INTRODUCTION

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Prostaglandin E₂ (PGE₂), a potent autacoid derived from the cyclooxygenase pathway of arachidonic acid metabolism, has been shown to increase the sensitivity of dorsal root ganglion (DRG) nociceptive neurons by activation of the cyclic AMP (cAMP)/protein kinase A (PKA) transduction cascade (Lopshire and Nicol 1998; Smith et al. 2000; Southall and Vasko 2001). The synthesis and release of PGE₂ in various visceral organs, including the lungs and airways, are elevated under pathophysiological conditions such as tissue injury or inflammation (Bley et al. 1998). Vagus nerves provide the primary afferent innervation of several visceral organs and play an important role in the initiation of visceral/viscerosomatic reflexes and the regulation of vegetative functions. However, the effect of PGE₂ on the excitability of vagal sensory neurons was not well characterized, and the signal transduction pathway involved was not fully understood. A recent study carried out in our laboratory showed that the sensitivities of vagal pulmonary C-fiber afferents to chemical stimuli, such as capsaicin, were enhanced by PGE₂ in anesthetized rats (Ho et al. 2000). However, whether the sensitizing effect is caused by a direct action of PGE₂ on sensory terminals of these afferents or an indirect effect on other target cells (e.g., smooth muscles) could not be determined in that study. Furthermore, the mechanisms underlying this sensitizing effect of PGE₂ on vagal chemosensitive neurons remain to be explored.

Intracellular calcium ([Ca²⁺]_i) is an important signal transduction molecule in neurons and plays a critical role in the control of neuronal membrane excitability (Kostyuk and Verkhratsky 1994), synaptic activity, and neurotransmitter release (Llinas et al. 1992; Robitaille et al. 1993). Transient changes in [Ca²⁺]_i (Ca²⁺ transient) are known to contribute to short- or long-term alterations in ion channels, gene expression, and neuronal survival (Ghosh and Greenberg 1995; Simpson et al. 1995). We reasoned that the PGE₂-induced sensitization of vagal chemosensitive neurons should lead to a greater degree of subthreshold depolarization of the neuronal membrane and/or a larger number of action potentials in response to a given level of chemical stimulation (Kwong and Lee 2002), which may then generate a higher level of Ca²⁺ influx via voltage-dependent Ca²⁺ channels (VDCCs). In addition, the PGE₂ sensitization may also evoke a larger influx of Ca²⁺ via certain ligand-gated cation channels, such as the vanilloid type 1 receptor (VR1) (Bevan and Szolcsanyi 1990; Caterina et al. 1997), in the neuronal membrane. In light of the background information and these unanswered questions described in the preceding text, this study aimed to determine whether PGE₂ enhances the chemical stimulation-induced increase in Ca²⁺ transients in isolated rat vagal sensory neurons and, if so, to determine if the cAMP/PKA signaling pathway is involved in this sensitizing effect of PGE₂.

	METHODS

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Isolation and culture of nodose and jugular ganglion neurons

Experiments were performed on young adult male Sprague-Dawley rats (150-200 g) that were anesthetized with 4% halothane in air and decapitated. The head was immediately immersed in ice-cold Hank's balanced salt solution (HBSS). Nodose and jugular ganglia were extracted under a dissecting microscope and placed in ice-cold Dulbecco's minimal essential medium/F12 (DMEM/F12) solution. Each ganglion was desheathed, cut into ~10 pieces, placed in 2 ml 0.125% type IV collagenase, and incubated in a humidified chamber for 1 h in 5% CO₂ in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min) and supernatant aspirated. The ganglion pellet was resuspended in 2 ml 0.05% trypsin and 0.53 mM EDTA in HBSS, incubated for 5 min, and centrifuged (150 g, 5 min). The ganglion pellet was then resuspended in 1 ml modified DMEM/F12 solution [DMEM/F12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/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% (wt/vol) bovine serum albumin to separate the cells from the myelin debris (McLatchie and Bevan 2001). The pellets of nodose and jugular ganglion cells were resuspended in the modified DMEM/F12 solution supplemented with 50 ng/ml 2.5S-nerve growth factor and plated onto eight poly-L-lysine-coated glass coverslips (4 coverslips for each type of ganglion) and then incubated (5% CO₂ balance air, 37°C) for 15-48 h before experiments.

In some experiments, sensory neurons innervating the lungs and airways were identified by retrograde labeling from the lungs (Christian et al. 1993; Kwong and Lee 2002) with the fluorescent neuronal tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Briefly, the rats were anesthetized with intraperitoneal pentobarbital sodium (45 mg/kg) and intubated with a polyethylene tube such that the tip rests in the trachea at the mid-cervical level. With the rat tilted head-up at ~30°, 0.25 ml of DiI (0.5 mg/ml) was instilled into the lungs twice separated by >5 min, and the animal was allowed to recover for 7-10 days to permit DiI to be transported back to the cell soma of respiratory 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 (3 times) with extracellular solution and allowed to de-esterify for 30 min before use. Ratiometric Ca²⁺ imaging was performed using a Zeiss fluorescence inverted microscope equipped with a variable filter wheel (Sutter Instruments) and digital CCD camera (Princeton Instruments). Dual images (340- and 380-nm excitation, 510-nm emission) were collected, and pseudocolor ratiometric images were monitored during the experiments by using the software Axon Imaging Workbench (Axon Instruments). 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 dd H₂0] and were prepared as follows: (- standard) 18 ml 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 (K_d; 225), the ratio values for the (-) and (+) concentration standards (R_min and R_max), and the fluorescence intensity at 380-nm excitation for the (-) and (+) concentration standards (Den_min and Den_max). [Ca²⁺]_i (in nM) was calculated according to the following equation described by Grynkiewicz et al. (1985)
Typical R_min, R_max, and Den_min/Den_max values were 0.59, 2.54, and 2.27, respectively.

Experimental protocols and data analysis

After the incubation period with fura-2 AM, the coverslip containing cells was mounted into a chamber (0.2 ml) placed on the stage of the microscope. All experiments were performed at room temperature (20-23°C). During the experiments, the cells were continuously perfused with an extracellular solution containing (in mM) 5.4 KCl, 136 NaCl, 1 MgCl₂, 1.8 CaCl₂, 0.33 NaH₂PO₄, 10 glucose, and 10 HEPES (pH 7.4). Nominally Ca²⁺-free extracellular solution was prepared by replacing CaCl₂ with equimolar amounts of MgCl₂. Pharmacological agents were perfused through the chamber by a gravity-fed valve-control system (VC-66CS, Warner); a complete change of bath solution occurred in 6 s.

Four study series were performed to determine the Ca²⁺ transients evoked in cultured vagal sensory neurons by four different chemical stimulants: 1) capsaicin (3 × 10⁸ to 10⁷ M), phenylbiguanide (PBG; 2×10⁶ to 5×10⁶ M), adenosine 5'-triphosphate (ATP; 5 × 10⁷ to 10⁶ M), and KCl (15 mM); all these chemical substances are known to activate single-unit C-fiber endings in vivo or cultured DRG nociceptive neurons in vitro (Lee and Lundberg 1994; Ralevic and Burnstock 1998); 2) the effects of PGE₂ pretreatment on the Ca²⁺ transients evoked by these chemical stimulants in vagal sensory neurons and, in a separate series, the subgroup of vagal neurons specifically innervating the respiratory tract; 3) the effect of forskolin (10⁶ M; 5 min), an activator of adenylyl cyclase, and 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP; 3 × 10⁶ M, 10 min), a membrane-permeable cAMP analogue, on the capsaicin-evoked Ca²⁺ transient; and 4) the effect of PGE₂, forskolin, and CPT-cAMP on chemical stimulation-evoked Ca²⁺ transients after pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), which is known to inhibit the activity of PKA.

KCl solution (final concentration: 100 mM) was perfused at the end of each experimental run to test for cell viability. To avoid tachyphylaxis of the neurons to the chemical stimulants, each coverslip was used for only one study series. The peak amplitude of the Ca²⁺ transient ([Ca²⁺]_i)-evoked by a certain chemical stimulant was measured as the difference between the 6-s average at peak and the 30-s average at baseline. The duration of the Ca²⁺ transient in response to a chemical stimulant was measured as the interval when the [Ca²⁺]_i exceeded and remained at >20% of its peak [Ca²⁺]_i at control.

Chemicals

DMEM/F12, trypsin-EDTA solution, and 2.5S-nerve growth factor were obtained from Gibco. Fura-2 AM and DiI were purchased from Molecular Probes. Collagenase, capsaicin, capsazepine, PBG, ATP, PGE₂, forskolin, CPT-cAMP, and H89 were obtained from Sigma. DiI was first dissolved and sonicated in ethanol at 25 mg/ml, then diluted with extracellular solution at final concentration of 0.25 mg/ml. Stock solution of capsaicin (10³ M) was prepared in a vehicle of 10% Tween80, 10% ethanol, and 80% extracellular solution; PGE₂ (5 × 10³ M), forskolin (5 × 10³ M), and CPT-cAMP (10² M) were dissolved in ethanol; capsazepine (10² M) and H89 (10² M) were dissolved in DMSO. These stock solutions were then diluted with the extracellular solution to yield the appropriate concentrations prior to application. No detectable effect of the vehicles of these chemical agents was found in our preliminary experiments.

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, pair-wise comparisons were made with a post hoc analysis (Newman-Keuls test). Data are reported as means ± SE. A P value <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The resting [Ca²⁺]_i averaged 81.2 ± 6.4 nM (n = 251). All neurons showed a rapid and reversible increase in [Ca²⁺]_i while depolarized by KCl solution (100 mM for 15 s; e.g., Fig. 1). The data from nodose and jugular ganglion neurons were pooled in this study because we did not find any significant difference in the responses to capsaicin (3 × 10⁸ to 10⁷ M; 15 s) between the vagal sensory neurons isolated from these two types of ganglia, either before (n = 31; P > 0.05) or after the PGE₂ pretreatment (n = 31; P > 0.05), in our preliminary studies.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Capsaicin-evoked Ca2+ transients in cultured vagal sensory neurons. A: the Ca2+ transient evoked by capsaicin (107 M; 15 s) was reproducible in the same neuron (nodose; diameter: 20 µm) after washout for 10 min. B: the capsaicin (107 M; 15 s)-induced Ca2+ transient was completely and reversibly blocked by pretreatment of capsazepine (105 M; 5 min), a selective antagonist of the vanilloid type 1 receptor (VR1) receptor in a jugular neuron (19 µm). C: the capsaicin (5 × 108 M; 15 s)-evoked Ca2+ transient was absent when the neuron (nodose; 26 µm) was superfused with nominally Ca2+-free solution. KCl solution (100 mM; 15 s) was applied to test cell viability at the end of the experiment. Cap, capsaicin; Capz, capsazepine; [Ca2+]i, intracellular concentration of Ca2+ in nM.

Effect of chemical stimulants on Ca²⁺ transient in cultured vagal sensory neurons

To investigate the effect of PGE₂, this study series was carried out first to characterize the control response to capsaicin and other chemical stimulants in cultured vagal sensory neurons. On application of capsaicin (3 × 10⁸ to 10⁷ M; 15 s), ~58% (110/189) of the neurons tested exhibited an increase in [Ca²⁺]_i, and the peak amplitude of the [Ca²⁺]_i exceeded 20% of that evoked by KCl (100 mM; 15 s) in the same neurons (e.g., Fig. 1); capsaicin sensitivity was found predominately in small- and medium-size neurons (<35 µM). Hence, only small- and medium-size neurons were selected for the later experiments.

The Ca²⁺ transient was reversible and reproducible even at high concentration of capsaicin (10⁷ M) when >10 min elapsed between two challenges (Fig. 1A). However, the responses between cells varied considerably (e.g., Fig. 1). The mean [Ca²⁺]_i evoked by capsaicin (3 × 10⁸ to 10⁷ M; 15 s) was 96.8 ± 16.9 nM (n = 110). After pretreatment with capsazepine (10⁵ M; 5 min), a selective antagonist of the VR1, capsaicin (5 × 10⁸ to 10⁷ M; 15 s)-evoked Ca²⁺ transient was completely blocked (n = 14), indicating that it was probably mediated through the VR1 (Fig. 1B). The increase in [Ca²⁺]_i in response to capsaicin (5 × 10⁸ M; 15 s) was abolished in nominally Ca²⁺-free medium (e.g., Fig. 1C; n = 7). Similarly, control responses of Ca²⁺ transient to PBG (2 × 10⁶  5 × 10⁶ M; 15 s), ATP (5 × 10⁷ to 10⁶ M; 15 s) and KCl (15 mM; 15 s) were also reversible, reproducible when tested after >10 min washout, and abolished in nominally Ca²⁺-free solutions; whereas pretreatment with capsazepine (10⁵ M; 5 min) did not have any detectable effect on the Ca²⁺ transients evoked by these three chemical stimulants (n = 13; P > 0.05); these experiments were carried out in separate groups of neurons (data not shown).

PGE₂ potentiation of the Ca²⁺ transients evoked by chemical stimulants

The Ca²⁺ transient evoked by capsaicin was greatly augmented by pretreatment with PGE₂; a representative example is shown in Fig. 2A. After a 5-min pretreatment with PGE₂ (3 × 10⁷ M), the peak [Ca²⁺]_i evoked by capsaicin (5 × 10⁸ M; 15 s) was elevated from a control response of 85.3 to 224.3 nM. This augmented response to capsaicin gradually declined after washout but remained higher than control 15 min later (116.8 nM). The group data showed that the capsaicin (3 × 10⁸ to 10⁷ M; 15 s)-induced Ca²⁺ transient was enhanced after the PGE₂ pretreatment (3 × 10⁷ M; 5 min) by almost fourfold (Fig. 2B; at control: 64.2 ± 11.2 nM; after PGE₂ pretreatment: 314.2 ± 36.8 nM; after washout: 201.2 ± 35.8; n = 38). After PGE₂ pretreatment, the response of Ca²⁺ transient to capsaicin rapidly declined but did not completely return to the baseline in a majority (26/38) of the cells. Instead it exhibited a sustaining and slowly declining second phase (e.g., Fig. 2A). Overall, the PGE₂ pretreatment increased significantly the duration of the Ca²⁺ transient (at control: 69.5 ± 15.7 s; after PGE₂ pretreatment: 321.5 ± 74.8 s; after washout: 134.1 ± 30.8 s; n = 38). This potentiating effect of PGE₂ as measured by the change in [Ca²⁺]_i was absent in nominally Ca²⁺-free extracellular solution (e.g., Fig. 2C; n = 8).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Capsaicin-evoked Ca2+ transient was potentiated after the prostaglandin E2 (PGE2) pretreatment in vagal sensory neurons. A: an experimental record illustrating that the effect of PGE2 (3 × 107 M; 5 min) pretreatment on the Ca2+ transient evoked by application of capsaicin (5 × 108 M; 15 s) in a jugular neuron (22 µm). B: group data showing that the potentiating effect of PGE2 on capsaicin (3 × 108 to 107 M; 15 s)-evoked Ca2+ transient (n = 38). C: an experimental record illustrating that the potentiating effect of PGE2 (3 × 107 M; 5 min) on the response to capsaicin (5 × 108 M; 15 s) was absent in nominally Ca2+-free solution in a nodose neuron (20 µm). D: a comparison of the potentiating effect of PGE2 pretreatment (3 × 107 M; 5 min) on the capsaicin (3 × 108 M; 15 s)-evoked Ca2+ transient between pulmonary [1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeled; ; n = 11] and nonpulmonary (unlabeled; ; n = 14) vagal sensory neurons matched in size from the same cultures. Data are means ± SE *P < 0.05 significantly different from the control response.

The potentiating effect of PGE₂ on pulmonary vagal sensory neurons identified by DiI labeling was investigated in a separate group of neurons. In a total of >5000 jugular and nodose neurons harvested and cultured from six rats that had received DiI instillation into their lungs 7 - 10 days earlier, ~16% of them were labeled with DiI. Based on our previous observation (Kwong and Lee 2002), this subgroup of vagal sensory neurons provided innervation specifically to the respiratory tract. Only a small fraction of these neurons were studied in this series due to the limitation of our experimental protocol; our results showed no significant difference in the capsaicin (3 × 10⁸ M; 15 s)-evoked Ca²⁺ transients between labeled neurons and nonlabeled ones matched in size from the same culture, either during control or after PGE₂ (Fig. 2D; DiI-labeled neurons: 43.9 ± 10.3 nM at control and 100.9 ± 21.4 after PGE₂ pretreatment; n = 11; unlabeled neurons: 54.2 ± 8.8 nM at control and 125.0 ± 27.2 nM after PGE₂ pretreatment; n = 14).

To determine whether the potentiating effect of PGE₂ was limited only to the response to capsaicin, we tested three other chemical agents: PBG, ATP, and KCl. Similarly, the Ca²⁺ transients evoked by PBG (2 × 10⁶ to 5 × 10⁶ M; 15 s), ATP (5 × 10⁷ to 10⁶ M; 15 s), and KCl (15 mM; 15 s) were also significantly potentiated after PGE₂ pretreatment (3 × 10⁷; 5 min; Figs. 3, A, C, and E). The PBG-evoked Ca²⁺ transient was enhanced to 2.4-fold (Fig. 3B; at control: 104.8 ± 19.1 nM; after PGE₂ pretreatment: 253.3 ± 62.1 nM; after washout: 136.7 ± 34.4; n = 12); the Ca²⁺ transient evoked by ATP was doubled after PGE₂ pretreatment (Fig. 3D; 77.0 ± 20.8 nM at control; 157.0 ± 36.7 nM after PGE₂ pretreatment; 107.4 ± 27.9 nM after washout; n = 11); whereas the Ca²⁺ transient evoked by KCl was increased to 163% after PGE₂ pretreatment (Fig. 3F; 49.5 ± 6.9 nM at control; 80.8 ± 11.0 nM after PGE₂ pretreatment; 49.7 ± 6.7 nM after washout; n = 11).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Pretreatment with PGE2 potentiated the Ca2+ transients evoked by phenylbiguanide (PBG), adenosine 5'-triphosphate (ATP), and KCl in vagal sensory neurons. A, C, and E: experimental records illustrating that the effect of PGE2 pretreatment (3 × 107 M; 5 min) on the Ca2+ transients evoked by application of PBG (3 × 106 M; 15 s; jugular; 25 µm), ATP (5 × 107 M; 15 s; nodose; 22 µm), and KCl (15 mM; 15 s; nodose; 20 µm), respectively. B, D, and F: group data showing the effect of PGE2 (3 × 107 M; 5 min) on the Ca2+ transients evoked by PBG (2 × 106 to 5 × 106 M; 15 s; n = 12), ATP (5 × 107 to 106 M; 15 s; n = 11), and KCl (15 mM; 15 s; n = 11), respectively. Data are means ± SE. *P < 0.05 significantly different from the control response.

Although PGE₂ pretreatment (3 × 10⁷ M; 5 min) did not consistently elevate the baseline of [Ca²⁺]_i, we observed a slow and small increase in [Ca²⁺]_i in 25 of 97 neurons tested; [Ca²⁺]_i usually reached a peak of <50 nM and then declined toward baseline even before termination of the PGE₂ perfusion (e.g., Fig. 3, A and E). In all of these 25 neurons, PGE₂ also elevated the Ca²⁺ transients evoked by chemical stimuli.

Forskolin and CPT-cAMP mimicked the potentiating effect of PGE₂

To determine a possible involvement of cAMP transduction cascade in the sensitizing effect of PGE₂, we investigated whether forskolin, an activator of adenylyl cyclase, and CPT-cAMP, a membrane permeable cAMP analogue, affected the capsaicin-evoked Ca²⁺ transient. Figure 4 clearly showed that pretreatment with forskolin (10⁶ M; 5 min) or CPT-cAMP (3 × 10⁶ M; 10 min) enhanced the capsaicin-evoked Ca²⁺ transient; after forskolin and CPT-cAMP, the capsaicin-induced Ca²⁺ transient was potentiated by 3.9-fold (Fig. 4B; at control: 50.2 ± 15.7 nM; after forskolin pretreatment: 195.9 ± 45.4 nM; after washout: 79.9 ± 22.2; n = 13) and 4.7-fold (Fig. 4D; at control: 71.2 ± 18.2 nM; after CPT-cAMP pretreatment: 334.4 ± 111.1 nM; after washout: 117.7 ± 27.5; n = 8), respectively. Interestingly, the effect of forskolin and CPT-cAMP showed a faster and more complete recovery than that after PGE₂ pretreatment after 15-min washout.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Forskolin and 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP) mimicked the effect of PGE2 on capsaicin-evoked Ca2+ transient in vagal sensory neurons. A and C: experimental records illustrating the effect of pretreatment with forskolin (106 M; 5 min; nodose; 24 µm) and CPT-cAMP (3 × 106 M, 10 min; nodose; 23 µm) on capsaicin (3 × 108 M; 15 s)-evoked Ca2+ transient, respectively. B and D: group data showing the effect of forskolin (106 M; 5 min; n = 13) and CPT-cAMP (3 × 106 M, 10 min; n = 8) on capsaicin (3 × 108 - 5 × 108 M; 15 s) -evoked Ca2+ transients, respectively. Data are means ± SE *P < 0.05 significantly different from the control response.

Effect of H89 on PGE₂, forskolin, and CPT-cAMP-induced potentiation

To determine whether the potentiating effect of PGE₂ was due to a direct action of cAMP or the activation of PKA by cAMP, we examined the effect of H89, a membrane-permeant PKA inhibitor, on the PGE₂-mediated enhancement of capsaicin response. As illustrated in Fig. 5, pretreatment with H89 (10⁵ M; 15-20 min) completely abolished the potentiating effect of PGE₂ (3 × 10⁷ M; 5 min) on capsaicin (3 × 10⁸ - 5 × 10⁸ M; 15 s)-evoked Ca²⁺ transient in vagal sensory neurons (Fig. 5B; 114.5 ± 26.3 nM at control and 118.6 ± 30.7 nM after H89 and PGE₂; n = 10; P > 0.05). Similarly, the potentiating effect of PGE₂ on the Ca²⁺ transients evoked by other chemicals stimulants (e.g., PBG, ATP, and KCl) was also abolished after H89 pretreatment (n = 14; P > 0.05). Furthermore, H89 (10⁵ M; 15-20 min) was equally effective to prevent the potentiation of the capsaicin (3 × 10⁸ - 5 × 10⁸ M; 15 s)-evoked Ca²⁺ transient by forskolin (10⁶ M; 5 min; Fig. 5D; 107.7 ± 38.5 nM at control and 98.5 ± 28.2 nM after H89 and forskolin; n = 9; P > 0.05) and CPT-cAMP (3 × 10⁶ M; 10 min; Fig. 5F; 122.9 ± 41.2 nM at control and 109.1 ± 35.7 nM after H89 and CPT-cAMP; n = 8; P > 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effect of N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) on the potentiating effect of PGE2, forskolin, and CPT-cAMP in vagal sensory neurons. A, C, and E: experimental records illustrating the effect of pretreatment with H89 (105 M; 15-20 min) on the potentiation of capsaicin (5 × 108 M; 15 s)-evoked Ca2+ transient by PGE2 (3 × 107 M; 5 min; jugular; 18 µm), forskolin (106 M; 5 min; nodose; 24 µm), and CPT-cAMP (3 × 106 M; 10 min; jugular; 20 µm), respectively. B, D, and F: group data showing the effect of H89 (105 M; 15-20 min) on the potentiation of capsaicin (3 × 108 - 5 × 108 M; 15 s)-induced Ca2+ transients by PGE2 (3 × 107 M; 5 min; n = 10), forskolin (106 M; 5 min; n = 9), and CPT-cAMP (3 × 106 M; 10 min; n = 8), respectively. Data are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our results have demonstrated that application of capsaicin reproducibly caused a rapid surge of [Ca²⁺]_i in small- and medium-size jugular and nodose neurons. Pretreatment with PGE₂ markedly and consistently potentiated the Ca²⁺ transient evoked by the same concentration of capsaicin. Similarly, PGE₂ pretreatment also markedly enhanced the Ca²⁺ transients induced by PBG, ATP, and KCl, three other known chemical stimulants of C neurons. Furthermore, this potentiating effect of PGE₂ could be mimicked by pretreatment with forskolin or CPT-cAMP and was completely abolished by pretreatment with H89. Therefore these results suggest that the sensitizing actions of PGE₂ on vagal sensory neurons are probably mediated through the cAMP/PKA pathway.

It is well documented that capsaicin excites a subset of mammalian sensory neurons. This excitation results from the activation of a ligand-gated nonselective cation channel with subsequent membrane depolarization and action potential generation (Bevan and Szolcsanyi 1990; Marsh et al. 1987; Oh et al. 1996). Indeed, such a channel, VR1, has been cloned (Caterina et al. 1997) and found to be expressed in various types of sensory neurons (Caterina et al. 1997; Helliwell et al. 1998; Tominaga et al. 1998). Our results demonstrated that capsaicin evoked a Ca²⁺ transient in small- and medium-size nodose and jugular neurons. The response was blocked by capsazepine, a selective antagonist of the VR1, and was dependent on the extracellular Ca²⁺. Furthermore, both the peak and the duration of Ca²⁺ transient evoked by capsaicin are substantially potentiated after the pretreatment with PGE₂ (Fig. 2, A and B). This potentiation occurs in vagal sensory neurons including the neurons specifically innervating airways and lungs (Fig. 2D). These results are therefore in agreement with our previous experiments that showed PGE₂-induced enhancement of pulmonary chemoreflex response and the single-unit C-fiber sensitivity (Ho et al. 2000; Lee and Morton 1995; Lee and Pisarri 2001). In those studies in intact animals, we were unable to determine whether PGE₂ was acting directly on the sensory terminals or through an intermediary effect on other cells (e.g., airway smooth muscle, degranulation of mast cells, etc.). In a parallel study using perforated patch-clamp recording in cultured nodose and jugular ganglion neurons, Kwong and Lee (2002) recently demonstrated that PGE₂ increases the sensitivity to chemical and electrical stimulations in small-diameter pulmonary vagal chemosensitive neurons, but the underlying mechanisms were not determined in that study. The present study has not only lent additional support to the conclusion that the sensitizing effect of PGE₂ is caused by a direct action on pulmonary C-fiber terminals but also provide the evidence demonstrating the involvement of intracellular cAMP/PKA pathway.

PGE₂ is synthesized and released in response to tissue injury, contributes to hyperalgesia, and is involved in the acute and chronic inflammatory reactions (Nicol et al. 1992; Vasko et al. 1994). All these wide-ranging biological actions of PGE₂ are mediated by membrane-bound prostanoid receptors (Coleman et al. 1994). Among the family of prostanoid receptors, the EP receptor has the highest affinity for PGE₂ based on the ligand-binding studies, and the presence of some of the subtypes of the EP receptor (e.g., EP₂, EP₃, and EP₄ receptors) on the sensory nerves is well documented (Coleman et al. 1994; Narumiya et al. 1999). Several species of heterotrimeric G proteins are known to be coupled to the EP receptors and participate in their signal transduction. For example, EP₂, EP_3B, EP_3C, and EP₄ receptors are coupled to G_s proteins, which on stimulation can activate adenylyl cyclase. Indeed, recent studies in rat DRG neurons have implicated that PGE₂-induced nociceptor sensitization is due to an increase of enzyme activity of adenylyl cyclase (England et al. 1996; Hingtgen et al. 1995; Lopshire and Nicol 1998; Smith et al. 2000). The resulting rise in the level of cAMP may then stimulate PKA, which in turn enhances the neuronal excitability by increasing the phosphorylation of certain ion channels. In the present study, the direct evidence in support of a role for the activation of PKA in the PGE₂ potentiation of the capsaicin-evoked Ca²⁺ transient is provided by the inhibition of the PGE₂ effect by H89, a membrane-permeant inhibitor of PKA (Fig. 5). Further support is provided by the observation that both forskolin and CPT-cAMP enhanced the capsaicin-evoked Ca²⁺ transient in a manner analogous to that produced by PGE₂ (Fig. 4). Moreover, the effect of both forskolin and CPT-cAMP was prevented after the pretreatment with H89.

Our results have shown that pretreatment with PGE₂ not only potentiated the neuronal response to capsaicin but also to other chemical stimulants, including PBG, ATP and KCl (Fig. 3). PBG is known to activate the serotonin type 3 (5HT₃) receptor, which belongs to the ligand-gated ion channel family and has been shown to be permeable to Ca²⁺ in sensory neurons (Moore et al. 1999; Yang et al. 1992). ATP is known to activate P2X purinoceptors that are coupled to nonselective cation channels and widely expressed in both the central and periphery nervous systems (Ralevic and Burnstock 1998). Seven subunits of the P2X receptor family (P2X₁ to P2X₇) have been identified and cloned; the Ca²⁺ permeability of cloned P2X channels was found to be relatively high but varied among the different subunits (Burnashev 1998). Thus the three chemical agents (capsaicin, PBG, and ATP) applied in this study are known to activate different ligand-gated ion channels. It has been reported that the ligand-gated ion channels, in general, are heteromeric proteins comprised of homologous subunits, each of which spans the membrane several times and contains a large intracellular loop that is mosaic of consensus sites for protein phosphorylation (Swope et al. 1992, 1999). Indeed, phosphorylation of ligand-gated ion channels is recognized as a potentially important mechanism for short- and long-term modulation of ion-channel function and may play an important role in synaptic plasticity and neuronal excitability (Smart 1997). Unlike the ligand-gated ion channel activators, KCl presumably evokes the Ca²⁺ transient in vagal sensory neurons mainly through the depolarization of these neurons and the subsequent activation of VDCCs. Increasing evidence shows that multiple types of VDCCs, including L (Fraser and Scott 1999; Sculptoreanu et al. 1993), N, and Q (Fukuda et al. 1996) types, can be modulated by PKA, probably via the action of A-kinase anchoring proteins (Fraser and Scott 1999; Gray et al. 1998).

It is important to note, however, that we do not know what proportion of the increase in [Ca²⁺]_i that occurred in response to chemical stimulants including capsaicin, PBG, and ATP is due to the influx of Ca²⁺ through the nonspecific cation channels described in the preceding text. Enhanced Ca²⁺ release from the intracellular stores and/or VDCCs may also contribute to the potentiation of these chemical stimulation-evoked responses after pretreatment with PGE₂. The argument against the possibility of intracellular Ca²⁺ release is that PGE₂-induced enhancement of chemical stimuli-evoked Ca²⁺ transients does not occur in nominally Ca²⁺-free medium (Fig. 2C). In general, activation of sensory neurons by these chemical stimulants is accompanied by membrane depolarization, resulting from the opening of nonselective cation channels and an increase in membrane permeability (Bevan and Szolcsanyi 1990; Oh et al. 1996). Membrane depolarization could subsequently activate VDCCs of these neurons. Therefore it seems reasonable to assume that PGE₂-induced potentiation of the Ca²⁺ transients evoked by these chemical stimulants is, at least in part, associated with a function of PKA-mediated phosphorylation of VDCCs, which may increase the channel availability (Kavalali et al. 1997) or modulate the channel properties (Dolphin 1991; Gross et al. 1990). This assumption is supported by our observation that PGE₂ are capable of potentiating the Ca²⁺ transient evoked by KCl. Further, on the basis of our results, we cannot rule out the possibility that activation of VDCCs or ligand-gated ion channels may initiate Ca²⁺ release from intracellular stores via a process of Ca²⁺-induced Ca²⁺ release (CICR) (Verkhratsky and Petersen 1998). Indeed, previous studies have provided direct evidence showing that CICR could be triggered solely by Ca²⁺ influx in DRG (Shmigol et al. 1995) and sympathetic neurons (Hua et al. 1993). In addition, it has been shown that CICR can be evoked by the action of a single action potential in dissociated nodose neurons (Cohen et al. 1997; Cordoba-Rodriguez et al. 1999).

In summary, our results demonstrate that PGE₂ potentiates the chemical-stimuli-evoked Ca²⁺ transients in cultured rat vagal sensory neurons. This sensitizing effect is probably mediated through the action of PGE₂ on certain subtypes of prostanoid receptors, which in turn activate the cAMP/PKA intracellular transduction pathway and increase the Ca²⁺ permeability in the neuronal membrane. However, the specific prostanoid receptor subtype(s) involved in these effects cannot be determined until more selective antagonists of these receptors become available.