Effect of Nociceptin in Acid-evoked Cough and Airway Sensory Nerve Activation in Guinea Pigs
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《美国呼吸和危急护理医学》
Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland
UCB Research, Inc., Cambridge, Massachusetts
ABSTRACT
Rationale: Nociceptin/orphanin FQ has been reported to inhibit capsaicin- and mechanically provoked cough in animal models, but the mechanism of this effect has not been elucidated.
Objectives: The objectives of this study were to determine whether nociceptin inhibits acid-evoked cough in conscious animals and to evaluate the mechanism of this effect.
Methods: We tested the effect of nociceptin on acid-induced cough in conscious guinea pigs and acid-induced nerve activation in airway-specific vagal sensory neurons using calcium imaging techniques and the gramicidin-perforated patch clamp technique.
Measurements and Main Results: Nociceptin (3 mg/kg, intraperitoneal) effectively inhibited acid-evoked cough in guinea pigs by nearly 70%. Acid (pH 5) increased intracellular free calcium in acutely dissociated vagal jugular ganglionic neurons. The acid-induced increase in intracellular calcium was inhibited by a selective transient receptor potential vanilloid-1 antagonist, 5-iodo-resiniferatoxin (1 μM, 80% reduction). The inhibitory effect of 5-iodo-resiniferatoxin on acid-induced increases in calcium was mimicked by nociceptin (0.1 μM). In gramicidin-perforated patch clamp recordings on airway-specific capsaicin-sensitive jugular ganglion neurons, acid (pH 5) induced two distinct inward currents. A transient current was evoked that was inhibited by amiloride and a sustained current was evoked that was inhibited by 5-iodo-resiniferatoxin. Nociceptin selectively inhibited only the sustained component of acid-induced inward current.
Conclusion: These results indicate that the inhibitory effect of nociceptin on acid-induced cough may result from a direct inhibitory effect on peripheral C-fiber activity caused by the selective inhibition of acid-induced transient receptor potential vanilloid-1 activation.
Key Words: acid airway sensory cough nociceptin/orphanin FQ transient receptor potential vanilloid-1
Inhaled citric acid is commonly used experimentally to evoke cough in humans and guinea pigs. Acid-evoked coughing may be directly relevant to the chronic coughing associated with gastroesophageal reflux disorders (1). In addition, a decrease in airway pH that is associated with asthma, and perhaps other airway inflammatory diseases, may contribute to coughing associated with these conditions (2). Both citric acid and capsaicin evoke cough in conscious guinea pigs by mechanisms that are inhibited by antagonists of the capsaicin receptor (transient receptor potential vanilloid-1 [TRPV1]) (3). We have found that the capsaicin-sensitive vagal nerve fibers innervating the larynx, trachea, and bronchus of guinea pigs are derived nearly exclusively from neurons situated in the jugular vagal ganglia (4). Accordingly, inhibiting the activity of these jugular vagal afferent nerve fibers in the airways is a logical strategy in the development of novel antitussive drugs.
The heptadecapeptide nociceptin, also known as orphanin FQ, is an endogenous agonist of the G protein–coupled opioid-like receptor-1 (NOP1, also known as ORL1). NOP1 receptors are expressed in capsaicin-sensitive vagal sensory nerves innervating airways, and nociceptin effectively inhibits tachykinin release from vagal C-fibers in guinea pig bronchi (5, 6). Intravenous administration of nociceptin also effectively inhibits in conscious animals cough reflexes evoked by mechanical perturbation of the airways or by inhalation of capsaicin (7, 8). Although nociceptin may act centrally to inhibit cough, observations that nociceptin can inhibit the function of vagal afferent nerves within the airway indicate that it may also inhibit cough through a peripheral mechanism of action (5, 6).
The aims of the current study were twofold. First, we set out to evaluate the efficacy of nociceptin in inhibiting acid-evoked cough in conscious animals. Second, experiments were designed to decipher the mechanisms by which nociceptin inhibits acid-induced responses in capsaicin-sensitive jugular vagal afferent neurons.
METHODS
In Vivo Cough
Unanesthetized male Hartley guinea pigs (425–475 g) were placed in individual Perspex cylindrical plethysmographs and exposed to citric acid aerosol (0.25 M, 0.5 ml/min) generated with an ultrasonic nebulizer (EMKA Technologies, Paris, France). The time of exposure to the citric acid was 2 min, followed by a further observation period of 9 min. Individual coughs were detected in three ways: (1) via a pressure transducer attached to the plethysmograph, (2) via a microphone placed inside the plethysmograph, and (3) via visual observation of the animal. Each animal was exposed to citric acid only once. Animals were injected intraperitoneally with nociceptin (1–3 mg/kg), 30 min before citric acid administration. Saline was used as the vehicle control.
Intracellular Free Calcium Measurements and Patch Clamp Recordings
The animals (300–400 g) were anesthetized, and DiI (1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate) solution was injected into the tracheal lumen 7 to 9 d before an experiment as previously described (9). The animals were killed (using CO2) and the right and left jugular ganglia were dissected and enzymatically dissociated with Diaspase (GIBCO; Invitrogen, Carlsbad, CA) and collagenase, using standard techniques. The dissociated jugular ganglionic neurons were transferred onto circular 25-mm glass coverslips coated with poly-D-lysine (0.1 mg/ml). The neurons were studied within 24 h of dissociation.
For intracellular calcium measurements, the neurons were loaded with Fura-2 AM (Invitrogen, Carlsbad, CA) and then, 20 min before experiment, superfused by infusion pump (8 ml/min) with 35°C Locke buffer (mM): NaCl, 136; KCl, 5.6; MgCl2, 1.2; CaCl2, 2.2; NaH2PO4, 1.2; NaHCO3, 14.3; dextrose, 10 (pH 7.3–7.4). Intracellular calcium measurements were performed under a microscope equipped for epifluorescence. A field of cells was monitored by sequential dual excitation (352 and 380 nm) and ratios of the images were converted to calcium concentration according to methods and parameters presented previously (10). The ratio images were acquired every 6 s.
Gramicidin-perforated whole cell patch clamp recordings were done in jugular neurons labeled from lungs and airways. The neurons labeled were identified by fluorescence microscopy (excitation filter, 540 nm; emission filter, 600 nm). To maintain intracellular signal pathways, a gramicidin-perforated whole cell patch clamp technique was employed. A pipette (1.5–3 M) was filled with pipette solution composed of the following (mM): KCl, 140; CaCl2, 1; MgCl2, 2; ethyleneglycol-bis- (-aminoethyl ether)-N,N'-tetraacetic acid, 11; dextrose, 10; titrated to pH 7.3 with KOH; 304 mOsm. Gramicidin was dissolved in dimethyl sulfoxide and mixed into the pipette solution to a final concentration of 1 μg/ml just before each recording. During the experiments, the cells were continuously superfused (6 ml/min) by gravity with Locke solution. All recordings were done at 35°C. The membrane potential of the cells was held at –60 mV.
Drugs and Data Analysis
Acidic phosphate buffer (pH 5) and physiologic phosphate buffer (pH 7.3) were modified from Locke solution by replacing bicarbonate with phosphate (Na2HPO4 and NaH2PO4) and then adjusting the sodium concentration with NaCl. Citric acid (0.25 M) was directly dissolved in 0.9% saline just before experiment. In the cough studies data were compared by one-way analysis of variance and Dunnett's post test. Electrophysiologic and calcium data were expressed as means ± SEM. A t test and Mann-Whitney rank sum test were used when appropriate.
RESULTS
Cough
In 14 animals, inhalation of citric acid evoked on average 15 to 20 coughs during the 11-min period of observation. Nociceptin (3 mg/kg, intraperitoneal) significantly inhibited this response by nearly 70% (Figure 1).
Acid-induced Increases in Cytosolic Calcium
The capsaicin-sensitive nerves in guinea pig large airways are derived from cell bodies situated in the jugular ganglia. We therefore addressed the hypothesis that nociceptin may inhibit cough by inhibiting the acid-induced activation of these nerves. Jugular neurons obtained from seven animals were studied. We successfully recorded changes in intracellular free calcium in approximately 15 to 20 neurons per coverslip. In our first series of experiments we found that 72 of 96 jugular neurons responded to acid (pH 5 for 2 min) with rapid increases in intracellular calcium concentration. All 72 of these acid-sensitive neurons responded to capsaicin added 5 min after acid application, whereas the 24 acid-insensitive neurons were found to be capsaicin insensitive. The calcium ionophore ionomycin (1 μM) was used to obtain a "maximal" response in the neurons. The peak acid-induced increase in intracellular calcium averaged 21 ± 7% of that obtained with ionomycin (n = 96); a trace of averaged calcium changes by acid application is shown in Figure 2A. In the second series of experiments we found that the TRPV1 antagonist 5-iodo-resiniferatoxin (I-RTX, 1 μM), reduced the acid-induced response to 4 ± 1% of the ionomycin response (n = 42; Figure 2B). These data indicate that acid causes increases in intracellular calcium in jugular neurons primarily through activation of TRPV1.
In the third series of experiments nociceptin was found to reduce the acid-induced increases in intracellular calcium in jugular capsaicin-sensitive neurons by 53% (Figure 2B). When only capsaicin-sensitive neurons were compared between the control and nociceptin-treated groups, the acidic buffer (pH 5 for 2 min) induced 28.9 ± 3.3 and 13.6 ± 2.4% of the ionomycin-induced intracellular calcium increase in the control group (n = 72) and nociceptin (0.1 μM, 5 min)-treated group (n = 42), respectively (p < 0.01, Mann-Whitney rank sum test).
Patch Clamp Recordings
Forty-five dissociated jugular neurons specifically labeled from the airways of 24 guinea pigs were studied in whole cell patch clamp recordings with a holding potential of –60 mV. In an attempt to maintain intracellular signals, including G protein–coupled signals, the gramicidin-perforated patch clamp technique was employed. Exposing neurons to pH 5 (10-s application) caused a slowly developing, sustained inward current in all capsaicin-sensitive neurons. The elapsed time to the peak inward current of the sustained response was 7.6 ± 1.0 s (n = 23). This sustained current was virtually abolished by I-RTX (1 μM) in all tested neurons (4 ± 4% of control response, n = 5), indicating the involvement of TRPV1 receptors (Figure 3A). In 6 of 23 neurons this slowly developing current was preceded by a rapidly developing and transient inward current in which the elapsed time to the peak of the inward current was 0.8 ± 0.1 s (n = 6). This acid-induced transient current was not inhibited by I-RTX (124 ± 51% of control response, n = 4; Figure 3B) but it was partially inhibited by a nonselective acid-sensing ion channel inhibitor, amiloride (10 μM), in two neurons (90 and 58% inhibition). Two consecutive acid applications with a 5-min interval showed only modest desensitization in sustained I-RTX–sensitive inward current (74 ± 14% of first response, n = 5). After the transient components were isolated by I-RTX pretreatment (for 2 min), the transient components were not desensitized by repeated acid applications (second response averaged 105 ± 33% of first response, n = 5).
Nociceptin (0.1 μM) did not overtly evoke an inward or outward current in voltage-clamped neurons. However, nociceptin effectively and selectively inhibited the I-RTX–sensitive sustained inward current induced by acid application (Figure 4A). When nociceptin (0.1 μM) was superfused over the neurons for 2 min before the second acid application, the sustained inward current was reduced to 20 ± 5% (n = 6) of the first response. This was significantly different from the natural desensitization (p < 0.01, t test; Figure 4B). In six experiments the effect of nociceptin on the rapid transient I-RTX–insensitive current (I-RTX was present throughout these experiments) was evaluated. In contrast to its effect on the slow sustained I-RTX–sensitive component of the acid response, nociceptin had no significant effect on the acid-induced I-RTX–insensitive transient current. In the presence of nociceptin the transient response averaged 107 ± 10% of the control response (p > 0.1, n = 6; Figure 4C).
DISCUSSION
The present results show that nociceptin effectively inhibits cough evoked by acid inhalation in guinea pigs. These results with acid-induced cough are consistent with the known inhibitory effects of nociceptin on capsaicin-induced cough in guinea pigs (7). It is known that cough evoked by inhalation of capsaicin or citric acid in conscious guinea pigs is blocked by TRPV1 antagonists (3, 11, 12). We have previously found that the TRPV1-containing vagal afferent nerves in the guinea pig larynx, trachea, and main bronchi (cough regions) are nearly exclusively nociceptors derived from the jugular vagal ganglia (4). We have also noted that nociceptin can directly inhibit tachykinin release from jugular C-fibers in guinea pig airways (5). We reasoned therefore that nociceptin may inhibit acid-induced cough by interfering with acid-induced activation of jugular afferent nerves. Acid stimulates action potential discharge in terminals of jugular afferent fibers in the airways via both TRPV1-dependent and TRPV1-independent mechanisms (13). The data presented here further support the hypothesis that the mechanism by which nociceptin inhibits acid-induced activation of airway-specific jugular neurons is by selectively inhibiting the TRPV1-dependent component of the response.
Acid evoked large increases in cytosolic calcium only in jugular neurons that were capsaicin sensitive (presumably cell bodies of C-fibers). This calcium response was nearly abolished by I-RTX, indicating that much of the response is secondary to TRPV1 activation. The observation that nociceptin effectively inhibited this acid-induced rise in free cytosolic calcium supports the hypothesis that nociceptin can lead to inhibition of acid-induced gating of TRPV1 channels. These results are in agreement with those of Jia and coworkers, who found that nociceptin similarly inhibited capsaicin-induced TRPV1-dependent increases in cytosolic calcium in isolated vagal sensory neurons (14). The findings are also in agreement with behavioral studies on monkeys, in which nociceptin was found to inhibit capsaicin-induced thermal nociception, presumably by interfering with TRPV1 function (15).
There are several mechanisms that may account for the inhibitory effect of nociceptin on acid-induced increases in cytosolic calcium. Nociceptin has been found to lead to inhibition of voltage-gated Ca2+ channels (16, 17). Others have reported that in medullary dorsal horn neurons and neurons in the raphe nucleus, nociceptin leads to activation of an inward-rectifying potassium current (18, 19). This could lead to a decrease in the amplitude or duration of the acid-induced membrane depolarization and consequent reduction in the calcium response. Consistent with this idea, tertiapin, an antagonist of the inward rectifier current, has been found to interfere with the inhibitory effect of nociceptin on capsaicin-induced increases in cytosolic calcium in nodose neurons (14).
It is difficult to draw firm conclusions regarding potential ionic mechanisms of nociceptin action by studying increasing cytosolic calcium alone. We therefore also addressed these issues with the gramicidin-perforated patch clamp recordings of airway-specific sensory neurons. Airway-labeled capsaicin-sensitive jugular neurons were voltage clamped at –60 mV to reduce the contribution of voltage-gated channels, including voltage-gated calcium channels and inward-rectifying potassium channels. Under these conditions we observed no overt changes in resting membrane current on nociceptin exposure. In these neurons acid evoked a strong TRPV1-dependent inward current and, in a subset of neurons, a TRPV1-independent inward current. The acid-induced TRPV1 activation is consistent with findings of others that acidification can lead to TRPV1 currents at 37°C (20–22). That acid caused TRPV1-dependent and TRPV1-independent inward currents is in keeping with our studies on jugular C-fiber nerve terminals within the airways (13, 23). That nociceptin significantly inhibited only the TRPV1 component of the acid-induced inward current reveals an important selectivity in its mechanisms of action. The results indicate that neither inhibition of voltage-gated calcium channels nor activation of inward rectifier channels is necessary for the observed inhibitory effects of nociceptin on acid-induced responses in airway C-fiber neurons.
We previously found that guinea pig vagal sensory neurons express NOP1 receptors (5), and Jia and coworkers have found that the inhibitory effect of nociceptin on capsaicin-induced calcium increases in guinea pig nodose neurons is blocked by a selective NOP1 receptor antagonist (14). The antitussive action of nociceptin in guinea pigs and cats was also found to be inhibited by a selective NOP1 receptor antagonist (7, 8). With these data in mind, we hypothesize that activation of NOP1 receptors with nociceptin leads to signaling events that selectively decrease the efficacy by which acid (and presumably other TRPV1 agonists) opens the TRPV1 channels. This would be consistent with the observation that nociceptin decreases acid-induced plasma extravasation in guinea pigs and rabbits by a prejunctional inhibition of sensory neuropeptide release (24, 25). Investigation of the signal transduction mechanism was beyond the scope of the present study, but it is noteworthy that nociceptin receptor activation has been found to be associated with decreases in cyclic AMP formation (26, 27). This may be informative in that increases in cyclic AMP are known to increase TRPV1 activity (28).
In contrast to the C-fiber–driven cough observed on acid inhalation, direct application of acid to the larynx or trachea, as might occur on aspiration, stimulates cough via activation of a unique capsaicin-insensitive vagal A "cough receptor" (29). It is not known whether nociceptin can directly inhibit acid-induced activation of A "cough fibers" or the A fiber–induced cough reflex. We have shown that acid activates these A fibers by a mechanism similar to the rapid TRPV1-independent component of the acid response in C-fiber neurons (13). As this was the component of the acid response that was unaffected by nociceptin in C-fiber neurons, it is tempting to speculate that nociceptin will be ineffective in inhibiting acid activation of the A cough fibers.
In summary, the results reveal that nociceptin effectively inhibits acid-evoked cough in conscious guinea pigs. We found that nociceptin can initiate a signaling pathway that leads to significant and selective inhibition of acid-evoked activation of TRPV1. This, in turn, results in decreases in acid-evoked action potential discharge in C-fiber terminals within the airway wall. This type of mechanism may underlie the well-known inhibitory effects of nociceptin on evoked cough reflexes in various animal models, and may provide important clues for those interested in developing effective peripherally acting antitussive agents.
FOOTNOTES
Originally Published in Press as DOI: 10.1164/rccm.200507-1043OC on October 20, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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UCB Research, Inc., Cambridge, Massachusetts
ABSTRACT
Rationale: Nociceptin/orphanin FQ has been reported to inhibit capsaicin- and mechanically provoked cough in animal models, but the mechanism of this effect has not been elucidated.
Objectives: The objectives of this study were to determine whether nociceptin inhibits acid-evoked cough in conscious animals and to evaluate the mechanism of this effect.
Methods: We tested the effect of nociceptin on acid-induced cough in conscious guinea pigs and acid-induced nerve activation in airway-specific vagal sensory neurons using calcium imaging techniques and the gramicidin-perforated patch clamp technique.
Measurements and Main Results: Nociceptin (3 mg/kg, intraperitoneal) effectively inhibited acid-evoked cough in guinea pigs by nearly 70%. Acid (pH 5) increased intracellular free calcium in acutely dissociated vagal jugular ganglionic neurons. The acid-induced increase in intracellular calcium was inhibited by a selective transient receptor potential vanilloid-1 antagonist, 5-iodo-resiniferatoxin (1 μM, 80% reduction). The inhibitory effect of 5-iodo-resiniferatoxin on acid-induced increases in calcium was mimicked by nociceptin (0.1 μM). In gramicidin-perforated patch clamp recordings on airway-specific capsaicin-sensitive jugular ganglion neurons, acid (pH 5) induced two distinct inward currents. A transient current was evoked that was inhibited by amiloride and a sustained current was evoked that was inhibited by 5-iodo-resiniferatoxin. Nociceptin selectively inhibited only the sustained component of acid-induced inward current.
Conclusion: These results indicate that the inhibitory effect of nociceptin on acid-induced cough may result from a direct inhibitory effect on peripheral C-fiber activity caused by the selective inhibition of acid-induced transient receptor potential vanilloid-1 activation.
Key Words: acid airway sensory cough nociceptin/orphanin FQ transient receptor potential vanilloid-1
Inhaled citric acid is commonly used experimentally to evoke cough in humans and guinea pigs. Acid-evoked coughing may be directly relevant to the chronic coughing associated with gastroesophageal reflux disorders (1). In addition, a decrease in airway pH that is associated with asthma, and perhaps other airway inflammatory diseases, may contribute to coughing associated with these conditions (2). Both citric acid and capsaicin evoke cough in conscious guinea pigs by mechanisms that are inhibited by antagonists of the capsaicin receptor (transient receptor potential vanilloid-1 [TRPV1]) (3). We have found that the capsaicin-sensitive vagal nerve fibers innervating the larynx, trachea, and bronchus of guinea pigs are derived nearly exclusively from neurons situated in the jugular vagal ganglia (4). Accordingly, inhibiting the activity of these jugular vagal afferent nerve fibers in the airways is a logical strategy in the development of novel antitussive drugs.
The heptadecapeptide nociceptin, also known as orphanin FQ, is an endogenous agonist of the G protein–coupled opioid-like receptor-1 (NOP1, also known as ORL1). NOP1 receptors are expressed in capsaicin-sensitive vagal sensory nerves innervating airways, and nociceptin effectively inhibits tachykinin release from vagal C-fibers in guinea pig bronchi (5, 6). Intravenous administration of nociceptin also effectively inhibits in conscious animals cough reflexes evoked by mechanical perturbation of the airways or by inhalation of capsaicin (7, 8). Although nociceptin may act centrally to inhibit cough, observations that nociceptin can inhibit the function of vagal afferent nerves within the airway indicate that it may also inhibit cough through a peripheral mechanism of action (5, 6).
The aims of the current study were twofold. First, we set out to evaluate the efficacy of nociceptin in inhibiting acid-evoked cough in conscious animals. Second, experiments were designed to decipher the mechanisms by which nociceptin inhibits acid-induced responses in capsaicin-sensitive jugular vagal afferent neurons.
METHODS
In Vivo Cough
Unanesthetized male Hartley guinea pigs (425–475 g) were placed in individual Perspex cylindrical plethysmographs and exposed to citric acid aerosol (0.25 M, 0.5 ml/min) generated with an ultrasonic nebulizer (EMKA Technologies, Paris, France). The time of exposure to the citric acid was 2 min, followed by a further observation period of 9 min. Individual coughs were detected in three ways: (1) via a pressure transducer attached to the plethysmograph, (2) via a microphone placed inside the plethysmograph, and (3) via visual observation of the animal. Each animal was exposed to citric acid only once. Animals were injected intraperitoneally with nociceptin (1–3 mg/kg), 30 min before citric acid administration. Saline was used as the vehicle control.
Intracellular Free Calcium Measurements and Patch Clamp Recordings
The animals (300–400 g) were anesthetized, and DiI (1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate) solution was injected into the tracheal lumen 7 to 9 d before an experiment as previously described (9). The animals were killed (using CO2) and the right and left jugular ganglia were dissected and enzymatically dissociated with Diaspase (GIBCO; Invitrogen, Carlsbad, CA) and collagenase, using standard techniques. The dissociated jugular ganglionic neurons were transferred onto circular 25-mm glass coverslips coated with poly-D-lysine (0.1 mg/ml). The neurons were studied within 24 h of dissociation.
For intracellular calcium measurements, the neurons were loaded with Fura-2 AM (Invitrogen, Carlsbad, CA) and then, 20 min before experiment, superfused by infusion pump (8 ml/min) with 35°C Locke buffer (mM): NaCl, 136; KCl, 5.6; MgCl2, 1.2; CaCl2, 2.2; NaH2PO4, 1.2; NaHCO3, 14.3; dextrose, 10 (pH 7.3–7.4). Intracellular calcium measurements were performed under a microscope equipped for epifluorescence. A field of cells was monitored by sequential dual excitation (352 and 380 nm) and ratios of the images were converted to calcium concentration according to methods and parameters presented previously (10). The ratio images were acquired every 6 s.
Gramicidin-perforated whole cell patch clamp recordings were done in jugular neurons labeled from lungs and airways. The neurons labeled were identified by fluorescence microscopy (excitation filter, 540 nm; emission filter, 600 nm). To maintain intracellular signal pathways, a gramicidin-perforated whole cell patch clamp technique was employed. A pipette (1.5–3 M) was filled with pipette solution composed of the following (mM): KCl, 140; CaCl2, 1; MgCl2, 2; ethyleneglycol-bis- (-aminoethyl ether)-N,N'-tetraacetic acid, 11; dextrose, 10; titrated to pH 7.3 with KOH; 304 mOsm. Gramicidin was dissolved in dimethyl sulfoxide and mixed into the pipette solution to a final concentration of 1 μg/ml just before each recording. During the experiments, the cells were continuously superfused (6 ml/min) by gravity with Locke solution. All recordings were done at 35°C. The membrane potential of the cells was held at –60 mV.
Drugs and Data Analysis
Acidic phosphate buffer (pH 5) and physiologic phosphate buffer (pH 7.3) were modified from Locke solution by replacing bicarbonate with phosphate (Na2HPO4 and NaH2PO4) and then adjusting the sodium concentration with NaCl. Citric acid (0.25 M) was directly dissolved in 0.9% saline just before experiment. In the cough studies data were compared by one-way analysis of variance and Dunnett's post test. Electrophysiologic and calcium data were expressed as means ± SEM. A t test and Mann-Whitney rank sum test were used when appropriate.
RESULTS
Cough
In 14 animals, inhalation of citric acid evoked on average 15 to 20 coughs during the 11-min period of observation. Nociceptin (3 mg/kg, intraperitoneal) significantly inhibited this response by nearly 70% (Figure 1).
Acid-induced Increases in Cytosolic Calcium
The capsaicin-sensitive nerves in guinea pig large airways are derived from cell bodies situated in the jugular ganglia. We therefore addressed the hypothesis that nociceptin may inhibit cough by inhibiting the acid-induced activation of these nerves. Jugular neurons obtained from seven animals were studied. We successfully recorded changes in intracellular free calcium in approximately 15 to 20 neurons per coverslip. In our first series of experiments we found that 72 of 96 jugular neurons responded to acid (pH 5 for 2 min) with rapid increases in intracellular calcium concentration. All 72 of these acid-sensitive neurons responded to capsaicin added 5 min after acid application, whereas the 24 acid-insensitive neurons were found to be capsaicin insensitive. The calcium ionophore ionomycin (1 μM) was used to obtain a "maximal" response in the neurons. The peak acid-induced increase in intracellular calcium averaged 21 ± 7% of that obtained with ionomycin (n = 96); a trace of averaged calcium changes by acid application is shown in Figure 2A. In the second series of experiments we found that the TRPV1 antagonist 5-iodo-resiniferatoxin (I-RTX, 1 μM), reduced the acid-induced response to 4 ± 1% of the ionomycin response (n = 42; Figure 2B). These data indicate that acid causes increases in intracellular calcium in jugular neurons primarily through activation of TRPV1.
In the third series of experiments nociceptin was found to reduce the acid-induced increases in intracellular calcium in jugular capsaicin-sensitive neurons by 53% (Figure 2B). When only capsaicin-sensitive neurons were compared between the control and nociceptin-treated groups, the acidic buffer (pH 5 for 2 min) induced 28.9 ± 3.3 and 13.6 ± 2.4% of the ionomycin-induced intracellular calcium increase in the control group (n = 72) and nociceptin (0.1 μM, 5 min)-treated group (n = 42), respectively (p < 0.01, Mann-Whitney rank sum test).
Patch Clamp Recordings
Forty-five dissociated jugular neurons specifically labeled from the airways of 24 guinea pigs were studied in whole cell patch clamp recordings with a holding potential of –60 mV. In an attempt to maintain intracellular signals, including G protein–coupled signals, the gramicidin-perforated patch clamp technique was employed. Exposing neurons to pH 5 (10-s application) caused a slowly developing, sustained inward current in all capsaicin-sensitive neurons. The elapsed time to the peak inward current of the sustained response was 7.6 ± 1.0 s (n = 23). This sustained current was virtually abolished by I-RTX (1 μM) in all tested neurons (4 ± 4% of control response, n = 5), indicating the involvement of TRPV1 receptors (Figure 3A). In 6 of 23 neurons this slowly developing current was preceded by a rapidly developing and transient inward current in which the elapsed time to the peak of the inward current was 0.8 ± 0.1 s (n = 6). This acid-induced transient current was not inhibited by I-RTX (124 ± 51% of control response, n = 4; Figure 3B) but it was partially inhibited by a nonselective acid-sensing ion channel inhibitor, amiloride (10 μM), in two neurons (90 and 58% inhibition). Two consecutive acid applications with a 5-min interval showed only modest desensitization in sustained I-RTX–sensitive inward current (74 ± 14% of first response, n = 5). After the transient components were isolated by I-RTX pretreatment (for 2 min), the transient components were not desensitized by repeated acid applications (second response averaged 105 ± 33% of first response, n = 5).
Nociceptin (0.1 μM) did not overtly evoke an inward or outward current in voltage-clamped neurons. However, nociceptin effectively and selectively inhibited the I-RTX–sensitive sustained inward current induced by acid application (Figure 4A). When nociceptin (0.1 μM) was superfused over the neurons for 2 min before the second acid application, the sustained inward current was reduced to 20 ± 5% (n = 6) of the first response. This was significantly different from the natural desensitization (p < 0.01, t test; Figure 4B). In six experiments the effect of nociceptin on the rapid transient I-RTX–insensitive current (I-RTX was present throughout these experiments) was evaluated. In contrast to its effect on the slow sustained I-RTX–sensitive component of the acid response, nociceptin had no significant effect on the acid-induced I-RTX–insensitive transient current. In the presence of nociceptin the transient response averaged 107 ± 10% of the control response (p > 0.1, n = 6; Figure 4C).
DISCUSSION
The present results show that nociceptin effectively inhibits cough evoked by acid inhalation in guinea pigs. These results with acid-induced cough are consistent with the known inhibitory effects of nociceptin on capsaicin-induced cough in guinea pigs (7). It is known that cough evoked by inhalation of capsaicin or citric acid in conscious guinea pigs is blocked by TRPV1 antagonists (3, 11, 12). We have previously found that the TRPV1-containing vagal afferent nerves in the guinea pig larynx, trachea, and main bronchi (cough regions) are nearly exclusively nociceptors derived from the jugular vagal ganglia (4). We have also noted that nociceptin can directly inhibit tachykinin release from jugular C-fibers in guinea pig airways (5). We reasoned therefore that nociceptin may inhibit acid-induced cough by interfering with acid-induced activation of jugular afferent nerves. Acid stimulates action potential discharge in terminals of jugular afferent fibers in the airways via both TRPV1-dependent and TRPV1-independent mechanisms (13). The data presented here further support the hypothesis that the mechanism by which nociceptin inhibits acid-induced activation of airway-specific jugular neurons is by selectively inhibiting the TRPV1-dependent component of the response.
Acid evoked large increases in cytosolic calcium only in jugular neurons that were capsaicin sensitive (presumably cell bodies of C-fibers). This calcium response was nearly abolished by I-RTX, indicating that much of the response is secondary to TRPV1 activation. The observation that nociceptin effectively inhibited this acid-induced rise in free cytosolic calcium supports the hypothesis that nociceptin can lead to inhibition of acid-induced gating of TRPV1 channels. These results are in agreement with those of Jia and coworkers, who found that nociceptin similarly inhibited capsaicin-induced TRPV1-dependent increases in cytosolic calcium in isolated vagal sensory neurons (14). The findings are also in agreement with behavioral studies on monkeys, in which nociceptin was found to inhibit capsaicin-induced thermal nociception, presumably by interfering with TRPV1 function (15).
There are several mechanisms that may account for the inhibitory effect of nociceptin on acid-induced increases in cytosolic calcium. Nociceptin has been found to lead to inhibition of voltage-gated Ca2+ channels (16, 17). Others have reported that in medullary dorsal horn neurons and neurons in the raphe nucleus, nociceptin leads to activation of an inward-rectifying potassium current (18, 19). This could lead to a decrease in the amplitude or duration of the acid-induced membrane depolarization and consequent reduction in the calcium response. Consistent with this idea, tertiapin, an antagonist of the inward rectifier current, has been found to interfere with the inhibitory effect of nociceptin on capsaicin-induced increases in cytosolic calcium in nodose neurons (14).
It is difficult to draw firm conclusions regarding potential ionic mechanisms of nociceptin action by studying increasing cytosolic calcium alone. We therefore also addressed these issues with the gramicidin-perforated patch clamp recordings of airway-specific sensory neurons. Airway-labeled capsaicin-sensitive jugular neurons were voltage clamped at –60 mV to reduce the contribution of voltage-gated channels, including voltage-gated calcium channels and inward-rectifying potassium channels. Under these conditions we observed no overt changes in resting membrane current on nociceptin exposure. In these neurons acid evoked a strong TRPV1-dependent inward current and, in a subset of neurons, a TRPV1-independent inward current. The acid-induced TRPV1 activation is consistent with findings of others that acidification can lead to TRPV1 currents at 37°C (20–22). That acid caused TRPV1-dependent and TRPV1-independent inward currents is in keeping with our studies on jugular C-fiber nerve terminals within the airways (13, 23). That nociceptin significantly inhibited only the TRPV1 component of the acid-induced inward current reveals an important selectivity in its mechanisms of action. The results indicate that neither inhibition of voltage-gated calcium channels nor activation of inward rectifier channels is necessary for the observed inhibitory effects of nociceptin on acid-induced responses in airway C-fiber neurons.
We previously found that guinea pig vagal sensory neurons express NOP1 receptors (5), and Jia and coworkers have found that the inhibitory effect of nociceptin on capsaicin-induced calcium increases in guinea pig nodose neurons is blocked by a selective NOP1 receptor antagonist (14). The antitussive action of nociceptin in guinea pigs and cats was also found to be inhibited by a selective NOP1 receptor antagonist (7, 8). With these data in mind, we hypothesize that activation of NOP1 receptors with nociceptin leads to signaling events that selectively decrease the efficacy by which acid (and presumably other TRPV1 agonists) opens the TRPV1 channels. This would be consistent with the observation that nociceptin decreases acid-induced plasma extravasation in guinea pigs and rabbits by a prejunctional inhibition of sensory neuropeptide release (24, 25). Investigation of the signal transduction mechanism was beyond the scope of the present study, but it is noteworthy that nociceptin receptor activation has been found to be associated with decreases in cyclic AMP formation (26, 27). This may be informative in that increases in cyclic AMP are known to increase TRPV1 activity (28).
In contrast to the C-fiber–driven cough observed on acid inhalation, direct application of acid to the larynx or trachea, as might occur on aspiration, stimulates cough via activation of a unique capsaicin-insensitive vagal A "cough receptor" (29). It is not known whether nociceptin can directly inhibit acid-induced activation of A "cough fibers" or the A fiber–induced cough reflex. We have shown that acid activates these A fibers by a mechanism similar to the rapid TRPV1-independent component of the acid response in C-fiber neurons (13). As this was the component of the acid response that was unaffected by nociceptin in C-fiber neurons, it is tempting to speculate that nociceptin will be ineffective in inhibiting acid activation of the A cough fibers.
In summary, the results reveal that nociceptin effectively inhibits acid-evoked cough in conscious guinea pigs. We found that nociceptin can initiate a signaling pathway that leads to significant and selective inhibition of acid-evoked activation of TRPV1. This, in turn, results in decreases in acid-evoked action potential discharge in C-fiber terminals within the airway wall. This type of mechanism may underlie the well-known inhibitory effects of nociceptin on evoked cough reflexes in various animal models, and may provide important clues for those interested in developing effective peripherally acting antitussive agents.
FOOTNOTES
Originally Published in Press as DOI: 10.1164/rccm.200507-1043OC on October 20, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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