Chloralose

This study was performed to determine whether activation of dopamine D^sub 2^-like receptors inhibits the hyperresponsiveness of pulmonary C fibers induced by inflammatory mediators such as prostaglandin E^sub 2^ (PGE^sub 2^). In anesthetized, open-chest rats, constant infusion of PGE^sub 2^ (1.5-4.5 [mu]g/kg per minute, 2 minutes) significantly enhanced the C-fiber response to capsaicin injection. At 20 minutes after pretreatment with quinpirole (3 mg/kg, intravenous), a D^sub 2^-like receptor agonist, the hyperresponsiveness to capsaicin of the same C fibers induced by PGE^sub 2^ infusion was markedly attenuated, and this inhibitory effect lasted for more than 90 minutes. The effect of quinpirole was dose dependent and was antagonized by pretreatment with domperidone (5 mg/kg, intravenous), a D^sub 2^-like receptor antagonist, administrated 10 minutes before the quinpirole injection. In a separate series of experiments, C-fiber responses to injections of phenyl biguanide and lactic acid and to constant-pressure lung inflation were augmented by PGE^sub 2^; these potentiating responses were also significantly reduced by quinpirole. Furthermore, the effect of quinpirole was equally effective in inhibiting the increase in excitability of pulmonary C fibers induced by alveolar hypercapnia or constant infusion of adenosine. In conclusion, these results clearly show that activation of the dopamine D^sub 2^-like receptors attenuates the hyperresponsiveness of pulmonary C fibers to both chemical stimuli and lung inflation.

Keywords: inflammation mediators; quinpirole; respiratory hypersensitivity; sensory

Morphologic and physiological studies show that vagal C-fiber afferents innervate all levels of the respiratory tract and play an important role in regulating airway functions under both physiological and pathophysiological conditions (1, 2). Stimulation of these C fibers is known to elicit diffuse and pronounced cardiopulmonary reflex responses (2, 3). The excitability of these afferents can be elevated by certain autacoids (4) that are released from various types of cells (e.g., epithelium, mast cell) in the vicinity of these sensory terminals during airway inflammatory reactions (5). Furthermore, increased excitability of the C-fiber afferents is believed to contribute to the pathogenesis of airway hyperresponsiveness (2, 6). One of these endogenous inflammatory mediators that has been shown to sensitize lung C-fiber afferents is prostaglandin E^sub 2^ (PGE^sub 2^) (3, 7, 8). PGE^sub 2^, a prostanoid derived from arachidonic acid metabolism through the enzymatic action of cyclo-oxygenase and PGE synthase, is released from a number of inflammatory cells in the lungs (5). Inhalation of PGE^sub 2^ aerosol enhances the sensitivity of the cough reflex elicited by capsaicin in humans (9), suggesting a sensitization of pulmonary C-fiber afferents. Indeed, studies in our laboratory have clearly demonstrated the sensitizing effect of PGE^sub 2^ on vagal pulmonary C neurons both in anesthetized rats (7, 8) and in isolated nodose and jugular neurons (10).

Dopamine is a potent neurotransmitter; its receptors can be divided into two subtypes, D^sub 1^- and D^sub 2^-like receptors, on the basis of their pharmacologic properties (11). The dopamine D^sub 2^-like receptor subfamily consists of three receptor subtypes: the D^sub 2^, D^sub 3^, and D^sub 4^ receptors (11); the D^sub 3^ and D^sub 4^ subtypes are less abundant than the D^sub 2^ subtype. Activation of dopamine D^sub 2^-like receptors is known to inhibit the activity of dopaminergic neurons in the ventral tegmental area of the brain (12). In addition, stimulation of D^sub 2^-like receptors has been shown to attenuate the afferent responses of carotid chemoreceptors to hypoxia (13). One study further reported that activation of D^sub 2^-like receptors reduces the histamine-induced activation of rapidly adapting receptors (RARs) in canine lungs (14). Indeed, the presence of D^sub 2^-like receptors has been demonstrated in rat sensory neurons (15) that project into airways (16). In light of this background information, the present study was performed to investigate whether activation of D^sub 2^-like receptors attenuates the hypersensitivity of pulmonary C-fiber afferents induced by endogenous inflammatory mediators such as PGE^sub 2^. Some of the preliminary results of this study have been previously reported in the form of an abstract (17).

METHODS

Animal Preparations

Male Sprague-Dawley rats (360-445 g) were anesthetized with an intraperitoneal injection of a-chloralose (100 mg/kg) and urethane (500 mg/kg). The procedures for surgical preparation of the animal and the methodology for obtaining physiological measurements are described in detail in our previous study (18). Briefly, the femoral vein and artery, and the left jugular vein, were cannulated to allow infusion of PGE^sub 2^, recording of the arterial blood pressure, and right atrial administration of pharmacologic agents, respectively. The trachea was cannulated and tracheal pressure (Ptr) was measured via a side port of the cannula. The rat was artificially ventilated by a respirator with a positive end-expiratory pressure of 3 cm H2O after the chest was opened to identify the locations of receptor endings; tidal volume (V^sub T^) and respiratory frequency were set at 8-10 ml/kg and 50 breaths/minute, respectively. The fiber activities (FA) of vagal afferents were measured by the conventional single-fiber recording technique as described previously (8, 18).

Experimental Design and Protocols

Two study series were performed.

Study series 1. To study whether D^sub 2^-like receptor activation altered the PGE^sub 2^-induced hyperresponsiveness of bronchopulmonary C fibers, the afferent responses to right atrial injection of capsaicin during PGE^sub 2^ were investigated before, and 20 and 90 minutes after, pretreatment with quinpirole (3 mg/kg, intravenous), an agonist of the D^sub 2^-like receptors. The dose and the protocol were determined in our preliminary studies. To determine the specificity of the D^sub 2^-like receptor involvement, the same protocol was repeated in a different group of animals, except that domperidone (5 mg/kg, intravenous), an antagonist of D^sub 2^-like receptors, was injected 10 minutes before the administration of quinpirole. This dose of domperidone has been reported to exert a complete blocking effect on D^sub 2^-like receptor-mediated depressions in cardiovascular responses in anesthetized rats (19). At least 30 minutes elapsed between the two PGE^sub 2^ infusions to ensure complete recovery from the effect of PGE^sub 2^. To determine whether the effect oi quinpirole was dose dependent, three doses of quinpirole [0 (vehicle), 1.5, and 3.0 mg/kg] were used; to avoid possible cumulative effects of quinpirole, each dose was administered to a separate group of rats.

Study series 2. To determine whether the effect of quinpirole on C fibers was limited to capsaicin as a stimulant, two other chemical stimulants of C-flber afferents were chosen: phenyl biguanide (PBG, 3-8 [mu]g/kg) and lactic acid (LA, 9-18 mg/kg) (20, 21). To avoid any sustained systemic effect of the injected chemicals, only one of these stimulants was studied in each animal. In addition, the possible effect of quinpirole on the PGE^sub 2^-induced hyperresponsiveness of C fibers to mechanical stimulation was also studied with constant-pressure lung inflation (30 cm H2O maintained for 10 seconds). Chemical or mechanical challenge was applied during the last 30 seconds of a 2-minute continuous infusion of PGE^sub 2^ (1.5-4.5 [mu]g/kg per minute).

To investigate the specificity and effectiveness of this inhibitory effect of quinpirole, similar experimental protocols were performed when the C-fiber hyperresponsiveness was produced by transient alveolar hypercapnia and by intravenous infusion of adenosine, both of which have been demonstrated to induce hypersensitivity of pulmonary C-fiber afferents; both protocols have been described in detail in our previous studies (22, 23).

Materials

Solutions of PGE^sub 2^, capsaicin, PBG, and LA were prepared as described in our previous studies (8, 21). Quinpirole solution (1 mg/ml) was prepared in isotonic saline. Domperidone (5 mg) was first dissolved in acetic acid (20 [mu]l) and then diluted with saline to a final concentration of 2.5 mg/ml before use.

Statistical Analysis

One-way analysis of variance was used for the statistical analysis. When the analysis of variance showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Fisher's least significant difference). A p value

RESULTS

A total of 66 vagal bronchopulmonary C-fiber afferents were studied in 54 anesthetized, open-chest rats. The distributions of locations of these afferents were as follows: 18, 23, 14, and 8 in the upper, middle, lower, and accessory lobes of the right lung, respectively. The precise locations of the remaining three fibers were not determined.

Study Series 1

During control, right atrial injection of capsaicin (0.25-1.0 [mu]g/kg; mean, 0.53 [mu]g/kg) evoked a mild but distinct discharge of C-fiber afferents ([Delta]FA, 2.1 + or - 0.4 impulses [imp]/second; n = 10). During PGE^sub 2^ infusion (1.5-4.5 [mu]g/kg per minute; mean, 3.1 [mu]g/kg per minute, 2 minutes), the afferent response induced by the same dose of capsaicin was greatly enhanced (e.g., Figure 1). Immediately after the administration of quinpirole (3 mg/kg, slow infusion for about 30 seconds), arterial blood pressure and heart rate decreased from baselines of 89 + or - 4 mm Hg and 304 + or - 11 beats/minute to lowest values of 55 + or - 3 mm Hg and 277 + or - 11 beats/minute, respectively. Both returned toward baseline (80 + or - 4 mm Hg and 305 + or - 10 beats/minute) after about 20 minutes. At the same time (about 20 minutes) after quinpirole pretreatment, the hyperresponsiveness of C-fiber afferents to capsaicin induced by PGE^sub 2^ was substantially attenuated, but was not completely abolished: [Delta]FA evoked by capsaicin during PGE^sub 2^ infusion was 8.9 + or - 1.8 imp/second before quinpirole, and 3.7 + or - 1.0 imp/second 20 minutes after quinpirole (n = 10, p 0.05).

The quinpirole-induced inhibitory effect on these afferents was dose dependent; a lower dose (1.5 mg/kg) showed a similar but less pronounced attenuating effect on the PGE^sub 2^-induced C-fiber hyperresponsiveness: [Delta]FA evoked by capsaicin during PGE^sub 2^ infusion were 9.1 + or - 1.6 imp/second before quinpirole, and 6.9 + or - 0.9 imp/second 20 minutes after quinpirole (n = 8; p 0.05); heart rate was 318 + or - 12 and 311 + or - 14 beats/minute before and immediately after quinpirole, respectively (n = 9; p > 0.05). The inhibitory effect of quinpirole on PGE^sub 2^-induced C-fiber hyperresponsiveness was also clearly antagonized by domperidone (Figures 1-3).

Study Series 2

Inhibition of C-fiber hyperresponsiveness by quinpirole was not limited to capsaicin as the stimulant; quinpirole (3 mg/kg) significantly attenuated the PGE^sub 2^-induced hyperresponsiveness of pulmonary C fibers to PBG (n = 8) and LA (n = 7) (Figure 4). Furthermore, quinpirole pretreatment (3 mg/kg) also significantly depressed the PGE^sub 2^-induced enhancement of C-fiber response to constant-pressure (Ptr = 30 cm H2O) inflation of the lung (n = 10) (Figures 5 and 6). These inhibitory effects of quinpirole lasted for more than 90 minutes (e.g., Figure 5) and were also prevented by domperidone pretreatment (5 mg/kg; n = 7) (Figures 5 and 6).

During a constant intravenous infusion of adenosine (40 [mu]g/kg per minute, 2 minutes), C-fiber response to capsaicin was significantly enhanced ([Delta]FA, 2.7 + or - 1.5 imp/second at control and 8.7 + or - 2.2 imp/second during adenosine), and this enhancement was markedly reduced 20 minutes after quinpirole administration ([Delta]FA, 3.7 + or - 0.3 imp/second; n = 3). Transient alveolar hypercapnia was induced by the method described in our report (23). Briefly, a CO2-enriched gas mixture (15% CO2, balance air) was administered via the respirator inlet for 30 seconds, and capsaicin challenge was made during the last 5 seconds of hypercapnia. A similar inhibitory effect of quinpirole was also found in hypercapnia-induced hyperresponsiveness to capsaicin in the three pulmonary C fibers tested: [Delta]FA: 5.2 + or - 2.1 imp/second at control; 16.7 + or - 5.8 imp/second during hypercapnia; 6.7 + or - 4.4 imp/second during hypercapnia after quinpirole.

DISCUSSION

Our results show that activation of D^sub 2^-like receptors by quinpirole attenuated the PGE^sub 2^-induced hyperresponsiveness of pulmonary C-fiber afferents to all three chemical agents tested in this study. This inhibitory effect was dose dependent and lasted for more than 90 minutes. The quinpirole-induced inhibition of C-fiber excitability was also found in the responses of these afferents to hyperinflation of the lung (Ptr = 30 cm H2O). Furthermore, this inhibitory effect was not limited to PGE^sub 2^ as the sensitizing agent; a similar inhibition was also observed in the C-fiber hyperresponsiveness induced by adenosine infusion and by transient alveolar hypercapnia. Studies have demonstrated that the sensitizing effect of PGE^sub 2^ on pulmonary C-fiber afferents is mediated through activation of E-prostanoid 2 (EP^sub 2^) receptors and other subtypes of prostanoid receptors coupled to the G^sub s^ proteins (10, 24). Activation of these EP receptors will then activate adenylyl cyclase and trigger the cyclic AMP/protein kinase A (PKA) transduction cascade, which in turn leads to an increase in membrane excitability. On the other hand, studies in our laboratory have shown that the hypersensitivities of pulmonary C fibers induced by transient alveolar hypercapnia and by adenosine infusion are mediated through the action of protons (23) and the activation of the adenosine A^sub 1^ receptor (22, 25), respectively. Furthermore, the sensitizing effect of alveolar hypercapnia on pulmonary C fibers is not affected by pretreatment with indomethacin, a cyclo-oxygenase inhibitor, suggesting that this effect is not mediated through the CO2-induced production of PGE^sub 2^ (26) (Q. Gu, J. Hong, and L. Y. Lee, unpublished data). Hence, the possibility that the inhibitory action of quinpirole is due to its direct modulatory effect on the prostanoid receptor can be ruled out. Instead, the attenuating effect of quinpirole is probably produced by a selective activation of D^sub 2^-like receptors, presumably located on the terminal membrane of these afferents (16).

Morphologic evidence shows that about 75% of the afferent fibers in the vagal branches innervating the respiratory tract are C fibers (1). Stimulation of these C-fiber afferents is known to elicit a number of important reflex responses involving the central nervous system and autonomic nervous system such as bronchoconstriction, hypersecretion of mucus, cough, airway mucosal edema, and bronchial vasodilation (2, 3). In addition, several sensory neuropeptides such as tachykinins are released locally from the C-fiber sensory terminals on stimulation and are known to produce local "axon reflex" responses, including bronchoconstriction, plasma extravasation, and edema of airway mucosa (27). Hence, sensitization of these afferents by certain locally released inflammatory mediators, such as PGE^sub 2^, may exaggerate the airway responses mediated by the central and local reflexes described above, which may in turn contribute to the pathogenesis of airway dysfunction during mucosal inflammation (28). On the basis of our findings in this study, activation of D^sub 2^-like receptors can, presumably, attenuate these augmented airway responses to a substantial extent. Indeed, an inhibitory effect of D^sub 2^-like receptor activation on plasma extravasation in the trachea and on neuropeptide release from airway sensory neurons induced by chemical stimulation has been reported (29, 30).

The cellular mechanism by which quinpirole attenuates the hypersensitivity of pulmonary C fibers is not well understood, but it is probably related to modulation of the membrane conductance as a result of activating D^sub 2^-like receptors. In fact, D^sub 2^-like receptor activation is known to trigger several intracellular events via the second-messenger pathways; for example, stimulation of D^sub 2^-like receptors inhibits adenyl cyclase activity via activation of the inhibitory G^sub i^ protein and depresses the activity of PKA (11, 31). The inhibition of PKA activity may in turn attenuate the neuronal excitability by decreasing the phosphorylation of certain ion channels. Indeed, D^sub 2^-like receptor activation is known to lead to reductions in voltage-dependent Ca^sup 2+^ currents (32, 33). Furthermore, stimulation of the D^sub 2^-like receptors has also been shown to modulate the hyperpolarization-activated current I^sub h^ (34, 35) and increase the K^sup +^ conductance (36, 37) in various types of neurons. These effects have been suggested to be involved in depression of the excitability of the vagal sensory axons (30, 38) and the dopaminergic neuron in the ventral tegmental area of the brain (12).

The possibility that quinpirole may produce an inhibitory effect by activating D^sub 2^-like receptors located on other cell types in airways should also be considered. For example, activation of D^sub 2^-like receptors on alveolar epithelium can increase lung liquid reabsorption via a mechanism that is independent of amiloride-sensitive Na^sup +^ transport (39). Because pulmonary edema is known to stimulate both RARs and pulmonary C fibers (40), these observations could imply that the inhibitory effect of D^sub 2^-like receptor activation on hyperresponsiveness of C fibers may be related to its modulation of lung fluid clearance. Indeed, PGE^sub 2^ has been suggested to act as an edematous mediator in the lung (41). However, the dose range of PGE^sub 2^ applied in this study was not expected to produce pulmonary edema. This assumption was based on the finding in our previous study that a similar dose of PGE^sub 2^ failed to alter the excitability of RARs (9); RARs are known to be more sensitive to change in the mechanical properties of the lung than are C fibers (2, 3). Furthermore, this inhibitory effect of quinpirole is also clearly effective under the conditions of transient alveolar hypercapnia and adenosine infusion, which are not expected to induce pulmonary edema. In addition, the presence of D^sub 2^-like receptors on the cell membrane of airway vagal sensory neurons has been reported (16). Taken together, these observations seem to suggest that the inhibitory effect of quinpirole on pulmonary C-fiber hyperresponsiveness is most likely mediated through its direct action on D^sub 2^-like receptors located on these afferent endings.

Intravenous injection of quinpirole caused immediate bradycardia and hypotension in anesthetized rats. However, these cardiovascular responses subsided almost completely 20 minutes later, while the quinpirole-induced inhibitory effect on pulmonary C-fiber hyperresponsiveness was still present, suggesting that the initial cardiovascular depression induced by quinpirole cannot account for its inhibitory effects on C-fiber excitability. To further determine the specificity of the involvement of D^sub 2^-like receptor activation, the same protocol was repeated in a different group of animals except that domperidone was administered 10 minutes before the quinpirole pretreatment. After domperidone pretreatment, the initial transient depression of cardiovascular responses was completely abolished, indicating that the dose of domperidone was sufficient to block D^sub 2^-like receptors. Moreover, domperidone pretreatment also effectively prevented the sustained inhibition of C-fiber hyperresponsiveness induced by quinpirole, which demonstrates the specificity of the D^sub 2^-like receptor involvement in attenuating C-fiber hyperresponsiveness.

In summary, results obtained in this study show that quinpirole attenuates the PGE^sub 2^-induced hyperresponsiveness of pulmonary C-fiber afferents to chemical stimulations and to lung inflation. This inhibitory effect was also evident when C-fiber hyperresponsiveness was induced by other chemical agents. The attenuating effect of quinpirole involves a selective activation of D^sub 2^-like receptors, which are presumably located on the sensory terminals of these afferents. A number of autacoids, including PGE^sub 2^, are known to increase the excitability of bronchopulmonary C fibers (3). In contrast, few endogenously released substances, to our knowledge, have been identified as attenuating the hypersensitivity of these afferents. Results obtained in this study seem to suggest that endogenous dopamine may induce a similar inhibitory effect on these afferents via the activation of D^sub 2^-like receptors. However, the significance of this finding with respect to C-fiber hyperresponsiveness under various physiological and pathophysiological conditions remains to be explored.

Acknowledgment: The authors are grateful to Dr. Jonathan Turner (AstraZeneca UK, Ltd.) for helpful suggestions and discussions, and to Mr. Robert Morton for technical assistance in this study.

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You Shuei Lin, Qihai Gu, and Lu-Yuan Lee

Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky

(Received in original form October 14, 2002; accepted in final form January 13, 2003)

Supported by a grant from the NHLBI (HL58686) and by research funds from AstraZeneca UK, Ltd.

Correspondence and requests for reprints should be addressed to Lu-Yuan Lee, Ph.D., Department of Physiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0298. E-mail: lylee@uky.edu

Am J Respir Crit Care Med Vol 167. pp 1096-1101, 2003

Originally Published in Press as DOI: 10.1164/rccm.200210-1171OC on January 16, 2003

Internet address: www.atsjournals.org

Copyright American Thoracic Society Apr 15, 2003
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