INTRODUCTION

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In addition to their established proteolytic roles, proteases such as trypsin and thrombin may also act as cell-signaling molecules via protease-activated receptors (PARs). This family of seven transmembrane G protein-coupled receptors currently includes four receptor subtypes (PAR1, PAR2, PAR3, and PAR4). PAR1, PAR3, and PAR4, but not PAR2, are activated by thrombin, whereas trypsin can activate PAR2 and PAR4 (1, 2). The enzymatic activation of PARs involves the proteolytic unmasking of an extracellular N-terminal receptor sequence that acts intramolecularly as a tethered receptor- activating ligand. PAR1, PAR2, and PAR4 can also be activated by exogenously applied short peptides, corresponding to sequences of their tethered ligands.

The roles played by PARs in the lung are yet to be fully elucidated, however, trypsin(ogen) and a variety of proteases with trypsin-like activity have recently been shown to be present in the airway epithelium (3) and may play a role in the control of airway function by directly activating airway smooth muscle PARs (6) or by the activation of epithelial PARs and the subsequent release of cyclooxygenase products (7). Additional roles for trypsin in the airways are unknown, but given the variety of tissue and cell types that express PARs (1) including neurons (8), trypsin clearly has the potential to play multiple signaling roles in the lung. We hypothesized that epithelial-derived trypsin may play a role in the airways by activating sensory C-fibers that lie within and beneath the airway epithelium (9). These sensory C-fibers not only participate in centrally mediated reflexes (12) but are also thought to release neurokinins via an axon reflex, leading to bronchoconstriction, mucus secretion, and neurogenic inflammation (13, 14). The aims of this study were to determine if trypsin could induce the release of neurokinins from sensory C-fibers that innervate guinea pig airways and to determine if this was associated with action potential generation in these fibers.

	METHODS

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Tissue Preparation

Male Hartley guinea pigs (200-400 g) were killed by asphyxiation with CO₂ and exsanguinated. For contraction studies the trachea and primary bronchi were removed and placed in a dissecting dish containing Krebs bicarbonate buffer solution (KBS) composed of (in mM) NaCl, 118; KCl, 5.4; NaH₂PO₄, 1.0; MgSO₄, 1.2; CaCl₂, 1.9; NaHCO₃, 25.0; dextrose, 11.1. For combined electrophysiological/contraction studies, the airways with intact right-side extrinsic innervation (including nodose and jugular ganglia) were removed and placed in a dissecting dish containing KBS. The cyclooxygenase inhibitor indomethacin (3 µM; Sigma Chemical Company, St. Louis, MO) was present in the KBS throughout all studies to prevent spontaneous and epithelial PAR-mediated (7) release of cyclooxygenase products.

Contraction Studies

The left and right primary bronchi were bisected into two rings each for paired experiments involving pharmacological treatment. The lower trachea was divided into four consecutive rings, each roughly three cartilage rings in width, for paired pharmacological treatments. In some studies the epithelium was removed by gentle rubbing of the luminal surface with a cotton-covered probe. Tracheal and bronchial tissue rings were placed in tissue baths then tied with silk surgical suture (Lukens Medical Corp., Rio Rancho, NM) to force-displacement transducers (FT03C, Grass Instrument Co., Quincy, MA) for recording of isometric tension on a Grass polygraph. Resting tension was set at 1 g. Tissue baths contained 3 ml of KBS, which was maintained at 37° C and bubbled with 95% O₂-5% CO₂ and replaced every 15 min during a 60-min equilibration period. Bovine trypsin (Worthington Biochemical Corp., Freehold, NJ), bovine thrombin (Sigma, St. Louis, MO), or mast cell tryptase purified from human skin (15, 16) was added to tissue baths in a cumulative manner. Also tested were recombinant tryptase (17) and recombinant tryptase stabilized with heparin. These proteases were added to tissue baths to give a final concentration of 100 nM. Stock concentrations of trypsin and tryptase were determined using their specific activities, which were 0.07 and 0.2 (µmol min¹) (nmol enzyme)¹, respectively, for hydrolysis of 1 mM benzoyl-Arg-p-nitroanilide in 0.2 M NaCl, 0.1 M MOPS, 9% dimethyl sulfoxide (DMSO). The molar extinction coefficient of nitroalinine was assumed to be 8800 (18). Thrombin concentration in stock solution was determined using its reported specific activity for hydrolysis of the substrate Tos-Gly-Pro-Arg-p-nitroalinine in 0.10 M Tricine buffer (19). Skin mast cell tryptase (40 µM) and recombinant tryptase (60 µM) were stored at 70° C in 2 M NaCl, 0.01 M MOPS, pH 6.8. Tryptase-heparin complexes were made by diluting stock tryptase 10-fold with a solution of 10 µM heparin (5 kD; Calbiochem, San Diego, CA). Complexes were frozen until use. The PAR1-activating peptide SLFFRN (Bachem Bioscience Inc., King of Prussia, PA) and the PAR2-activating peptide SLIGRL (Bachem) were added to tissue baths to give the concentrations indicated.

In some studies, isolated tissues were pretreated for 45 min with vehicle, or the NK₁ receptor-selective antagonist SR 14033 (1 µM; Zeneca Inc., Wilmington, DE) and the NK₂ receptor-selective antagonist SR 48968 (1 µM; Zeneca). Neuropeptide depletion studies were performed as previously described (20) by addition of capsaicin (10 µM; Sigma) to the tissue bath, which caused a large increase in tension (approximately 60% of that produced by 100 mM BaCl₂), which returned to baseline within 90 min. Following pretreatment with vehicle or antagonists or depletion of neuropeptides, cumulatively increasing concentrations of trypsin (3-100 nM) were added to the tissue bath. Only one concentration-effect curve to trypsin was constructed in each tissue. In studies using soybean trypsin inhibitor (Sigma), trypsin (100 nM) was preincubated with soybean trypsin inhibitor (500 µg/ml) for 30 min at 37° C prior to addition to tissue that had also been pretreated (30 min) with soybean trypsin-inhibitor (500 µg/ml). Some tissues were preincubated in the presence or absence of the bradykinin B₂ receptor-selective antagonist HOE140 (1 µM; Hoechst AG, Frankfurt, Germany) or the neuronal sodium channel blocker tetrodotoxin (1 µM; Sigma) for 30 min prior to the addition of trypsin (100 nM).

To investigate the possibility that trypsin could evoke adrenergic or nonadrenergic, noncholinergic relaxations, guinea pig bronchial preparations were precontracted with the muscarinic cholinoceptor agonist methacholine (1 µM) to give a contraction approximately 70-80% of maximum. To determine whether these preparations were capable of exhibiting nerve-mediated relaxation, electrical field stimulation (30 Hz, 1 ms duration, for 10 s) was delivered by choloridized silver wire electrodes placed on opposite sides of the bronchus. Current across the bronchus was delivered as rectangular pulses by a Grass model S48 stimulator (Grass Instruments, Quincy, MA) connected to a current-monitoring stimulus splitter (Stimu-Splitter II; MedLab, Fort Collins, CO). Stimulation intensity was 24 V.

Combined Electrophysiological/Contraction Studies

Connective tissue was carefully trimmed away from the trachea, larynx, and right bronchus leaving the vagus, superior laryngeal, and recurrent nerves, including nodose and jugular ganglia, intact. A longitudinal cut was made to open the larynx, trachea, and bronchus along the ventral surface. Airways were then pinned, mucosal surface up, to a Sylgard-lined Perspex chamber. The right nodose and jugular ganglia, along with the rostralmost vagus and superior laryngeal nerves, were gently pulled through a small hole into an adjacent compartment of the same chamber for recording of single fiber activity. Both compartments were superfused with KBS gassed with 95% O₂-5% CO₂. The temperature was maintained at 37° C with a flow rate of 6-8 ml/ min¹. This method is a modification of that described elsewhere (21).

Extracellular recordings were performed by manipulating a fine aluminosilicate glass microelectrode pulled using a Flaming/Brown micropipette puller (Sutter Instrument Company, Novato, CA) and filled with 3 M sodium chloride near neuronal cell bodies in jugular ganglion. The filled electrode was placed into an electrode holder and connected directly to a headstage (A-M Systems, Everett, WA). A return electrode of silver-silver chloride wire and a silver-silver chloride pellet ground were placed in the perfusion fluid of the recording chamber and attached to the headstage. The recorded signal was amplified (A-M Systems) and filtered (low cut-off = 0.3 kHz; high cut-off = 1 kHz) and the resultant activity was displayed on an oscilloscope (TDS 340, Tektronix, Beaverton, OR) and a model TA240S chart recorder (Gould, Valley View, OH). The data were stored on digital tape (DT-120 RA; Sony Corporation, Tokyo, Japan) for off-line analysis on a Macintosh computer using the software program The NerveOfIt (PHOCIS, Baltimore, MD).

Single fiber activity in the airway was discriminated by placing a concentric electrical stimulating electrode on the recurrent laryngeal nerve, through which the majority of fibers enter the trachea (21). The recording electrode was placed within the ganglion and manipulated until single unit activity was detected. When electrically evoked action potentials were seen, the stimulator was switched off and the trachea and bronchi were gently brushed with a von Frey filament. Mechanically sensitive receptive fields were revealed when a burst of action potentials was elicited in response to von Frey filament stimulation. Conduction velocities were determined by electrically stimulating the receptive field and monitoring the time elapsed between the shock artifact and the recorded action potential. This delay was divided by the distance between the receptive field and the recording electrode to yield a conduction velocity. Only mechanically sensitive neurons with conduction velocities less than 1 m/s were studied. These nerve fibers had little or no activity at rest; if spontaneous activity exceeded 1 action potential/s, the fiber was not studied further.

Isometric tension recording of tracheal smooth muscle tone was monitored during the course of electrophysiological experiments. Two incisions (3 mm each) approximately 5 mm apart were made on the right side of the trachea just above the level of bronchial bifurcation. The resulting "flap" of trachea was secured with silk surgical suture to a Grass model FT03C force-displacement transducer under 500 mg of resting tension. Changes in isometric tension were displayed on a TA240S chart recorder.

The sensitivity of identified airway afferent C-fibers to trypsin was determined by superfusing KBS containing trypsin through the compartment containing the tracheal-bronchial preparation, at a rate of 6-8 ml/min. During this period, afferent activity in identified C-fibers and tracheal smooth muscle tone was monitored. In all cases trypsin caused a contraction and superfusion with KBS containing trypsin was maintained until the contraction reached a stable plateau at which time 200 µl of KBS containing capsaicin (1 µM) was applied directly to the receptive field of the C-fiber. Tracheal smooth muscle responses to trypsin were expressed in terms of the maximum contraction evoked by histamine (10 µM), which was superfused through the compartment containing the tracheal-bronchial preparation at the end of the experiment.

Data Analysis

Data were expressed as arithmetic mean ± SEM. Contraction in guinea pig isolated tracheal and bronchial preparations was expressed as a percentage of contraction induced by 100 mM BaCl₂ (Ba²⁺ Max) or 10 µM histamine added at the end of the experiment. The responses of sensory C-fibers were presented as the peak frequency (highest number of action potentials recorded in 1 s) of arrival of action potentials at cell bodies in the jugular ganglion. Peak frequency data were compared using ANOVA, followed by a Student's nonpaired t test.

Adjacent segments of bronchi responded similarly to trypsin, however, we noted considerable intraanimal variation. For example, the maximum trypsin-induced contractile response of the bronchi isolated from 15 guinea pigs ranged from 10 to 70% of Ba²⁺ Max. For this reason, contraction studies in the presence or absence of pharmacological pretreatments (i.e., Neurokinin receptor-selective antagonists, depletion of neurokinins with capsaicin, tetrodotoxin, or HOE140) were in all cases preformed in tissues from adjacent sections of bronchus from the same animal. As control and treated isolated tracheal or bronchial preparations were from adjacent sections of airway, Student's paired t test was used to compare differences between the contractions in the control and treated tissues. In experiments in which SLIGRL or mast cell tryptase did not evoke a response, trypsin was added at the end of the experiment as a positive control. In all cases, contractions of superfused tracheal preparations used in combined contraction/electrophysiological experiments responded to 100 nM trypsin with a contraction 50% or greater than that evoked by a high concentration of histamine (10 µM).

	RESULTS

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Contraction Studies

Trypsin evoked sustained, concentration-dependent contractions of guinea pig isolated bronchial and tracheal smooth muscle preparations (Figures 1A and 1B). The spasmogenic action of trypsin (100 nM) was prevented (n = 4) by preincubation (30 min) with soybean trypsin inhibitor (500 µg/ml). Trypsin (100 nM) did not induce relaxations of bronchial preparations precontracted with methacholine (1 µM; n = 3). However, electrical field stimulation of adrenergic and nonadrenergic, noncholinergic relaxant nerves within these preparations evoked a 20 ± 5% (n = 3) relaxation of the methacholine-induced contraction.

View larger version (12K): [in this window] [in a new window]   Figure 1.   (A) Typical isometric tension recordings showing that trypsin-induced (closed circles, 100 nM) contractions of guinea pig isolated tracheal rings were sustained (upper trace) and were reversed by the combined presence of the NK1 receptor-selective antagonist SR 14033 and the NK2 receptor-selective antagonist SR 48968 (closed triangle, 3 µM each). (B) Concentration-effect curves demonstrating the relative potency of trypsin (closed circles, n = 7) and mast cell tryptase (closed squares, n = 4) in guinea pig isolated bronchial smooth muscle. The PAR2-activating peptide SLIGRL (open square: SLIGRL-OH, n = 4; SLIGRL-NH2, n = 3) was used at a single high concentration. Data are mean ± SEM.

The PAR2 tethered ligand sequence SLIGRL (100 µM) did not evoke contraction of guinea pig isolated bronchus. This did not appear to be due to limited penetration of SLIGRL as in two experiments in preparations from which the epithelium had been removed 1 mM SLIGRL did not evoke a contractile response; however, in both these tissues trypsin (100 nM) evoked a contraction. Human skin mast cell tryptase (1-100 nM) was not an effective spasmogen of guinea pig isolated bronchus (Figure 1B). We considered the possibility that this may be due to the rapid, spontaneous inactivation of tryptase that occurs at physiological temperatures and salt concentrations. As highly sulfated polysaccharides such as heparin can greatly slow spontaneous inactivation of mast cell tryptase (15), we tested the ability of mast cell tryptase stabilized with heparin to evoke contraction. In two of two isolated airway preparations tested, recombinantly expressed human skin mast cell tryptase (100 nM) stabilized with heparin did not evoke contraction; however, both these tissues responded to trypsin (100 nM).

As shown in Figure 1A, trypsin-evoked contractions were reversed by combined application of the NK₁ receptor-selective antagonist SR 14033 (3 µM) and the NK₂ receptor-selective antagonist SR 48968 (3 µM). Moreover, combined pretreatment with SR 14033 (1 µM) and SR 48968 (1 µM) markedly reduced trypsin evoked responses (Figure 2). Similarly, trypsin was a weak spasmogen in bronchial preparations that had been pretreated with capsaicin (Figure 2), an agent known to deplete functional pools of neurokinins in sensory C-fibers.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Concentration-effect curves for trypsin in guinea pig isolated bronchus pretreated with vehicle (closed circles, n = 7), a combination of the NK1 receptor-selective antagonist SR 140333 and the NK2 receptor-selective antagonist SR 48968 (1 µM each, closed triangles, n = 7), or capsaicin (10 µM, closed squares, n = 7). Data are mean ± SEM. *p < 0.05 compared with vehicle.

Thrombin was a weak spasmogen of guinea pig isolated airway smooth muscle (Figure 3). At concentration of up to 30 nM, thrombin caused no contraction. At a concentration of 300 nM, 300-fold greater than its reported EC₅₀ for activation of PARs in guinea pig myenteric neurons (7), thrombin evoked a contraction only 10 ± 6% (n = 3) of Ba²⁺ Max. Contractions to thrombin were similar in epithelium-intact and epithelium-denuded preparations (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Concentration-effect curves for thrombin and the PAR1 activating peptide SFLLRN in guinea pig isolated bronchus. Thrombin was an equally effective spasmogen in isolated bronchial rings with intact (open squares, n = 3) and denuded epithelium (open circles, n = 3). SFLLRN-induced contractions in guinea pig isolated bronchus pretreated with vehicle (closed circles, n = 5), a combination of the NK1 receptor-selective antagonist SR 140333 and the NK2 receptor-selective antagonist SR 48968 (1 µM each, closed triangles, n = 4) or capsaicin (10 µM, closed squares, n = 4). Data are mean ± SEM.

The PAR1 selective agonist SLFFRN caused concentration-dependent contractions of guinea pig bronchial preparations. In contrast to trypsin-evoked contractions, SLFFRN-evoked contractions were not inhibited by pretreatment with a combination of SR 14033 (1 µM) and SR 48968 (1 µM) or capsaicin (Figure 3).

Contractions evoked by 100 nM trypsin (28 ± 9% of Ba²⁺ Max, n = 6) were not significantly different than those in preparations that had been preincubated with 1 µM of the bradykinin B₂ receptor-selective antagonist HOE140 (20 ± 8% of Ba²⁺ Max, n = 6). In two paired experiments, contractions to 100 nM trypsin were similar in preparations that had been preincubated in the absence (15 and 22% of Ba²⁺ Max) or presence of 1 µM of the neuronal sodium channel blocker tetrodotoxin (17 and 20% of Ba²⁺ Max).

Combined Electrophysiological/Contraction Studies

Five mechanically sensitive, slowly conducting afferent C-fibers (conduction velocity 0.68 ± 0.07 m/s) whose receptive fields were located in the trachea or primary bronchi were studied. The low level of resting activity recorded from the cell bodies of these C-fibers (Figure 4B) was not significantly elevated by superfusion of the tracheal-bronchial preparation with KBS containing trypsin (100 nM). However, trypsin (100 nM) was an effective spasmogen of superfused tracheal preparations (Figure 4A), inducing a contraction equal to 56 ± 2% (n = 5) of that induced by 10 µM histamine. Although trypsin did not stimulate action potential discharge from any of the sensory C-fibers we recorded from, capsaicin (200 µl of a 1µM solution) applied to the receptive field of these neurons evoked a burst of action potentials in all five experiments (Figures 4A and 4B).

View larger version (10K): [in this window] [in a new window]   Figure 4.   The traces shown in (A) are from an experiment in which smooth muscle tone and the arrival of action potentials at the cell bodies of slowly conducting C-fibers, whose receptive fields were located in the trachea or main bronchus, were recorded simultaneously. The upper trace shows an isometric tension recording of tracheal smooth muscle tone during superfusion with KBS containing trypsin (100 nM). The lower trace shows a recording made at the same time with an extracellular electrode placed in the jugular ganglion, near the cell body of a slowly conducting C-fiber. Once a stable level of contraction was reached, capsaicin (200 µl of 1 µM) was applied to the receptive field of the C-fiber and evoked a burst of action potentials. The data in (B) show the mean ± SEM (n = 5) peak frequency of action potentials arriving at cell bodies in jugular ganglia prior to (baseline) and during application of trypsin (100 nM) or capsaicin (200 µl of 1 µM). *p < 0.05 compared with baseline.

	DISCUSSION

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The major conclusion of this study was that trypsin can evoke contraction of guinea pig airways by releasing neurokinins from sensory nerves that innervate airway smooth muscle. This action of trypsin appeared to occur independently of action potential generation in sensory C-fibers whose receptive fields were identified in the trachea or main bronchus.

Previous studies have shown that both NK₁ and NK₂ receptors are involved in the contractile response of guinea pig airway smooth muscle to endogenous neurokinins (22). In the present study, we found that contractions to trypsin were markedly attenuated in the combined presence of NK₁ and NK₂ receptor-selective antagonists, indicating trypsin-induced contractions were mediated by NK receptors. However, trypsin does not appear to directly activate airway smooth muscle NK receptors, as depletion of neurokinins from sensory nerves within the airways with capsaicin also markedly reduced the ability of trypsin to evoke contractile responses. Thus, a finding of the current study is that trypsin can induce guinea pig airway smooth muscle contraction, apparently by stimulating the release of neurokinin peptides from sensory neurons within the airways.

It has been postulated that the release of neurokinins within the airways occurs physiologically by an axon reflex, where stimulation of a C-fiber ending results in action potentials that in addition to traveling orthodromically toward the central nervous system also travel antidromically via peripheral branches to effect the release of neurokinins (13). As trypsin appeared to evoke the release of neurokinins from capsaicin-sensitive fibers, we hypothesized that trypsin would also evoke orthodromically traveling action potentials in these neurons. However, we did not observe an increase in the frequency of arrival of action potentials at the cell bodies of C-fibers (whose receptive fields were located in the trachea or primary bronchus) prior to or during contraction to trypsin. These findings suggest that trypsin can evoke the release of neurokinin transmitters from C-fibers without stimulating the sensory function of these nerves. In this respect, trypsin differs markedly from other stimuli of vagal afferents such as capsaicin and bradykinin, which not only evoke release of neurokinins from sensory neurones within guinea pig airways (22, 23) but also evoke action potentials in C-fibers (current study; 24, 25).

An assumption made in this study was that the airway C-fibers whose cell bodies we recorded from are typical of all C-fibers in the airway. Because of this, we cannot definitively rule out the possibility that there is a small population of neuropeptide-containing fibers that innervate airway smooth muscle and respond to trypsin not only by releasing neuropeptides, but also by evoking action potentials. However, this seems unlikely given the apparent uniformity of tracheal and bronchial afferent C-fibers in this preparation with respect to their pharmacological and physiological properties and neuropeptide content (11, 21). Our observation that tetrodotoxin, at concentrations that block conduction of orthodromic action potentials in guinea pig tracheal C-fibers (data not shown), did not abolish trypsin-evoked contractions is consistent with our hypothesis that trypsin causes release of neurokinins independently of action potential generation.

Bradykinin, a metabolite of the kallikrein-kinin system, has multiple actions in the airways, including the ability to stimulate sensory nerves (24) and evoke the release of neurokinins (23). It has been known for some time that trypsin is capable of catalyzing the formation of bradykinin (26). However it is unlikely this effect accounted for the ability of trypsin to release neurokinins in guinea pig bronchus as bradykinin is known to evoke action potentials in C-fibers that innervate guinea pig trachea/bronchus (24). Moreover, trypsin-induced contractions were not inhibited by the bradykinin B₂ receptor-selective antagonist HOE140, although we and others have previously shown that this antagonist inhibits the ability of bradykinin to stimulate sensory C-fibers that innervate guinea pig trachea (24, 25).

Steinhoff and coworkers (27) have recently reported that the amino acid sequence corresponding to the PAR2 tethered ligand (SLIGRL) can evoke the release of peptide transmitters from peripheral terminals of rat spinal sensory neurons that innervate bladder and atrium. As PAR2 is known to be activated by trypsin (1, 2), we investigated the possibility that trypsin-induced, neurokinin-mediated contraction of guinea pig airway smooth muscle involved a protease-activated receptor. We found that trypsin-evoked contractions were inhibited by soybean trypsin inhibitor, indicating that enzymatic activity was required to achieve a response, as is PAR2 activation by trypsin (27). However, the PAR2-activating peptide SLIGRL did not evoke contraction in isolated bronchial preparations. The inactivity of SLIGRL does not appear to be due to a general inactivity of this peptide in guinea pig tissues as SLIGRL evokes responses in a variety of guinea pig tissue types (7, 8, 28, 29). Tryptase, a mast cell protease with trypsin-like hydrolytic specificity, also did not evoke contraction in the current study, but was previously shown to activate PAR2 (27, 30). Combined, the lack of effect of SLIGRL and mast cell tryptase would argue against the involvement of PAR2 in the release of neurokinins in guinea pig bronchus. Although we cannot explain the differences between the study of Steinhoff and coworkers (27) and our findings, they may reflect a differential expression of PAR2 in spinal sensory neurons and vagal sensory neurons and/or a difference in the expression of a PAR2 in sensory neurons of rats and guinea pigs.

In addition to activating PAR2, trypsin is also known to be an effective activator of PAR4, as is thrombin, which can also activate PAR1 and PAR3 (1, 2). We found that thrombin did not evoke a substantial contractile response in guinea pig bronchial preparations, even in those from which the epithelium had been removed. In addition, contractions to the PAR1-activating peptide SFLLRN did not appear to involve the release of neurokinins, as they were not inhibited by NK receptor-selective antagonists or by depletion of neurokinins with capsaicin. Thrombin- and SFLLRN-induced contractions may be mediated directly by airway smooth muscle PAR receptors (6). Regardless, given the limited efficacy of thrombin, the lack of effect of SLIGRL, and the insensitivity of SFLLRN-induced contractions to neurokinin receptor antagonists it would appear that neither PAR1, PAR2, PAR3, nor PAR4 (as defined by the current classification scheme) mediates trypsin-induced release of neurokinins from sensory nerves innervating guinea pig airway smooth muscle. Whether the spasmogenic action of trypsin in guinea pig bronchus involves a novel PAR or is mediated by some other pathway remains to be resolved. A definitive answer as to the role of PARs in the release of neurokinins within guinea pig airways awaits the development of PAR subtype-selective antagonists.

In addition to nonadrenergic, noncholinergic contractile pathways (i.e., sensory C-fibers), the guinea pig bronchus is also known to be innervated by parasympathetic, cholinergic nerves that mediate contraction (33) and sympathetic, adrenergic nerves and parasympathetic nonadrenergic, noncholinergic pathways that can elicit relaxation (33, 34). Our observation that there was little contraction remaining in tissues treated with capsaicin or NK receptor-selective antagonists provides indirect evidence that trypsin does not evoke the release of the spasmogenic neurotransmitter acetylcholine from cholinergic nerves. Although trypsin can evoke PAR-mediated cyclooxygenase-dependent relaxations of isolated airway smooth muscle preparations from a variety of species including humans (7, 35), trypsin was not able to relax precontracted guinea pig bronchial preparations in the presence of indomethacin (current study), suggesting that trypsin cannot promote the release of neurotransmitters from adrenergic (34) or nonadrenergic, noncholinergic relaxant nerves (33) that innervate guinea pig bronchial smooth muscle. Combined, these observations suggest that the action of trypsin to evoke neurotransmitter release in guinea pig airways is selective for neurokinin-containing sensory nerves.

In conclusion, the results from this study support the hypothesis that trypsin activates a mechanism allowing for local release of sensory neurokinins from afferent C-fibers and that this release occurs independently of the sensory function of these nerves. It is currently unknown if there is a physiological or pathophysiological role for this pathway, or what stimuli within the airways, if any, utilize this mechanism. One possibility is that trypsin itself may be the endogenous activator of this mechanism as trypsin(ogen) is present within the airway epithelium (3, 7), which is innervated by neurokinin-containing sensory neurons (11, 36). mRNA for trypsin(ogen) has also been localized to various inflammatory cell types such as macrophages, monocytes, and lymphocytes (3). However, it remains to be determined if epithelium- and inflammatory cell-derived trypsin can promote the release of neurokinins from these nerves.