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J Appl Physiol 102: 1545-1555, 2007. First published December 14, 2006; doi:10.1152/japplphysiol.01010.2006
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Epinephrine enhances the sensitivity of rat vagal chemosensitive neurons: role of beta3-adrenoceptor

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

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

Submitted 10 September 2006 ; accepted in final form 7 December 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This study was carried out to determine whether epinephrine alters the sensitivity of rat vagal sensory neurons. In anesthetized rats, inhalation of epinephrine aerosol (1 and 5 mg/ml, 3 min) induced an elevated baseline activity of pulmonary C fibers and enhanced their responses to lung inflation (20 cmH2O, 10 s) and right atrial injection of capsaicin (0.5 µg/kg). In isolated rat nodose and jugular ganglion neurons, perfusion of epinephrine (3 µM, 5 min) alone did not produce any detectable change of the intracellular Ca2+ concentration. However, immediately after the pretreatment with epinephrine, the Ca2+ transients evoked by chemical stimulants (capsaicin, KCl, and ATP) were markedly potentiated; for example, capsaicin (50 nM, 15 s)-evoked Ca2+ transient was increased by 106% after epinephrine (P < 0.05; n = 11). The effect of epinephrine was mimicked by either BRL 37344 (5 µM, 5 min) or ICI 215,001 (5 µM, 5 min), two selective beta3-adrenoceptor agonists, and blocked by SR 59230A (5 µM, 10 min), a selective beta3-adrenoceptor antagonist, whereas pretreatment with phenylephrine ({alpha}1-adenoceptor agonist), guanabenz ({alpha}2-adrenoceptor agonist), dobutamine (beta1-adrenoceptor agonist), or salbutamol (beta2-adrenoceptor agonist) had no significant effect on capsaicin-evoked Ca2+ transient. Furthermore, pretreatment with SQ 22536 (100–300 µM, 15 min), an adenylate cyclase inhibitor, and H89 (3 µM, 15 min), a PKA inhibitor, completely abolished the potentiating effect of epinephrine. Our results suggest that epinephrine enhances the excitability of rat vagal chemosensitive neurons. This sensitizing effect of epinephrine is likely mediated through the activation of beta3-adrenoceptor and intracellular cAMP-PKA signaling cascade.

stress; vagal afferents; fura-2; adenylate cyclase; cAMP-dependent protein kinase

EPINEPHRINE IS THE PRIMARY CATECHOLAMINE secreted by the adrenal medulla in response to various physiological stresses (41). The physiological functions of epinephrine are mediated through the activation of cell membrane {alpha}- and beta-adrenoceptors, both of which belong to the superfamily of seven-transmembrane G-protein-coupled receptors (13). Vagus nerves provide the primary afferent innervation of a broad spectrum of visceral organs and play an important role in the initiation of visceral/viscerosomatic reflexes and the regulation of vegetative functions (24, 29). Numerous studies have identified the presence of beta-adrenoceptors on the vagus nerve in both rats and humans (23, 28). Although a sensitizing effect of epinephrine on nociceptors was recently reported (21), whether epinephrine can modulate the excitability of vagal sensory neurons is largely unknown.

The aims of the present study were 1) to determine whether epinephrine alters the excitability of pulmonary vagal afferents in vivo and the chemosensitivity of isolated vagal sensory neurons in vitro; 2) if so, to identify the specific subtype of adrenoceptors subserving the effect of epinephrine; and 3) to investigate the involvement of cAMP-PKA intracellular transduction cascade in mediating this effect. Since intracellular Ca2+ is known to play a critical role in regulating a diverse range of cellular processes such as neuronal membrane excitability, neurotransmitter release, synaptic plasticity, cell proliferation, and gene transcription (3), fura-2-based ratiometric Ca2+ imaging was employed to determine the sensitivity of these vagal sensory (nodose and jugular ganglion) neurons isolated from adult Sprague-Dawley rats. Three chemical agents (capsaicin, KCl, and ATP) known to stimulate vagal sensory neurons (12) by activating different ion channels were used as the chemical stimuli in the Ca2+ imaging study.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The procedures described below 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 were also approved by the University of Kentucky Institutional Animal Care and Use Committee.

In Vivo Study

Recording of single-fiber activity of pulmonary afferents.   Sprague-Dawley rats (280–455 g; n = 31) were initially anesthetized with an intraperitoneal injection of {alpha}-chloralose (100 mg/kg) and urethane (500 mg/kg) dissolved in a borax solution (2%); supplemental doses of these anesthetics were injected intravenously to maintain abolition of pain reflexes. A short tracheal cannula was inserted after a tracheotomy, and tracheal pressure was measured (Validyne MP 45-28, Northridge, CA) via a side port of the tracheal cannula. The expiratory outlet of the respirator was placed under 3 cmH2O pressure to maintain a near-normal functional residual capacity. Tidal volume and frequency were set at 8–10 ml/kg and 50 breaths/min, respectively. One femoral artery was cannulated for recording the arterial blood pressure (ABP). For right-atrial injection of capsaicin, the left jugular vein was cannulated, and a catheter was advanced until its tip was positioned just above the right atrium. The volume of each bolus injection was 0.15 ml, which was first injected into the catheter (dead space, ~0.2 ml) and then flushed into the circulation by an injection of 0.4 ml of saline. Body temperature was maintained at ~36°C by means of a heating pad placed under the animal. At the end of the experiment, the animal was killed by an intravenous injection of KCl.

Vagal pulmonary afferents can be broadly classified into three major types, based on the criteria established by previous investigators (33): slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and pulmonary C-fibers. These pulmonary afferents were identified using the procedures described previously (14), and the fiber activity (FA) was measured by the conventional method of single-fiber recording. Briefly, right cervical vagus nerve was sectioned as far rostrally as possible, and the caudal end of the cut vagus was placed on a small dissecting platform and immersed in a pool of mineral oil. A thin filament was teased away from the desheathed nerve trunk and placed on a miniature platinum-iridium electrode. Action potentials were amplified, monitored by an audio monitor, and displayed on an oscilloscope. The thin filament was further split until the action potentials arising from a single unit were electrically isolated. FA, tracheal pressure, ABP, and heart rate were analyzed with an online data acquisition system (Biocybernetics TS-100, Taipei, Taiwan).

Experimental protocols.   To study the effect of epinephrine on pulmonary C-fiber afferents, the single-unit responses to lung inflation (20 cmH2O, 10 s) and to capsaicin (0.5 µg/kg) were investigated before and 2, 10, and 20 min after the administration of epinephrine. To minimize systemic effects, epinephrine was delivered into the airways by aerosol; during the delivery of the epinephrine, the ultrasonic nebulizer (Lumiscope 6610; Lumiscope, East Brunswick, NJ) that generated the aerosol was connected between the outlet of the respirator and the tracheal tube to prevent contamination of the breathing circuit by the residual epinephrine. The mass mean diameter of aerosol and the total volume of solution delivered over 3 min under our experimental setting were estimated to be 3–4 µm and 0.05–0.06 ml, respectively. To determine whether the effect of epinephrine was dose dependent, three doses of epinephrine were used in different groups of rats: 0 mg/ml (vehicle: 5 mg/ml ascorbic acid in saline), 1 mg/ml (low concentration), and 5 mg/ml (high concentration). No more than two doses of epinephrine were administrated in each animal. Because of the long-lasting effect of the high concentration of epinephrine, responses were also measured at 60 min after the aerosol challenge. Identical protocols were followed to study the effect of epinephrine on the responses of SARs and RARs to lung inflation (20 cmH2O, 10 s) in a separate group of rats.

In Vitro Study

Isolation and culture of nodose and jugular ganglion neurons.   Because subtypes of adrenoceptors are known to be expressed in different cell types in the airways and their individual involvements cannot be differentiated in the in vivo preparation, the following experiments were performed in isolated vagal sensory neurons to identify the receptor subtypes subserving the effect of epinephrine. Young adult male Sprague-Dawley rats (150–220 g; n = 35) were anesthetized with 4% halothane and decapitated. The head was immediately immersed in ice-cold Hank's balanced salt solution. Nodose and jugular ganglia were extracted under a dissecting microscope and placed in ice-cold Dulbecco's minimal essential medium/F-12 (DMEM/F12) solution. Each ganglion was desheathed, cut into ~10 pieces, placed in 0.125% type IV collagenase, and incubated for 1 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min) and supernatant aspirated. The cell pellet was resuspended in 0.05% trypsin in Hanks' balanced salt solution for 5 min and centrifuged (150 g, 5 min); the pellet was then resuspended in a modified DMEM/F12 solution [DMEM/F12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µM MEM nonessential amino acids] and gently triturated with a small bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 g, 8 min) through a layer of 15% bovine serum albumin to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/F12 solution supplemented with 50 ng/ml 2.5S nerve growth factor, plated onto poly-L-lysine-coated glass coverslips, and then incubated overnight (5% CO2 in air at 37°C).

In a subset of experiments, sensory neurons innervating the lungs and airways were identified by retrograde labeling from the lungs (22) with the fluorescent neuronal tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Briefly, rats (140–180 g) were anesthetized with intraperitoneal injection of pentobarbital sodium (40 mg/kg) and intubated with a polyethylene catheter (PE-150) with its tip positioned in the trachea above the thoracic inlet. DiI was initially sonicated and dissolved in ethanol, diluted in saline (1% ethanol, vol/vol), and then instilled into the lungs (0.2 mg/ml; 0.2 ml x 2) with animal's head tilted upward at ~30°C. The animal was allowed to recover for 7–10 days to permit DiI to be transported back to the cell soma of pulmonary vagal sensory neurons.

Intracellular Ca2+ measurement.   Intracellular Ca2+ was monitored using the fluorescent Ca2+ indicator fura 2-AM. Cells were loaded with 5 µM fura 2-AM for 30 min at 37°C, then rinsed (x3) with extracellular solution and allowed to deesterify for at least 30 min before use. Ratiometric Ca2+ imaging was performed using a fluorescence inverted microscope (Axiovert 100; Carl Zeiss, Thornwood, NY) equipped with a variable filter wheel (Lambda 10-2; Sutter Instrument, Novato, CA) and a digital charge-coupled device camera (Princeton Instruments, Trenton, NJ). Dual images (340- and 380-nm excitation, 510-nm emission) were collected and pseudocolor ratiometric images monitored during the experiments by using the software Axon Imaging Workbench (Axon Instruments, Union City, CA). The imaging system was standardized with a two-point calibration, using a Ca2+-free standard (–) and a Ca2+-saturated standard (+). Both standards contained 11 µM fura 2 [44 µl of 10 mM fura 2 Penta K+ salt, 8 ml of 20 mM HEPES-Na (pH 7.4), 32 ml of H2O] and were prepared as follows: (– standard) 18 ml of fura 2, 1.98 ml of 10 mM EGTA-Na (pH 7.6); (+ standard) 18 ml fura 2, 1.98 ml of 10 mM CaCl2. The parameters used for the two-point calibration include: the dissociation constant of fura-2 (Kd; 225), the ratio values for the – and + concentration standards (Rmin and Rmax), and the denominator wavelength intensities for the – and + concentration standards (Denmin and Denmax). The intracellular Ca2+concentration ([Ca2+]i; in nM) was estimated according to the following equation described by Grynkiewicz et al. (11): [Ca2+]i = Kd[(R – Rmin)/(Rmax – R)](Denmin/Denmax). Typical Rmin and Rmax values were 0.586 and 2.54, respectively.

Experimental protocols.   Following the incubation period with fura 2-AM, the coverslip containing vagal sensory neurons was mounted onto a chamber (0.2 ml) placed on the stage of the microscope. During the experiments, the entire chamber was continuously perfused with the standard extracellular solution (containing in mM: 5.4 KCl, 136 NaCl, 1 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES; pH 7.4) or with standard extracellular solution containing various pharmacological and chemical agents by a gravity-fed valve-control system (VC-66CS; Warner Instruments, Hamden, CT). At the onset of each test, the perfusing solution was switched from one to another (e.g., from extracellular solution to epinephrine or from epinephrine to capsaicin) and delivered via the common outlet of a manifold operated by the valve-control system. The perfusion was kept at a constant rate of ~2 ml/min; a complete change of bath solution occurred in 6 s. KCl solution (100 mM) was perfused at the end of each experimental run to test for cell viability. All experiments were performed at room temperature (20–23°C).

Three study series were performed to determine 1) the effect of epinephrine on the Ca2+ transients evoked by low doses of chemical stimulants including capsaicin, KCl, and ATP; 2) the adrenoceptor subtype(s) potentially involved in the effect of epinephrine; and 3) the possibility of the involvement of cAMP-PKA transduction cascade.

Chemicals.   DMEM/F12, trypsin and 2.5S-nerve growth factor were obtained from Invitrogen (Calsbad, CA). Fura 2-AM and DiI were purchased from Molecular Probes (Eugene, OR). Urethane, {alpha}-chloralose, borax, collagenase, capsaicin, KCl, ATP, epinephrine, ascorbic acid, phenylephrine ({alpha}1-adrenoceptor agonist), guanabenz ({alpha}2-adrenoceptor agonist), dobutamine (beta1-adrenoceptor agonist), salbutamol (beta2-adrenoceptor agonist), BRL 37344 (beta3-adrenoceptor agonist), ICI 215,001 (beta3-adrenoceptor agonist), SR 59230A (beta3-adrenoceptor antagonist), SQ 22536 (adenylate cyclase inhibitor), and H89 (PKA inhibitor) were obtained from Sigma (St. Louis, MO).

In the Ca2+ imaging study, stock solution of capsaicin (1 mM) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% extracellular solution; H89 (10 mM) was dissolved in dimethyl sulphoxide; epinephrine (20 mM) was dissolved in a mixture of extracellular solution with equivalent amount of ascorbic acid before each experiment and was kept on ice in subdued lighting conditions. These stock solutions were then diluted with the extracellular solution to yield the appropriate concentration before application. All other chemicals were prepared daily in extracellular solution before applications. The concentrations of BRL 37344 and SR 59230A used have been reported to effectively and selectively activate and antagonize, respectively, the rat beta3-adrenoceptors (18). The concentration of ICI 215,001 was selected based on a concentration-response relationship demonstrating that this concentration produces ~75% of beta3-adrenoceptor-mediated maximal relaxing effect in guinea pig ileum strips while showing no significant agonist activities on guinea pig atrium beta1- and trachea beta2-adrenoceptors (39). The concentrations of SQ 22536 and H89 used have been shown to be effective in selectively inhibiting adenylate cyclase and PKA, respectively, in various cell types (12, 34).

In the single-fiber recording study, desired concentrations of the pharmacological agents were prepared in a similar manner, except that isotonic saline, instead of extracellular solution, was used as vehicle.

Statistic Analysis

A one- or two-way repeated-measures ANOVA was used for the statistical analysis. When results of the ANOVA showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Newman-Keuls test). Data are reported as means ± SE. A P value of <0.05 was considered significant difference.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In Vivo Study

Effect of inhalation of epinephrine aerosol on rat vagal pulmonary afferents.   A total of 25 pulmonary C-fiber afferents were studied in 17 anesthetized, open-chest rats. The distribution of locations of these receptors was as follows: 5 in the upper lobe, 11 in the middle lobe, 8 in the lower lobe, and 1 in the accessory lobe of the right lung.

Inhalation of epinephrine aerosol at both low and high concentrations markedly enhanced the pulmonary C-fiber response to lung inflation (Figs. 1 and 2). This potentiating effect sustained a substantially longer duration after the high concentration of epinephrine was administered.


Figure 1
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  		Fig. 1. Experimental records illustrating the effect of aerosolized epinephrine (Epi) on pulmonary C-fiber responses to lung inflation (20 cmH2O, 10 s) in 3 anesthetized, open-chest rats. A: treatment with vehicle aerosol (5 mg/ml ascorbic acid in saline); receptor location: middle lobe of the right lung; body weight: 410 g. B: treatment with low concentration of Epi aerosol (1 mg/ml); receptor location: right lower lobe; body weight: 350 g. C: treatment with high concentration of Epi aerosol (5 mg/ml); receptor location: right lower lobe; body weight: 380 g. From top to bottom: before, 2 min, 10 min, and 20 min after aerosol inhalation (duration = 3 min). Epi, epinephrine; conc., concentration; AP, action potential; Ptr, tracheal pressure; ABP, arterial blood pressure

 

Figure 2
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  		Fig. 2. Time course of the effect of aerosolized Epi on afferent activity of pulmonary C-fibers in anesthetized, open-chest rats. A–C: 3-min treatments with aerosol of vehicle (5 mg/ml ascorbic acid in saline; n = 12), low concentration of Epi (1 mg/ml; n = 18), and high concentration of Epi (5 mg/ml; n = 10), respectively. Top: baseline fiber activities (FA). Middle: responses to lung inflation (20 cmH2O for 10 s). Bottom: responses to right-atrial injections of capsaicin (Cap; 0.5 µg/kg). FA at each time point was averaged over a 20-s interval for each fiber. {Delta}FA was measured as the difference in fiber activity between the 10-s average during lung inflation or 2-s average after Cap injection, and the 10-s average at the baseline in each fiber. No more than two doses of Epi were administered in each animal. Data are means ± SE. *Significantly different from the corresponding control (P < 0.05).

 
In comparison, a significant potentiating effect on C-fiber response to capsaicin was only found after the challenge with high concentration of epinephrine (Fig. 2, bottom). In addition, inhalation of epinephrine aerosol increased the baseline activity of the pulmonary C-fibers in a dose-dependent manner (Fig. 2, top). This stimulatory effect of epinephrine had a rapid (<2 min) onset and returned slowly to control 10 and 60 min after deliveries of the low and high concentrations of epinephrine, respectively.

Inhalation of low concentration of epinephrine aerosol did not cause any significant change in either ABP or heart rate, whereas high concentration of epinephrine induced an intense hypertension ({Delta}ABP = 37.5 mmHg; n = 8) during aerosol delivery, but this hypertensive response was short lasting (compared with the effect of epinephrine on C-fiber sensitivity) and completely returned to control in <10 min after termination of aerosol delivery.

In contrast, we did not find any detectable effect of epinephrine aerosol on the activity of either SARs or RARs. For example, the average FA of SARs in response to lung inflation did not change significantly after termination of aerosolized epinephrine treatment (pre-aerosol control: 91.0 ± 29.1 imp/s; 2 min after epinephrine: 127.3 ± 34.1 imp/s; P = 0.15; n = 8). Furthermore, a similar pattern of response was found after the delivery of vehicle (5 mg/ml ascorbic acid in saline) in the same group of receptors (pre-aerosol control: 95.4 ± 32.3 imp/s; 2 min after saline: 102.3 ± 29.7 imp/s; P = 0.55; n = 8). Similarly, epinephrine did not cause any detectable effect on the average FA response of RARs to lung inflation (n = 5).

In Vitro Study

Epinephrine potentiated the chemical stimulation-evoked Ca2+ transients in rat vagal sensory neurons.   Consistent with what we have demonstrated in a previous study (12), application of capsaicin (50 nM, 15 s) evoked a reversible Ca2+ transient in small- and medium-size (diameter <35 µm) nodose and jugular ganglion neurons. As shown in Fig. 3A, epinephrine pretreatment (3 µM, 5 min) alone did not have any significant effect on the basal [Ca2+]i but dramatically increased the peak Ca2+ transient evoked by capsaicin. The group data shown in Fig. 3C illustrated that the capsaicin-induced Ca2+ transient was significantly increased after the epinephrine pretreatment by 106% (control: 55.8 ± 12.6 nM; after epinephrine: 115.1 ± 24.7 nM; washout: 75.2 ± 15.7; P < 0.05; n = 11). Pretreatment with the vehicle of epinephrine had no detectable effect on the capsaicin-induced Ca2+ transient (P = 0.81; n = 8) (Fig. 3, B and C).


Figure 3
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  		Fig. 3. Epi potentiates capsaicin-evoked Ca2+ transient in isolated rat vagal sensory neurons. A: experimental record illustrating that the Ca2+ transient evoked by Cap (50 nM, 15 s) was potentiated after pretreatment with Epi (3 µM, 5 min). B: pretreatment with vehicle of Epi had no effect on Cap (50 nM, 15 s)-evoked Ca2+ transient. C: group data showing the effects of Epi (n = 11) and its vehicle (n = 8) on capsaicin-evoked Ca2+ transient. Data are means ± SE. Cap, capsaicin; [Ca2+]i, intracellular concentration of Ca2+ in nM; {Delta}[Ca2+]i, the peak amplitude of Ca2+ transient measured as the difference between the 4-s average at peak and the 30-s average at baseline. *Significantly different from the corresponding control (P < 0.05). {dagger}Significant difference between the corresponding data obtained from pretreatments with Epi and its vehicle (P < 0.05).

 
The potentiating effect of epinephrine was not limited only to respond to capsaicin, the Ca2+ transient evoked by KCl (15 mM, 15 s) was also significantly potentiated after pretreatment with epinephrine (3 µM, 5 min) (Fig. 4). The KCl-evoked Ca2+ transient was elevated after epinephrine pretreatment in both capsaicin-sensitive (control: 83.3 ± 9.7 nM; after epinephrine: 130.5 ± 20.1 nM; washout: 96.3 ± 18.2; P < 0.05; n = 6) and capsaicin-insensitive neurons (control: 86.4 ± 8.3 nM; after epinephrine: 126.5 ± 16.5 nM; washout: 92.6 ± 14.9; P < 0.05; n = 9) (Fig. 4D). In addition, pretreatment with same dose of epinephrine potentiated the ATP (0.3 µM, 15 s)-evoked Ca2+ transient from a control level of 102.1 ± 11.6 nM to 143.5 ± 9.8 nM after the treatment (P < 0.05; n = 8). We should point out that the responses to these chemical stimuli and also the potentiating effect of epinephrine varied considerably among different cells (e.g., Figs. 3 and 4).


Figure 4
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  		Fig. 4. Epi potentiates KCl-evoked Ca2+ transient in isolated rat vagal sensory neurons. Data are means ± SE. A: experimental record illustrating that the Ca2+ transient evoked by KCl (15 mM, 15 s) was potentiated after pretreatment with Epi (3 µM, 5 min). B: pretreatment with vehicle of Epi had no effect on KCl (15 mM, 15 s)-evoked Ca2+ transient. C: group data showing the effects of Epi (n = 8) and its vehicle (veh; n = 7) on KCl-evoked Ca2+ transient. *Significantly different from the corresponding control (Con) (P < 0.05). {dagger}Significant difference between the corresponding data obtained from pretreatments with Epi and its vehicle (P < 0.05). D: group data showing the effect of Epi on KCl-evoked Ca2+ transient in capsaicin-sensitive (Cap+; n = 6) and capsaicin-insensitive (Cap–; n = 9) neurons. *Significantly different from the corresponding control (P < 0.05).

 
The potentiating effect of epinephrine on pulmonary vagal sensory neurons identified by DiI labeling was investigated in separate groups of neurons. Our results showed no significant difference in the capsaicin (50 nM, 15 s) and KCl (15 mM, 15 s)-evoked Ca2+ transients between labeled neurons and nonlabeled ones matched in size from the same cultures, either during control or after epinephrine pretreatment (Fig. 5).


Figure 5
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  		Fig. 5. Comparison of the potentiating effect of Epi between pulmonary (DiI labeled) and nonpulmonary (unlabeled) rat vagal sensory neurons matched in size from the same cultures. A: effect of Epi (3 µM; 5 min) on the Cap (50 nM; 15 s)-evoked Ca2+ transient (DiI labeled: n = 10; unlabeled: n = 11). B: effect of Epi (3 µM; 5 min) on the KCl (15 mM; 15 s)-evoked Ca2+ transient (DiI labeled: n = 8; unlabeled: n = 9). Data are means ± SE. *Significantly different from the corresponding control (P < 0.05).

 
Selective activation of {alpha}1-, {alpha}2-, beta1-, or beta2-adrenoceptor did not alter the sensitivity of rat vagal sensory neurons.   Because epinephrine has affinity for both {alpha}- and beta-adrenoceptors, our first approach was to determine whether activation of either or both of these major types of adrenoceptor could mimic the potentiating effect of epinephrine. As shown in Fig. 6, pretreatment with neither phenylephrine (5 µM, 5 min; P = 0.74; n = 12), an {alpha}1-adrenoceptor agonist, nor guanabenz (5 µM, 5 min; P = 0.35; n = 11), an {alpha}2-adrenoceptor agonist, altered the vagal sensory neuron responses to capsaicin (50 nM, 15 s) (Fig. 6, A and B). Similarly, pretreatment with either beta1-adrenoceptor agonist dobutamine (5 µM, 5 min; P = 0.23; n = 10) or beta2-adrenoceptor agonist salbutamol (5 µM, 5 min; P = 0.16; n = 10) had no significant effect on capsaicin (50 nM, 15 s)-evoked Ca2+ transient (Fig. 6, C and D).


Figure 6
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  		Fig. 6. Effects of {alpha}1-, {alpha}2-, beta1-, and beta2-adrenoceptor agonists on the capsaicin-evoked Ca2+ transient in rat vagal sensory neurons. A–D: effects of pretreatments with phenylephrine ({alpha}1-adrenoceptor agonist; 5 µM, 5 min; n = 12), guanabenz ({alpha}2-adrenoceptor agonist; 5 µM, 5 min; n = 11), dobutamine (beta1-adrenoceptor agonist; 5 µM, 5 min; n = 10), and salbutamol (beta2-adrenoceptor agonist; 5 µM, 5 min; n = 10), respectively, on the Ca2+ transient evoked by Cap (50 nM, 15 s). Data are means ± SE.

 
The effect of epinephrine was mediated through beta3-adrenoceptor in rat vagal sensory neurons.   As illustrated in Fig. 7A, pretreatment with BRL 37344 (5 µM, 5 min), a selective beta3-adrenoceptor agonist, produced a similar potentiating effect as epinephrine. The group data showed that capsaicin (50 nM, 15 s)-evoked Ca2+ transient was significantly increased from the control level of 97.5 ± 13.0 nM to 204.4 ± 39.9 nM after BRL 37344 (P < 0.05; n = 17) (Fig. 7B). Pretreatment with another selective beta3-adrenoceptor agonist ICI 215,001 (5 µM, 5 min) also significantly potentiated the capsaicin-evoked Ca2+ transient but to a lesser extent (control: 86.3 ± 15.6 nM; after ICI 215,001: 127.7 ± 19.9 nM; P < 0.05; n = 13) (Fig. 7, C and D). Furthermore, pretreatment with SR59230A (5 µM, 10 min), a selective beta3-adrenoceptor antagonist, completely abolished the potentiating effect of epinephrine on the Ca2+ transients evoked by both capsaicin (P = 0.23; n = 14) and KCl (P = 0.24; n = 15) (Fig. 8), whereas pretreatment with SR 59230A alone did not affect the sensory neuron responses to these two chemical stimulants (P = 0.88; n = 12).


Figure 7
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  		Fig. 7. Effects of beta3-adrenoceptor agonists on the capsaicin-evoked Ca2+ transient in rat vagal sensory neurons. A and C: experimental records illustrating the effects of pretreatments with BRL 37344 (beta3-adrenoceptor agonist; 5 µM, 5 min) and ICI 215,001 (beta3-adrenoceptor agonist; 5 µM, 5 min), respectively, on the Ca2+ transient evoked by Cap (50 nM, 15 s). B and D: group data showing the effects of pretreatments with BRL 37344 (n = 17) and ICI 215,001 (n = 13), respectively, on the capsaicin-evoked Ca2+ transient. Data are means ± SE. *Significantly different from the control response (P < 0.05).

 

Figure 8
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  		Fig. 8. Effect of SR 59230A on the potentiation of capsaicin- and KCl-evoked Ca2+ transients by Epi in rat vagal sensory neurons. A and C: experimental records illustrating the effect of pretreatment with SR 59230A (5 µM, 10 min), a beta3-adrenoceptor antagonist, on the potentiation of capsaicin- (Cap; 50 nM, 15 s) and KCl (15 mM, 15 s)-evoked Ca2+ transients by Epi (3 µM, 5 min), respectively. B and D: group data showing the effect of SR 59230A (SR) on the potentiating effect of Epi; n = 14 and 15 in B and D, respectively. Data are means ± SE.

 
Adenylate cyclase inhibitor abolished the effect of epinephrine in rat vagal sensory neurons.   To investigate whether the activation of adenylate cyclase and subsequent increase of intracellular cAMP is critical to the potentiating effect of epinephrine, we tested the effect of SQ22536, a membrane-permeable adenylate cyclase inhibitor. As shown in Fig. 9, pretreatment with SQ22536 (100 µM, 15 min) completely prevented the potentiating effect of epinephrine on capsaicin-evoked Ca2+ transient (P = 0.32; n = 8) (Fig. 9, A and B). In addition, pretreatment with SQ22536 also prevented the effect of epinephrine on the Ca2+ transient induced by KCl (P = 0.44; n = 16) (Fig. 9, C and D).


Figure 9
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  		Fig. 9. Effect of SQ 22536 on the potentiating effect of Epi in rat vagal sensory neurons. A and C: experimental records illustrating the effect of pretreatment with SQ 22536 (100 µM in A, 300 µM in C; 15 min), an adenylate cyclase inhibitor, on the potentiation of Cap- (50 nM; 15 s) and KCl (15 mM, 15 s)-evoked Ca2+ transients by Epi (3 µM, 5 min). B and D: group data showing that the effect of SQ 22536 (SQ) on the potentiating effect of Epi; n = 8 and 16 in B and D, respectively. Data are means ± SE.

 
Inhibition of PKA prevented the epinephrine-induced potentiation in rat vagal sensory neurons.   To determine whether the potentiating effect of epinephrine was due to the cAMP activation of PKA, we examined the effect of H89, a membrane-permeable PKA inhibitor, on the epinephrine-mediated enhancement of chemical stimulation-induced Ca2+ transient. Pretreatment with H89 (3 µM, 15 min) completely abolished the potentiating effect of epinephrine on the Ca2+ transients evoked by both capsaicin (P = 0.87; n = 10) and KCl (P = 0.51; n = 9) (Fig. 10), whereas the vehicle of H89 had no significant effect.


Figure 10
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  		Fig. 10. Effect of H89 on the potentiation of capsaicin- and KCl-evoked Ca2+ transients by Epi in rat vagal sensory neurons. A and B: experimental records illustrating the effects of pretreatments with H89 (3 µM, 15 min), a membrane permeable PKA inhibitor, and its vehicle, respectively, on the potentiation of Cap (50 nM, 15 s)-evoked Ca2+ transient by Epi (3 µM, 5 min). C: group data showing the effects of H89 (n = 10) and its vehicle (n = 8) on the potentiating effect of Epi on the Ca2+ transient evoked by Cap (50 nM, 15 s). D: group data showing the effects of H89 (n = 9) and its vehicle (n = 9) on the potentiating effect of Epi on the Ca2+ transient evoked by KCl (15 mM, 15 s). Data are means ± SE. *Significantly different from the corresponding response to Cap or KCl alone (P < 0.05). {dagger}Significant difference between the corresponding data obtained from pretreatments with H89 and its vehicle (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
beta-Adrenoceptors are among the most extensively characterized members of the G-protein-coupled receptors. Three different subtypes of beta-adrenoceptor (beta1, beta2, and beta3) have been characterized on the basis of biochemical, functional, and receptor-cloning studies. Although the fourth adrenoceptor (beta4) has been reported in heart muscle (17), it is now recognized that this newly proposed beta-adrenoceptor most likely represents only a particular state of the beta1-adrenoceptor (25). Functional assignment of beta-adrenoceptor subtypes suggests that beta1-adrenoceptor is the predominant subtype regulating heart rate and contractility, although all three beta-adrenoceptors appear to be involved in the cardiac stimulation (17). beta2-Adrenoceptor is the predominant subtype mediating the vascular smooth muscle-relaxant properties (32). beta3-Adrenoceptor was initially identified in adipose tissue, and its presence has been recently demonstrated in a variety of tissues including blood vessel (32), gastrointestinal tract (9), urinary bladder smooth muscle (43), and retinal endothelial cell (37). In the present study, our results have demonstrated that, in anesthetized, open-chest, and artificially ventilated rats, inhalation of epinephrine aerosol substantially enhanced pulmonary C-fiber responses to lung inflation and capsaicin injection (Figs. 1 and 2). Our data have also shown that pretreatment with epinephrine significantly potentiated chemical stimulation-evoked Ca2+ transient in isolated rat vagal sensory neurons including those specifically innervating the lungs (Figs. 3–5). In addition, the potentiating effect of epinephrine was mimicked by both BRL 37344 and ICI 215,001, two selective beta3-adenoceptor agonists (Fig. 7), and abolished by SR 59230A, a selective beta3-adrenoceptor antagonist (Fig. 8), whereas pretreatment with none of the {alpha}1-, {alpha}2-, beta1-, and beta2-adrenoceptor agonists had any significant effect on capsaicin-evoked Ca2+ transient (Fig. 6). Taken together, these results strongly suggest that activation of beta3-adrenoceptor mediates the sensitizing actions of epinephrine on these vagal sensory neurons, although additional evidence obtained from immunohistochemistry and/or molecular cloning will be required to further confirm the expression of this specific beta-adrenoceptor in these neurons.

All three beta-adrenoceptor subtypes are known to be coupled to the Gs-protein, leading to the activation of adenylate cyclase and accumulation of the second messenger cAMP, which has been demonstrated both in native tissues and in reconstitution systems (13, 38). It has been proposed that epinephrine, acting on nociceptors, produces mechanical hyperalgesia through three different signaling pathways involving 1) cAMP-dependent PKA; 2) epsilon isozyme of PKC; and 3) extracellular signal-regulated kinases 1 and 2 (1, 20). It has been demonstrated that the latter two pathways may also be initiated by the accumulation of cAMP (8, 35). However, recent pharmacological and electrophysiological studies have reported the presence of beta-adrenoceptor-mediated cAMP-independent signaling pathways (38); for example, the beta3-adrenoceptor-induced relaxation of gastrointestinal smooth muscle has been proposed to be mediated through the activation of a delayed rectified K+ channel (15). In the present study, our results suggest that intracellular cAMP-PKA transduction cascade is substantially involved in, if not solely responsible for, the sensitizing effect of epinephrine in rat vagal sensory neurons, since pretreatment with either adenylate cyclase inhibitor SQ 22536 (Fig. 9) or PKA inhibitor H89 (Fig. 10) completely prevented the potentiating effect of epinephrine in these neurons.

We have recently demonstrated that capsaicin evoked a Ca2+ transient in small- to medium-size rat vagal sensory neurons; the response was mediated through transient receptor potential vanilloid receptor type 1 (TRPV1) and was dependent on the extracellular Ca2+ (12). TRPV1, a member of the transient receptor potential ion channel superfamily (27), is a polymodal nonspecific cation channel expressed predominantly in unmyelinated (C) fibers and is activated not only by capsaicin but also by noxious heat, protons, anandamide, and lipoxygenase metabolites (4, 16). Therefore, TRPV1 may act as a thermal and chemical transducer and contribute to neurogenic inflammation (4). It has been demonstrated that cAMP-dependent PKA activation can enhance capsaicin-induced inward current and Ca2+ transient in dorsal root ganglion neurons (36) as well as in vagal sensory neurons (12, 22). Indeed, recent studies have successfully delineated several candidate PKA-phosphorylation sites in TRPV1, through which PKA may mediate the sensitization of this channel as well as prevent its desensitization (2).

ATP, another chemical stimulant used in this study, is known to activate the ligand-gated P2X3 purinoceptor that is coupled to nonselective cation channel and widely expressed in both the central and peripheral nervous systems (31). PKA-dependent sensitization of TRPV1 and P2X3 may increase the permeability of these channels to cations such as Ca2+ and Na+, which can lead to cell membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels (VDCCs). On the other hand, VDCCs are also the potential phosphorylation targets of PKA activation, which may increase the channel availability (19) or modulate the channel properties (10). This assumption is supported by our observation that epinephrine also potentiates the KCl-evoked Ca2+ transient (Fig. 4), a response resulting from the cell membrane depolarization and subsequent activation of VDCCs. We cannot compare the extent of the potentiation of epinephrine on these three chemical stimulants (capsaicin, ATP, and KCl) because a full range of the dose responses of these activators was not established in this study. Our in vitro study was carried out in cultured neurons, and the responses were recorded from the neuronal soma. Hence, we could not determine the specific types of afferent terminals of these individual isolated neurons, although we have purposely selected the small- to medium-size neurons for this study. In fact, in the experiments testing the responses to ATP and KCl, we did not exclude capsaicin-insensitive neurons because our previous studies indicated that ~40% of small- to medium-size (diameter of <35 µm) nodose and jugular ganglion neurons prepared under similar conditions do not exhibit any sensitivity to capsaicin (6, 42). More importantly, our in vivo single-fiber recording data clearly indicate that the sensitizing effect of epinephrine is found exclusively in C-fiber afferents and not in SARs or RARs in the rat lungs. In addition, activation of TRPV1, P2X3, or VDCCs may initiate Ca2+ release from intracellular stores via a process of Ca2+-induced Ca2+ release (40). Indeed, previous studies have provided direct evidence that Ca2+-induced Ca2+ release can be triggered solely by Ca2+ influx in various sensory neurons including dorsal root (36) and nodose (7) ganglion neurons. On the basis of our results, we cannot rule out the possibility that increased Ca2+-induced Ca2+ release may have also contributed to the enhanced chemical stimulation-evoked Ca2+ transients after the epinephrine pretreatment.

The present study was carried out using two different experimental approaches. We used the in vivo single-fiber recording to determine the effect of epinephrine in intact animals and the in vitro Ca2+ imaging to identify the subtypes of adrenoceptor mediating this effect in isolated vagal sensory neurons. Whether there are differences in the receptor concentrations and the sensitivities of these receptors between the sensory terminal and the soma of these neurons remains to be determined. Nevertheless, our study has shown that inhalation of epinephrine aerosol markedly enhanced pulmonary C-fiber sensitivity in anesthetized rats and that epinephrine pretreatment significantly potentiated chemical stimulation-evoked Ca2+ transient in isolated rat vagal sensory neurons; these results are in general agreement with the previous findings from other investigators (1, 5, 21, 28). Under the conditions of acute stress, the systemic plasma concentration of epinephrine increases dramatically in various species, including humans (41). Intradermal administration of epinephrine has been shown to produce mechanical and thermal hyperalgesia (5, 21), which may be explained by a direct sensitizing effect of epinephrine on primary nociceptive afferents (1, 21). It has been recently reported that neural discharge in vagal afferent fibers is significantly increased by elevations of peripheral concentrations of epinephrine in rats (28).

The primary afferents innervating various visceral organs are mainly conducted through vagus nerves. It is known that the chemosensitive vagal afferent endings can be stimulated by various endogenous substances, such as H+, adenosine, and serotonin (24, 29), and by various inhaled irritants such as cigarette smoke, SO2, ozone, and also lung expansion (24). Under normal physiological conditions, these stimuli (endogenous chemicals, inhaled irritants, and lung expansion) may not generate a significant stimulatory effect on the vagal afferent terminals. However, when the excitability of these afferents is upregulated by epinephrine, as shown in the present study, their stimulation thresholds will be lowered (e.g., Fig. 1B), which may then lead to afferent activation. In addition, some of these endogenous activators (e.g., H+) are known to be secreted at high concentrations under acute stressful conditions (e.g., strenuous exercise), during which the circulating level of epinephrine is also expected to increase substantially (30). Therefore, the consequent augmented visceral/viscerosomatic reflexes may, presumably, contribute to the adaptive responses of these visceral organs to the acute stresses and play a role in maintenance of homeostasis under these conditions.

In summary, our results show that inhalation of epinephrine aerosol induced a pronounced increase in the sensitivity of pulmonary C-fibers to lung inflation and right atrial injection of capsaicin. Furthermore, pretreatment with epinephrine markedly and consistently potentiated the chemical stimulation-evoked Ca2+ transients in isolated rat vagal sensory neurons. The effect of epinephrine was mimicked by either BRL 37344 or ICI 215,001, two specific beta3-adrenoceptor agonists, and blocked by SR 59230A, a selective beta3-adrenoceptor antagonist. In contrast, pretreatment with none of {alpha}1-, {alpha}2-, beta1-, or beta2-adrenoceptor agonists had any significant effect. Furthermore, pretreatment with either adenylate cyclase inhibitor SQ 22536 or PKA inhibitor H89 completely blocked the potentiating effect of epinephrine. Taken together, these results suggest that epinephrine upregulates the sensitivity of rat vagal chemosensitive neurons; this sensitizing effect is probably mediated through the beta3-adrenoceptor and the subsequent activation of intracellular cAMP-PKA transduction cascade.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-58686 and HL-67379 (L.-Y. Lee), and grants from the American Lung Association (Q. Gu). Q. Gu is a Parker B. Francis Fellow in Pulmonary Research.


   ACKNOWLEDGMENTS
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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
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REFERENCES
 
Current address of Y. S. Lin: Department of Physiology, Taipei Medical University, Taipei, Taiwan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L.-Y. Lee, Dept. of Physiology, Univ. of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536-0298 (e-mail: lylee{at}uky.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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