Prostaglandin E 2 Enhances Chemical and Mechanical Sensitivities of Pulmonary C Fibers in the Rat

Prostaglandin E 2 Enhances Chemical and Mechanical Sensitivities of Pulmonary C Fibers in the Rat CHING-YIN HO, QIHAI GU, JU-LUN HONG, and LU-YUAN LEE Department of Physiology, University of Kentucky, Lexington, Kentucky; and Department of Otolaryngology, Veterans General Hospital and National Yang-Ming University, Taipei, Taiwan, Republic of China It has been recently reported that pulmonary reflex responses to injection or inhalation challenge of capsaicin are enhanced by exogenous Prostaglandin E 2 (PGE 2 ). The present study was carried out to determine whether PGE 2 enhances the stimulatory effects of chemical stimulants and lung inflation on vagal pulmonary C fibers, and if so, whether the excitabilities of other types of lung afferents are also augmented by PGE 2. In anesthetized, open-chest rats, administration of PGE 2 (1.5 g/kg/min for 2 min) did not significantly change the baseline activity of vagal pulmonary C fibers, but it markedly enhanced the stimulatory effects of both low (0.25 g/kg) and high doses (0.5 g/kg) of capsaicin on these fibers. Similarly, potentiating effects of PGE 2 were found on the pulmonary C-fiber responses to injections of lactic acid and adenosine, although considerable variability existed in the degrees of potentiation between the different stimulants. Furthermore, PGE 2 infusion also significantly enhanced the C-fiber response to constantpressure lung inflation (tracheal pressure [Pt] 30 cm H 2 O). In contrast, PGE 2 did not alter the responses of either slowly adapting pulmonary receptors or rapidly adapting pulmonary receptors to lung inflation. In summary, these results show that the sensitivity of pulmonary C-fiber afferents to both mechanical and chemical stimuli is enhanced by PGE 2, suggesting that endogenous release of this autocoid may play a part in the airway irritation and dyspneic sensation associated with airway inflammation. It is known that bronchopulmonary C-fiber afferents play an important role in the regulation of various airway functions during both physiological and pathological conditions (1 3). Prostaglandin E 2 (PGE 2 ), an inflammatory mediator derived from the cyclooxygenase pathway of arachidonic acid metabolism, is released from several types of cells in the lungs (e.g., epithelial cells, macrophages) during various airway inflammatory reactions (4). It has been well documented that PGE 2 enhances the excitability of somatic nociceptors, the counterpart of bronchopulmonary C-fiber endings in the peripheral tissue, by lowering their threshold to various mechanical and chemical stimuli (5 8). Consistent with these findings, Lee and Morton have recently reported that PGE 2 augments the pulmonary chemoreflex responses elicited by intravenous injection of capsaicin, a potent and selective stimulant of pulmonary C fibers (9). In addition, inhalation of PGE 2 aerosol has been shown to increase the sensitivity of the capsaicin-induced cough reflex in healthy human subjects (10). All these observations seem to suggest a potentiating effect of PGE 2 on the sensitivity of pulmonary C-fiber endings to capsaicin; indeed, preliminary observation made in a small number of pulmonary C fibers seems to support such a notion (9). However, (Received in original form October 15, 1999 and in revised form January 28, 2000) Supported by Grants NIH HL40369 and HL58686 (U.S.A.) to L. Y. Lee, and NSC88-2314-B075-015 (R.O.C.) to C. Y. Ho. 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@pop.uky.edu Am J Respir Crit Care Med Vol 162. pp 528 533, 2000 Internet address: www.atsjournals.org whether the potentiating effect of PGE 2 is also present in the responses of pulmonary C fibers to other chemical stimulants and to mechanical stimulation is not known. Furthermore, question still remains as to whether this modulatory function of PGE 2 is limited only to this particular type of lung afferent. In light of these unanswered questions, the present study was carried out to determine if PGE 2 enhances the stimulatory effects of chemical stimulants and lung inflation on pulmonary C-fiber afferents; and if so, whether administration of the same dose of PGE 2 alters the excitabilities of other types of pulmonary vagal afferents, namely, the slowly adapting pulmonary receptor (SAR) and the rapidly adapting pulmonary receptor (RAR). METHODS The following procedures were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and also were approved by the University of Kentucky Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (290 to 470 g) were anesthetized with intraperitoneal injection of -chloralose (100 mg/kg; Sigma Chemical, St. Louis, MO) and urethane (500 mg/kg; Sigma) dissolved in a 2% borax solution; supplemental doses of the same anesthetics were given, whenever necessary, to maintain abolition of pain reflex elicited by paw-pinch. Femoral artery and vein were cannulated for recording arterial blood pressure (ABP) and for administration of PGE 2 or anesthetics, respectively. The left jugular vein was also cannulated with the tip of the catheter positioned slightly above the right atrium for bolus injections of chemical stimuli. Body temperature was maintained at approximately 36 C throughout the experiment with a servo temperature controller and a heating pad placed under the animal. The trachea was cannulated, and tracheal pressure (Pt) was measured (MP 45-28; Validyne, Northridge, CA) via a side port of the tracheal cannula. The rats were artificially ventilated with a respirator (model 7025; UGO Basile, Comerio-Varese, Italy); tidal volume (VT) and respiratory frequency were set at 8 to 10 ml/kg and 44 breaths/ min, respectively. After a midline thoracotomy, both vagus nerves were ligated just above the diaphragm to eliminate afferent signals arising from lower visceral organs. The opening of the thorax was covered by a sheet of polyethylene film to keep the lung moist, and the expiratory outlet of the respirator was placed under 3 cm H 2 O pressure to maintain a near-normal FRC. The right cervical vagus nerve was separated from the carotid artery and sectioned as rostrally as possible. The caudal end of the cut vagus nerve was placed on a small dissection platform and immersed in a trough of mineral oil held by tethered cervical skin; its nerve sheath was then removed. With the aid of a dissecting microscope and fine-tip forceps, a thin filament was teased away from the nerve trunk and placed on a platinum iridium hook electrode. Action potentials were amplified (P511K; Grass, Quincy, MA), monitored by an audio monitor (Grass AM8RS), and displayed on an oscilloscope (model 2211; Tektronix, Beaverton, OR). The thin filament was further split until the afferent activity from a single unit was electrically isolated. Because vagal pulmonary C fibers usually have a sparse ( 0.5 imp/s) and irregular baseline discharge, hyperinflation of lung (3 to 4 VT) was used as the first step in searching for these fibers. Once the afferent activity of a single unit was identified by hyperinflation, capsaicin (0.5 g/kg) was injected via jugular venous catheter into the right atrium. Only the fibers that responded to capsaicin

Ho, Gu, Hong, et al.: PGE 2 Enhances Excitability of Pulmonary C Fibers 529 within 1 s after the injection were studied. SARs and RARs were identified initially by their distinct phasic discharge synchronous with the respirator cycles, and single units of these receptors were further classified by their adaptation indexes (AI) in response to lung inflation (11) (SARs: AI 70%; RARs: AI 70%). The signals of the afferent activities, Pt, and Pa were recorded on a Thermal Writer (model TW11; Gould, Cleveland, OH) and on a VCR (model 500H; Pacer/Vetter, Los Angeles, CA). Fiber activities (FA) were analyzed (TS-100; Biocybernetics, Taipei, Taiwan) later by a computer for each 0.5-s interval. The responses to intravenous injections of chemical stimulants and to lung inflation were studied in each pulmonary C fiber. Three chemical agents were chosen for this study: capsaicin (0.25 and 0.5 g/kg), lactic acid (90 and 180 g/kg), and adenosine (150 and 300 g/kg). To avoid any sustained systemic effects of the injected stimulants and the difficulties of maintaining the recorded signal from the same fiber for more than 1 h, only two doses of each of these stimulants were tested in each fiber; the low dose chosen for each of these stimulants was near the threshold dose established from our previous studies, and it was doubled for the high dose. Only one chemical stimulant was tested in each fiber. To allow the baseline fiber activity to return to control levels and to avoid any accumulated effect, we waited approximately 15 min between injections. Constant-pressure lung inflation was applied by inflating the lung with a constant air flow (12 ml/s) until Pt reached 15 and 30 cm H 2 O, respectively, and was maintained at that pressure for 10 s after turning off the respirator. In general, SARs and RARs in the rat lung are not sensitive to chemical stimuli, as shown in our earlier study (12), and therefore only the response to lung inflation was tested in these two types of lung afferents. Two minutes before each injection or inflation challenge, the rat s lung was hyperinflated (3 VT) to avoid any pulmonary atelectasis. To establish the reproducibility in each animal, the response to either chemical stimulant or lung inflation was tested at least twice during control. The response was then tested again during a 2-min slow constant-rate (0.5 ml/min) infusion of PGE 2 (1.5 g/kg/min) via the other venous catheter; chemical or mechanical challenge was applied 90 s after the beginning of infusion for ABP to reach a steady state and for measuring any change in baseline fiber activity induced by the PGE 2 infusion. The same challenge was repeated approximately 20 min after the termination of PGE 2 infusion in approximately 45% of the C fibers studied to determine if the effect of PGE 2 was reversible. In addition, to determine whether the potentiating effect of PGE 2 on pulmonary C-fiber excitability was dose-dependent, in a separate group of rats we tested the fiber responses to capsaicin (0.5 g/kg) and to lung inflation (30 cm H 2 O) during infusion of the vehicle and two different doses of PGE 2 (0.75 and 1.5 g/kg/min) in random sequence in each of six pulmonary C fibers. At the end of the experiment, conduction velocities of approximately 56% of the vagal afferent fibers were measured; the intrathoracic segment of the right vagus nerve was isolated as caudally as possible, and a pair of stimulating electrodes was then placed under the vagus nerve cranially to the exit of its pulmonary branches to deliver rectangular constant-current pulse (duration: 1 ms; intensity: 0.3 to 3.0 ma) generated by a pulse generator (model A310; WPI, Sarasota, FL) and a stimulus isolation unit (WPI A360R-C). The exact distance between stimulating and recording electrodes for calculating the conduction velocity was measured postmortem. Finally, the general locations of all fibers were identified by their responses to gently pressing the lungs with a saline-wetted cotton Q-tip or a blunt-ended glass rod. Animals were killed after the experiment by an intravenous injection of KCl. Stock solution of PGE 2 (Sigma; 100 g/ml) was prepared in ethanol and kept in 0.1-ml aliquot at 70 C. Stock solution of capsaicin (Sigma; 200 g/ml) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% saline, and that of lactic acid (Sigma; 500 g/ml) and adenosine hemisulfate salt (Sigma; 10 mg/ml) were prepared by distilled water and saline, respectively. Solution of the desired concentration on the basis of the animal s body weight was prepared daily with isotonic saline dilution, and the volume of each bolus injection of these agents was kept at 0.2 ml. Unless mentioned otherwise, a two-way analysis of variance (ANOVA) was used for the statistical analysis. One factor was the treatment effect of PGE 2 ; the other factor was the effect of chemical stimulants (e.g., capsaicin) or lung inflation. When the two-way ANOVA showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Fisher s least significant difference). Data are reported as means SEM. A p value less than 0.05 was considered significant. RESULTS A total of 103 pulmonary C fibers, 12 RARs and 14 SARs were studied in 66 anesthetized, open-chest rats. The average conduction velocities measured in these fibers were 0.93 0.04 m/s (n 46), 13.8 1.6 m/s (n 12), and 15.7 1.2 m/s (n 14) for pulmonary C fibers, RARs, and SARs, respectively. Pulmonary C fibers had either very low and irregular or no baseline activity during eupneic breathing (0.01 0.00 impulses/second [imp/s]) (Figures 1 and 2). Only the receptors whose locations were identified in the lung structures at the end of the experiment were included for the data analysis in this study. During PGE 2 infusion, the baseline ABP decreased slightly from 89.8 4.9 mm Hg during the control period to 82.5 5.1 mm Hg at the end of infusion, and returned to control immediately ( 20 s) after the termination of infusion. Heart rate and peak Pt did not change significantly during the infu- Figure 1. Experimental records illustrating responses to rightatrial injections of capsaicin of a pulmonary C fiber arising from an ending in the right lower lobe of an anesthetized, open-chest rat (361 g). Conduction velocity of this fiber was 0.93 m/s. Upper panels (A, B), control responses; lower panels (C, D), responses during a constant infusion of PGE 2 (1.5 g/ kg/min, intravenously). Left panels (A, C), responses to bolus injections of low dose of capsaicin (0.25 g/kg); right panels (B, D), responses to high dose of capsaicin (0.5 g/kg). Injectate (0.2 ml) was first slowly injected into the catheter (dead space: 0.3 ml) and then flushed into the right atrium (at the arrow) as a bolus with saline. Approximately 15 min elapsed between injections. Bradycardia and hypotension immediately after the capsaicin injection were reflex responses that could be abolished by sectioning the left vagus nerve (data not shown). AP action potentials; Pt tracheal pressure; BP arterial blood pressure.

530 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 162 2000 sion. During PGE 2 infusion, there was no significant change in the average baseline activity of pulmonary C fibers (Figure 2). However, the stimulatory effect of the same dose of capsaicin on these fibers was markedly enhanced by PGE 2 ; for example, FA, the difference between peak FA (2-s average) and baseline FA (10-s average), in response to the low dose of capsaicin (0.25 g/kg) was 0.72 0.24 imp/s at control and remained unchanged during the repeated challenge (0.64 0.19 imp/s), but the response increased threefold during the PGE 2 infusion (2.09 0.52 imp/s; n 27, p 0.01) (Figure 2). Similarly, a potentiating effect of PGE 2 was found in the response to the higher dose of capsaicin (0.5 g/kg) in the same C fibers ( FA: 4.56 0.75 imp/s at control; 9.13 1.34 imp/s during PGE 2 ). The fiber responses increased in both peak activity and the duration of firing (Figures 1 and 2), and returned to the control level in all the fibers tested approximately 20 min later (n 14). Furthermore, the PGE 2 -induced augmentation of pulmonary C fiber responses to capsaicin was highly reproducible in the six fibers in which the administration of the same dose of PGE 2 was repeated 20 to 35 min later; the first PGE 2 infusion increased response of FA to 0.5 g/kg of capsaicin from 3.79 0.66 imp/s to 7.12 1.01 imp/s, which was not any different (p 0.05) from that induced by the second PGE 2 infusion ( FA: 4.32 0.97 imp/s at control; 8.80 1.48 imp/s during PGE 2 ). PGE 2 potentiated these responses in a dose-dependent manner; higher dose of capsaicin increased both responses to capsaicin and to lung inflation, whereas low dose only potentiated the response to lung inflation but not to capsaicin (Figure 3). In contrast, infusion of PGE 2 vehicle did not cause any potentiating effect on C-fiber response to either capsaicin or lung inflation (Figure 3). Responses of pulmonary C fibers to other chemical stimulants were also elevated during PGE 2 infusion, though considerable variability existed in the degrees of potentiation between these different stimulants (Figure 2). PGE 2 significantly increased the peak responses of C fibers to both low dose (90 g/kg) ( FA 1.43 0.47 imp/s at control; 4.50 0.89 imp/s during PGE 2 ; n 24, p 0.01) and high dose of lactic acid (180 g/kg) ( FA 7.87 0.96 imp/s at control; 10.20 0.99 imp/s during PGE 2 ; n 24, p 0.05). Similarly, PGE 2 increased the C-fiber peak responses to both low dose (150 g/kg) ( FA 2.76 0.63 imp/s at control; 5.96 0.67 imp/s during PGE 2 ; n 20, p 0.01) and high dose of adenosine (300 g/ kg) ( FA 3.79 0.70 imp/s at control; 6.34 0.79 imp/s during PGE 2, n 20, p 0.01). There was a delay of 4 to 11 s in the pulmonary C-fiber response to right atrial injection of either dose of adenosine (Figure 2). In contrast, bolus injection of the same volume of isotonic saline did not stimulate any of the C fibers tested (n 4) either before or during infusion of PGE 2. Constant-pressure lung inflation at Pt 15 cm H 2 O did not cause any detectable stimulation of pulmonary C fibers at control, and the lack of a significant stimulatory effect of lowpressure inflation on these afferents persisted even during Figure 2. Effect of PGE 2 on pulmonary C-fiber responses to injections of chemical stimulants and to lung inflation in anesthetized, open-chest rats. FA was measured in 0.5-s intervals. Capsaicin (n 27), lactic acid (n 24), or adenosine (n 20) was injected as a bolus (0.2 ml) into the right atrium at time zero. Constant-pressure lung inflation (n 45) was maintained for 10 s (between the two arrows). Open circle, control response; closed circle, response during PGE 2 infusion. Left panels, low level of stimulus; right panels, high level. Data represent means SE. Figure 3. Average pulmonary C-fiber responses to capsaicin injection and to lung inflation before (control; open bars) and during infusion of two different doses of PGE 2 (closed bars) in six anesthetized, openchest rats. In the response to capsaicin (0.5 g/kg), FA represents the difference between the peak FA (averaged over 2-s intervals) and the baseline FA (averaged over 10-s intervals). In the response to lung inflation (30 cm H 2 O), FA represents the difference between FA during the 10-s inflation and the baseline FA (each averaged over a 10-s interval). Data at the PGE 2 dose of 0.00 g/kg/min represent responses obtained during infusion of the PGE 2 vehicle. Data represent means SE. *p 0.05, compared with the control response (one-way ANOVA).

Ho, Gu, Hong, et al.: PGE 2 Enhances Excitability of Pulmonary C Fibers 531 Figure 4. Representative experimental records illustrating receptor and cardiovascular responses to constantpressure (30 cm H 2 O) lung inflation at control (upper panels) and during PGE 2 infusion (lower panels) in anesthetized, open-chest rats. Panel A: a pulmonary C fiber arising from an ending in the right upper lobe (body weight: 395 g). Conduction velocity of this fiber was 0.78 m/s. Panel B: a RAR located in the right upper lobe (body weight: 380 g); conduction velocity, 25.0 m/s. Panel C: a SAR located in the right middle lobe (body weight: 370 g); conduction velocity, 16.7 m/s. AP action potentials; Pt tracheal pressure; BP arterial blood pressure. PGE 2 infusion (p 0.06). However, the response to inflation pressure of 30 cm H 2 O was markedly potentiated (Figure 2); FA, the difference between FA during inflation (10-s average) and baseline FA (10-s average), was 0.89 0.18 imp/s at control, and increased to 2.19 0.28 imp/s during PGE 2 infusion (p 0.01; n 45). All the SARs and a majority (92%) of the RARs exhibited distinct phasic baseline activity, which was synchronous with the respirator cycles (e.g., Figure 4). The phasic discharge was present during the expiratory phase in nine of 12 RARs (e.g., Figure 4), during the inspiratory phase in two receptors, and no phasic baseline activity in the remaining one. The discharge increased but ceased rapidly ( 2 s) when a hyperinflation of the lung was induced and maintained for 10 s, whereas it sustained when prolonged (10-s) lung deflation was produced by turning off the respirator and exposing the expiratory line to atmospheric pressure (Figure 5). In sharp contrast, infusion of PGE 2 at the same dose did not cause any change in the afferent responses of RARs to either lung inflation or deflation (Figures 4 and 5; n 12). Similarly, PGE 2 failed to alter the afferent response of SARs to lung inflation (Figures 4 and 6; n 14). DISCUSSION These results show that infusion of PGE 2 markedly enhanced the excitability of pulmonary C fibers to all three chemical agents tested in this study without significant elevation of the baseline activity of these fibers. This augmenting effect of PGE 2 on the C-fiber response was not limited to chemical stimulation; PGE 2 also clearly augmented the responses of these afferents to hyperinflation of the lung (Pt 30 cm H 2 O). Furthermore, the effect of PGE 2 on pulmonary C-fiber excitability was readily reversible and also reproducible in the same fibers tested. In sharp contrast, the same dose of PGE 2 did not cause any detectable change in the response of either RARs or SARs to hyperinflation of the lung, and hence, this potentiating effect of PGE 2 on pulmonary afferent sensitivity seems to be present only in the nonmyelinated C fibers. The cellular mechanisms underlying the PGE 2 -induced hypersensitivity of pulmonary C-fiber afferent endings are not totally understood, but they are probably related to changes in the membrane conductance, or to the resting membrane potential mediated by the activation of prostanoid receptors located on the membrane of the nerve terminals (13), or to both phenomena. Although the type or subtype of prostanoid receptors mediating the sensitizing effect of PGE 2 on pulmonary C fibers observed in this study has not been identified, the EP 2, EP 3A, EP 3B, and EP 4 receptors that have high affinity for PGE 2 based upon the ligand-binding studies are known to be present on the sensory nerves and to mediate the activation of these endings by PGE 2 (13, 14). It has been well documented that PGE 2 increases the sensitivity of nociceptors in the skin and skeletal muscles (5 8). For example, local administration of PGE 2 has been shown to lower the activation threshold of C-fiber endings in these tissues to various mechanical and chemical stimuli (6 8, 15). Recent studies in rat dorsal root ganglion sensory neurons have suggested that PGE 2 -induced sensitization is caused by an increase of the enzyme activity of adenylyl cyclase and the resulting rise in the concentration of intracellular cyclic adenosine monophosphate (AMP), because a similar effect can be also produced by forskolin, a direct activator of adenylyl cyclase (16 18). Cyclic AMP can then stimulate protein kinase A (PKA), which in turn increases the phosphorylation of ion channels (16 18). Indeed, administration of a PKA inhibitor, WIPTIDE, markedly attenuated the PGE 2 -mediated hyperalgesic effect (19). Additional evidence indicates that this PKA- Figure 5. Effect of PGE 2 on RAR responses to lung inflation and deflation in anesthetized, open-chest rats. FA was measured in 0.5-s intervals. Constant-pressure (Pt 30 cm H 2 O) lung inflation was maintained for 10 s (between the two arrows, panel A). Lung deflation was induced by turning off the respirator and exposing the expiratory line to atmospheric pressure for 10 s (between the two arrows, panel B). Open circle, control response; closed circle, response during PGE 2 infusion. Data represent means SE of all 12 receptors from nine rats.

532 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 162 2000 Figure 6. Effect of PGE 2 on SAR responses to lung inflation in anesthetized, open-chest rats. FA was measured in 0.5-s intervals. Constant-pressure (Pt 30 cm H 2 O) lung inflation was maintained for 10 s (between the two arrows). Open circle, control response; closed circle, response during PGE 2 infusion. Data represent means SE of all 14 receptors from eight rats. dependent increase in dorsal root C-neuron sensitivity induced by PGE 2 is mediated through increased phosphorylation of the tetrodotoxin-resistant sodium channel (18, 20). Whether the same pathway holds true for the PGE 2 -induced hypersensitivity of pulmonary C neurons remains to be determined. Three chemical stimulants with entirely different chemical structures and pharmacological properties were chosen for testing pulmonary C-fiber sensitivity in this study. Capsaicin is known for its potency and selectivity in activating C-fiber endings (3, 12). Lactic acid is a major product of anaerobic tissue metabolism and has recently been shown to cause a consistent and rapidly reversible stimulatory effect on pulmonary C fibers (21). Adenosine is produced during hypoxia in virtually all cell types through the degradation of ATP, and is also the drug of choice for the treatment of patients suffering from supraventricular tachycardia; its potent stimulatory effect on pulmonary C fibers has been recently reported (22). The degree of potentiation by PGE 2 did vary considerably among the responses to these three stimulants (Figure 2). The largest increase occurred in the response to capsaicin, whereas the response to the high dose (180 g/kg) of lactic acid was only slightly enhanced (Figure 2), probably because at this high dose the peak response of pulmonary C fiber has already approached its maximal activity even before the administration of PGE 2. Nonetheless, the degree of variation should not be a surprise considering the fact that these different chemical agents activate pulmonary C fibers through different transduction mechanisms. Despite all these differences, the stimulatory effects of both high and low doses of all three chemical agents on pulmonary C fibers were elevated by the administration of PGE 2. Furthermore, in a limited number of trials, we have found that PGE 2 also enhanced, to a varying degree, the responses of pulmonary C fibers to intravenous injections of other chemical stimulants, including phenylbiguanide (2 g/ kg), nicotine (3 g/kg), and formic acid (50 g/kg) (Q. Gu, R. F. Morton, J. L. Hong, and L.-Y. Lee; unpublished data). There is a latency of 4 to 11 s in the C-fiber response to adenosine, both before and during the PGE 2 infusion (Figure 2), which is very different from the responses to capsaicin or lactic acid. In our previous studies (22, 23) we established clear evidence that this exceptionally long latency was caused by the slow action of adenosine. What may account for this latency is not known, but we suggest that it may involve one or two of the possible mechanisms. Adenosine may act on other types of cells in the lung (e.g., mast cells) and trigger the release of inflammatory mediators (e.g., histamine) (24), which can in turn stimulate C-fiber endings. On the other hand, we cannot dismiss the possibility that adenosine may exert a direct stimulatory effect on the C-fiber endings. We have shown that the action of adenosine on these nerve endings is mediated through the activation of A 1 receptors (22, 23), and this subtype of purinoceptors in the nervous system is known to be coupled with several types of G proteins which upon activation can initiate different second-messenger pathways and modulate the conductance of ion channels in the neuronal membrane (25). It is, therefore, possible that the long latency of the afferent response is related to the process mediating these intracellular signal transduction pathways. Whatever the cause, it is noteworthy that the low dose of adenosine administered in this study (150 g/kg) is lower than the therapeutic dose (approximately 200 g/kg, intravenous bolus) for the treatment of supraventricular tachycardia in patients. It is well documented that stimulation of pulmonary C-fiber afferents elicits a number of reflex responses mediating through the cholinergic pathway, such as bronchoconstriction, airway hypersecretion, and bronchial vasodilatation (3). Activation of these afferents is also believed to evoke dyspneic sensation, tachypnea, and coughing (1, 3). Furthermore, a number of neuropeptides (e.g., tachykinins and calcitonin gene related peptide) are synthesized in the cell bodies of pulmonary C fibers and released locally from the sensory endings upon stimulation, which may lead to the development of neurogenic inflammation (26 28). Hence, on the basis of our finding in this study, we suggest that PGE 2 may play a part in enhancing the sensitivities of these endings and in evoking the airway irritation and dyspneic sensation during various airway inflammatory reactions. Coleridge and coworkers have previously demonstrated that a bolus injection of PGE 2 into the right atrium at higher doses (5 to 10 g/kg) significantly increased the baseline activity of pulmonary and bronchial C fibers in dogs (29). However, a possible change in chemical and mechanical sensitivities of these afferents was not investigated in their study. Our results obtained in the present study show that PGE 2 infused continuously at a lower dose, which induced minimal systemic effects, did not cause any change in the baseline activity of pulmonary C fibers but markedly enhanced their sensitivities to chemical stimulants and to lung inflation. These findings jointly have provided an electrophysiological basis that seems to support several previous reports describing the reflex effects of PGE 2 on breathing. For example, inhalation of aerosolized PGE 2 elicits coughs and retrosternal soreness without significant change in baseline airway resistance (30, 31), and augments the dyspneic sensation during exercise (31) in healthy human subjects. In addition, inhaled PGE 2 enhances the sensitivity of the cough reflex elicited by capsaicin in humans (10) and potentiates the chemoreflex responses to capsaicin and phenylbiguanide, another potent stimulant of pulmonary C fibers, in anesthetized rats (9). 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