that almost all the ischaemia-sensitive cardiac sympathetic afferents are located on the epicardial surface (Pan & Longhurst, 1995; Pan et al. 1999; Pan & Chen, 2002).

RTX is an ultrapotent analogue of capsaicin and is capable of inducing rapid degeneration of VR1-expressing afferent neurons and fibres in adult rats (Szallasi & Blumberg, 1989; Khan et al. 2002; Pan et al. 2003). Like capsaicin, RTX treatment activates VR1 receptors to elicit an immediate nociceptive response (Szallasi & Blumberg, 1989). Following this acute effect, there is a prolonged desensitization of VR1 receptors and degeneration of VR1-expressing primary sensory neurons in adult rats (Szallasi & Blumberg, 1989; Khan et al. 2002; Pan et al. 2003). It has been well documented that thermal nociception is mediated by capsaicin-sensitive afferents and VR1 receptors (Meller et al. 1992; Caterina et al. 2000; Khan et al. 2002). For this reason, we measured paw withdrawal latency in response to a noxious thermal stimulus as an indicator of substantial depletion of capsaicin-sensitive afferent fibres by RTX. Furthermore, we confirmed that RTX depletes VR1-expressing afferent neurons and fibres in dorsal root ganglia and the heart using an immunocytochemical technique. Thus, RTX is an important experimental tool to determine the role of capsaicin-sensitive afferent neurons in the cardiac sympathetic reflex in our study.

Sensory signals triggering chest pain and associated sympathetic reflex responses during myocardial ischaemia are conveyed by thinly myelinated Aδ-fibres and unmyelinated C-fibres that travel in cardiac sympathetic afferents (White, 1957; Brown, 1967; Baker et al. 1980; Pan et al. 1999; Li & Pan, 2000; Pan & Chen, 2002). These cardiac sympathetic afferent fibres project to the dorsal horn of the upper thoracic spinal cord through the stellate ganglia and sympathetic chain (Kuo et al. 1984; Pan & Longhurst, 1995; Li & Pan, 2000; Pan & Chen, 2002). Myocardial ischaemia produces various metabolites including bradykinin (Pan et al. 2000). It has been shown that bradykinin activates kinin B2 receptors on cardiac sensory afferents to evoke a sympathoexcitatory reflex (Pan & Longhurst, 1995; Veelken et al. 1996; Tjen-A-Looi et al. 1998; Pan & Chen, 2002). While direct single-unit recording studies have identified ischaemia-sensitive afferents as thinly myelinated Aδ-fibres and unmyelinated C-fibres, the phenotypes of the sensory neurons and afferent fibres involved in ischaemic cardiac pain and the sympathetic reflex are not clear. VR1 receptors are primarily expressed in unmyelinated fibres and about 30 % of myelinated afferent fibres in the sciatic nerve (Ma, 2002). Thus, not all Aδ- and C-fibre afferents are capsaicin sensitive. In the present study, epicardial bradykinin or capsaicin produced a large increase in RSNA and blood pressure in vehicle-treated rats. In contrast, in rats treated with RTX to deplete capsaicin-sensitive afferents, the sympathoexcitatory response elicited by epicardial bradykinin or capsaicin was completely eliminated. To ensure that the sympathetic vasomotor tone remained intact in RTX-treated rats, we injected a GABAA receptor antagonist, bicuculline, through an intracerebroventricular catheter as a positive control in this protocol. We observed that bicuculline produced a profound increase in RSNA and blood pressure, suggesting that the sympathetic outflow remained intact in RTX-treated rats. These data strongly suggest that capsaicin-sensitive afferent nerves are essential for the initiation of the cardiogenic sympathetic reflex. Since epicardial bradykinin failed to elicit the sympathoexcitatory reflex in RTX-treated rats, both kinin B2 and VR1 receptors are probably expressed on capsaicin-sensitive afferent nerve endings on the heart. Therefore, these results provide important new information regarding the phenotype of the afferent neurons involved in the cardiac sympathetic reflex.

Recent studies suggest that bradykinin may interact with VR1 receptors in cultured dorsal root ganglia and HEK293 cells (Shin et al. 2002; Sugiura et al. 2002). In this regard, bradykinin may activate VR1 receptors through protein kinase C-dependent inositol 1,4,5-trisphosphate (IP3) disinhibition of VR1 receptors (Thayer et al. 1988; Premkumar & Ahern, 2000) and 12-lipoxygenase products (Hwang et al. 2000; Shin et al. 2002). Indeed, Sugiura et al. (2002) have shown that bradykinin increases heat-induced inward currents in a dose-dependent manner, and this response is blocked with a VR1 antagonist, capzasapene, and a protein kinase C inhibitor. However, Schultz & Ustinova (1998) have previously shown that capsazepine does not attenuate cardiac vagal afferent discharge in response to epicardial bradykinin application. Because the lack of the cardiogenic sympathetic reflex elicited by bradykinin in RTX-treated rats could be explained by the action of bradykinin through VR1 receptors, we further determined the role of cardiac VR1 receptors in bradykinin-induced cardiac sympathetic responses. In this study, we used the newly discovered specific VR1 receptor antagonist iodo-RTX, which is much more potent than capsazepine (Wahl et al. 2001). We found that iodo-RTX completely blocked the capsaicin-elicited cardiogenic reflex but had no significant effect on bradykinin-elicited sympathoexcitatory responses. Thus, VR1 receptors do not contribute significantly to bradykinin-elicited cardiogenic sympathoexcitatory responses. This finding strongly suggests that VR1 receptors do not play an important role in the action of bradykinin at the nerve endings of cardiac sympathetic afferents. Nevertheless, it should be emphasized that the effects of bradykinin on the cardiogenic sympathetic reflex are linked to cardiac afferents that possess VR1 receptors. Taken together, the data from this study provide important functional evidence demonstrating the potential role of capsaicin-sensitive afferents in the cardiac sympathetic reflex and chest pain caused by myocardial ischaemia.

Acknowledgments

This study was supported by National Institutes of Health grants HL60026 and HL04199. H.L.P. was a recipient of an Independent Scientist Award supported by the National Institutes of Health during the course of this study.

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