Bradykinin (BK) activates sensory nerves and causes hyperalgesia. Transient receptor potential A1 (TRPA1) is expressed in sensory nerves and mediates cold, mechanical, and chemical nociception. TRPA1 can be activated by BK. TRPA1 knockout mice show impaired responses to BK and mechanical nociception. However, direct evidence from sensory nerve terminals is lacking. This study aims to determine the role of TRPA1 in BK-induced visceral mechanical hypersensitivity. Extracellular recordings of action potentials from vagal nodose and jugular neurons are performed in an ex vivo guinea pig esophageal-vagal preparation. Peak frequencies of action potentials of afferent nerves evoked by esophageal distension and chemical perfusion are recorded and compared. BK activates most nodose and all jugular C fibers. This activation is repeatable and associated with a significant increase in response to esophageal distension, which can be prevented by the B2 receptor antagonist WIN64338. TRPA1 agonist allyl isothiocyanate (AITC) activates most BK-positive nodose and jugular C fibers. This is associated with a transient loss of response to mechanical distensions and desensitization to a second AITC perfusion. Desensitization with AITC and pretreatment with TRPA1 inhibitor HC-030031 both inhibit BK-induced mechanical hypersensitivity but do not affect BK-evoked activation in nodose and jugular C fibers. In contrast, esophageal vagal afferent Aδ fibers do not respond to BK or AITC and fail to show mechanical hypersensitivity after BK perfusion. This provides the first evidence directly from visceral sensory afferent nerve terminals that TRPA1 mediates BK-induced mechanical hypersensitivity. This reveals a novel mechanism of visceral peripheral sensitization.
- transient receptor potential A1
- visceral hypersensitivity
abnormal esophageal sensations, such as heartburn and esophageal-related noncardiac chest pain, are common complaints (12). They result from noxious stimuli (mechanical, chemical, and thermal stimuli) that are transduced into action potentials by esophageal primary sensory afferents and transmitted to the central nervous system via both spinal and vagal pathways (27, 28, 31, 32, 33, 40). These esophageal sensations are enhanced after tissue injury or inflammation through mechanisms that involve both peripheral and central sensitization of sensory inputs, resulting in visceral hypersensitivity (22, 13). Peripheral sensitization plays an important role in this process but is less understood.
Visceral sensory afferent nerves provide gastrointestinal sensory inputs in both physiological and noxious ranges. Those that sense noxious stimuli are referred to as nociceptors (17, 37). Gastrointestinal primary afferent nociceptive nerves usually are polymodel with their cell bodies situated not only in dorsal root ganglion (DRG) but also in nodose and jugular ganglia, with their nerve terminals distributed in the peripheral organs. These nociceptive nerves can transduce mechanical, chemical, and thermal stimuli from nerve terminals and transmit impulses via spinal and vagal pathways to the central nervous system to induce nociception. Under certain conditions, such as tissue damage or inflammation, these processes are enhanced by peripheral and central sensitization and result in visceral hyperalgesia (13, 22).
Certain mediators have been proved to sensitize visceral afferent nociceptive nerves. Among them, bradykinin (BK) is well studied (36). BK is cleaved from the plasma precursor kininogen by kininogenase during inflammation or tissue injury. BK, via its G protein-coupled receptor, not only activates sensory afferent nerves but also causes an increase in their excitability to other stimuli. This process involves multiple intracellular signaling pathways which lead to interactions with downstream ion channels (15). The exact role of those ion channels involved in visceral nociceptor sensitization is still to be determined. Several transient receptor potential (TRP) ion channels have recently been identified on sensory nerves to involve nociception transduction (35). Transient receptor potential A1 (TRPA1), a new member of the TRP ion channel family, is expressed in sensory nerves and mediates cold, mechanical, and chemical nociception (3, 7, 16, 34). TRPA1 can be activated by pungent compounds and BK (3, 16). TRPA1 knockout mice show impaired responses to BK and mechanical nociception and to BK-induced thermal or somatic mechanical hyperalgesia (4, 19). These suggest a role for TRPA1 in BK-induced mechanical hypersensitivity. These studies were performed on disassociated sensory neurons and animal behavior models for somatic pain. Direct electrophysiological evidence from sensory afferent nerve terminals, especially from innervated visceral organs, is lacking.
Visceral sensory afferent nerves are derived from two distinct embryonic tissues: cranial placodes and neural crest (2, 11). Placode-derived sensory nerves have their neuronal cell bodies in vagal nodose ganglia. Neurocrest-derived sensory neurons are located in vagal jugular (supranodose) ganglia and spinal DRG. The guinea pig is the smallest laboratory mammal in which nodose and jugular ganglia can be readily distinguished. This allows the study of nerves derived from the two embryonic origins in one preparation. We have developed an ex vivo guinea pig esophageal-vagal preparation for extracellular single-fiber recording to study esophageal vagal sensory afferent activity. In this model, we have shown that vagal nociceptive-like nerve fibers in the guinea pig esophagus are derived from both nodose and jugular ganglia and include nodose C fibers and jugular C/Aδ fibers, with distinctive responses to mechanical distension and chemical stimulation by P2X and TRPV1-receptor agonists (39). We also demonstrated that mediators released by esophageal mast cell degranulation sensitize esophageal sensory afferent nodose C fibers but not Aδ and induce mechanical hypersensitivity to esophageal distension (38).
In the present study, we tested the hypothesis that esophageal vagal sensory afferent nerve fiber subtypes respond differently to BK and to a TRPA1 agonist and that TRPA1 mediates BK-induced mechanical hypersensitivity in esophageal nociceptive subtype afferent nerves. Our data support the hypothesis that BK selectively activates esophageal nodose and jugular unmyelinated C fibers, and most of them also respond to the TRPA1 agonist allyl isothiocyanate (AITC). BK activation in these subtype C fibers is associated with a mechanical hypersensitivity to esophageal distension. This increase can be prevented by a B2 antagonist, attenuated by TRPA1 desensitization, and inhibited by a TRPA1 inhibitor. These results provide the first evidence directly from visceral sensory afferent C fiber nerve terminals that TRPA1 mediates BK-induced mechanical hypersensitivity.
MATERIALS AND METHODS
Male Hartley guinea pigs (Hilltop Laboratory Animals, Scottsdale, PA) weighing 100–300 g were used. All experiments were approved by the Pennsylvania State University Animal Care and Use Committee.
Extracellular single-fiber recordings from the vagal afferent nerves innervating the esophagus.
The esophageal-vagal preparation was set up and extracellular single-nerve fiber recordings were performed as previously described (38, 39). The esophagus and trachea were dissected with intact bilateral extrinsic vagal innervation (including jugular and nodose ganglia). The tissue was pinned in a small Sylgard-lined Perspex chamber filled with Krebs solution (35°C) [KBS, composed of (in mM) 118 NaCl, 5.4 KCl, 1.0 NaH2PO4, 1.2 MgSO4, 1.9 CaCl2, 25.0 NaHCO3, and 11.1 dextrose; gassed with 95% O2-5% CO2]. The chamber has two compartments: the esophagus with attached trachea (to support the recurrent laryngeal nerves) and the vagus were pinned in the tissue compartment, and the rostral aspect of the vagus nerves including the nodose and jugular ganglia were pinned in the recording compartment. The two compartments were separated by a silicone grease plug and were separately superfused with KBS (pH 7.4; 35°C; 4–6 ml/min). Polyethylene tubing was inserted 3–5 mm into the cranial and caudal esophagus and secured for perfusion.
The pressure in the fluid (KBS)-filled esophagus was measured with a differential pressure transducer connected in series to the esophagus and recorded simultaneously with neural activity by the chart recorder (TA240S, Gould, Valley View, OH). Isobaric esophageal distension for 20 s with an intraluminal pressure of 10–100 mmHg separated by at least 60 s was used to determine the distension pressure-nerve activity relationship of an esophageal afferent fiber. Distension with a pressure of 10 or 60 mmHg (20 s) was routinely used to assess the viability and mechanical responsiveness of an afferent fiber during experiments.
Extracellular recordings were performed by using an aluminosilicate glass microelectrode (pulled with a Flaming-Brown micropipette puller, Sutter Instrument, Novato, CA) filled with 3 M sodium chloride (electrode resistance 2 MΩ). The microelectrode was placed into either a nodose or jugular ganglion with an electrode holder connected directly to the headstage (A-M Systems, Everett, WA). A return electrode of silver-silver chloride wire and earthed silver-silver chloride pellet were placed in the perfusion fluid of the recording compartment. The recorded signal was amplified (Microelectrode AC amplifier 1800, A-M Systems) and filtered (low cutoff, 0.3 kHz; high cutoff, 1 kHz), and the resultant activity was displayed on an oscilloscope (TDS 340, Tektronix, Beaverton, OR) and chart recorder. The data were stored and analyzed on a Macintosh computer using the software TheNerveOfIt (sampling frequency 33 kHz; PHOCIS, Baltimore, MD) and further processed by use of spreadsheet software (Microsoft Excel 2004 for Mac).
To identify a vagal afferent nerve fiber projecting to the esophagus, the recording electrode was positioned in the nodose ganglion (left or right) by use of a micromanipulator. A distension-sensitive unit was identified when esophageal distension (with a rapid increase in intraluminal pressure to 100 mmHg for 5 s) evoked an action potential discharge. The serosal surface of the esophagus was then searched with a punctate mechanical probe (Von Frey hair, 1 mN, filament diameter <0.5 mm) applied to the tissue, with a firm probe (outside diameter ∼1 mm) having already been inserted into the esophageal lumen. A mechanosensitive receptive field was located when the punctate stimulus evokes a discharge of action potentials with waveforms identical to the action potentials evoked by distension. The receptive field was then stimulated electrically (pulse duration = 1 ms, frequency = 1 Hz) via a concentric electrode inserted into the esophagus with the tip positioned at the site of the mechanosensitive receptive field. The initial voltage (100 V) was gradually reduced to the lowest voltage (threshold voltage) at which each stimulation pulse was followed by a single action potential (30–90 V for most afferent nerve fibers recorded). The waveforms of the electrically evoked action potentials are identical to those induced by distension and the punctate mechanical stimulus. Conduction time was measured as the time between the stimulation pulse and the action potential (visualized by oscilloscope). Variability of conduction time (during the train stimulation, 10 Hz, 10 s) of less than 3 ms indicates direct electrical stimulation (indirect activation of a unit by electrically evoked muscle contractions is readily discernable by high variability >20 ms of conduction time). Conduction velocity was calculated by dividing the length of the approximated nerve pathway (from the recorded nodose and jugular neurons to the mechanosensitive receptive field) by conduction time. The nerve fiber is considered a C fiber if it conducts action potentials at <1 m/s.
The chemicals diluted in KBS solution were delivered to the esophagus in the external perfusate for 10–20 min. The nerve activity (action potential discharge) are monitored continuously and analyzed in 1-s bins (yielding the number of action potentials in each second, Hz). The “peak of action potentials (Hz)” (y-axis labels in all the figures) is defined as the peak of frequency of action potentials. The response to the particular stimulus is considered positive when the stimulus-evoked action potential discharges with a peak frequency of at least 3 Hz (in the fibers with no baseline activity) or a peak frequency at least three times the frequency of baseline activity. The time to onset of the response is defined as the time elapsed between adding the drug to the tissue to the onset of the action potential discharge. The compounds used in the experiment included BK, α,β-methylene-ATP, AITC, WIN 64338 (all from Sigma-Aldrich, MO), and HC-030031 (Chembridge, CA). The stock solutions of BK (10 mM), α,β-methylene-ATP (10 mM), WIN64338 (10 mM), and HC-030031 (10 mM) were stored at −20 and 4°C, respectively, and diluted in KBS to final concentration on the day of use.
In one series of studies, the responses of esophageal vagal afferents to BK and TRPA1 agonist (AITC) were characterized in consecutively recorded nodose C fibers (n = 30), jugular C fibers (n = 13), and nodose Aδ fibers (n = 20). These vagal afferents can be divided as BK responsive (BK-responsive), responsive to both BK and AITC (BK-responsive/AITC-responsive), and responsive to BK but not to AITC (BK-responsive/not AITC-responsive). The esophageal afferents mechanoexcitabilities to esophageal distension at pressures of 10, 30, and 60 mmHg were then compared before and after BK perfusion for 30 min among BK-responsive, BK-responsive/AITC-responsive, and BK-responsive/not AITC-responsive nodose and jugular C fibers. Esophageal afferents mechanoexcitabilities were also compared before and after AITC perfusion in BK-responsive/AITC-responsive nodose and jugular C fibers.
In another series of studies, only BK-responsive/AITC-responsive nodose and jugular C fibers were selected to study the mechanism of BK-induced mechanical hypersensitivity. First, the effects of WIN64338, a BK B2 receptor antagonist, on BK-induced activation and on BK-induced mechanical hypersensitivity were studied in nodose (n = 5) and jugular (n = 8) C fibers. Second, the effect of AITC-induced TRPA1 desensitization on BK-induced mechanical hypersensitivity was studied in esophageal nodose (n = 5) and jugular (n = 4) C fibers. Third, the effect of TRPA1 inhibitor HC-030031 on BK-induced mechanical hypersensitivity was determined in nodose (n = 9) and jugular (n = 7) C fibers.
The distension-evoked nerve response was quantified as the peak frequency of the action potential discharge within the 20-s distension period. Chemical perfusion-evoked response was quantified as the peak frequency of action potential discharge within 10–30 min from the start of the response after the spontaneous activity (if present) was subtracted. The peak frequencies (Hz) of the action potential discharges were presented as means ± SE and compared by paired t-test. For all experiments the significance was defined as P < 0.05.
A total of 101 esophageal vagal afferent nerve fibers were selected and analyzed in the present study. The average conduction velocity of nodose C fibers was 0.61 ± 0.02 m/s (n = 49), that of jugular C fibers was 0.64 ± 0.03 m/s (n = 32), and that of nodose Aδ fibers was 2.5 ± 0.1 m/s (n = 20).
BK evokes action potentials in esophageal afferent C fibers.
BK (10 μM) perfusion in tissue compartment for 30 min evokes action potential discharges in most nodose (28 of 32) and all jugular (13 of 13) C fibers. The peak discharge rate of action potentials averaged 4.7 ± 0.5 Hz in nodose C fibers (compared with baseline activity 1.27 ± 0.23, P < 0.01, n = 28) (Fig. 1, A and E) and 4.2 ± 0.5 Hz in jugular C fibers (compared with baseline activity 0.31 ± 0.17, P < 0.01, n = 13) (Fig. 2A). The activations started after 1–5 min perfusion with BK and were inactivated 1–3 min after washout of BK from the tissue compartment. Both nodose (n = 15) and jugular (n = 5) C fibers responded to a second perfusion of BK after 30 min perfusion with KBS, with a similar response to that following initial perfusion of BK (Figs. 1B and 2B).
AITC evokes action potentials in esophageal afferent C fibers.
Perfusion of the TRPA1 agonist AITC (1 mM, 30 min) induced a robust activation of responses in most nodose (15 of 18) and jugular (5 of 7) C fibers. The peak discharge rate averaged 9.1 ± 1.1 Hz in nodose C fibers (compared with baseline activity 1.1 ± 0.2, P < 0.01, n = 15) (Fig. 1, C and F) and 6 ± 2 Hz in jugular C fibers (compared with baseline activity 0.8 ± 0.49, P < 0.05, n = 5) (Fig. 2C). Unlike the latency of response to BK perfusion, AITC activated both nodose and jugular C fibers immediately on perfusion into the tissue compartment. The activation effects persisted in the presence of AITC and lasted 5–10 min after washout of AITC. AITC perfusion for the second time (1 mM, 30 min) also evoked action potentials, but the response was significantly decreased compared with those following the first AITC perfusion in both nodose (3 ± 0.41 vs. 9.75 ± 1.75 Hz, P < 0.05, n = 4) and jugular (2.67 ± 1.2 vs. 6.67 ± 1.67 Hz, P < 0.05, n = 3) (Figs. 1D and 2D) neurons. This indicates that 30-min perfusion of AITC desensitizes both nodose and jugular C fibers' responses to the second perfusion of AITC.
BK but not AITC induces mechanical hypersensitivity in esophageal afferent C fibers.
In 23 BK-responsive nodose C fibers, the responses to esophageal distension were compared before and after BK perfusion. Following activation by BK perfusion for 30 min, the peak discharge rates evoked by esophageal distension were significantly increased from 3.2 ± 0.3, 6.3 ± 0.5, and 9.8 ± 0.8 to 7.7 ± 0.8, 11 ± 1.2, and 14.4 ± 1.5 at distension pressures of 10, 30, and 60 mmHg, respectively (P < 0.01, n = 23) (Fig. 3A). A similar mechanical hypersensitivity was seen in all 13 BK-positive response jugular C fibers after BK perfusion, with esophageal distension-evoked peaks of action potentials significantly increased from 2 ± 0.38, 4 ± 0.56, and 6.2 ± 0.53 before BK perfusion to 5.5 ± 0.77, 8.6 ± 1.3, and 11.9 ± 1.65 at distension pressures of 10, 30, and 60 mmHg, respectively (P < 0.01, n = 13) (Fig. 3C). Such mechanical hypersensitivity only lasts in the presence of BK perfusion and returns back to the baseline activities after washout of BK for 5–10 min (data not shown).
Esophageal distension-evoked action potentials were also compared before and after AITC perfusion (1 mM, 30 min) in 12 nodose and 6 jugular BK-responsive/AITC-responsive C fibers. In contrast to BK-induced mechanical hypersensitivity, AITC perfusion was associated with a loss of response to esophageal distension in most tested nodose (11 of 12) and jugular (5 of 6) C fibers. This loss of response started 5–10 min after AITC-evoked activation and persisted in the presence of AITC and for 5–10 min after AITC was washed away. After 10-min perfusion with KBS, esophageal distension-evoked action potentials were similar to those recorded pre-AITC perfusion in nodose (P > 0.05, n = 12) and jugular (P > 0.05, n = 6) C fibers (Fig. 3, B and D).
To determine the role of TRPA1 in BK-induced mechanical hypersensitivity, the effect of BK perfusion on the mechanoexcitability of BK-responsive/AITC-responsive and BK-responsive/not AITC-responsive esophageal afferent C fibers were compared. All studied BK-responsive/AITC-responsive nodose (n = 7) and jugular (n = 5) C fibers displayed mechanical hypersensitivity after BK perfusion. In contrast, in BK-responsive/not AITC-responsive nodose (n = 2) and jugular (n = 2) C fibers, BK perfusion did not induce mechanical hypersensitivity (data not shown) (Table 1). This may indicate that BK-induced mechanical hypersensitivity requires the presence of TRPA1 on the nerve terminals.
BK B2 receptor antagonist WIN64338 inhibits BK-evoked action potentials and prevents BK-induced mechanical hypersensitivity in esophageal afferent C fibers.
Having demonstrated that BK not only evokes action potentials but also induces mechanical hypersensitivity in both nodose and jugular C fibers, we next tested the hypothesis that these effects are initiated by direct activation of the BK B2 receptor on esophageal sensory afferent nerve terminals. Esophageal vagal C fibers displaying a positive response to BK perfusion (10 μM, 30 min) were first identified. This was followed by a 30-min wash with KBS. Esophageal distension-evoked action potentials were then recorded as control. After a 30-min perfusion with WIN64338 (10 μM, 30 min), BK was perfused for 30 min in the presence of WIN64338. Esophageal distension was repeated, and evoked action potentials were recorded and compared with control. All the studied C fibers were confirmed to respond to AITC at the end of each study. If there was no response to AITC, they were not included for analysis. Pretreatment with the BK B2 receptor antagonist WIN64338 significantly inhibited BK-evoked action potentials in both nodose (P < 0.01, n = 5) and jugular (P < 0.01, n = 8) C fibers (Fig. 4, A and C). Pretreatment with WIN64338 also prevented BK-induced mechanical hypersensitivity in both nodose (compared with control, P > 0.05, n = 4) and jugular (compared with control, P > 0.05, n = 8) C fibers (Fig. 4, B and D).
Desensitization of esophageal afferent C fiber by TRPA1 agonist AITC prevents BK-induced mechanical hypersensitivity.
We have observed that TRPA1 agonist AITC perfusion not only evoked action potentials in esophageal afferent C fibers but also desensitized their response to a second perfusion of AITC. Thus we tested the hypothesis that desensitization of TRPA1 in esophageal afferent C fibers by AITC perfusion would prevent BK-induced mechanical hypersensitivity. After perfusion with AITC (1 mM, 30 min) and washout with KBS for 30 min, esophageal distension-evoked action potentials were recorded in esophageal afferent C fibers as control. Then BK perfusion-evoked action potentials and esophageal distension-evoked action potentials at the end of 30 min of perfusion with BK were recorded. All esophageal afferent C fibers were confirmed to be desensitized to a second AITC perfusion at the end of each study. Consistent with the results from the first series of studies, AITC perfusion evoked robust action potentials in both nodose (n = 5) and jugular (n = 4) C fibers. This was followed by a transient loss of response to esophageal distension that was sustained in the presence of AITC. A response to esophageal distension was restored after a 10-min washout of AITC. After washout of AITC for 30 min, BK perfusion (10 μM, 30 min) also evoked action potentials that were significantly increased over the baseline activities in both nodose and jugular C fibers (Fig. 5, A and C). Esophageal distension-evoked action potentials in the presence of BK were not changed compared with those before the BK perfusion in both nodose (n = 5) and jugular (n = 4) C fibers (Fig. 5, B and D). Compared with the first perfusion of AITC, the second perfusion (1 mM, 30 min) evoked action potentials that were significantly decreased in both nodose (P < 0.05, n = 5) and jugular (P < 0.01, n = 4) C fibers (Fig. 5, A and C). This confirms that TRPA1 was desensitized by the first AITC perfusion over the study period and that TRPA1 desensitization prevents BK-induced mechanical hypersensitivity.
TRPA1 inhibitor HC030031 inhibits BK-induced mechanical hypersensitivity in esophageal afferent C fibers.
To further confirm the role of TRPA1 in BK-induced mechanical hypersensitivity, the effect of a TRPA1 inhibitor on BK-induced mechanical hypersensitivity was studied. HC-030031 is a newly identified potent and selective TRPA1 inhibitor. HC-030031 blocked calcium response and inward currents elicited by AITC in hTRPA1-expressing cells in a concentration-dependent manner and greatly attenuated pain-related flinching in vivo (23). HC-030031 was used in the present study to determine the role of TRPA1 in BK-induced mechanical hypersensitivity. The studied afferent C fibers were first perfused with HC-030031 (10 μM, 30 min) and then esophageal distension-evoked action potentials were recorded as control. BK was then perfused for 30 min. By the end of the perfusion and in the presence of BK, esophageal distensions were repeated and evoked action potentials were recorded and compared with the control. All afferent C fibers were confirmed to respond to AITC at the end of the study. Perfusion with HC-030031 did not change baseline activity (spontaneous discharges) and did not affect BK-evoked action potentials in both nodose (HC-030031 vs. BK+HC-030031: 0.7 ± 0.4 vs. 4.7 ± 1.3 Hz, P < 0.05, n = 9) and jugular (HC-030031 vs. BK+HC-030031: 0.4 ± 0.3 vs. 4.0 ± 0.2 Hz, P < 0.05, n = 7) C fibers. Following the perfusion of HC-030031, esophageal distension-evoked action potentials at pressures of 10, 30, and 60 mmHg were not significantly different before and after 30-min perfusion of BK in the presence of HC-030031 in both nodose (P > 0.05, n = 9) and jugular (P > 0.05, n = 7) C fibers (Fig. 6). This indicates that pretreatment with TRPA1 inhibitor prevents BK-induced mechanical hypersensitivity in these nodose and jugular C fibers.
Nodose Aδ fiber responses to BK and AITC perfusion.
TRPA1 expression has been reported mainly on small diameter sensory neurons from DRG, trigeminal, and nodose ganglia with their unmyelinated C fibers innervating peripheral tissues. In this last part of the study, the effects of BK or AITC perfusion on action potentials and the mechanoexcitability to esophageal distension in nodose Aδ fibers were studied. In contrast to esophageal afferent C fibers, BK perfusion (10 μM, 30 min) did not evoke action potentials in all tested nodose Aδ fibers (compared with baseline activity, P > 0.05, n = 20), and AITC perfusion (1 mM, 30 min) failed to evoke action potentials in all tested nodose Aδ fibers (compared with baseline activity, P > 0.05, n = 9) (Fig. 7A). Esophageal distension evoked action potentials at pressures of 10, 30, and 60 mmHg that were not changed after perfusion with BK for 30 min compared with control (P > 0.05, n = 16) (Fig. 7B). All studied Aδ fibers were confirmed to respond to α,β-methylene-ATP (data not shown), a P2X3 receptor agonist that is used as a tool to indicate that the recorded nerve terminals are exposed to drug perfusion.
BK is an important mediator released locally in the sites of inflammation and tissue injury. BK not only directly activates sensory afferents but also subsequently sensitizes their responses to other stimuli, thus playing an important role in peripheral nerve sensitization (36). It has been reported that BK activates sensory afferent nerves innervating different parts of the gastrointestinal tract, including the esophagus (28, 33), jejunum (6, 21), and colon (5, 30). An increased response to mechanical stimulation (probing) has been described in pelvic colonic afferents following the perfusion of BK (5). Intraesophageal instillation of BK enhances responsiveness of upper thoracic spinal neurons to esophageal distension (28). But the mechanism of this BK-induced mechanical hypersensitivity is still unclear. To explore this mechanism in our esophageal-vagal preparation, we first defined the responses of different esophageal vagal afferent nerve subtypes to BK. In the opossum esophagus, BK has been shown to activate esophageal sympathetic afferent wide-dynamic range and high-threshold mechanonociceptors via B2 receptor but not to directly activate the vagal low-threshold mechanoreceptor in esophageal sensory afferent nerves (32). In agreement with this observation, the present study in the guinea pig esophagus shows that the esophageal nodose Aδ fiber, which is considered similar to the low-threshold mechanoreceptor, does not respond to BK perfusion. Sensory afferent neurons from both vagal jugular (supranodose) ganglia and spinal DRG are neurocrest-derived sensory neurons. We speculated that the subtypes of esophageal vagal afferent neurons and nerve fibers, especially those from jugular ganglion, were similar to sympathetic afferents and spinal neurons and would response to BK (5, 28, 32). The present study provides new evidence that both esophageal vagal nodose and jugular C fibers are activated by BK perfusion, which resemble the previously described sympathetic afferent wide-dynamic range and high-threshold mechanonociceptors (32). One explanation for this difference is that different animal species (such as opossum and guinea pig) may have a different population of vagal sensory afferent nerve subtypes and that the different embryonic origin of these subtypes of sensory afferents may determine their distinct functions. We also show that this repeatable activation in vagal nodose and jugular C fibers is inhibited by BK B2-receptor antagonist, which is consistent with previous studies showing that BK activated gastrointestinal sensory afferents directly through B2-receptor on the nerve endings (4, 6, 21, 30, 31). That BK increased the responses of colonic afferent fibers to probing and enhanced responsiveness of upper thoracic spinal neurons to esophageal distension indicated that BK might potentiate mechanoexcitabilities of these sensory afferents and spinal neurons (5, 28). We further demonstrated in the esophagus that activation of vagal nodose and jugular C fibers by BK was associated with a significant increase in response to esophageal distension, which was also inhibited by BK B2-receptor antagonist, indicating that the potentiated mechanoexcitability was initiated directly via the B2-receptor from the nerve endings. These findings extend our understanding of these previously unrecognized esophageal vagal sensory afferent subtypes that are not only activated but also sensitized by BK directly via the B2 receptor on nerve terminals.
TRPA1 has been identified on small-diameter sensory neurons from dorsal root (34), trigeminal (16), and nodose ganglia (25). Many studies using disassociated sensory neurons and animal behavior models have clearly demonstrated that TRPA1 is involved in cold, chemical, and mechanical nociception. A recent study clearly revealed that TRPA1 is expressed in mice nodose neurons labeled retrogradely from the lung and that the TRPA1 agonist cinnamaldehyde activated these neurons and vagal afferent C fibers (26). Our study demonstrates that TRPA1 agonist AITC selectively activates esophageal vagal nodose and jugular C fibers, but not nodose Aδ fibers. This activation is associated with a transient loss of response to mechanical distension and desensitization to the second AITC perfusion. This study provides the first direct evidence from visceral sensory afferent nerve ending that TRPA1 agonist selectively activates nociceptive sensory nerves, which have a small-diameter neuron cell body situated in nodose and jugular ganglia and unmyelinated C fiber nerve terminal innervating the peripheral tissues. Esophageal vagal afferent nerve subtypes respond to chemical stimuli differently. We have shown that the TRPV1 agonist capsaicin activates both nodose and jugular C fibers, but not nodose Aδ fibers, and the P2X3 receptor agonist α,β-methylene-ATP only activates nodose C and Aδ fibers, but not jugular C fibers (39). We have previously defined the esophageal vagal sensory afferent nerves as nociceptive-like nerves on the basis of their conduction velocity (fitting in C-fiber range), response to noxious mechanical (>30 mmHg distension pressure) and chemical (capsaicin) stimuli (39). In the present study, TRPA1 agonist AITC selectively activated esophageal vagal nodose and jugular C fibers, but not nodose Aδ fibers, which is the similar subpopulation that respond to TRPV1 agonist capsaicin. This is supported by previous morphological studies showing that the majority of nociceptive DRG sensory neurons that express TRPA1 also express TRPV1 (18, 34). Interestingly, we also found that most BK-positive response nodose and jugular C fibers also display a positive response to AITC. Moreover, BK-induced mechanical hypersensitivity is only observed in those nodose and jugular C fibers that display positive responses to both BK and AITC. Those C fibers only responsive to BK but not to AITC are not associated with mechanical hypersensitivity after BK perfusion. This suggests that TRPA1 is essential for BK-induced mechanical hypersensitivity.
BK activates and sensitizes sensory nerves through G protein-coupled receptor-mediated PLC and PLA signaling pathways, which subsequently interact with downstream ion channels to change the excitability of sensory nerves. The exact role of which ion channel is involved in BK-induced sensitization is still to be determined. Among them, TRPA1 is a likely candidate. BK evoked an immediate current response in CHO cells, which expressed mTRPA1 and were transiently transfected with B2 receptor, but not in controls, which included CHO cells, TRPA1 cells, and B2R-only-expressing cells (3). In a TRPA1-deficient mouse model (4), BK-evoked calcium influx from trigeminal neurons was significantly decreased, and TRPA1−/− mice displayed an impaired response to BK-induced thermal hyperalgesia. Another TRPA1-knockout mouse model (19) was shown to be much less sensitive to BK injected in a paw. BK-induced mechanical hypersensitivity to von Frey stimulation was absent in TRPA1 knockout mice, compared with a fivefold decrease in the threshold in wild-type mice. These two TRPA1 knockout mice studies strongly indicated that TRPA1 is involved in mediating BK-induced thermal and mechanical hyperalgesia. The present study provides new evidence from visceral sensory nerve endings to support the important role of TRPA1 in BK-induced mechanical hypersensitivity in visceral sensory afferent nerves. First, BK-induced mechanical hypersensitivity only occurs in sensory afferents that respond to both BK and TRPA1 agonist AITC, but not in those displaying a positive response only to BK and not to AITC. Second, desensitizing TRPA1 with AITC attenuates BK-induced mechanical hypersensitivity. Third, pretreatment with the TRPA1 inhibitor HC-030031 prevents BK-induced mechanical hypersensitivity.
TRPA1 can be activated not only by a variety of noxious stimuli including pungent compounds such as AITC (16), but also by BK (3). But the underlying mechanism might be different. AITC, like other irritants, directly activates TRPA1 by covalent modification of cysteine and lysine residues within the NH2-terminal cytoplasmic domain of the channel protein (14, 20). BK, on the other hand, may activate TRPA1 via its G protein-coupled receptor-mediated PLC pathway to induce an increase in intracellular calcium by release from calcium stores or by calcium influx through TRPV1 (3, 4), which then activates the NH2-terminal EF-hand calcium-binding domain of the TRPA1 channel (9). The different mechanisms of TRPA1 activation induced by BK and AITC might explain why in our study AITC inhibitor only inhibited BK-induced mechanical hypersensitivity but not BK-evoked activation itself. This may also indicate that BK activates and sensitizes sensory nerve through two independent mechanisms. AITC-evoked activation underwent tachyphylaxis in TRPA1-transfected HEK cells (8), TRPA1/TRPV1-transfected CHO cells, sensory neurons from trigeminal ganglion (1), and lumbar spinal wide-dynamic range neurons (24). This desensitization effect may act through Ca2+-independent AITC-directed inhibition of TRPA1 channel or capsaicin-induced Ca2+-dependent PIP2 depletion (1, 29). Given that AITC-induced TRPA1 desensitization acts at a similar site as that by which HC-030031 acts to inhibit AITC activation of TRPA1, it is not surprising that, in the present study, both AITC-induced TRPA1 desensitization and HC-030031 act similarly to inhibit BK-induced mechanical hypersensitivity in nodose and jugular C fibers. In addition, we observed that perfusion with the TRPA1 agonist AITC itself does not induce mechanical hypersensitivity in either nodose or jugular C fibers. This is similar to the reported findings that perfusion with the TRPA1 agonist AITC did not increase the responses to cutaneous von Frey stimuli recorded from lumbar spinal wide-dynamic range neurons (24). Also, increases in either the number of DRG neurons or the expression of TRPA1 only lead to thermal but not mechanical hypersensitivity in cutaneous C fiber nociceptors in a transgenic mouse model that overexpresses artemin, a neuronal survival factor, in skin keratinocytes (ART-OE mice) (10). That AITC perfusion causes a transient loss in response to esophageal distension in nodose and jugular C fibers is similar to our previous observation that the TRPV1 agonist capsaicin induces a long-lasting loss of response to esophageal distension and chemical stimulation and that the P2X3 agonist α,β-methylene-ATP also induces a transient loss of response to esophageal distension (unpublished observation). These observations may indicate the involvement of these ion channels in visceral mechanical sensory transduction.
In summary, our data provide the first evidence that TRPA1 plays a critical role in BK-induced mechanical hypersensitivity of visceral sensory nerve terminals. This novel mechanism in visceral peripheral sensitization may contribute to visceral hypersensitivity in functional gastrointestinal disorders.
This study is supported by a grant from the Investigator Sponsored Studies Program of AstraZeneca.
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