INTRODUCTION

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VAGAL PRIMARY AFFERENT NEURONS [nodose ganglion neurons (NGNs)] isolated from normal adult guinea pigs fail to respond electrophysiologically to exogenously applied tachykinins [e.g., substance P (SP) or neurokinin A]. This suggests that NGNs lack electrically competent neurokinin receptors: either tachykinin receptors are not present, or they are functionally uncoupled from effector ion channels. Under pathological conditions (e.g., allergic airway inflammation in vivo), however, the majority of NGNs express functional tachykinin receptors (14). Functional tachykinin receptors can also be expressed in vitro after antigenic activation of nodose ganglion mast cells: ~80% of NGNs are depolarized by SP (25). Pharmacological characterization of these newly expressed SP responses indicated that they are mediated by neurokinin-2 (NK₂) tachykinin receptors (25). This upregulation or "unmasking" of functional NK₂ tachykinin receptors that follows allergic inflammation in vitro is independent of new protein synthesis, suggesting that NK₂ tachykinin receptors are present but nonfunctional in control NGNs.

On antigenic activation, mast cells release a host of preformed (e.g., histamine, serotonin, and heparin) and de novo synthesized mediators [e.g., prostanoids and leukotrienes (5)]. Any of these inflammatory mediators acting alone, or in combination, may underlie the unmasking of functional NK₂ receptors. Previously, our laboratory has screened a number of mast cell-derived mediators for their ability to unmask functional NK₂-receptor responses by using acutely isolated NGNs (13). These studies demonstrated that exogenous serotonin [5-hydroxytryptamine (5-HT)] application could mimic the effects produced by allergic inflammation. After incubation of isolated NGNs with 5-HT (10 µM) for as little as 5 min, ~65% of the NGNs were depolarized by subsequently applied SP, acting via NK₂ receptors. Pharmacological analysis revealed that the unmasking effect of 5-HT in isolated NGNs was potent (EC₅₀ = 14 nM), was mediated by 5-HT₃ receptors, and required an intracellular calcium-nitric oxide signaling cascade.

In the present work, we ask whether 5-HT is also critical for NK₂-receptor unmasking in response to in vitro mast cell activation in the intact nodose ganglion. Our results illustrate that the percentage of SP-responsive NGNs was significantly reduced (to ~20%) when ganglia were challenged in the presence of a 5-HT₃-receptor antagonist. Incubation with a cocktail of 5-HT-receptor antagonists did not further diminish antigen-induced NK₂-receptor unmasking. These results suggest that endogenous 5-HT release and 5-HT₃-receptor activation are critical for the unmasking of SP responses provoked by in vitro allergenic activation of mast cells within nodose ganglia.

	METHODS

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Immunization of animals. Adult male Hartley guinea pigs (200-250 g; Charles River, Wilmington, MA) were actively immunized with ovalbumin [chicken egg albumin (OVA); Sigma Chemical, St. Louis, MO], as described previously (26). The animals were killed by asphyxiation with CO₂ as approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore. Nodose ganglia were dissected bilaterally and placed in ice-cold (4°C) Locke solution (composition in mM: 136 NaCl, 5.6 KCl, 1.2 MgCl₂, 2.2 CaCl₂, 14.3 NaHCO₃, 1.2 NaH₂PO₄, and 10 dextrose) equilibrated with 95% O₂-5% CO₂, pH 7.2-7.4.

In vitro antigenic challenge. Ganglia were prewarmed to 37°C in Locke solution for 10 min and then transferred to a Locke solution (37°C) containing 10 µg/ml antigen (OVA) for 15 min (23). To test the involvement of various inflammatory mediators, one of each pair of nodose ganglia (chosen randomly) was incubated with either an enzyme inhibitor or receptor antagonists 15 min before and during the OVA challenge. Ganglia were treated with either tropisetron [ICS 205-930, 1 µM (18) or Y-25130, 1 or 10 µM (14)] to block 5-HT₃ receptors; a combination of methiothepin [1 µM (9)], RS-39604 [1 µM (9)], and Y-25130 (1 µM) to nonselectively block metabotropic 5-HT receptors (19); pyrilamine (1 µM) and burimamide (50 µM) to inhibit H₁, H₂, and H₃ histamine receptors (8); indomethacin (3 µM), a cyclooxygenase inhibitor, to prevent prostaglandin synthesis; or ZD-2138 (3 µM), a 5'-lipoxygenase inhibitor, to suppress peptidoleukotriene synthesis (4). The other ganglion of the pair was treated in the same fashion, except vehicle was substituted for drug. After challenge with OVA, ganglia were returned to Locke solution (37°C) for at least 45 min before enzymatic dissociation.

Tissue preparation. Acutely dissociated neurons were prepared enzymatically as described (14) and maintained at 37°C for at least 8 h before recording. Nerve growth factor was not present in the culture medium.

Electrophysiological recording. Intracellular recording, micropipette fabrication, data acquisition, and data analysis were accomplished after the procedures previously described (14). Neurons were accepted for study only if they showed a stable resting membrane potential (E_m; less than or equal to 50 mV) and had action potentials overshooting 0 mV. Agonists were superfused over isolated neurons for 30-60 s. The temperature of the Locke solution flowing through the recording chamber was maintained at 33-35°C.

Preparation of drug solutions and sources. Drug solutions were prepared daily from concentrated (10 mM) stock solutions stored at 20°C. ZD-2138 was a gift from Zeneca (Wilmington, DE), and burimamide was a gift from Smith Kline Beecham (Philadelphia, PA). 3-Tropanyl-indole-3-carboxylate hydrochloride (ICS 205-930; tropisetron) was obtained from Research Biochemicals (Natick, MA). N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dihydro- 2H-1,4-benzoxazine-8-carboxamide (Y-25130 hydrochloride), methiothepin, and RS-39604 were procured from Tocris Cookson (Ballwin, MO). All other reagents were acquired from Sigma Chemical.

Data analysis. Data are expressed as means ± SE. Student's two-tailed t-test was utilized to assess significant differences between calculated means; P  0.05 was considered significant. The z-test was utilized to compare differences in the percentage of neurons responding to SP after various treatments. Statistical analysis was performed by using Sigmastat software (Jandel Scientific, San Rafael, CA).

	RESULTS

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Antigenic activation in nodose ganglia induces functional tachykinin receptors. NGNs obtained from nodose ganglia removed from control or from actively immunized unchallenged guinea pigs rarely (<5%) show measurable membrane potential or membrane current changes to bath-applied SP (13, 14, 25). However, after antigen challenge of nodose ganglia isolated from actively immunized guinea pigs, 53 of 89 (60%) of the NGNs were depolarized an average of 13 ± 1.2 mV by bath-applied SP (100 nM; Fig. 1). The SP-induced membrane depolarization was accompanied by a 34 ± 4.0% decrease in membrane input resistance (R_in) in 72% of the NGNs in which it was monitored (n = 26 of 36 neurons). In the remaining 28%, there was either no change (n = 5) or an 11 ± 2.8% increase in R_in (n = 5). When NGNs were voltage clamped to their E_m (approximately 60 mV), SP evoked an inward current averaging 955 ± 241.7 pA (n = 9) accompanied by a 55 ± 27.2% increase in membrane conductance. Resting membrane properties (E_m, 64 ± 0.9 mV and R_in, 39 ± 4.2 M; n = 62) in NGNs from allergenically activated ganglia were not significantly different from those recorded previously in control NGNs [E_m, 63 ± 0.7 mV and R_in, 39 ± 2.3 M; n = 156 (14)].

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Responses produced by substance P (SP) in nodose neurons isolated from nodose ganglia challenged with ovalbumin (OVA) in the presence or absence of 5-hydroxytryptamine (5-HT3)-receptor antagonists. A1: in a nodose neuron from a chick OVA-challenged ganglion, a 50-s bath application of SP (100 nM; horizontal bar) evoked a membrane depolarization. Downward deflections are electronic voltage transients elicited by hyperpolarizing current pulses (100 pA, 300 ms, 0.6 Hz); the magnitude of these transients reflects membrane input resistance (Rin). The SP-induced depolarization was accompanied by a decrease in Rin. The resting membrane potential (Em) and Rin were 62 mV and 40 M, respectively. A2: a nodose neuron from the contralateral nodose ganglion OVA challenged in the presence of tropisetron (1 µM) does not respond to SP. Resting Em and Rin were 76 mV and 50 M, respectively. Sixteen percent of neurons from tropisetron-treated ganglia responded to SP (see Table 1). B1: electrophysiological responses to SP in nodose neurons from another animal. SP (100 nM, 40 s) depolarizes a nodose neuron isolated from the OVA-challenged ganglion to action potential threshold. Resting Em was 60 mV; resting Rin was 20 M. Electronic voltage transients were elicited by hyperpolarizing current pulses (200 pA, 300 ms, 0.6 Hz). B2: the contralateral ganglion was incubated with Y-25130 (1 µM; a 5-HT3-receptor antagonist chemically distinct from tropisetron) 15 min before and during OVA challenge. SP produces no measurable changes in electrophysiological properties of neurons isolated from this ganglion. Resting Em and Rin were 72 mV and 30 M, respectively. Nineteen percent of neurons from Y-25130-treated ganglia responded to SP (see Table 1).

5-HT₃ receptors in intact ganglia are required for the unmasking of SP responses after allergenic activation. The role of 5-HT, acting via 5-HT₃ receptors, in antigen-induced unmasking of SP responses was investigated by incubating ganglia with receptor antagonists 15 min before and during antigen (OVA) challenge. The percentage of nodose neurons depolarized by SP was significantly reduced when ganglia were challenged in the presence of the 5-HT₃-receptor antagonist tropisetron (1 µM; P < 0.001). Bath-applied SP evoked a membrane depolarization in 16% of nodose neurons from ganglia treated with tropisetron, compared with 64% in neurons from contralateral antigen-challenged ganglia (Table 1). The average amplitude of the SP-induced depolarization in the small percentage of neurons that responded after tropisetron treatment (11 ± 2.4 mV, n = 6) did not differ significantly (P = 0.566) from contralateral OVA-challenged ganglia (13 ± 1.8 mV, n = 18; Table 1).

Histamine does not contribute to the unmasking of SP responses after in vitro allergic inflammation. Histamine is one of many mast cell-derived mediators released on specific antigen challenge of nodose ganglia (23). Histamine-receptor antagonists (H₁, H₂, and H₃) were utilized to examine whether histamine-receptor activation contributes to the unmasking of SP responses evoked by antigenic activation. Incubation with a combination of pyrilamine (1 µM, H₁-receptor antagonist) and burimamide (50 µM, at this concentration both H₂ and H₃ receptors are blocked) did not significantly reduce the percentage of SP-responsive NGNs or the average amplitude of the SP-induced depolarization (P = 0.492 and 0.668, respectively; Table 1). Fifty percent of NGNs from ganglia that were antigen challenged in the presence of histamine-receptor antagonists were depolarized 14 ± 3.0 mV (n = 9) by SP. Sixty-two percent of NGNs from contralateral ganglia challenged in the absence of antagonists were depolarized an average of 17 ± 2.5 mV (n = 13) by SP.

De novo synthesized prostanoids and peptidoleukotrienes do not contribute to allergen-induced unmasking of SP responses. A number of inflammatory mediators are synthesized de novo on antigenic activation of mast cells. The contribution of some of these lipid mediators to antigen-induced unmasking of SP responses was examined by using enzyme antagonists. As described above, indomethacin (3 µM, a cyclooxygenase inhibitor) was ineffective in reducing the percentage of SP-responsive NGNs observed after OVA challenge of nodose ganglia. These results suggest that prostanoids do not play a role in the unmasking of functional tachykinin receptors in NGNs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our principal finding is that the unmasking of NK₂ tachykinin receptors in nodose neurons (NGNs) by allergic inflammation in the nodose ganglia in vitro requires activation of 5-HT₃ receptors. Both tropisetron and Y-25130, two chemically distinct 5-HT₃-receptor antagonists, substantially reduced the percentage of NGNs expressing NK₂ receptors after specific antigen challenge. SP application does not evoke measurable membrane potential changes in NGNs from naive animals or from immunized, but nonantigen challenged, animals. Subsequent to OVA challenge of the ganglia, SP depolarizes 59% of nodose neurons. In the presence of 5-HT₃-receptor antagonists, NK₂ receptors are unmasked in only ~20% of NGNs. Thus it appears that 5-HT₃-receptor activation mediates two-thirds of the NK₂-receptor unmasking observed after antigenic challenge. The mediator(s) responsible for upregulation of NK₂ receptors in the remaining 20% of NGNs is unknown. However, it appears from the present work that histamine, prostanoids, and peptidoleukotrienes are not essential for the functional expression of tachykinin receptors after acute allergic inflammation.

Endogenous source of 5-HT. Given that 5-HT₃-receptor activation is essential for NK₂-receptor unmasking in the majority of NGNs, release of 5-HT on antigenic activation is requisite. There are several potential sources of 5-HT in the nodose ganglion, including mast cells, platelets, and NGNs themselves. Most reports suggest that guinea pig mast cells do not contain appreciable levels of 5-HT (17). However, the EC₅₀ for 5-HT-induced unmasking of tachykinin receptors is ~15 nM (13). Therefore, mast cell 5-HT content could be relatively low but still achieve a concentration in the ganglionic extracellular space [0.4 ml/g (6)] sufficient to unmask NK₂ receptors. One group (12) has reported that the concentration of 5-HT in trigeminal ganglion mast cells of the guinea pig was 66 nmol/kg wet wt. If nodose ganglion mast cells contain similar levels of 5-HT and antigen challenge releases ~15% of granular-stored 5-HT (23), then the 5-HT concentration in the ganglion could reach 25 nM. This estimate is approximately two times the EC₅₀ for 5-HT-induced NK₂-receptor unmasking in isolated NGNs (13). Another possibility is that antigen-mediated activation of ganglionic mast cells evokes 5-HT release indirectly, through a multicellular cascade involving platelets. Antigenic activation of mast cells elicits the release of a number of lipid-derived mediators, including platelet-activating factor (PAF) (24). PAF can mobilize 5-HT from platelets and thus may evoke NK₂-receptor unmasking. It is noteworthy that incubation of nodose ganglia isolated from nonimmunized guinea pigs with PAF (0.1-1 µM, 30 min) resulted in the unmasking of SP responses in ~40% of neurons (n = 15 of 39 neurons; unpublished observations). However, the actions of exogenously applied PAF were not blocked by tropisetron (1 µM), suggesting that PAF-induced unmasking of SP responses may not be mediated through 5-HT. Furthermore, PAF (1 µM) also induced SP responses in 50% (n = 3 of 6) of acutely isolated NGNs (i.e., under conditions in which platelets are absent), suggesting that this unmasking effect may be either through PAF receptors on NGNs or a nonselective lipid effect.

Other inflammatory mediators involved in antigen-induced NK₂-receptor unmasking. In the absence of 5-HT₃-receptor activation, allergic inflammation in the nodose ganglion still elicits unmasking in ~20% of neurons. Incubation with a prostaglandin mixture (PGD₂, PGE₂, PGF₂, PGI₂; 1 µM each) unmasked depolarizing SP responses in ~20% of acutely isolated nodose neurons (13), suggesting that prostanoids may mediate the remainder of NK₂-receptor unmasking. However, it does not appear that endogenously released prostaglandins contribute to antigen-induced unmasking of SP responses in intact ganglia. When cyclooxygenase was used to inhibit the synthesis of prostanoids during antigenic activation of the ganglia, there was no measurable reduction in the percentage of neurons with SP responses. It will be interesting to learn whether selective prostaglandin-receptor antagonists prevent prostaglandin-induced unmasking in isolated NGNs.

Functional importance of unmasking tachykinin receptors in vagal afferents. After allergic airway inflammation in vivo, profound changes occur in airway-projecting guinea pig NGNs. A phenotypic switch in neurotransmitter type occurs wherein NGNs projecting to the airway begin expressing tachykinin mRNA and protein (3, 11), and NGNs develop newly functional NK₂ receptors that evoke depolarizing responses (14). To date, these inflammation-induced changes have been characterized only in the somata of the NGNs; whether a similar upregulation of the tachykininergic systems occurs at peripheral and central nerve terminals of these afferents remains undetermined. It is well documented that tachykinins are released from primary sensory nerve cell bodies (10, 15, 22). In guinea pig trigeminal ganglia, KCl, capsaicin, or nerve impulses release SP, and unilateral orofacial inflammation greatly increased both basal and evoked SP release from the inflamed side (15). Thus inflammation-induced unmasking of functional NK₂ receptors may allow excitatory autoreceptor signaling and/or permit extrasynaptic communication between primary sensory somata. The existence of chemical communication between dorsal root somata is well documented (1), and similar chemical "cross talk" may also exist between NGNs (unpublished observations). Whether the tachykininergic system contributes to cross talk remains to be tested.