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J Physiol Volume 514, Number 1, 111-124, January 1, 1999
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The Journal of Physiology (1999), 514.1, pp. 111-124
© Copyright 1999 The Physiological Society

Serotonin unmasks functional NK-2 receptors in vagal sensory neurones of the guinea-pig

Kimberly A. Moore, Glen E. Taylor and Daniel Weinreich

University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, 685 W. Baltimore Street, Baltimore, MD 21201-1559, USA

Received 22 June 1998; accepted after revision 29 September 1998.

  ABSTRACT
Top
Abstract
Introduction
 Methods
 Results
 Discussion
 References

  1. The regulation of substance P (SP) responsiveness in acutely isolated nodose neurones from adult guinea-pigs was investigated using standard intracellular recording techniques.

  2. In control neurones, SP produced no measurable electrophysiological effects. However, following incubation with serotonin (5-HT, 10 µM), 64 % of neurones were depolarized by 10 ± 0·6 mV (n = 84 of 132 neurones) by SP (100 nM). 5-HT-induced SP responses were inhibited by SR48968 (100 nM, n = 6), a neurokinin 2 (NK-2) receptor antagonist, but were unaffected by CP99,994 and SR142801, NK-1 and NK-3 receptor antagonists (n = 3 each), respectively.

  3. 5-HT-induced unmasking of SP responses was maximal within 5 min. Increasing the 5-HT incubation time up to 120 min did not increase the mean response amplitude or the percentage of SP responsive neurones (P = 0·611 and 0·867, respectively).

  4. 5-HT-induced unmasking of SP responses was dose dependent (EC50 = 14 nM). A 5-HT3 receptor agonist CPBG (1 µM), mimicked the unmasking effects of 5-HT (n = 10 of 19 neurones), while 5-CT (10 µM), a non-selective 5-HT agonist devoid of action at 5-HT3 receptors, did not (n = 18). ICS205-930 (1 µM), a 5-HT3 receptor antagonist, completely blocked the 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones).

  5. In 68 % of the neurones tested, bath-applied 5-HT (10 µM) evoked a 178 ± 29·5 nM increase in [Ca2+]i (n = 16), which was blocked by nominally zero [Ca2+]o (n = 4) or by ICS205-930 (1 µM, n = 4). Nodose neurones incubated with 5-HT in the presence of nominally zero [Ca2+]o did not respond to SP (n = 12 of 13 neurones) in Locke solution containing normal [Ca2+]o, indicating that the 5-HT-mediated elevation of [Ca2+]i is required for unmasking of SP responses. Calmidazolium (100 nM), a calmodulin inhibitor, inhibited the unmasking effects of 5-HT (n = 5 of 5 neurones).

  6. Incubating neurones with the nitric oxide (NO) donors papaNONOate (1 mM, 15-30 min) or SNAP (50 µM, 30-60 min) unmasked depolarizing SP responses in 71 % and 45 % of the neurones studied, respectively. L-NMMA (30 µM), a NO synthase inhibitor, blocked 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones).

  7. In sum, these results suggest that stimulation of 5-HT3 receptors activates an intracellular signalling cascade that couples calcium-calmodulin and NO activation to NK-2 receptor unmasking in sensory neurones.
  INTRODUCTION
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Abstract
 Introduction
Methods
 Results
 Discussion
 References

Mast cells are a rich source of numerous biologically diverse mediators. They are distributed throughout the connective tissues of the body, primarily near blood vessels and peripheral nerves, and are abundant beneath the epithelial surfaces of the skin, gastrointestinal tract and lungs (Galli, 1990), facilitating exposure to exogenous antigens. Upon antigenic activation, mast cells release a plethora of preformed and de novo synthesized mediators. Preformed, or granule-stored, mediators are best exemplified by biogenic amines (histamine and serotonin), proteolytic enzymes, zinc and heparin, while the newly synthesized mediators include prostaglandins, leukotrienes and many other lipid-derived factors. In addition, murine (Gordon et al. 1990) and human (Gibbs et al. 1997) mast cells can rapidly release cytokines, such as tumour necrosis factor-alpha and interleukin-4.

Inflammation and the biochemical sequelae that follow, including mast cell activation, evoke a number of neuroplastic changes in primary sensory neurones in vitro and in situ. These alterations range from modulation of electrical membrane properties (Undem et al. 1993; Riccio et al. 1996) to increased expression of the tachykinins substance P (SP) and neurokinin A (Hanesch et al. 1993; Neumann et al. 1996; Fischer et al. 1996). Recently, we have reported that allergen-induced mast cell activation in isolated vagal sensory ganglia evokes a long-lasting (days) expression of functional neurokinin 2 (NK-2) receptors (Weinreich et al. 1997).

In most cases, the inflammatory mediator(s) initiating neuroplastic changes in sensory neurones are unknown. Any of the mast cell-derived mediators, acting alone or in combination, may contribute to NK-2 tachykinin receptor unmasking in nodose neurones. In addition, it is possible that mast cell-released mediators are activating non-neuronal cells (e.g. macrophages, endothelial cells or glial cells) which in turn generate intermediaries essential for neuroplastic changes. To determine which, if any, mast cell-derived mediators are capable of unmasking functional NK-2 receptors, we have incubated acutely isolated adult nodose neurones with known inflammatory mediators. Our results indicate that serotonin (5-HT), acting on the 5-HT3 receptor, is sufficient to reveal functional NK-2 receptors in isolated nodose ganglion neurones. Furthermore, 5-HT-induced unmasking of NK-2 receptors requires calcium- calmodulin-dependent activation of nitric oxide.

  METHODS
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Abstract
 Introduction
 Methods
Results
 Discussion
 References

Tissue preparation

Adult male Hartley guinea-pigs (250-500 g; Charles River, Wilmington, MA, USA) 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 (mM): 136 NaCl, 5·6 KCl, 1·2 MgCl2, 2·2 CaCl2, 14·3 NaHCO3, 1·2 NaH2PO4 and 10 dextrose, equilibrated with 95 % 02-5 % CO2, pH 7·2-7·4. Acutely dissociated neurones were prepared enzymatically as described by Jafri et al. (1997), then resuspended in Leibovitz L-15 medium (Gibco BRL) containing 10 % (v/v) fetal bovine serum (FBS; JRH Biosciences, Lexena, KS, USA). Cell suspensions (0·15 ml) were transferred onto circular polylysine (0·1 mg ml-1 poly-D-lysine; Sigma)-coated glass coverslips (Bellco, Vineland, NJ, USA) in a 24-well culture plate. Neurones were maintained at 37°C for at least 8 h prior to treatment with inflammatory mediators. Inflammatory mediators were diluted in Leibovitz L-15 containing 10 % FBS and added to the culture wells for 1 h at 37°C unless otherwise noted. Subsequently, coverslips were transferred to a recording chamber and superfused with Locke solution at 35-37°C. Neurones were tested for electrophysiological responses to SP 10-120 min later. Control neurones were subjected to the same manipulations in the absence of inflammatory mediators.

Electrophysiological recording

Intracellular recording micropipettes were fabricated from aluminosilicate capillary glass (1·0 mm o.d., 0·68 mm i.d.; Sutter Instrument Co.) on a Flaming-Brown P-97 micropipette puller (Sutter). Microelectrodes had resistances of 40-100 MOmega when backfilled with 3 M KCl. Current- and voltage-clamp recordings were made with an Axoclamp-2A amplifier (Axon Instruments) in discontinuous mode (sample rate, 5 kHz; filtered at 0·3-3 kHz); the head stage voltage was monitored continuously to ensure that the sampled voltage reached steady state. Current and voltage signals were viewed on-line with an oscilloscope and digitized with a Neurocorder (Neurodata Instruments Inc., Delaware Water Gap, PA, USA) for storage on videocassette for off-line analysis. Membrane input resistance was monitored by measuring the magnitude of electrotonic voltage transients produced by 100 pA hyperpolarizing current pulses (300 ms). Neurones were accepted for study only if they showed a stable resting membrane potential ( -50 ± 2 mV) and had action potentials overshooting 0 mV. Data acquisition and analysis of electrophysiological data were performed using pCLAMP 6.2 software in conjunction with a Digidata 1200 interface (Axon Instruments).

[Ca2+]i measurement

To measure [Ca2+]i, dissociated neurones plated on 25 mm coverslips were incubated with 1 µM fura-2 AM for 75 min at room temperature (22-24°C) as described previously (Cohen et al 1997). After incubation, the coverslip was placed in the recording chamber and superfused with Locke solution. Fura-2 fluorescence measurements were performed with a DeltaScan illumination system (Photon Technology International (PTI), South Brunswick, NJ, USA) coupled to an IM35 microscope (Carl Zeiss Inc.) through a fibre-optic cable. Each neurone under study was alternately illuminated with 340 and 380 nm light. The fluorescence emission, after passing through a 450 nm bandpass filter, was measured by a photomultiplier tube (PMT). The PMT output was digitized and stored for subsequent analysis. Instrument control, data acquisition and analysis were performed using FELIX 1.1 software (PTI).

[Ca2+]i calibration

Values of [Ca2+]i were derived using the ratio method of Grynkiewicz et al. (1985). All fura-2 fluorescence records were corrected for background fluorescence by subtracting the light intensity measured from neurones depleted of fura-2 by digitonin permeabilization (Kao, 1994). [Ca2+]i was calculated using the equation of Grynkiewicz et al. (1985):

[Ca2+]i = Kd × [(R - Rmin)/(Rmax - R)] × [(Sf2)/(Sb2)],

where R is the ratio F340/F380 and Rmin and Rmax are the minimum and maximum values of the ratio, attained at zero and saturating calcium concentrations, respectively. F340 is the fluorescence emitted by the dye when excited at 340 nm and F380 is the fluorescence emitted by the dye when excited at 380 nm. Sf2/Sb2 is the ratio of fluorescence intensities for calcium-free and calcium-bound indicator measured with 380 nm excitation. Rmin, Rmax and Sf2/Sb2 were determined from five acutely dissociated neurones used specifically for calibration purposes.

Recording chamber and drug delivery

For electrophysiological recording or calcium imaging from isolated neurones, a custom recording chamber that provides superfusion of the coverslip with Locke solution via a gravity flow system was employed. The superfusate level was lowered to 50 µm above the surface of the neurones with an adjustable aspirator to minimize electrode stray capacitance. The chamber was mounted on the stage of a compound microscope (ausJENA) equipped with Hoffman optics (× 400) to allow direct visualization of neurones for intracellular impalement or on the stage of an inverted microscope (Zeiss IM35) equipped with a × 40 oil-immersion objective (Zeiss Fluar; numerical aperture, 1·3) for fluorescence measurements.

Reservoirs containing various drugs were connected to the inflow line of the recording chamber with three-way valves that could rapidly divert the source of superfusion from the main reservoir. Agonists were superfused over isolated neurones for 20-60 s. When receptor antagonists were used, neurones were superfused with these reagents for at least 2 min before addition of agonists. The temperature of the Locke solution flowing through the recording chamber was maintained at 33-35°C, unless noted otherwise.

Drug solutions

Drug solutions were prepared daily from concentrated ( 10 mM) stock solutions stored at -20°C. (+)-(2S,3S)-3-(2-methoxybenzyl-amino)-2-phenylpiperidine (CP99,994) was provided by Pfizer, Inc. (Groton, CT, USA). (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR48968) was a gift from Zeneca (Wilmington, DE, USA). Calmidazolium, 1-(m-chlorophenyl)-biguanide hydrochloride (CPBG), 3-tropanyl-indole-3-carboxylate hydrochloride (ICS205-930; tropisetron) and S-nitroso-N-acetylpenicillamine (SNAP) were obtained from Research Biochemicals Inc. (Natick, MA, USA), 5-carboxamidotryptamine (5-CT) from Tocris Cookson, Inc. (Ballwin, MO, USA), 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) from Pfaltz & Bauer (Waterbury, CT, USA), nerve growth factor (NGF 2·5 s and 7 s) from Alomone Laboratories (Jerusalem, Israel), papaNONOate from Alexis Biochemicals (San Diego, CA, USA) and fura-2 from Molecular Probes (Eugene, OR, USA). All other reagents were purchased from Sigma.

Data analysis

Data are expressed as means ± S.E.M. Student's two-tailed t test and one-way analysis of variance (ANOVA) were 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 neurones responding to SP following various treatments. Statistical analysis was performed using Sigmastat software (Jandel Scientific, San Rafael, CA, USA). Curve fitting and figure construction were accomplished using Origin (Microcal Software Inc., Northampton, MA, USA). A semilogarithmic plot of the concentration-response relationship was iteratively fitted using the four-parameter logistic equation:

Y = ((max - min)/(1 + x/EC50)b) - min,

where Y is the SP depolarization in response to x concentration of 5-HT, max and min are the maximum and minimum SP responses, EC50 is the 5-HT concentration at the half-maximal SP response, and b is the Hill coefficient.

  RESULTS
Top
Abstract
 Introduction
 Methods
Results
Discussion
 References

Serotonin selectively unmasks tachykinin receptors

As observed in intact nodose ganglia, the resting membrane properties of acutely isolated nodose neurones from control guinea-pigs are not measurably affected by exogenously applied SP (0·1-10 µM; Weinreich et al. 1997). In sharp contrast, following a 5-120 min pre-incubation with 10 µM 5-HT, 84 of 132 (64 %) isolated nodose neurones were depolarized a mean of 10 ± 0·6 mV by a subsequent bath application of SP (100 nM; Figs 1Aa and B , 2 and 6). In 88 % of neurones in which membrane input resistance (Rin) was monitored, a 48 ± 4·4 % (n = 35) decrease in Rin accompanied the SP-induced depolarization. In the remaining 12 %, there was either no change (2 %) or a small increase (10 %) in Rin. In 18 % of the SP-responsive neurones, the SP-induced membrane depolarization induced action potentials (Fig. 1B). When neurones were voltage-clamped to their resting potentials (-65 mV), SP produced an inward current that averaged 248 ± 53·0 pA (n = 8) and was accompanied by a 42 ± 10·7 % increase in membrane conductance in seven of eight neurones (Fig. 1Ab). The resting membrane potential (-65 ± 1·3 mV; n = 56) and membrane input resistance (57 ± 6·1 MOmega; n = 55) following pre-incubation with 5-HT were not significantly different from values recorded from control neurones incubated in the absence of 5-HT. To rule out the possibility that 5-HT was acting in concert with another mediator present in Leibovitz L-15 media or FBS to elicit SP responses, neurones were incubated with 5-HT in Locke solution. Two of four neurones incubated with 5-HT in Locke solution for 1 h were subsequently depolarized 8 ± 2 mV by SP.

To determine whether the SP response expressed following exposure to 5-HT was similar to that unmasked by allergen challenge in the intact nodose ganglion, we estimated the reversal potential (Vrev) for the SP response. The amplitude of the steady-state holding current was measured at varying pre-set membrane potentials in the presence and absence of SP. As shown in Fig. 1C, the slope of the current-voltage (I-V ) relationship was increased in the presence of SP, reflecting an increased membrane conductance. The control and SP I-V curves for this neurone intersected at -16 mV. The mean Vrev value for the SP response was -27 ± 3·4 mV (range, -15 to -40 mV; n = 9). A depolarizing SP response accompanied by an increase in membrane conductance and a reversal potential value of -27 mV suggests that SP may activate a non-selective cation conductance. This Vrev value is comparable with that estimated for SP responses evoked by allergen challenge in nodose ganglia (-21 ± 4·9 mV; range, -15 to -31 mV; n = 3; P = 0·437).

We screened the capacity of a variety of other inflammatory mediators to unmask functional tachykinin receptors. Unlike 5-HT, incubating nodose neurones for 30-60 min with ATP (10 µM), bradykinin (1 µM), DMPP (a nicotinic agonist, 10 µM), gamma-amino-n-butyric acid (GABA, 10 µM), heparin (100 µg ml-1), histamine (10 µM) and NGF 2·5 s or 7 s (100 ng ml-1), in a variety of combinations, did not unmask SP responses (Table 1). Following incubation with a mixture of prostaglandins (PGD2, PGE2, PGF2alpha and PGI2, 1 µM each) or 50 mM KCl for 45-90 min, 18 % of neurones were depolarized by SP (Table 1).

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    Figure 1. Changes in electrical membrane properties produced by SP in acutely isolated nodose neurones following 5-HT treatment

    Aa, depolarizing response produced by SP (100 nM, 30 s), recorded in a nodose neurone pre-incubated with 5-HT (10 µM, 60 min). In this and subsequent figures, the horizontal bar represents the time of drug application. Downward deflections are electrotonic voltage transients elicited by hyperpolarizing current pulses (100 pA, 300 ms, 0·6 Hz); the magnitude of these transients is a measure of membrane input resistance (Rin). The SP-induced membrane depolarization was accompanied by a decreased Rin. The resting membrane potential (Vm) was -72 mV; resting Rin was 70 MOmega. b, inward current in response to SP (100 nM, 30 s), recorded in the same nodose neurone voltage clamped to -70 mV. Downward current deflections were elicited by hyperpolarizing voltage step commands (10 mV, 300 ms, 0·6 Hz) to monitor membrane conductance (Gm). The SP-induced inward current was accompanied by an increased Gm. B, membrane potential change produced by SP recorded in another nodose neurone previously incubated with 5-HT (1 µM, 60 min). In 18 % of the neurones pre-incubated with 5-HT (1-10 µM, 5-120 min), the SP-induced membrane depolarization was sufficient to reach spike threshold; action potential amplitudes are truncated. Downward deflections are electrotonic voltage transients as described in Aa. Resting Vm was -62 mV; Rin was 50 MOmega. C, steady-state I-V relationship in the presence (fullcir) and absence (cir) of 100 nM SP. The neurone was voltage clamped to -60 mV and subjected to a series of voltage step commands (250 ms) before and during bath application of SP. Steady-state currents were recorded and plotted against the step potentials. The reversal potential (Vrev, dotted line) was taken as the point at which the two curves intersect (i.e. where the difference current is zero).

Table 1. Effects of various treatments on the substance P responsiveness in acutely isolated nodose neurones of the guinea-pig

Treatment Concentration Neurones tested Responsive neurones Responders (%) Mean response (mV)
Control - 58 0 0 n.a.
ATP 10 µM 17 0 0 n.a.
Bradykinin 1 µM 11 0 0 n.a.
DMPP 10 µM 17 0 0 n.a.
GABA 10 µM 6 0 0 n.a.
Heparin 100 µg ml-1 6 0 0 n.a.
Histamine 10 µM 5 0 0 n.a.
KCl 50 mM 11 4 1817 ± 10 (2)
18 -8 ± 3 (2)
NGF 2·5 s/7 s 100 ng ml-1 6 0 0 n.a.
PGD2, PGE2, PGF2alpha, PGI2 1 µM each 11 2 18 6 ± 1·1
papaNONOate 1 mM 7 5 71 12 ± 4·7
Serotonin 10 µM 132 84 64 10 ± 0·6
SNAP 50 µM 38 2245 11 ± 1•8 (17)
13 -7 ± 1·2 (5)
Nodose neurones were incubated with a variety of mediators or signalling molecules at 37 °C. Subsequently, membrane potential changes were measured following bath application of SP (100 nM, 30-60 s). In control nodose neurones, SP did not elicit any measurable changes in resting membrane properties (Weinreich et al. 1997). Following incubation with 5-HT, papaNONOate or SNAP, SP elicited membrane potential changes in the majority of neurones studied. A prostaglandin mixture (PGD2, PGE2, PGF2alpha and PGI2) or KCl elicited SP responses in a small percentage of neurones. Mean response values are given as means ± S.E.M. When two types of responses were elicited by SP, the number of neurones depolarized or hyperpolarized is indicated in parentheses next to the average response amplitude. n.a. designates not applicable. DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide (a nicotinic agonist); NGF, nerve growth factor; papaNONOate, a NO donor; SNAP, S-nitroso-N-acetylpenicillamine (a NO donor).

Pharmacological characterization of the tachykinin receptors unmasked by serotonin

Endogenous tachykinins activate three distinct neurokinin receptors (NK-1, NK-2 and NK-3) with differential specificity (Maggi, 1995). We have pharmacologically characterized the neurokinin receptor subtype unmasked by 5-HT using selective non-peptide neurokinin receptor antagonists. SR48968 (100 nM), a selective NK-2 receptor antagonist (Emonds-Alt et al. 1992), reversibly abolished the SP-induced depolarization (n = 6; Fig. 2). In contrast, CP99,994 (100 nM), a selective NK-1 receptor antagonist (McLean et al. 1993), did not significantly inhibit the SP-mediated depolarization (11 ± 0·9 vs. 11 ± 1·9 mV before and during antagonist application (n = 3), Fig. 2). Additionally, the unmasked SP-induced depolarizations were unaffected by an NK-3 receptor antagonist (13 ± 1·7 vs. 12 ± 3·3 mV before and during treatment with 100 nM SR142801 (Oury-Donat et al. 1995), respectively; n = 3). Taken together, these pharmacological results suggest that the tachykinin receptor unmasked by 5-HT, like that unmasked antigenically, is of the NK-2 receptor subtype.

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    Figure 2. Effect of tachykinin receptor antagonists on SP-induced membrane depolarization in a 5-HT treated nodose neurone

    Following pre-incubation with 5-HT (10 µM, 20 min), bath application of SP (100 nM, 40 s) produced a membrane depolarization. Downward deflections are electrotonic voltage transients to monitor Rin, as described in Fig. 1Aa. In this neurone, SP produced little change in Rin. The SP-induced membrane depolarization was unaffected by CP99,994 (100 nM, 5 min), a specific NK-1 receptor antagonist. Subsequent application of SR48968 (100 nM, 4 min), a specific NK-2 receptor antagonist, blocked the SP response. After a 15 min wash with drug-free Locke solution, the SP-induced depolarization partially recovered. The resting membrane potential was -71 mV and Rin was 30 MOmega.

Time course of serotonin-induced unmasking of NK-2 receptors

To gain insight into the process by which 5-HT unmasks NK-2 receptors, we determined how rapidly 5-HT induces the expression of SP responses (Fig. 3). After a 5 min incubation with 10 µM 5-HT (the earliest time point examined), three of four neurones tested showed a measurable membrane depolarization (11 ± 2·4 mV) in response to bath-applied SP (100 nM). Incubating nodose neurones with 10 µM 5-HT for longer time periods (15-120 min) did not significantly enhance the magnitude of the SP-induced depolarization or the percentage of SP responsive neurones (P = 0·611 and 0·867, respectively; Fig. 3). Thus, the 5-HT-mediated unmasking of SP responses follows a relatively rapid time course.

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    Figure 3. Time course of 5-HT-induced unmasking of SP responses in acutely isolated nodose neurones

    Following a 5 min pre-incubation with 5-HT (10 µM), three of four neurones tested responded electrically to bath-applied SP (100 nM, 30-60 s). The mean SP response amplitude and the percentage of neurones responding to SP was not affected by increasing the 5-HT pre-incubation time up to 2 h (P = 0·611 and 0·867, respectively). The zero time point represents neurones that were not pre-incubated with 5-HT. fullcir, mean SP response (± S.E.M.) of the number of neurones indicated near the symbol. square, percentage of neurones depolarized by SP following varying times of pre-incubation with 5-HT.

Role of protein synthesis in unmasking and maintaining NK-2 receptors

Since 5-HT-induced expression of functional NK-2 receptors occurs within 5 min, it seems unlikely that new protein synthesis underlies this phenomenon. Nonetheless, we tested the role of protein synthesis by treating nodose neurones with cycloheximide (100 µg ml-1) 60 min prior to and during a 15 min incubation with 10 µM 5-HT. This protocol inhibits protein synthesis in the guinea-pig nodose ganglia by 96 % as measured by incorporation of [3H]leucine into protein (Weinreich et al. 1997). Within 30 min of incubation with 5-HT under these conditions, SP depolarized the membrane potential by 8 ± 2·7 mV in five of nine neurones studied. The depolarizing responses in these neurones were not significantly different from those recorded in neurones treated with 5-HT alone (P = 0·434). Additionally, the percentage of nodose neurones responding to bath-applied SP did not differ significantly between the two groups (56 % vs . 64 %; z = 0·125, P = 0·901). We also examined whether protein synthesis was necessary to maintain unmasked SP responses. Nodose neurones were treated with cycloheximide (100 µg ml-1) 60 min prior to and during a 60 min incubation with 5-HT. Under these conditions, none of the 11 neurones tested revealed a detectable (> 2 mV) membrane potential change in response to bath-applied SP. Long-term incubation with cycloheximide does not block 5-HT-evoked membrane potential changes. Following a 2·5 h incubation with cycloheximide, 5-HT elicited a 19 ± 3·8 mV depolarization in 63 % of the neurones studied (n = 5 of 8). Taken together, these results suggest that protein synthesis is not required for the unmasking of NK-2 receptors but there may be a critical window during which protein synthesis is essential for stabilization of the unmasked receptors.

Pharmacology of the serotonin receptor mediating unmasking

Currently, seven classes and a total of 14 subtypes of serotonin receptor have been characterized based upon pharmacological and molecular biological criteria (Hoyer & Martin, 1997). To determine whether the unmasking of NK-2 receptors by 5-HT is receptor mediated, we have derived the EC50 value from the 5-HT concentration-SP response relationship. We have also employed a battery of pharmacological reagents to define the profile of the receptor subtype mediating 5-HT-induced changes in membrane properties and the unmasking of NK-2 receptors.

The concentration-response relationship was examined by incubating neurones with varying concentrations of 5-HT for 30-60 min and measuring the peak amplitude of the SP (100 nM)-induced depolarization. Normalization of the depolarizing responses was not necessary because of the reproducibility of responses from neurone to neurone. 5-HT produced a concentration-dependent increase in the SP response that saturated at 100 nM 5-HT with a peak SP-induced depolarization of 10·5 mV (Fig. 4). A semilogarithmic plot of the peak SP-induced depolarization versus 5-HT concentration was well fitted (chi2 = 0·221) by a sigmoidal relationship that varied over 2 orders of magnitude (Fig. 4). The estimated EC50 value for 5-HT-induced unmasking of SP responses was 14 ± 16·4 nM with a Hill coefficient of 2·6. A saturating concentration-response relationship with an EC50 in the nanomolar range implies that the unmasking of NK-2 responses by 5-HT is receptor mediated.

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    Figure 4. Relationship between 5-HT concentration and the peak amplitude of the SP-induced membrane depolarization

    Semilogarithmic plot of the concentration-response relationship between 5-HT concentration and the amplitude of unmasked SP responses. Neurones were pre-incubated for 1 h with varying concentrations of 5-HT then tested for changes in membrane potential in response to bath-applied SP (100 nM, 30-60 s). Each data point represents the mean (± S.E.M.) SP-induced depolarization for the number of neurones indicated next to the data point. The continuous line represents a logistical fit to the data (chi2 = 0·221). The EC50 value was 14 ± 16·4 nM 5-HT with a Hill coefficient of 2·6.

In 65 % of acutely isolated guinea-pig nodose neurones, bath application of 10 µM 5-HT evokes a 15 ± 1·9 mV membrane depolarization accompanied by a 50 ± 7·6 % decrease in input resistance (n = 24; Fig. 5A and B). In a small population of neurones (3 of 24), the 5-HT-induced depolarization had two components, possibly reflecting the activation of multiple receptor subtypes. As illustrated in Fig. 5A and B, both types of 5-HT-induced depolarization were completely inhibited by ICS205-930 (1 µM; n = 7), a 5-HT3 receptor antagonist (Richardson et al. 1985). Unfortunately, the relatively weak affinity of ICS205-930 in guinea-pig nodose neurones compared with other species (-log of the dissociation constant (pKb) = 7·8 vs. 11·0 in rabbit; Fozard, 1992) precludes the use of nanomolar concentrations of this antagonist. The depolarizing effects of 5-HT can also be mimicked by bath application of 1 µM CPBG, a selective 5-HT3 receptor agonist (Hoyer et al. 1994), suggesting the presence of functional 5-HT3 receptors in guinea-pig nodose neurones (n = 3, data not shown).

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    Figure 5. Effect of a selective 5-HT3 receptor antagonist on the 5-HT-induced membrane depolarization and elevation of [Ca2+]i in acutely isolated nodose neurones

    A, current-clamp recording of a depolarizing response produced by 5-HT (10 µM, 20 s) from a neurone with a resting membrane potential of -62 mV. Downward deflections reflect Rin as described in Fig. 1Aa. 5-HT induced a decrease in Rin from a resting value of 40 MOmega. The 5-HT-induced depolarization was abolished by ICS205-930 (1 µM, 2 min), a selective 5-HT3 receptor antagonist. After a 7 min wash with drug-free Locke solution, the 5-HT-induced depolarization recovered. B, a biphasic depolarizing response to 5-HT accompanied by a decrease in Rin recorded in another neurone. Biphasic depolarizations were observed in 13 % of the neurones studied. Both phases of the 5-HT-induced depolarization were abolished by ICS205-930 (1 µM, 4 min) and partially recovered following a 10 min wash with drug-free Locke solution. The resting membrane potential was -75 mV; Rin was 80 MOmega. C, in a third neurone, superfusion of 5-HT (10 µM, 40 s) induced an elevation of intracellular calcium that was reversibly abolished by ICS205-930 (1 µM, 3 min). After a 20 min wash with drug-free Locke solution, a subsequent application of 5-HT again produced an elevation of intracellular calcium. Resting [Ca2+]i was 40 nM.

To determine whether 5-HT3 receptors play a role in 5-HT-evoked unmasking of NK-2 receptors, we incubated nodose neurones with 1 µM CPBG. Following a 60 min incubation with CPBG, 100 nM SP induced a 16 ± 3·2 mV depolarization in 10 of 19 neurones (Table 2 and Fig. 6C). The membrane depolarization was accompanied by a 33 ± 4·2 % decrease in input resistance in all but one neurone. In contrast, nodose neurones treated with 5-CT (10 µM, 60 min), an agonist active at most 5-HT receptors but without action at 5-HT3 receptors (Hoyer et al. 1994), were not measurably affected (< 2 mV) by 100 nM SP (n = 18; Table 2 and Fig. 6B). Further evidence for the role of 5-HT3 receptors in the unmasking of NK-2 receptors was obtained by treating neurones with a 5-HT3 receptor antagonist. Incubating isolated nodose neurones with 1 µM ICS205-930 for 15 min prior to and during treatment with 5-HT (1 µM, 60 min) completely prevented the expression of SP responses (n = 10; Table 2 and Fig. 6D).

As illustrated in Fig. 5, SP responses unmasked by CPBG were longer in duration than those unmasked by 5-HT. Following incubation with 5-HT, the majority (87 %) of SP responses paralleled the duration of the SP application. In contrast, following incubation with CPBG, the SP-induced depolarization outlasted the SP application in the majority of neurones studied (71 %). The basis for this difference was not pursued.

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    Figure 6. Effect of 5-HT agonists and antagonists on the expression of SP responses in nodose neurones of the guinea-pig

    A, following pre-incubation of an acutely isolated neurone with 5-HT (10 µM, 5 min), bath-applied SP (100 nM, 60 s) evoked a membrane depolarization. Downward deflections reflect changes in Rin, as described in Fig. 1Aa. The SP-induced membrane depolarization was accompanied by a decrease in Rin. The decreased Rin was not due to activation of voltage-gated channels because when the membrane potential was depolarized to approximately the same membrane potential elicited by SP, Rin was relatively unaffected. Resting Vm was -74 mV and Rin was 70 MOmega. B, in another neurone, a 40 min pre-incubation with a relatively non-selective 5-HT agonist without action at 5-HT3 receptors, 5-CT (5-carboxamidotryptamine, 10 µM), failed to induce a SP response. Resting Vm was -76 mV and Rin was 60 MOmega. C, when a third neurone was pre-incubated for 45 min with a 5-HT3 receptor agonist, CPBG (1-(m-chlorophenyl)-biguanide hydrochloride, 10 µM), SP caused a membrane depolarization accompanied by a decrease in Rin. In general, the durations of CPBG-induced SP depolarizations were longer than SP-induced depolarizations elicited by 5-HT (see Results). Resting Vm was -52 mV and Rin was 49 MOmega. D, following pre-incubation with 5-HT in the presence of ICS205-930 (1 µM each for 60 min), SP application did not elicit a response. Resting Vm was -75 mV and Rin was 40 MOmega. Downward voltage deflections in traces A-D were elicited by 100 pA rectangular hyperpolarizing pulses.

Temperature dependence of serotonin-mediated NK-2 receptor unmasking

In all the studies described thus far, neurones were always incubated with 5-HT at physiological temperature (37°C). Under these conditions, bath-applied SP evoked a measurable membrane depolarization in 64 % of the neurones. In order to test the effects of temperature on 5-HT-mediated unmasking of NK-2 receptors, we incubated neurones with 5-HT at room temperature (22-24°C) for 60-120 min and tested for SP responses at 37°C. 5-HT (10 µM) still elicited a membrane depolarization at room temperature (21 ± 2·7 mV, n = 8 of 9 neurones); however, following incubation with 5-HT at room temperature, SP never evoked a measurable change in membrane potential or input resistance (n = 15; Table 2). The temperature dependence of 5-HT-induced unmasking of NK-2 receptors most likely reflects the involvement of a metabolically labile second messenger(s).

Role of intracellular calcium in serotonin-induced unmasking of NK-2 receptors

5-HT3 receptors are known to permeate Ca2+ ions (Peters et al. 1992), which may be essential for the expression of functional NK-2 tachykinin receptors. To examine whether 5-HT3 receptors in nodose neurones can evoke changes in intracellular calcium levels, we measured [Ca2+]i using the ratiometric calcium indicator, fura-2. The baseline level of [Ca2+]i in these neurones was 117 ± 18·0 nM (n = 34). At 37°C, bath-applied 5-HT (10 µM) evoked a 178 ± 29·5 nM increase of [Ca2+]i (n = 16; Fig. 5C) in 68 % of the neurones tested. At room temperature, 5-HT also elicited a transient elevation of [Ca2+]i in six of nine neurones tested (60 %), albeit smaller (58 ± 14·9 nM) than that measured at 37°C. 5-HT-evoked elevations of [Ca2+]i were abolished by the 5-HT3 receptor antagonist ICS205-930 (1 µM, n = 4; Fig. 5C), which was analogous to both 5-HT-induced changes in electrical membrane properties and 5-HT-induced NK-2 receptor unmasking.

Several experimental protocols were utilized to determine the source of the 5-HT-induced elevation of [Ca2+]i (DeltaCa2+). When [Ca2+]o was reduced to nominally zero concentration, the 5-HT-evoked DeltaCa2+ was reduced to 13 ± 12·5 nM (n = 4), suggesting that the DeltaCa2+ is mediated by flux through membrane channels. To distinguish Ca2+ influx through the 5-HT3 receptor channel from influx via voltage-dependent calcium channels (VDCCs), we blocked VDCCs with 100 µM Cd2+ and measured the 5-HT-induced DeltaCa2+. The 5-HT-induced DeltaCa2+ was not significantly affected by Cd2+ (n = 4, P = 0·1613), suggesting that Ca2+ influx through VDCCs does not contribute to the DeltaCa2+. Finally, to confirm that release from intracellular Ca2+ stores does not contribute to the DeltaCa2+, we utilized 2,5-di(t-butyl)hydroquinone (DBHQ, an endoplasmic reticulum Ca2+-ATPase inhibitor; Inesi & Sagara, 1994) to deplete intracellular Ca2+ stores. In the presence of 10 µM DBHQ, the 5-HT-evoked DeltaCa2+ was 70 ± 13·9 % greater in amplitude than under control conditions (n = 2). This suggests that release from intracellular calcium stores does not contribute to generation of the 5-HT-induced DeltaCa2+ but that endoplasmic reticulum Ca2+-ATPases are involved in reducing elevated levels of [Ca2+]i. Taken in aggregate, these results indicate that the 5-HT-evoked DeltaCa2+ is generated by flux through 5-HT3 receptor channels, rather than flux through VDCCs or calcium release from intracellular stores.

To examine whether changes in [Ca2+]i are critical for 5-HT-evoked unmasking of NK-2 receptors, we incubated nodose neurones with 5-HT in the presence of nominally zero [Ca2+]o. Only 1 of 13 nodose neurones incubated with 5-HT (10 µM, 20-75 min) in the absence of [Ca2+]o was depolarized (17 mV) in response to SP (Table 2). It is noteworthy that although both ATP (10 µM) and bradykinin (10 µM) elevated [Ca2+]i (Delta = 1140 ± 377 nM, n = 12 and 377 ± 145 nM, n = 6, respectively), they did not cause the unmasking of functional NK-2 receptors. Thus, it appears that elevated [Ca2+]i alone is not sufficient to unmask NK-2 receptors.

Many intracellular effects of calcium are mediated through calmodulin. It is therefore possible that activation of 5-HT3 receptors triggers calmodulin. To test this possibility, we incubated neurones with 5-HT (10 µM) and calmidazolium (100 nM), a known calmodulin inhibitor (Moore & Handy, 1997). Calmidazolium inhibited the NK-2 receptor unmasking effect of 5-HT (n = 5; Table 2). From this series of experiments, we can infer that an elevation of [Ca2+]i in conjunction with calmodulin is essential for the unmasking of NK-2 receptors.

Table 2. Role of 5-HT3 receptors, [Ca2+]i and NO in 5-HT-mediated unmasking of NK-2 tachykinin receptors in nodose neurones

Treatment Neurones tested Responsive neurones Responders (%) Mean response (mV)
5-HT (10 µM, 37 °C) 132 84 64 10 ± 0·6
5-HT (10 µM, 24 °C) 15 0 0 n.a.
5-CT (10 µM) 18 0 0 n.a.
CPBG (1 µM) 19 10 53 16 ± 3·2
5-HT + ICS205-930 (1 µM) 10 0 0 n.a.
5-HT + zero [Ca2+]o 13 1 8 17
5-HT + calmidazolium (100 nM) 5 0 0 n.a.
5-HT + L-NMMA (30 µM) 10 0 0 n.a.
Acutely isolated nodose neurones were incubated with 5-HT, or 5-HT receptor agonists, under a variety of conditions. Subsequently, membrane potential changes in response to bath-applied SP (100 nM, 30-60 s) were recorded. 5-HT or a 5-HT3 receptor agonist unmasked SP responses. 5-HT-induced unmasking of SP responses was inhibited by nominally zero [Ca2+]o, calmidazolium or L-NMMA. When antagonists were used, neurones were incubated with the antagonist for 15 min prior to and during incubation with 5-HT. Where not stated, the 5-HT concentration was 10 µM and incubations were performed at 37 °C. Mean response values are given as means ± S.E.M. n.a. designates not applicable. 5-CT, 5-carboxamidotryptamine (a non-selective 5-HT receptor agonist without action at 5-HT3 receptors); CPBG, 1-(m-chlorophenyl)-biguanide hydrochloride (a 5-HT3 receptor agonist); ICS205-930, a 5-HT3 receptor antagonist; calmidazolium, a calmodulin inhibitor; L-NMMA, N-6-methyl-L-arginine (a NOS inhibitor).

Role of NO in serotonin-induced unmasking of tachykinin receptors

Several studies have linked activation of 5-HT3 receptors to the generation of nitric oxide (NO) (Reiser, 1992). The above results reveal that activation of 5-HT3 receptors evokes an elevation of [Ca2+]i that is necessary for the unmasking of NK-2 receptors. Elevated [Ca2+]i can activate calmodulin which, in turn, can stimulate nitric oxide synthase (NOS), thus promoting the formation of NO. To examine whether NO is capable of unmasking NK-2 receptors, control nodose neurones were incubated with two different NO donors, papaNONOate and SNAP. PapaNONOate (1 mM, 15-60 min) unmasked depolarizing SP responses (12 ± 4·7 mV, n = 5) in 71 % of the neurones studied (Table 1 and Fig. 7A). Similarly, pre-incubation with SNAP (50 µM, 30-60 min) elicited SP responses in 58 % (22 of 38) of the neurones tested. Of those that responded, 17 were depolarized (11 ± 1·8 mV; Table 1 and Fig. 7B) by SP; the remainder were hyperpolarized (-7 ± 1·2 mV, n = 5; Table 1). The nature of the hyperpolarizing SP responses was not investigated further (cf. Jafri & Weinreich, 1996). The percentage of neurones depolarized by SP following incubation with papaNONOate and SNAP, as well as the mean SP response amplitude, did not differ significantly from neurones incubated with 5-HT. If NO mediates 5-HT-induced unmasking of NK-2 receptors then N-6-methyl-L-arginine (L-NMMA), a NOS inhibitor (Moore & Handy, 1997), should abolish the unmasking effect of 5-HT. Incubation of nodose neurones with L-NMMA (30 µM) 15 min prior to and during treatment with 5-HT (10 µM, 60 min) prevented unmasking of NK-2 receptors; none of 10 neurones tested responded to SP (Table 2 and Fig. 7C). To test whether calmidazolium (100 nM) or L-NMMA (30 µM) disrupted 5-HT3 receptor function, we examined 5-HT-induced changes in membrane potential following a 30-75 min incubation with both of these antagonists. 5-HT still depolarized the membrane by 14 ± 0·8 mV (n = 5 of 6 neurones), suggesting that calmidazolium and L-NMMA do not interfere with 5-HT3 receptor function. Furthermore, calmidazolium and L-NMMA did not block SP responses unmasked by 5-HT. Four of nine neurons treated with both calmidazolium and L-NMMA following 5-HT (10 µM, 15 min) were depolarized 6 ± 0·9 mV by SP. Together, these results suggest that calmidazolium and L-NMMA are selective in their action. We propose that the following signalling cascade couples 5-HT3 receptor activation to NK-2 receptor unmasking: (1) 5-HT3 receptor activation evokes an influx of Ca2+, and (2) calcium-dependent calmodulin activates NOS, thus promoting the synthesis of NO. How NO 'unmasks' functional NK-2 receptors remains unresolved.

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    Figure 7. Effects of nitric oxide donors and a nitric oxide synthase inhibitor on the SP responsiveness of acutely isolated nodose neurones

    A, SP application (60 s) subsequent to a 60 min pre-incubation with papaNONOate (1 mM), a NO donor, elicited a membrane depolarization accompanied by a decreased Rin (upper trace). Action potential activity is evident on the rising phase of the depolarizing response. When the same neurone is voltage clamped to -62 mV (lower trace), SP evoked an inward current. Downward deflections reflect Gm as described in Fig. 1Ab. The inward current was accompanied by an increased Gm. Resting Vm was -62 mV. B, following a 60 min pre-incubation with another NO donor, 50 µM SNAP (S-nitroso-N-acetylpenicillamine), bath-applied SP (100 nM, 20 s) evoked a membrane depolarization. Downward deflections reflect Rin, as described in Fig. 1Aa, which was decreased by SP. Resting Vm was -61 mV. C, inhibition of nitric oxide synthase by 30 µM L-NMMA (N-6-methyl-L-arginine) prior to and during 5-HT pre-incubation (10 µM, 60 min) prevented the unmasking of SP responses. Resting Vm was -67 mV. The calibration in B also applies to C.

  DISCUSSION
Top
Abstract
 Introduction
 Methods
Results
Discussion
References

Tachykinin receptor agonists do not produce measurable electrophysiological changes in nodose neurones of adult guinea-pigs (Weinreich et al. 1997). Our principal finding in the current work is that nanomolar concentrations of 5-HT acting through 5-HT3 receptors are capable of unmasking functional NK-2 tachykinin receptors.

Pharmacological characterization of the unmasking phenomenon

The vast majority of 5-HT receptors are coupled to second messenger signalling cascades via G-proteins (Fozard, 1992; Hoyer & Martin, 1997). The one exception is the ionotropic 5-HT3 receptor, which is directly coupled to a non-selective cation channel. Numerous investigators have established the presence of 5-HT3 receptor binding sites, as well as 5-HT3 receptor-mediated electrical membrane responses, in nodose ganglion neurones of several species (Zhuo et al. 1997 and references therein). Consistent with previous reports, in guinea-pig nodose neurones 5-HT elicits a membrane depolarization associated with an increased membrane conductance, most likely a non-selective cation conductance, and an elevation of [Ca2+]i. The 5-HT-induced membrane depolarization and elevation of intracellular calcium are mimicked by a 5-HT3 agonist, CPBG (1 µM; authors' unpublished observations) and blocked by a 5-HT3 antagonist, ICS205-930 (1 µM).

Using 5-HT agonists and antagonists, we have demonstrated that the 5-HT3 receptor subtype mediates the unmasking of NK-2 receptors. CPBG mimics the NK-2 receptor unmasking effects of 5-HT while 5-CT, a relatively non-specific 5-HT agonist devoid of activity at 5-HT3 receptors (Hoyer et al. 1994), was ineffective. The unmasking effects of 5-HT were blocked by ICS205-930, corroborating the role of 5-HT3 receptors. Interestingly, the EC50 for 5-HT-induced unmasking of NK-2 receptors in guinea-pig nodose neurones (14 nM) is well below the EC50 for the 5-HT3 receptor-mediated membrane depolarization (10 µM) in guinea-pigs (Butler et al. 1990). Though sub-micromolar concentrations of 5-HT do not elicit a measurable membrane depolarization in guinea-pig nodose neurones, 10-100 nM 5-HT does elicit an elevation of [Ca2+]i (n = 4; authors' unpublished observations). These data suggest that 5-HT-induced changes in [Ca2+]i, which are essential for the unmasking of NK-2 receptors, may not be accurately reflected by the 5-HT-evoked membrane depolarization.

An intracellular signalling cascade couples 5-HT3 receptor activation to NK-2 receptor unmasking

Activated 5-HT3 receptors in nodose neurones are permeable to Ca2+ ions. Indeed, the 5-HT-mediated elevation of [Ca2+]i and 5-HT-evoked unmasking of NK-2 receptors are both dependent upon [Ca2+]o. ATP and bradykinin also induce increases of [Ca2+]i similar to or greater than those produced by 5-HT, yet these autacoids never unmasked functional NK-2 receptors. It seems unlikely, therefore, that an elevation of [Ca2+]i alone is sufficient to evoke the expression of functional NK-2 receptors. The absence of 5-HT-induced unmasking of SP responses at room temperature also suggests the involvement of other second messengers.

Alternatively, since the fura-2 measurements of [Ca2+]i in the current work track global changes in [Ca2+]i, it is conceivable that activation of 5-HT3 receptors may give rise to compartmentalized increases of [Ca2+]i that control NK-2 receptor expression. In this regard, it is interesting to note that, unlike the 5-HT-induced rise of [Ca2+]i, the ATP response is not significantly attenuated by the removal of [Ca2+]o (authors' unpublished observations), suggesting that it is mediated by release from intracellular stores. The lack of SP responses following pre-incubation with ATP as compared with 5-HT suggests that the source of the intracellular calcium transient may be critical for effective unmasking of NK-2 receptors.

A number of reports have suggested that 5-HT3 receptors are coupled to activation of NOS, the calcium-calmodulin-dependent enzyme responsible for NO synthesis (Reiser, 1992). Several lines of pharmacological data support a connection between calcium-calmodulin-dependent activation of NO and 5-HT-induced unmasking of SP responses. We observed that calmidazolium inhibited the NK-2 receptor unmasking effects of 5-HT. Furthermore, bathing neurones with papaNONOate or SNAP, which are NO donors, induced unmasking of SP responses, while application of L-NMMA, a NOS inhibitor, prevented 5-HT-induced unmasking of SP responses. There are numerous mechanisms by which NO could unmask functional NK-2 receptors. NO can activate guanylyl cyclase to promote the formation of cGMP and the subsequent activation of cGMP-dependent protein kinase. cGMP-dependent protein kinase may act directly or indirectly to awaken silent NK-2 receptors. Alternatively, NO could stimulate S-nitrosylation of the NK-2 receptors, transforming them to a functional state. Further studies are required to distinguish between these mechanisms.

Endogenous source of 5-HT in the nodose ganglia

One potential source of 5-HT in the nodose ganglion is mast cells. Most studies indicate that guinea-pig mast cells do not contain significant levels of 5-HT (Parratt & West, 1957). However, one group has reported that mast cells in the guinea-pig trigeminal ganglia display 5-HT immunoreactivity and possess measurable quantities of 5-HT and its precursor, 5-hydroxytryptophan (66 and 109 nmol kg-1, respectively; Lehtosalo et al. 1984). Assuming that 5-HT levels in guinea-pig nodose ganglion mast cells are similar to those in the trigeminal ganglion and that the entire mast cell pool of 5-HT could be released, it is theoretically possible that the mean 5-HT concentration in the ganglionic extracellular space (0·4 ml g-1; Garthwaite, 1979) could reach 165 nM. Mast cells in the guinea-pig nodose ganglion only release 16 % of their granular mediators upon antigenic activation (Undem et al. 1993). Nonetheless, even at 16 % release, mast cell-derived 5-HT would be sufficient to unmask NK-2 receptors, given that the estimated EC50 value for unmasking is 14 nM.

Nodose neurones themselves represent an additional source of 5-HT in the nodose ganglia. 5-HT exists in nodose ganglion neurones but not in the satellite cells surrounding them (Gaudin-Chazal et al. 1981, 1982; Nosjean et al. 1990). Using immunohistochemical techniques, the expression of 5-HT in cat nodose ganglia appears ubiquitous, though there is wide variability in neuronal staining intensity (Gaudin-Chazal et al. 1982). During allergic inflammation, mast cell-released mediators are known to depolarize nodose neurones (Undem & Weinreich, 1993; Undem et al. 1993), which may promote the release of neuronal 5-HT. Indeed, Fueri et al. (1984) have reported depolarization and calcium-dependent somatic release of 5-HT from feline nodose ganglia. The unmasking of SP responses by KCl (50 mM), observed in some experiments (Table 1), may reflect somatic release of 5-HT.

Platelets represent another source of endogenous 5-HT in the nodose ganglion. It is well known that immunological activation of mast cells results in the release of numerous granule-bound proteases (Schwartz, 1994). An antigen- antibody reaction that releases proteases can promote the liberation of 5-HT from platelets (Humphrey & Jacques, 1955). The final result of this multicellular cascade would be the unmasking of neuronal NK-2 receptors. Interestingly, platelets have been implicated in human inflammatory pain (Schmelz et al. 1997).

Physiological implications of 5-HT-induced NK-2 receptor unmasking

The pain-producing effects of 5-HT have been well documented (Keele & Armstrong, 1964). Like many inflammatory mediators, 5-HT can produce pain either through direct activation of afferent neurones or through mechanisms that 'sensitize' afferents to pain-producing autacoids (Beck & Handwerker, 1974). 5-HT decreases the activation threshold of cutaneous afferent neurones, making neurones hyper-responsive to other autacoids (e.g. bradykinin) following exposure to 5-HT (Beck & Handwerker, 1974; Lang et al. 1990). These sensitizing actions of 5-HT are relatively short-lived, lasting on the order of 5-10 min.

In contrast to 5-HT-induced sensitization of somatosensory afferents, which has been well-documented, much less is known about the analogous phenomenon in visceral afferents. It has been established that 5-HT sensitizes vagal afferent C-fibres through blockade of spike frequency adaptation (Christian et al. 1989); whether this contributes to visceral nociception is not yet known. Though 5-HT is believed to be involved in the generation of visceral pain associated with migraine headaches, myocardial infarction and inflammatory bowel disease (Meller & Gebhart, 1992; Sanger, 1992), the cellular mechanisms remain unresolved.

5-HT-induced unmasking of functional NK-2 receptors provides a mechanism for long-lasting sensitization of visceral afferent neurones, one that may play a role in visceral nociception. A short exposure to 5-HT (minutes) can induce the expression of functional NK-2 receptors that persists for many hours. Control nodose neurones do not express functional tachykinin receptors, therefore their electrical membrane properties would be unaffected by endogenously released SP. However, following either allergic inflammation (Weinreich et al. 1997) or exposure to exogenously applied (and perhaps endogenously released) 5-HT, SP evokes a measurable membrane depolarization that can reach action potential threshold. Preliminary data reveal that the 5-HT3 antagonist ICS205-930 blocks allergic inflammation-induced unmasking of functional NK-2 receptors in intact, isolated nodose ganglia. Thus allergic inflammation, perhaps via 5-HT, sensitizes nodose neurones to another pain-producing autacoid, SP.

In conclusion, it is well recognized that long-lasting neuroplastic changes can occur following calcium influx via ligand-gated receptors. For example, long-term potentiation (LTP) of synaptic transmission is dependent upon calcium flux through NMDA receptors. In an analogous fashion, unmasking of functional NK-2 receptors is dependent upon calcium flux through 5-HT3 receptors in vagal afferents of the guinea-pig, a long-lived phenomenon that may coincide with neurogenic inflammation. As with LTP, NK-2 receptor induction is independent of protein synthesis, while the longevity of both phenomena requires new protein synthesis.

  REFERENCES
Top
Abstract
 Introduction
 Methods
Results
Discussion
References

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Acknowledgements

The authors wish to thank John Kadavil and Tony Gover for assistance with some of the fluorescence experiments and Drs Brendan Canning and Michael Gold for critical reading of an earlier version of this manuscript. This work was supported by NINDS grant NS22069 to D. W. and Neuroscience Training Grant NS07375 to K. A. M.

Corresponding author

D. Weinreich: University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, Room 522B, Health Science Facility, 685 W. Baltimore Street, Baltimore, MD 21201-1559, USA.

Email: dweinrei{at}umaryland.edu




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