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. 1998 Jul 15;510(Pt 2):633–641. doi: 10.1111/j.1469-7793.1998.633bk.x

Endogenous bradykinin activates ischaemically sensitive cardiac visceral afferents through kinin B2 receptors in cats

Stephanie C Tjen-A-Looi 1, Hui-Lin Pan 1, John C Longhurst 1
PMCID: PMC2231043  PMID: 9706010

Abstract

  1. Activity of ischaemically sensitive cardiac visceral afferents during myocardial ischaemia induces both angina and cardiovascular reflexes. Increased production of bradykinin (BK) and cyclo-oxygenase products (i.e. prostaglandins (PGs)) occurs during myocardial ischaemia. However, the role of these agents in activation of ischaemically sensitive cardiac afferents has not been established. The present study tested the hypothesis that BK produced during ischaemia activates cardiac afferents through kinin B2 receptors.

  2. Single-unit activity of cardiac afferents innervating the left ventricle was recorded from the left thoracic sympathetic chain (T1–T4) of anaesthetized cats. Ischaemically sensitive cardiac afferents were identified according to their response to 5 min of myocardial ischaemia. The mechanism of BK in activation of ischaemically sensitive cardiac afferents was determined by injection of BK (1 μg kg−1 i.a.), des-Arg9-BK (1 μg kg−1 i.a., a specific kinin B1 receptor agonist), kinin B2 receptor antagonists: HOE140 (30 μg kg−1 i.v.) and NPC-17731 (40 μg kg−1 i.v.), cyclo-oxygenase inhibition with indomethacin (5 mg kg−1 i.v.) and NPC-17731 (40 μg kg−1 i.v.) after pretreatment with indomethacin (5 mg kg−1 i.v.).

  3. We observed that BK increased the discharge rate of all eleven ischaemically sensitive cardiac afferents from 0.39 ± 0.12 to 1.47 ± 0.37 impulses s−1 (P < 0.05). Conversely, des-Arg9-BK did not significantly increase the activity of eleven ischaemically sensitive fibres (0.58 ± 0.02 vs. 0.50 ± 0.18 impulses s−1). HOE140 significantly attenuated the response of twelve afferents to ischaemia (0.61 ± 0.22 to 1.85 ± 0.5 vs. 0.53 ± 0.16 to 1.09 ± 0.4 impulses s−1). NPC-17731, another kinin B2 receptor antagonist, had similar inhibitory effects on six other ischaemically sensitive cardiac afferents (0.35 ± 0.14 to 1.19 ± 0.29 vs. 0.22 ± 0.08 to 0.23 ± 0.07 impulses s−1). Indomethacin significantly reduced the responses of seven afferents to ischaemia (0.35 ± 0.13 to 1.89 ± 0.48 vs. 0.40 ± 0.10 to 0.76 ± 0.24 impulses s−1). Indomethacin also significantly reduced the responses of six ischaemically sensitive cardiac afferents to BK (2.65 ± 1.23 to 1.2 ± 0.51 impulses s−1). In six cats pretreated with indomethacin, NPC-17731 attenuated the impulse activity of six ischaemically sensitive cardiac afferents (0.39 ± 0.12 to 1.0 ± 0.3 vs. 0.26 ± 0.14 to 0.48 ± 0.20 impulses s−1).

  4. This study demonstrates that BK produced during ischaemia contributes to stimulation of ischaemically sensitive cardiac visceral afferents through activation of kinin B2 receptors. Furthermore, BK stimulates ischaemically sensitive cardiac visceral afferents through a mechanism that is, at least in part, independent of cyclo-oxygenase activation.

Myocardial ischaemia is associated with both cardiovascular reflex responses and chest pain. The heart is innervated by sympathetic and vagal sensory nerve endings (White, 1957; Malliani, 1982; Longhurst, 1984). Activation of cardiac visceral sympathetic afferents reflexly evokes cardioexcitatory and vasopressor responses including increases in arterial blood pressure, heart rate, and myocardial contractility (Peterson & Brown, 1971; Malliani et al. 1972; Tjen-A-Looi et al. 1997). Cardiac visceral afferent fibres that travel in sympathetic nerves through the stellate ganglion and sympathetic chain to the spinal cord, constitute the main pathway for cardiac nociceptors (Baker et al. 1980; Malliani, 1982; Meller & Gebhart, 1992). These afferent fibres with sensory receptors in the ventricles, which are associated with cardiovascular reflexes, respond to mechanical and chemical stimuli (Purtock et al. 1977; Baker et al. 1980; Malliani, 1982; Huang et al. 1995a, b). However, the stimuli responsible for increasing visceral afferent activity during cardiac ischaemia are largely unknown.

Myocardial ischaemia produces a number of metabolites, including lactic acid, bradykinin (BK), prostaglandins (PGs), adenosine and reactive oxygen species, among others (Kimura et al. 1973; Hashimoto et al. 1977; Berger et al. 1977; Hirsh et al. 1981; Meller & Gebhart, 1992; Grill et al. 1992). Some of these chemical stimuli may either sensitize or directly activate sympathetic cardiac afferents following application to the epicardial surface or injection into the coronary artery (Brown, 1967; Staszewska-Barczak et al. 1976; Baker et al. 1980; Pal et al. 1989; Nganele & Hintze, 1990). Although it has been hypothesized that increased production of certain metabolites during myocardial ischaemia contributes to excitation of primary cardiac visceral afferents (Baker et al. 1980; Meller & Gebhart, 1992), little evidence is available about the role of specific metabolites during ischaemia. We have previously shown that free radicals, but not adenosine, play a role in afferent activation during myocardial ischaemia (Huang et al. 1995a; Pan & Longhurst, 1995).

It has been suggested that BK is a chemical mediator of cardiac pain (Baker et al. 1980). It is produced during cardiac ischaemia (Kimura et al. 1973; Hashimoto et al. 1977) and binds to two distinct receptors: kinin B1 and B2. Cardiovascular reflex responses induced by epicardial application of BK can be blocked by kinin B2, but not kinin B1, receptor antagonists (Staszewska-Woolley & Woolley, 1989), suggesting that the former receptor subtype is most important in activation of cardiac sensory nerves. In this regard, our laboratory has shown that BK is produced during mesenteric ischaemia and contributes to stimulation of ischaemically sensitive abdominal visceral afferents through kinin B2 receptors (Pan et al. 1994).

We previously have also shown that PGs are capable of sensitizing abdominal visceral afferents and thereby enhance the response to abdominal ischaemia and BK (Pan et al. 1994). PGs also enhance BK-induced cardiac-cardiovascular reflexes (Staszewska-Barczak et al. 1976). However, the effect of PGs on ischaemically sensitive cardiac afferents is less clear. Nerdrum et al. (1986) suggested an interaction between BK and PGE1 in stimulation of cardiac afferents. In this regard, application of PGE1 to the epicardial surface of the heart increases the response of chemosensitive nerve endings to BK (Nerdrum et al. 1986).

With the above considerations in mind, further experiments were undertaken to test the hypothesis that endogenously produced BK stimulates ischaemically sensitive cardiac afferents and that kinin B2 receptor blockade decreases the discharge activity of these afferent endings. In addition, experiments were undertaken to test whether the effect of BK on cardiac afferents during myocardial ischaemia is dependent upon the production of PGs. These data have been published in a preliminary form (Tjen-A-Looi et al. 1996).

METHODS

Surgical preparation

All experiments were performed on cats of either sex (2.3–5.9 kg). Surgical and experimental protocols used in this study were approved by the Animal Use and Care Committee at the University of California, Davis. Anaesthesia was induced with ketamine (20- 30 mg kg−1i.m.) and maintained with α-chloralose (40–60 mg kg−1i.v.). The trachea was intubated and respiration was maintained artificially (model 661, Harvard Apparatus, Ealing, South Natick, MA, USA). A femoral artery and vein were cannulated for measurement of pressure and administration of fluids and drugs, respectively. The left carotid artery was cannulated with a catheter (PE-90 tubing, Intramedic Clay Adams), which was passed retrogradely into the left ventricle (LV) for administration of BK and des-Arg9-BK. In some animals, a catheter (PE-160) was introduced through the appendage into the left atrium for administration of BK agonists. Arterial blood pressure was measured with a pressure transducer (Statham P23 ID, Gould, Cleveland, OH, USA) connected to the arterial catheter.

At the end of the experiments, animals were killed with saturated KCl solution injected into the circulation under deep anaesthesia. Deep anaesthesia was ensured by giving an additional dose of α-chloralose (50 mg kg−1) 5 min before administration of the KCl solution.

A mid-line sternotomy was performed, and the first to seventh left ribs and the left lung were removed. An occlusion cuff was placed around the descending thoracic aorta. Fascia overlying the left paravertebral sympathetic chain from T2 to T6 was removed. The sympathetic chain then was draped over a mirror platform and covered with warm mineral oil. Small nerve filaments were teased gently from the chain between T2 and T5 with the use of an operating microscope (Zeiss, Germany), and the rostral end was placed across a recording electrode. One pole of the recording electrode was earthed with a cotton thread to the animal. The recording electrode was attached to a high-impedence probe (Grass Instruments, Quincy, MA, USA). The signal was amplified (model P511 preamplifier, Grass) and processed through an audio amplifier (model AM8B audio monitor, Grass) and an oscilloscope (model 549, Tektronix, Beavertown, OR, USA). The signal was recorded on a physiograph (model TA 4000, Gould) and with a computer for off-line analysis using an EGAA program (RC Electronics, Santa Barbara, CA, USA) to allow data analysis and construction of histograms.

The location and conduction latency of each afferent nerve ending was confirmed by electrical stimulation through an electrode placed on the afferent's receptive field in the wall of the ventricle (Pan et al. 1995). Conduction distance was estimated from the receptive field along the course of the inferior cardiac nerve through the left stellate ganglion to the recording electrode on the sympathetic chain. C and Aδ fibre afferents were classified as those with conduction velocities (CVs) of < 2.5 and 2.5–30 m s−1, respectively (Huang et al. 1995a; Pan et al. 1995).

Myocardial ischaemia was induced by constricting the coronary artery supplying the receptive field of cardiac ventricular afferents with a thread placed around the vessel (Huang et al. 1995a; Pan et al. 1995). Ischaemia was confirmed by observing a regional change in the colour of the myocardium. Our laboratory has shown previously that this observation is closely correlated to lactic acid production as indicated by a local reduction in tissue pH (Pan et al. 1996). Afferents were considered to be ischaemically sensitive if their discharge frequency during 5 min of myocardial ischaemia was increased and sustained at least twofold above baseline activity. All sensory endings of ischaemically sensitive afferents included in this study were located in the left ventricle.

Arterial blood gases were analysed every hour during the search for cardiac afferents as well as before and after each period of myocardial ischaemia with a Radiometer blood gas analyser (model ABL 3) and were maintained within physiological limits (PO2 >100 mmHg, PCO2 28–35 mmHg, pH 7.35–7.45). When necessary, arterial PO2 was increased by increasing the inspired O2 supply; pH was corrected by administering NaHCO3 (1 M i.v.) and/or adjusting ventilation. Body temperature was maintained at 36–38°C with a circulating-water heating pad and a heat lamp.

Experimental protocols

Effects of BK receptor agonists on afferent activity

Bradykinin (Sigma) and des-Arg9-BK (Sigma) were dissolved in 0.9 % NaCl, diluted to a final concentration of 10 μg ml−1, and injected into the left atrium through a catheter in a volume of 1 ml. Just before ischaemia BK and des-Arg9-BK were tested in a random order on the same afferents.

Effect of kinin B2 receptor antagonists on afferent activity

D-Arg [Hyp3,Thi5,D-Tic7,Oic8]-BK) (HOE140; Hoest-Roussel Pharmaceuticals, Somerville, NJ, USA) (Rhaleb et al. 1992; Wirth et al. 1991) and D-Arg [Hyp3,trans-4-propoxy-D-proline (transpropyl)7,Oic8]-BK (NPC-17731; Nova Pharmaceuticals, Baltimore, MD, USA) (Burch & Kyle, 1992) were dissolved in 0.9 % NaCl to achieve a concentration of 1 mg ml−1. Antagonists were injected 15 min before the second period of ischaemia. In a different group of animals, myocardial ischaemia was repeated at an interval of 30 min to determine whether the afferent response was reproducible.

Effect of cyclo-oxygenase products on afferent activity

Indomethacin (Sigma) was dissolved in 8.4 % (w/v) sodium bicarbonate solution, and injected in a volume of 10 ml. Indomethacin was injected 15 min before the second period of ischaemia or before the second application of BK (i.a.). A third group of animals was pretreated with indomethacin. After an ischaemically sensitive cardiac afferent was identified, NPC-17731 was injected 15 min before the second period of ischaemia, and ischaemia was repeated. The responses of ischaemically sensitive cardiac afferents were compared in the indomethacin-treated animals by comparing activity during ischaemia in pre-NPC and post-NPC periods.

Data analysis

The discharge frequencies of afferents were averaged (and expressed in impulses s−1) during 5 min of the pre-ischaemia control period, 5 min of cardiac ischaemia, and 2 min during immediate re-perfusion. Responses of ischaemically sensitive cardiac afferents to BK and des-Arg9-BK were averaged during the entire period of response defined as the time when sustained activity exceeded baseline activity by 10 %. Baseline activity was determined over a 2–3 min control period, just prior to ischaemia. Data were presented as means ±s.e.m. The discharge frequencies of ischaemically sensitive cardiac afferents in response to BK agonists were compared using Student's paired t test. Differences in responses of ischaemically sensitive cardiac afferents to repeated ischaemic stimuli with or without the application of kinin B2 receptor antagonists or indomethacin, and to repeated exogenous BK stimuli with application of indomethacin were analysed using repeated measures analysis of variance on ranks, followed when necessary by Student-Newman-Keuls post hoc test. Differences were considered to be significant when P < 0.05.

RESULTS

Effects of BK receptor agonists on afferent activity

Responses of ischaemically sensitive cardiac afferents to BK (1 μg kg−1) and the selective kinin B1-receptor agonist des-Arg9-BK (1 μg kg−1) were studied in eleven animals. Pan et al. (1994) have showed that there are reproducible responses of ischaemically sensitive visceral afferents to repeated application of BK. The discharge frequencies of an individual afferent in response to BK and des-Arg9-BK are displayed in Fig. 1. The cardiac afferent activity was increased after intracardiac injection of BK, whereas injection of des-Arg9-BK did not alter the discharge rate of the afferent (Fig. 1A and B). The discharge frequencies of the same ischaemically sensitive cardiac afferent during control, cardiac ischaemia, and reperfusion are shown in Fig. 1C and D. The mean discharge frequencies of eleven ischaemically sensitive cardiac afferents upon application of BK and des-Arg9-BK are displayed in Fig. 1A. Similar to the individual afferent described above, injection of BK into the left atrium significantly activated ischaemically sensitive cardiac afferents (from 0.39 ± 0.12 to 1.47 ± 0.37 impulses s−1, P < 0.05), whereas no afferents were responsive to des-Arg9-BK (0.58 ± 0.02 vs. 0.50 ± 0.18 impulses s−1, P > 0.05).

Figure 1. Ischaemically sensitive cardiac afferent responses to BK agonists.

Figure 1

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A, group mean discharge frequencies of ischaemically sensitive cardiac afferent (n = 11) responses to administration of BK (▪) and des-Arg9-BK (×) (control, □). *Administration of BK resulted in responses that were significantly different (P < 0.05) from responses after injection of des-Arg9-BK. Error bars indicate s.e.m. and the comparisons are based on Student's paired t test. B, raw data displays of the discharge activity from an ischaemically sensitive cardiac afferent fibre (CV = 0.62 m s−1) that was stimulated after injection of BK but not des-Arg9-BK. C, histogram showing the frequency of the action potentials of the same cardiac afferent as in B during 5 min control (-5 to 0 min), 5 min ischaemia (0–5 min) and 2 min reperfusion (5–7 min). D, raw data displays during periods i-iii as indicated in C.

Effects of kinin B2 receptor antagonists on afferent activity

The effect of the kinin B2 antagonist HOE140 (30 μg kg−1i.v.) was tested on twelve afferents. This dose of HOE140 has been shown to successfully block the response to BK in a previous study (Pan et al. 1994). The activity of an ischaemically sensitive cardiac afferent before and after injection of HOE140 is displayed in Fig. 2. Figure 2A and C shows the discharge rate of the afferent before treatment with HOE140 during control, myocardial ischaemia and reperfusion. The discharge frequency of the ischaemically sensitive cardiac afferent decreased during myocardial ischaemia after application of HOE140 (Fig. 2B and D). HOE140 significantly decreased the responses of twelve ischaemically sensitive cardiac afferents (Table 1). Thus, comparing afferent activity before with that after application of HOE140 we observed a 41 % attenuation of the response to myocardial ischaemia.

Figure 2. Responses of an ischaemically sensitive cardiac afferent to kinin B2 receptor antagonist.

Figure 2

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A and B, histograms showing the frequency of the action potentials from an ischaemically sensitive cardiac afferent (CV = 0.95 m s−1) before and after application of HOE140, respectively, during 5 min control (-5 to 0 min), 5 min ischaemia (0–5 min), and 2 min reperfusion (5–7 min). C, raw data recordings of activity of the afferent before administration of HOE140 during periods i and ii as indicated in A. D, raw data recordings of the activity of the afferent after HOE140 application during periods i and ii as indicated in B.

Table 1.

Response of cardiac afferents (in impulses s−1) to repeated myocardial ischaemia before and after BK2 receptor, cyclo-oxgenase and combined blockade

Preblockade Postblockade
Control Ischaemia Control Ischaemia
No intervention (6) 0.67 ± 0.47 1.19 ± 0.53 0.81 ± 1.03 1.22 ± 1.56
HOE140 (12) 0.61 ± 0.22 1.85 ± 0.5 0.53 ± 0.16 1.09 ± 0.4 *
NPC-17731 (6) 0.35 ± 0.14 1.19 ± 0.29 0.22 ± 0.08 0.23 ± 0.07 *
Indomethacin (7) 0.35 ± 0.13 1.89 ± 0.48 0.40 ± 0.10 0.76 ± 0.24 *
Indomethacin-pretreated NPC-17731 (6) 0.39 ± 0.12 1.0 ± 0.30 0.26 ± 0.14 0.48 ± 0.2 *
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Number of afferents is indicated in parentheses.

*

Significant (P < 0.05) decrease in afferent responses comparing pre- to postblockade using repeated measures of analysis of variance and Student-Newman-Keuls post hoc test

The responses of a structurally dissimilar kinin B2 receptor antagonist NPC-17731 (40 μg kg−1i.v.) was studied in six additional afferents. This dose of NPC-17731 has previously been shown to successfully block the response to BK (Pan et al. 1994). The effect of NPC-17731 on the afferent response to ischaemia is shown in Table 1. NPC-17731 decreased the afferent activity of six ischaemically sensitive cardiac afferents during myocardial ischaemia by 79 %. After application of NPC-17731 the discharge frequency was not significantly increased during ischaemia. Repeated myocardial ischaemia, separated by a 30 min recovery period, demonstrated consistent responses of the cardiac afferents (n = 6) (Table 1). In a previous study (Pan et al. 1995) we reported similar reproducible responses to repeated myocardial ischaemia.

Effect of cyclo-oxygenase products on afferent activity

The effect of indomethacin (5 mg kg−1i.v.) was examined in seven ischaemically sensitive cardiac visceral afferents in seven animals during the repeated ischaemia protocol, and on six afferents in five animals during repeated application of BK. We have previously shown that this dose of indomethacin effectively inhibits the response of visceral afferents to stimulation with endogenous PGs (Longhurst et al. 1991). Indomethacin significantly (P < 0.05) attenuated (59 %) the response of seven cardiac afferents during myocardial ischaemia (Table 1). Indomethacin also significantly reduced the response of six ischaemically sensitive cardiac afferents following application of BK (from 2.65 ± 1.23 to 1.2 ± 0.51 impulses s−1, P < 0.05, Fig. 3). The effect of indomethacin (5 mg kg−1i.v.) was tested on six ischaemically sensitive cardiac visceral afferents in six indomethacin-pretreated animals. Responses of an afferent before and after injection of NPC-17731 (40 μg kg−1i.v.) in indomethacin-pretreated animals are displayed in Fig. 4. Figure 4A and C shows the discharge frequency of the afferent before injection of NPC-17731 during control, myocardial ischaemia and reperfusion. The discharge frequency of the ischaemically sensitive cardiac afferent decreased during myocardial ischaemia after treatment with NPC-17731 (Fig. 4B and D). NPC-17731 significantly attenuated the responses of six ischaemically sensitive cardiac afferents. Thus, the afferent response to the myocardial ischaemia after application of NPC-17731 was attenuated by 52 % in indomethacin-pretreated animals. After application of NPC-17731, the discharge frequency of afferents during ischaemia was not significantly different from baseline during control (Table 1).

Figure 3. The effect of indomethacin on discharge activity of six ischaemically sensitive cardiac afferents after BK application.

Figure 3

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Group data of ischaemically sensitive cardiac afferents responsive to application of BK (i.a.) before and after administration of indomethacin. □, control; ▪, after bradykinin; * Afferent responses to BK were significantly different (P < 0.05) comparing pre- to postblockade. † Application of BK increased the afferent responses significantly compared with spontaneous baseline afferent activity. Error bars indicate s.e.m. and comparisons were made with repeated measures of analysis of variance followed by Student-Newman-Keuls post hoc test.

Figure 4. Responses of an ischaemically sensitive cardiac afferent to NPC-17731 in indomethacin-pretreated animals.

Figure 4

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A and B, histograms showing the frequency of the action potentials from an ischaemically sensitive cardiac afferent (CV = 0.88 m s−1) pretreated with indomethacin before and after application of NPC-17731, respectively, during 5 min control (-5 to 0 min), 5 min ischaemia (0–5 min), and 2 min reperfusion (5–7 min). C, raw data recordings of activity of the afferent before administration of NPC-17731 during periods i and ii as indicated in A. D, raw data recordings of the activity of the afferent after NPC-17731 application during periods i and ii as indicated in B.

Receptive fields of cardiac afferents

Most of the ischaemically sensitive cardiac afferents were located in the anterior and posterior base of the left ventricle (Fig. 5). Some were identified near the left circumflex and the left anterior descending coronary vessels. The conduction velocity for most ischaemically sensitive cardiac afferents ranged from 0.39 to 2.5 m s−1. One afferent in the group of fibres tested with NPC-17731 had a conduction velocity of 6.7 m s−1. Another afferent in the group of fibres from indomethacin pre-treated animals had a conduction velocity of 8.8 m s−1.

Figure 5. Locations of the receptive fields of ischaemically sensitive cardiac afferents on the epicardial surface of the left ventricle.

Figure 5

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The locations of the receptive fields of the afferents identified during different treatments are indicated by different symbols as follows: □, BK and des-Arg9-BK (n = 11); ▪, HOE140 (n = 12); *, NPC-17731 (n = 6); •, NPC-17731 following indomethacin pretreatment (n = 6); ▴, repeated ischaemia (n = 6); ▵, BK following administration of indomethacin (n = 6); ^, indomethacin (n = 7).

DISCUSSION

The present study demonstrates, for the first time, the role of kinin B2 receptors in the stimulation of cardiac afferents during regional myocardial ischaemia. We observed that intracardiac injection of BK, but not des-Arg9-BK (a specific kinin B1-receptor agonist), significantly increased the activity of ischaemically sensitive cardiac afferents. Furthermore, the discharge activity of sympathetic cardiac afferents during ischaemia was attenuated significantly after treatment with either of two structurally dissimilar kinin B2 receptor antagonists. Finally, we found that cyclooxygenase products were not required for the BK-induced increase in activity seen for most ischaemically sensitive cardiac afferents. Therefore, the present study demonstrates that endogenous BK activates ischaemically sensitive cardiac afferents during myocardial ischaemia through the stimulation of kinin B2 receptors. In addition, BK primarily activates ischaemically sensitive cardiac afferents through a mechanism that is independent of cyclo-oxygenase products.

Bradykinin generally has been considered to be a potential chemical mediator in cardiac pain (Baker et al. 1980). Kimura et al. (1973) showed an increase in the concentration of BK in the coronary sinus within 2–5 min following occlusion of the left anterior descending coronary artery in dogs. Hashimoto et al. (1977) also detected an increase in BK concentration within 2 min in the coronary sinus following less severe cardiac ischaemia by occlusion of the anterior descending branch of the left coronary artery. Furthermore, exogenous BK has been shown to stimulate cardiac visceral afferent nerve endings (Nishi et al. 1977; Nerdrum et al. 1986). Conscious dogs respond with a decrease in blood pressure to large doses of BK administered into the coronary artery, but do not display pain-related behaviour (Pagani et al. 1986). In a similar study in humans (Schaefer et al. 1996) we have demonstrated that administration of exogenous BK into the coronary artery causes arterial hypotension and atypical (for angina) chest pain in patients both with and without coronary artery disease. However, the role of endogenous BK in activation of sympathetic ischaemically sensitive cardiac afferents has not been studied. To produce ischaemia sufficient to induce a rise in BK concentration we occluded for 5 min a coronary vessel supplying the area of the myocardium innervated by afferents. The present study demonstrates that exogenous and endogenous BK stimulate cardiac afferents that are ischaemically sensitive. Together these data suggest that BK may play a role in stimulating cardiac afferents during ischaemia and the subsequent reflex response. It is unclear if BK is responsible for cardiac pain (i.e. angina pectoris), although our data show that BK is capable of stimulating cardiac C fibres in spinal pathways that probably could transmit nociceptive responses during myocardial ischaemia.

Prostaglandins have been shown to sensitize cutaneous nociceptors (Ferreira, 1972), as well as ischaemically sensitive abdominal visceral afferents (Longhurst & Dittman, 1987). We previously have demonstrated that BK activates the cyclo-oxygenase system (Pan et al. 1994), which, in turn, augments afferent activity during abdominal ischaemia (Longhurst & Dittman, 1987; Longhurst et al. 1991). Bradykinin stimulates phospholipase A2 to generate arachidonic acid (Rang et al. 1991). Arachidonic acid formation leads to the synthesis of cyclo-oxygenase products, i.e. PGs. Prostaglandins have been shown to augment afferent activity during abdominal ischaemia (Longhurst & Dittman, 1987; Longhurst et al. 1991; Pan et al. 1994). Elevated plasma concentrations of PGs have also been found in patients with unstable angina and during myocardial ischaemia (Berger et al. 1977; Hirsh et al. 1981). Nerdrum et al. (1986) demonstrated that cyclo-oxygenase products are of importance in sensitizing cardiac afferents to the action of exogenous BK. However, the interaction between endogenous BK and PGs in activation of sympathetic cardiac afferent nerve endings during myocardial ischaemia is still unknown. The present study has shown that indomethacin reduced the visceral afferent responses to either ischaemia or BK and that inactivation of kinin B2 receptors further decreased the responses of ischaemically sensitive cardiac afferents in cats pretreated with indomethacin (5 mg kg−1). We previously have shown that this dose of indomethacin effectively inhibits the response of visceral afferents to stimulation with endogenous PGs (Longhurst et al. 1991). Only a slight decrease in arterial blood pressure was noted after indomethacin was administered. Thus, endogenous BK activates cardiac afferents during myocardial ischaemia through a mechanism that, in part, is independent of PGs.

During myocardial ischaemia all afferents studied were found to be partially independent of the activation of PGs. In this regard, we observed that the kinin B2 receptor antagonist substantially decreased the cardiac afferent responses in all six animals pretreated with indomethacin. Conversely, in our time control study repeated ischaemia yielded consistent responses from the afferents. In contrast to our observations in the heart, activation of the cyclo-oxygenase system in the abdominal region is a necessary part of the BK-related response for the majority (80 %) of abdominal visceral afferents during ischaemia (Pan et al. 1994). Similar to the results from Pan et al. (1994), we observed that ischaemically sensitive cardiac afferents are more difficult to locate in indomethacin-pretreated animals. Overall, however, the present study shows that cardiac visceral afferents are less dependent upon the action of cyclo-oxygenase products than abdominal visceral afferents during ischaemia.

Bradykinin binds to two receptor subtypes: BK1 and BK2. Through activation of BK1 and BK2 receptors, BK stimulates a large number of cell types including fibroblasts, endothelial cells, macrophages, kidney cells and neurons (Bhoola et al. 1992). Moreover, BK is known to stimulate visceral chemosensitive nerve endings (Lew & Longhurst, 1986). The application of BK to the epicardium of the heart excites some, but not all, unmyelinated and myelinated afferent endings of the spinal sympathetic nerves (Baker et al. 1980). Increased sympathetic spinal afferent impulse activity causes reflex tachycardia and increases in blood pressure (Uchida & Murao, 1974; Staszewska-Barczak et al. 1976; Baker et al. 1980). The reflex cardiovascular responses of BK applied to the epicardium are either abolished or attenuated by a BK2 receptor antagonist (Staszewska-Woolley & Woolley, 1989). Furthermore, as we have shown, the increase in impulse activity of sympathetic cardiac afferents during myocardial ischaemia is significantly reduced by HOE140 and NPC-17731, both BK2 receptor antagonists. Therefore, we have provided neurophysiological data showing that the stimulating effect of endogenous BK during myocardial ischaemia on ischaemically sensitive cardiac afferents is mediated by activation of BK2 receptors.

In conclusion, the results of this study show that BK stimulates cardiac afferents through the activation of BK2 receptors during myocardial ischaemia. Unlike abdominal visceral afferents, stimulation by BK of cardiac afferents during ischaemia appears less dependent upon an intact cyclo-oxygenase system.

Acknowledgments

This study was supported by NIH grants HL52165-01, HL36527, HL07682-04. H.-L. P. was a research fellow of the American Heart Association, California Affiliate. S. C. T.-A.-L. is currently a postdoctoral fellow of the National Institutes of Health.

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