Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 μM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP3) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca2+ stores) but not in the Ca2+-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP3, and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca2+ are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.
- cardiac afferents
- dorsal root ganglia
- transient receptor potential vanilloid type 1
primary afferent neurons innervating the heart transmit sensory signals generated by cardiac sensory receptors (2, 6, 29, 45). The cardiac sensory afferents and their cell bodies in the dorsal root ganglion (DRG) are generally considered to be the essential pathways for transmission of cardiac nociception to the dorsal horn of the upper thoracic spinal cord (21, 45). Increased production of certain metabolites, such as protons and bradykinin, during myocardial ischemia has been proposed to contribute to excitation of cardiac nociceptors (2, 6, 28, 31). In this regard, there is a significant increase in bradykinin release in the cardiac interstitial fluid during ischemia (30). Furthermore, bradykinin plays an important role in stimulation of cardiac afferents during myocardial ischemia (29, 38). However, it is not fully known how bradykinin activates and/or sensitizes cardiac nociceptors.
A long-onset latency is associated with the effect of bradykinin on cardiac spinal afferents (28, 31, 38), suggesting that a cascade of intracellular signaling events is involved in the stimulating effect of bradykinin. Various signaling pathways have been suggested to mediate the stimulating effect of bradykinin on cutaneous sensory neurons (12, 17, 32, 34). For example, activation of bradykinin B2 receptors results in stimulation of phospholipase C (PLC), followed by a transient increase in inositol 1,4,5-trisphosphate (IP3) production, which can depolarize cells associated with an increase in intracellular Ca2+ and a large acceleration of spike frequency (12). Transient receptor potential vanilloid type 1 (TRPV1) channels have been proposed to be another potential effector of bradykinin on primary sensory neurons (34). However, the distribution of many important molecules, such as TRPV1, P2X receptors, voltage-gated ion channels, and protein kinase C (PKC), varies among the different subtypes of DRG neurons innervating the somatic and visceral tissues (14, 16, 25, 43, 47, 48, 50). The signaling mechanisms involved in the stimulating effect of bradykinin on cardiac DRG neurons are little known. We have shown that afferent nerve endings innervating the surface of the rat heart possess TRPV1 (49). Iodoresiniferatoxin, a selective antagonist of TRPV1, attenuates both bradykinin- and ischemia-induced firing of cardiac spinal afferent nerves (29). Furthermore, the effect of intrapericardial application of bradykinin on spinal cord sensory neurons is dependent on TRPV1-containing afferent fibers (33). Although bradykinin increases intracellular Ca2+ and 12-lipoxygenase metabolites in DRG neurons (4, 34, 37), their respective roles in bradykinin stimulation of cardiac nociceptors remain poorly understood. In the present study, we used cardiac DRG neurons as the cell model to determine the contribution of TRPV1, 12-lipoxygenase, and intracellular Ca2+ to excitation of cardiac nociceptors by bradykinin.
Retrograde labeling of cardiac DRG neurons.
Male Sprague-Dawley rats (6–8 wk old; Harlan, Indianapolis, IN) were used in this study. Rats were anesthetized with pentobarbital sodium (60 mg/kg ip), placed in a supine position, and mechanically ventilated with a rodent animal respirator (Life Science Instruments, Woodland Hills, CA). A limited left thoracotomy was performed to expose the heart. A fluorescent retrograde tracer, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate (DiI; 60 mg/ml; Molecular Probes, Eugene, OR), was injected (10 μl in 2–3 injections) into the anterior ventricular wall of the heart. After injection, the ribs were approximated, the thoracic cavity was evacuated, and the incision was closed in layers. The animals were returned to their cages for 4–5 days to permit the retrograde tracer to be transported to DRG neurons. Rats were treated prophylactically with an antibiotic (enrofloxacin, 5 mg/kg sc daily for 3 days) and analgesic (buprenorphine, 0.2–0.5 mg/kg sc every 12 h for 2 days). All procedures conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of The University of Texas M. D. Anderson Cancer Center.
Isolation of DRG neurons.
Rats were reanesthetized with pentobarbital sodium (60 mg/kg ip) and then rapidly decapitated. Because the afferent fibers innervating the heart have their cell bodies located in the DRG from T1 to T6 (21), the thoracic segments (T1–T6) of the vertebrate column were dissected. The DRGs were quickly removed and transferred immediately into Dulbecco's modified Eagle's medium (DMEM; GIBCO, Carlsbad, CA). After removal of attached nerves and surrounding connective tissues, the DRGs were placed in a flask containing 5 ml of DMEM in which trypsin (type I, 0.5 mg/ml; Sigma, St. Louis, MO) and collagenase (type IA, 1 mg/ml; Sigma) had been dissolved. After incubation at 34°C in a shaking water bath for 30 min, soybean trypsin inhibitor (type II-s, 1.25 mg/ml; Sigma) was then added to stop trypsin action. The cell suspension was centrifuged (500 rpm, 5 min) to remove the supernatant and replenished with DMEM. Cells were then plated onto a 35-mm culture dish containing poly-l-lysine (50 μg/ml; Sigma)-precoated coverslips and kept for at least 30 min before electrophysiological recordings. Because neurons following prolonged neuronal culture produce neurites, recordings were made within 6 h after dissociation to keep the experiment as similar to in vivo conditions as possible.
Recordings of action potentials were performed using the whole cell current-clamp method as we described previously (48). The recording electrodes with a resistance of ∼2 MΩ were pulled from GC150TF-10 glass capillaries (inner diameter 1.17 mm, outer diameter 1.5 mm; Harvard Apparatus, Holliston, MA) with a micropipette puller (Sutter Instrument, Novato, CA) and fire-polished (DMF1000; World Precision Instruments, Sarasota, FL). All experiments were performed at room temperature. Whole cell recordings from DiI-labeled DRG neurons were made using an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany) under visual control by using a combination of fluorescence illumination and differential interference contrast (DIC; ×20–40) optics on an inverted microscope (Olympus Optical, Tokyo, Japan). Images of cells were taken with a charge-coupled device camera and displayed on a video monitor. Briefly, DiI-labeled neurons were first identified with the aid of fluorescence illumination (Fig. 1). A tight gigaohm seal was subsequently obtained in the selected neuron under DIC. Cells were recorded using whole cell current-clamp at 0 pA. The intracellular solution contained (in mM) 124 KCl, 2 MgCl2, 13.2 NaCl, 1 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Na-GTP (pH 7.2 adjusted with KOH, osmolarity 300 mosM). The bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH, osmolarity 320 mosM).
Before the effect of bradykinin was determined, the baseline membrane potential and firing frequency of DiI-labeled DRG neurons were recorded for 30 s. Because most DRG neurons are “silent” (48), a series of currents (0–200 pA with 10-pA increments, 200-ms duration) was injected to elicit action potentials. This procedure took 60 s to perform. We then applied 1 μM bradykinin for 90 s to examine the effect of 1 μM bradykinin on DiI-labeled DRG neurons. The cells with increased frequency and/or decreased threshold following bradykinin application were considered bradykinin sensitive. Subsequently, the spontaneous or current-elicited firing was determined every 2 min after bradykinin application. Since the effect of bradykinin reached the peak in 3–4 min after its application, data recorded at 7–9 min after bradykinin application was discontinued were used as the washout control.
The drug solution was delivered to the recording chamber by gravity. Most chemicals were dissolved in distilled water at 1,000 times the final concentration. Iodoresiniferatoxin, thapsigargin, BAPTA-AM, and baicalein were initially dissolved in DMSO to make the stock solution. The stock solutions were further diluted in the extracellular solution just before use and held in a series of independent syringes connected to an array of corresponding fused silica columns. The exchange of solutions was achieved rapidly by shifting the tubes horizontally with a micromanipulator. The distance from the column mouth to the cell examined was ∼100 μm. Cells in the recording chamber were continuously bathed in extracellular solution. Drugs and chemicals were purchased from Sigma-Aldrich, except iodoresiniferatoxin (Tocris, Bristol, UK) and DiI (Molecular Probes).
The action potential and membrane potential were analyzed using MiniAnalysis software (Synaptosoft, Fort Lee, NJ). The action potential threshold was defined as the lowest current injected that elicited an action potential with an overshoot. The firing frequency was calculated as the maximal number of action potentials elicited during the period of current injection. Data are means ± SE. All comparisons between means were tested for significance using Student's paired t-test or repeated-measures ANOVA. P < 0.05 was considered to be statistically significant.
A total of 72 rats was used in this study. The average number of DiI-labeled cells recovered was 147 ± 27 per animal in seven rats in our study. The size of DiI-labeled DRG neurons ranged from 15 to 35 μm in diameter. Spontaneous action potentials were recorded or action potentials were elicited by injection of a series of depolarizing currents (from 0 to 200 pA in 10-pA increments for 200 ms). In all DiI-labeled DRG neurons tested, ∼70.1% (132/186) responded to 1 μM bradykinin, showing a decrease in the threshold firing by injected currents and/or an increase in the spontaneous firing frequency. This effect was observed in 10–30 s and reached the peak at ∼3–4 min after bradykinin treatment. A total of 78 DiI-labeled DRG neurons that responded to bradykinin were included in the following eight protocols. Other cells that either responded to bradykinin but failed to complete the entire protocol (n = 54) or showed no response to 1 μM bradykinin (n = 54) were not included in this study.
Effect of bradykinin on DiI-labeled DRG neurons.
We first tested the response of DiI-labeled DRG neurons to repeated applications of bradykinin. Application of 1 μM bradykinin for 90 s significantly increased the frequency of action potentials from 2.2 ± 0.4 to 4.2 ± 0.5/200 ms, whereas the threshold decreased from 63.3 ± 16.8 to 38.7 ± 8.8 pA (Fig. 2). The bradykinin application also depolarized the membrane potential (from −58.9 ± 2.6 to −54.4 ± 2.7 mV, P < 0.05). In 3 of 12 DiI-labeled cells, bradykinin triggered the firing (n = 2) or increased the frequency of spontaneous action potentials (n = 1) without current injection. In another nine silent cells, the frequency was increased and/or less current injection was required after bradykinin application (Fig. 2). We then treated these neurons with 1 μM bradykinin again after washout for 8–10 min following the first application. The response of bradykinin was evoked reproducibly in the same 12 cells. Following the second application of bradykinin, the frequency of action potentials was increased from 2.5 ± 0.4 to 4.3 ± 0.6/200 ms, and the threshold was decreased from 66.6 ± 15.6 to 33.3 ± 10.5 pA (Fig. 2). However, 1 μM bradykinin did not produce any currents in all labeled DRG neurons at a holding potential of −60 mV in our study.
Role of TRPV1 in the effect of bradykinin on cardiac DRG neurons.
Work in our laboratory (29) has shown that both bradykinin- and ischemia-induced firing of cardiac afferent nerves are attenuated by iodoresiniferatoxin, a highly specific and cell-permeable TRPV1 antagonist (44, 46). We next determined the role of TRPV1 in bradykinin-induced stimulation/sensitization in DiI-labeled DRG neurons. After washout of the initial effect of bradykinin on 15 bradykinin-sensitive neurons, 300 nM iodoresiniferatoxin was applied for 2 min. Iodoresiniferatoxin alone did not produce any effect on the excitability of DRG neurons. In the presence of 300 nM iodoresiniferatoxin, the second application of 1 μM bradykinin did not significantly increase the frequency of action potentials or the threshold (n = 15, Fig. 3).
Role of 12-lipoxygenase in the effect of bradykinin on cardiac DRG neurons.
The 12-lipoxygenase products produced by bradykinin have been considered as the endogenous TRPV1 agonist to activate TRPV1 channels in primary sensory neurons (34). We thus examined the effect of bradykinin before and 5 min after application of baicalein, a specific 12-lipoxygenase inhibitor (8), in another nine DiI-labeled DRG neurons. Compared with the initially excitatory effect of bradykinin recorded in the absence of baicalein, bath perfusion of 10 μM baicalein significantly attenuated the effect of bradykinin on the excitability of the DiI-labeled DRG cells (Fig. 4). Following the second application of bradykinin, the frequency of action potentials was increased from 2.4 ± 0.4 to 3.1 ± 0.5/200 ms, and the threshold was decreased from 55.6 ± 13.2 to 46.7 ± 13.3 pA (n = 9, Fig. 4).
Role of extracellular and intracellular Ca2+ in the effect of bradykinin on cardiac DRG neurons.
The TRPV1 channel is highly permeable to Ca2+ (9). Bradykinin also substantially increases intracellular Ca2+ in DRG neurons (4). We therefore determined the role of intracellular Ca2+ in the excitatory effect of bradykinin on cardiac DRG neurons. BAPTA-AM is a potent, membrane-permeable Ca2+ chelator (40). Following the washout of the initial effect of bradykinin on eight DiI-labeled DRG neurons, 30 μM BAPTA-AM was perfused for 5 min before the effect of 1 μM bradykinin was reexamined. In all eight bradykinin-sensitive neurons tested, 1 μM bradykinin failed to alter significantly the threshold (70 ± 17.7 to 67.5 ± 17.3 pA) and frequency (from 2.8 ± 0.4 to 2.8 ± 0.4/200 ms) of action potentials following application of 30 μM BAPTA-AM (Fig. 5A).
To determine the role of extracellular Ca2+ in the excitatory effect of bradykinin on the cardiac DRG neurons, we examined the effect of bradykinin in the Ca2+-free external solution. Under this condition, application of 1 μM bradykinin still significantly increased the firing activity and decreased the threshold in eight DiI-labeled neurons (Fig. 5, B–D).
We also assessed the role of extracellular Ca2+ in the stimulatory effect of capsaicin on the cardiac DRG neurons. In another seven DiI-labeled DRG neurons, 1 μM capsaicin profoundly increased the firing activity and membrane depolarization in the Ca2+-free solution (Fig. 6).
Bradykinin activates kinin B2 receptors to cause a transient increase in IP3 production (11, 12). Both the TRPV1 and IP3 can cause the Ca2+ release from the endoplasmic reticulum (ER) (19, 20). Subsequently, we determined whether the Ca2+ release from the ER contributes to the effect of bradykinin. Thapsigargin (10 μM), a specific Ca2+-ATPase inhibitor to deplete intracellular Ca2+ stores (36), was applied for 5 min in 12 DiI-labeled bradykinin-sensitive DRG neurons. Interestingly, thapsigargin itself depolarized the membrane potential from −52.1 ± 2.5 to −47.0 ± 2.1 mV and excited all the cells tested (Fig. 7). This effect lasted for 3–5 min. After washout of the initial effect of thapsigargin for 7 min, cells were again tested with 1 μM bradykinin. The stimulatory effect of bradykinin on all 12 DiI-labeled DRG neurons was abolished after thapsigargin treatment (Fig. 7).
Role of IP3 in the effect of bradykinin on cardiac DRG neurons.
An increase in IP3 production can release the Ca2+ from the ER (20). We further determined whether IP3 produced by bradykinin contributes to the stimulating effect of bradykinin on cardiac DRG neurons. 2-Aminoethyl diphenylborinate (2-APB; 100 μM), a membrane-permeable antagonist of IP3 receptors (24), was used in eight DiI-labeled bradykinin-sensitive DRG neurons. After the initial effect of bradykinin was tested, cells were treated with 2-APB for 5 min. Notably, 2-APB alone caused initial excitation in six of eight labeled DRG neurons. This effect declined over time and disappeared 20–40 s after washout. The stimulating effect of 1 μM bradykinin on cardiac DRG neurons was significantly attenuated by 2-APB (Fig. 8). Following the second application of bradykinin, the frequency of action potentials was increased from 2.9 ± 0.6 to 4.0 ± 0.9/200 ms, and the threshold was decreased from 71.3 ± 17.0 to 57.5 ± 13.3 pA (n = 8, Fig. 8).
Lack of bradykinin-elicited currents in DiI-labeled DRG neurons.
To further determine whether bradykinin produces a current at different holding potentials, we tested the effect of bradykinin in six additional DiI-labeled DRG neurons. In all of these six cells examined, application of 1 μM bradykinin increased the firing activity but did not produce any current at a holding potential from −60 to 20 mV (Fig. 9, A and B). On the other hand, bath application of 1 μM capsaicin readily elicited an inward current in these cells (Fig. 9B).
This is the first study investigating the signaling mechanism responsible for the excitatory effect of bradykinin on cardiac sensory neurons. We found that bradykinin increased the firing frequency and lowered the threshold for generation of action potentials in cardiac DRG neurons labeled by a retrograde tracer. Iodoresiniferatoxin, a highly specific TRPV1 antagonist, attenuated the effect of bradykinin. Furthermore, the excitatory effect of bradykinin on cardiac DRG neurons was significantly reduced by baicalein, a selective inhibitor of 12-lipoxygenase. We also observed that the excitatory effect of bradykinin on cardiac DRG neurons was abolished by treatment with BAPTA-AM or thapsigargin, but not in the Ca2+-free extracellular solution. In addition, 2-APB, a specific IP3 antagonist, attenuated the stimulating effect of bradykinin on cardiac DRG neurons. Thus this study provides new information that 12-lipoxygenase products and TRPV1 channels play an important role in excitation of cardiac nociceptors by bradykinin. The increase in the intracellular Ca2+, through TRPV1 channels and IP3 receptors, is largely responsible for stimulation/sensitization of cardiac nociceptors by bradykinin.
Bradykinin receptors are constitutively expressed in DRG neurons (5, 22). Bradykinin can consistently increase the firing of cardiac spinal afferent fibers (28, 38). Also, blocking of kinin B2 receptors reduces the response of cardiac spinal afferents to myocardial ischemia (38). Previous studies have shown that it takes more than 5–10 s before the excitatory effect of bradykinin on cardiac spinal afferents can be detected (29, 38). Although a cascade of intracellular signaling events may be involved, the signaling mechanisms for the stimulatory effect of bradykinin on cardiac nociceptors are not clear. We thus used cardiac DRG neurons as the cell model to explore the signaling mechanisms involved in the bradykinin effect on cardiac nociceptors. In this study, bradykinin sensitized or excited cardiac DRG neurons with a slight depolarization of membrane potentials. Although almost all cardiac afferent fibers respond vigorously to bradykinin applied to the heart (29, 38), we observed a relatively smaller effect of bradykinin on the membrane potential of DiI-labeled DRG neurons in this study. In the bipolar DRG neurons, their soma has a much larger capacitance than the hillock and axon region. As a result, a small change in the membrane potential in the soma by bradykinin may reflect a greater depolarization at the site of afferent nerve endings.
Several ion channels may serve as the final effector following bradykinin receptor activation. In isolated neonatal DRG neurons, bradykinin evokes an inward current attributed to the opening of Na+ channels in a PKC-dependent manner (7). Furthermore, TRPV1, an ion channel that serves as the molecular target for capsaicin, also has been proposed to be another potent effector of bradykinin (34). TRPV1 is mainly located on small-sized DRG neurons and is considered an important sensor for noxious heat (9, 39). The primary sequence of TRPV1 predicts many putative phosphorylation sites for a variety of protein kinases, most notably PKC (26). It is well known that PKC can enhance TRPV1-mediated responses in DRG neurons (3, 10, 26, 32, 42). Work in our laboratory (49) has shown that the afferent nerve endings innervating the surface of the rat heart possess TRPV1. Our group (29) also has shown that the TRPV1 antagonist iodoresiniferatoxin attenuates both bradykinin- and ischemia-induced firing of cardiac spinal afferent nerves. In the present study, iodoresiniferatoxin significantly reduced the stimulatory effect of bradykinin on cardiac DRG neurons. These data provide further evidence that TRPV1 plays an important role in the effect of bradykinin on cardiac nociceptors.
Furthermore, the 12-lipoxygenase metabolites of arachidonic acids may be the endogenous agonist for TRPV1 (34) and may be involved in the bradykinin effect on cutaneous nociceptors (1). In this regard, bradykinin can stimulate synthesis of 12-hydroxyeicosatetraenoic acid, which by itself is an immediate metabolite of 12-lipoxygenase and one of the potent lipid agonists for TRPV1 (17, 34). We found that baicalein, a selective 12-lipoxygenase inhibitor (8), significantly reduced the effect of bradykinin on DiI-labeled DRG neurons. Also, Ca2+ released from the intracellular stores may be necessary to activate 12-lipoxygenase. This is because the activity of PLA2, the enzyme using free arachidonic acids as the substrate to produce cyclooxygenase and lipoxygenase products (23), is regulated by intracellular Ca2+ (13). Thus it is possible that 12-lipoxygenase products can activate or sensitize TRPV1 channels present in cardiac DRG neurons following activation of bradykinin receptors.
Because TRPV1 is highly permeable to Ca2+ (9, 39), activation of TRPV1 induces substantial Ca2+ influx into the DRG cells. The increase in intracellular Ca2+ caused by bradykinin has been demonstrated in DRG neurons (4, 37). In our study, application of BAPTA-AM, a rapid Ca2+ chelator (40), abolished the effect of bradykinin on DiI-labeled DRG neurons. Hence, the rise in intracellular Ca2+ appears to be the key event in bradykinin-induced excitation of cardiac DRG neurons. Although these data suggest the importance of intracellular Ca2+ in the effect of bradykinin on cardiac nociceptors, they do not discriminate the sources for the increase in intracellular Ca2+. It has been demonstrated that TRPV1 is located on both the plasma membrane and ER (19, 27). Consequently, we further examined the relative importance of extracellular Ca2+ and intracellular Ca2+ stores in the effect of bradykinin on cardiac DRG neurons. We observed that the stimulatory effect on DiI-labeled DRG neurons was not significantly reduced in the Ca2+-free extracellular solution. Also, stimulation of TRPV1 with capsaicin caused a profound excitation of DiI-labeled DRG neurons in the Ca2+-free solution. These findings suggest that extracellular Ca2+ is not essential for the excitatory effect of bradykinin on cardiac DRG neurons. However, we found that depletion of intracellular Ca2+ with thapsigargin, a highly specific inhibitor of Ca2+-ATPases (36), abolished the bradykinin effect on cardiac DRG neurons. Interestingly, thapsigargin alone excited DRG neurons with slight membrane depolarization, which is likely caused by an initial release of Ca2+ to the cytoplasm from the ER. It has been shown that thapsigargin increases intracellular anandamide in a Ca2+-dependent manner (41). It is possible that thapsigargin may activate TRPV1 to excite DRG neurons. Stimulation of kinin B2 receptors also results in activation of PLC, followed by a transient increase in IP3 production (11, 12), which can release the Ca2+ from the ER. In our study, 2-APB, a selective IP3 receptor antagonist (24), significantly reduced the stimulatory effect of bradykinin on cardiac DRG neurons. Because 2-APB can briefly induce the Ca2+ release (35), this can explain the initial excitatory effect of 2-APB on cardiac DRG neurons. Therefore, the excitatory effect of bradykinin on cardiac DRG neurons is probably mediated largely by Ca2+ released from the IP3- and TRPV1-sensitive ER.
Bradykinin-produced currents have been reported in only a small proportion (<13%) of thoracic and lumbar DRG neurons (34). It is possible that cardiac DRG neurons may possess different membrane properties compared with other subtypes of DRG neurons. Notably, bradykinin did not produce any current in bradykinin-sensitive DRG neurons at a holding potential from −60 to 20 mV in our study. Interestingly, it has been shown that the bradykinin-induced inward current appears to require a direct contact between DRG neurons and nonneuronal satellite cells (15). This could explain why the inward current was not observed in isolated cardiac DRG neurons in our study. Because the afferent nerve endings are largely inaccessible to the recording electrode, it is technically impossible to manipulate the cellular environment within the afferent terminals. It is not clear to what extent the ion channels or kinin receptors present on the soma of DRG cells can reflect those located at the afferent terminals. Therefore, cautions must be taken in extrapolating the finding of this study on cardiac DRG neurons. However, several lines of evidence suggests that the ion channels and receptors on the DRG neurons are similar to those at the nerve terminals. For instance, bradykinin can activate DRG neurons, afferent nerves, or the dorsal root (18). Also, both bradykinin receptors and TRPV1 channels are expressed in both the DRG soma and cardiac afferent terminals (49). Thus cardiac DRG neurons are the relevant and useful cell model to study the intracellular signaling pathways mediated the effect of bradykinin.
In summary, this study provides new evidence that TRPV1 and 12-lipoxygenase products activated by bradykinin play an important role in excitation/sensitization of cardiac DRG neurons. The increase in the intracellular Ca2+ released from the IP3- and TRPV1-sensitive ER seems critical for stimulation/sensitization of cardiac nociceptors by bradykinin. Bradykinin also activates tetrodotoxin-resistant Na+ channels in primary afferent fibers in a PKC-dependent manner (7). These findings are important for our understanding of the signaling mechanisms responsible for excitation of cardiac nociceptors by bradykinin.
This study was supported by National Heart, Lung, and Blood Institute Grants HL-60026 and HL-77400.
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