INTRODUCTION

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Nitric oxide (NO) produced and released from different cells of the respiratory tract regulates the tone of airway smooth-muscle cells. Thus, NO released from nonadrenergic, noncholinergic nerves relaxes the airways of several mammals including humans (1). Furthermore, various mediators produce a bronchorelaxant effect via NO released from the airway epithelium (2). Epithelium-dependent relaxation of tracheal-tube preparations by histamine (3), endothelins (4), and high concentrations of K⁺ (5) is mediated by release of NO. Of particular interest for the present study is the observation that the moderate bronchoconstriction induced by aerosolized bradykinin (BK) in anesthetized guinea pigs is markedly potentiated by inhibition of the L-arginine (L-Arg) NO-pathway (6). NO synthase (NOS) inhibitors have been found to reduce the epithelium-dependent relaxation produced by BK in guinea pig bronchial strips (7), and to change epithelium-dependent relaxation of tracheal-tube preparations into a contraction (8).

Recently, we provided evidence that exposure for 10 min or more to cold air mixed with nebulized 0.9% saline (13° C in the trachea) causes an inflammatory response in rodent airways, consisting of plasma extravasation (9) and bronchoconstriction (10). Both plasma extravasation and bronchoconstriction induced by cold-air inhalation are mediated by the release of kinins and tachykinins. In particular, cold-air-induced bronchoconstriction was completely blocked by a tachykinin NK₂-receptor antagonist and partly inhibited by a kinin B₂- receptor antagonist (10).

In the present study we examined the possibility that endogenous NO affects bronchoconstriction induced by cold-air inhalation in guinea pigs. We investigated the effect of NOS inhibitors on the inhalation of a dose of cold air (5 min) that did not affect airway tone per se. Because we found that this inhalation of cold air after inhibition of the L-Arg NOS pathway caused significant bronchoconstriction, we addressed the following questions: (1) Are kinins and tachykinins involved in the bronchoconstriction induced by short lasting inhalation of cold air; and (2) Is bronchorelaxant NO released by kinins, tachykinins, or cold air alone. To answer these questions, we studied the ability of selective antagonists of the BK B₂ and tachykinin NK₂ receptor to modulate bronchial smooth-muscle tone after inhalation of cold air. We also investigated the ability of BK and of the selective tachykinin NK₂-receptor agonist, [Ala⁸]neurokinin A (4) ([Ala⁸]NKA [4-10]) (11) to increase cyclic 3',5'-guanosine monophosphate (cGMP) levels in strips of guinea pig trachealis muscle. Our results indicate that kinins released in the airways following exposure to cold air stimulate the release of tachykinins, which contract the airway smooth muscle and also cause the release of NO, which inhibits this bronchoconstrictor effect of tachykinins.

	METHODS

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Animals

Male Hartley guinea pigs (Simonsen Laboratories, Inc., Gilroy, CA) weighing 300 to 350 g at the time of housing were used in this study. They were kept in a temperature-controlled environment with standard laboratory food and water freely available.

Exposure to Cold Air

Guinea pigs were anesthetized with sodium pentobarbital (45 mg/kg intraperitoneally; Anthony Product Corp., Arcadia, CA) and artificially ventilated with a tracheal cannula, using a constant-volume ventilator (Model 683; Harvard Apparatus Co., Inc., South Natick, MA) set at a frequency of 90 breaths/min. The VT was adjusted to maintain normal arterial blood gases as described previously (12). A cannula with a conical tip and an ID of 1 mm was inserted for 1 to 3 mm between the cricoid cartilage and the first tracheal ring. Air was passed through a coiled tubing immersed in ground dry ice before reaching the cannula (9). The temperature of cold air recorded at the distal end of the cannula prior to insertion in the trachea was 10° C when the room temperature was 26° C. To maintain a constant humidification of inhaled air, cold air was inhaled with a mist of nebulized 0.9% saline (Pulmo-Sonic 25; DeVillbiss, Somerset, PA). The nebulized 0.9% saline was mixed with cold air after the air had passed through the coiled tubing immersed in dry ice. The temperature of the tracheal cannula was measured before and after each experimental trial to monitor constancy of the air temperature throughout the study. Saline aerosol was also given to the animals inhaling room air.

Measurement of Total Pulmonary Resistance

Airflow was monitored continuously with a pneumotachograph (A. Fleisch, Medical Inc., Richmond, VA) connected to a differential pressure transducer (Model DP45; Validyne Engineering Corp., Northridge, CA). A fluid-filled polyethylene catheter was introduced into the esophagus to measure the esophageal pressure as an approximation of pleural pressure. Intratracheal pressure was measured with a polyethylene catheter inserted into a short tube connecting the tracheal cannula to the pneumotachograph. The transpulmonary pressure (defined as the pressure difference between the intratracheal and the esophageal pressures) was measured with a differential pressure transducer (Model DP7; Valydine Engineering Corp.). Output signals representing transpulmonary pressure and airflow were amplified with an amplifier (Model CD19; Validyne Engineering Corp.) and recorded on a polygraph recorder (Model 1508 B Visicorder; Honeywell, Inc., Denver, CO). Total pulmonary resistance (RL) was calculated as previously described (12). The right jugular vein and the left carotid artery were cannulated to permit administration of drugs and withdrawal of blood samples for arterial blood-gas measurement.

Measurement of Arterial Blood Pressure

A polyethylene catheter (ID = 0.8 mm, length = 2.5 cm; Angiocath, Deseret Medical) was inserted into the left carotid artery and connected to a pressure transducer (Model 1270A; Hewlett-Packard) for measurement of arterial pressure. The amplified signal from the transducer (Module M2102B; Electronics for Medicine) was displayed continuously on a video monitor (Model OM; Electronics for Medicine) and recorded with an oscillographic recorder (Model DASH-8; Astro-Med). Heart rate was derived from the pressure pulse signal through a cardiotachometer coupler.

Experimental Design

Guinea pigs received atropine (1.4 µmol/kg intravenously) 15 min before the exposure to cold air. The NOS inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg intravenously) or N^G-monomethyl- L-arginine (L-NMMA, 50 mg/kg intravenously) or their respective inactive enantiomers N^G-nitro-D-arginine methyl ester (D-NAME, 50 mg/kg intravenously) or N^G-monomethyl-D-arginine (D-NMMA, 50 mg/kg intravenously) were administered 10 min before the exposure to cold air. L-Arg (500 mg/kg intravenously) or D-arginine (D-Arg, 500 mg/kg intravenously) were administered 5 min before the exposure to cold air. The tachykinin NK₂-receptor antagonist, SR 48968 (13) (0.3 µmol/kg intravenously) and the kinin B₂-receptor antagonist HOE 140 (D-Arg⁰-[Hyp³,This⁵,D-Tic⁷,Oic⁸]-BK) (14) (0.1 µmol/kg intravenously), were injected 15 min before the exposure to cold air. The doses of SR 48968 and used in the study had previously been shown to selectively block tachykinin NK₂ (13, 15) and kinin B₂ (14, 16) receptors in guinea pigs in vivo. We also examined the effect of L-NAME (50 mg/kg intravenously) and D-NAME (50 mg/kg intravenously) on the increase in RL induced by aerosol administration of capsaicin (10 breaths) or BK (40 breaths). In these experiments L-NAME and D-NAME were administered 10 min before the beginning of the capsaicin or BK aerosol.

cGMP Assay

Strips (10 to 20 mm²) of trachealis muscle were placed in an aerated Krebs solution with the following composition (mM): NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, and glucose 8.3. Experiments were performed with slight modifications according to a recently reported procedure (8). After an equilibration period of 30 min at 37° C, the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 1 mM) was added for 5 min. After addition of the stimuli for 2 min, the tissues were frozen in liquid nitrogen. Experiments were performed three times in triplicate. Samples were homogenized in 10% ice-cold trifluoracetic acid (TFA) and centrifuged at 10,000 × g. Pellets were dissolved in 0.5 M NaOH and the protein content was measured with the method described by Bradford (17). The supernatant was acidified and extracted three times with ether to remove TFA. After neutralization, the samples were added to the assay buffer and acetylated as described previously (18). cGMP was measured with an enzymatic immunoassay, using acetylcholinesterase-cGMP and reading the absorbance at 415 nm with a spectrophotometer (18). The cGMP antiserum did not cross react with BK (10 µM) or [Ala⁸]NKA (4) (1 µM).

Drugs

L-Arg, D-Arg, L-NAME, D-NAME, L-NMMA, D-NMMA, capsaicin, and atropine were obtained from Sigma Chemical Co. (St. Louis, MO). Capsaicin was dissolved in a solution containing 10% ethanol, 5% Tween 80, and 85% of 0.9% NaCl. Further dilution was made in 0.9% saline. All the other drugs were dissolved in 0.9% saline. SR 48968 {(S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl] benzamide} was kindly provided by Dr. X. Emonds-Alt (Sanofi Recherche, Montpellier, France). HOE 140 was a kind gift of Dr. K. J. Wirth (Hoechst AG, Frankfurt, Germany). HOE 140, [Ala⁸]NKA (4) (Bachem, Switzerland), and SR 48968 (10 mM) were dissolved in dimethyl sulfoxide (DMSO), and further dilutions were made in 0.9% saline. BK (Bachem) was dissolved in 0.9% saline.

Statistical Analysis

All data are expressed as mean ± SEM. Statistical comparisons were made through one-way analysis of variance (ANOVA) and Dunnett's test, or bilateral paired or unpaired Students t tests when appropriate. In all cases, a value of p < 0.05 was considered significant.

	RESULTS

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Bronchoconstriction Induced by Cold Air

After a stabilization period of 30 min, baseline RL remained stable for at least 2 h during exposure to room air. Inhalation of cold air increased RL in the airways in a time-dependent manner: exposure to cold air for 3 or 5 min failed to increase RL significantly; significant peak increases in RL were evident after a 10-min exposure or after a 15-min exposure to cold air (Figure 1). Intravenous injection of isotonic saline (0.9% NaCl), L-NAME (50 mg/kg intravenously), D-NAME (50 mg/kg intravenously), L-NMMA (50 mg/kg intravenously), or D-NMMA (50 mg/kg intravenously) did not change the baseline value of RL (Table 1). Exposure to cold air for 5 min did not produce a significant increase in RL during the 30 min of recording as compared with the response induced by inhalation of room air for 5 min (Figure 2a). After pretreatment with L-NAME, but not with D-NAME, inhalation of cold air for 5 min caused a marked increase in RL (Figure 2b). Pretreatment with L-Arg but not with D-Arg abolished the increase in RL induced by inhalation of cold air for 5 min in guinea pigs pretreated with L-NAME (Figure 2c). The peak increase in RL above baseline induced by cold-air inhalation for 5 min after pretreatment with L-NMMA (50 mg/kg intravenously, n = 5) (0.22 ± 0.03 cm H₂O/ml/s) was significantly different (p < 0.01) from the increase in RL observed after pretreatment with D-NMMA (50 mg/kg intravenously, n = 5) (0.06 ± 0.01 cm H₂O/ml/s).

View larger version (59K): [in this window] [in a new window]   Figure 1.   Effect of inhalation of cold air (hatched columns) on the maximum increase in total pulmonary resistance (RL) in guinea pigs anesthetized, artificially ventilated, and pretreated with atropine (1.4 µmol/kg intravenously). Baseline (open column) values indicate RL after inhalation of room air mixed with nebulized 0.9% saline for 15 min. Inhalation of cold air for 3 min and 5 min did not increase RL above control, but longer periods of exposure increased RL significantly. Values are mean ± SEM of five experiments. *p < 0.001 versus control (room air alone for 15 min).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Effect on the increase in total pulmonary resistance (RL) of (a) Inhalation of cold air for 5 min (open circles) and of room air for 5 min (solid circles). (b) Inhalation of cold air for 5 min after pretreatment with L-NAME (50 mg/kg intravenously, open circles) or D-NAME (50 mg/kg intravenously, solid circles). (c) Inhalation of cold air for 5 min after pretreatment with L-NAME (50 mg/kg intravenously) and L-Arg (500 mg/kg intravenously, solid circles) or L-NAME (50 mg/kg intravenously) and D-Arg (500 mg/kg intravenously, open circles). Anesthetized and artificially ventilated guinea pigs were preteated with atropine (1.4 µmol/kg intravenously). Each point is the mean ± SEM of at least five experiments. *p < 0.05 versus respective solid circles.

Pretreatment with the tachykinin NK₂-receptor antagonist SR 48968 (0.3 µmol/kg intravenously) blocked completely the bronchoconstriction produced by inhalation of cold air for 5 min after pretreatment with L-NAME (Figure 3a). The kinin B₂-receptor antagonist HOE 140 (0.1 µmol/kg intravenously) also abolished the increase in RL observed after exposure to cold air and after pretreatment with L-NAME (Figure 3b). Inhalation of cold air for 5 min did not affect baseline RL. Baseline RL was also not affected by inhalation of cold air and after pretreatment with HOE 140 (0.1 µmol/kg intravenously) (Table 2). However, pretreatment with SR 48968, (0.3 µmol/kg intravenously) and inhalation of cold air caused a significant relaxation of baseline RL that was maximum 5 min after the beginning of the inhalation (Table 2).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effect of (a) the tachykinin NK2-receptor antagonist, SR 48968 (0.3 µmol/kg intravenously, solid circles) and (b) the kinin B2-receptor antagonist HOE 140 (0.1 µmol/kg intravenously, solid circles) or (a and b, open circles) their respective vehicles on the increase in total pulmonary resistance (RL) induced by inhalation of cold air for 5 min. Anesthetized and artificially ventilated guinea pigs were pretreated with atropine (1.4 µmol/kg intravenously) and with L-NAME (50 mg/kg intravenously). Each point is the mean ± SEM of five experiments. *p < 0.05 versus respective vehicles.

Bronchoconstriction Induced by Bradykinin and Capsaicin

The increase in RL above baseline in response to a dose of capsaicin (10 µM, 10 breaths) that produced a half-maximal response was not statistically different in guinea pigs pretreated with L-NAME (0.27 ± 0.03 cm H₂O/ml/s, n = 5) from those pretreated with D-NAME (0.22 ± 0.02 mm H₂O/ml/s, n = 5). The peak increase in RL above baseline induced by a dose of BK (1 mM, 40 breaths) that produced a half-maximal response after pretreatment with L-NAME was markedly greater (0.63 ± 0.07 cm H₂O/ml/s, n = 5) than the increase in RL induced by BK administered after pretreatment with D-NAME (0.21 ± 0.02 cm H₂O/ml/s, n = 5, p < 0.01).

Cardiovascular Measurements

Baseline heartbeat frequency and arterial blood pressure were not changed 10 min after administration of D-NAME (50 mg/ kg intravenously) or D-NMMA (50 mg/kg intravenously) (Table 1). Ten minutes after the injection of L-NAME (50 mg/kg intravenously) or L-NMMA (50 mg/kg intravenously), baseline heartbeat frequency was unchanged (Table 1). However, a significant increase in mean arterial blood pressure (MABP) was observed 10 min after L-NAME and after L-NMMA injection (Table 1).

cGMP Measurement

Baseline cGMP in strips of trachealis muscle with intact epithelium was 2.15 ± 0.31 pmol/g protein (n = 8). Exposure to BK increased cGMP content of the tracheal strips in a concentration-dependent manner (Table 3). In contrast, [ Ala⁸]- NKA (4) (0.1 to 10 µM) did not cause a significant increase in baseline cGMP (Table 3). The kinin B₂-receptor antagonist HOE 140 (1 µM), but not the tachykinin NK₂-receptor antagonist SR 48968 (1 µM) (both added 15 min before BK), inhibited the increase in cGMP induced by BK (1 µM) (Table 3).

	DISCUSSION

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Cold-air inhalation for periods longer than 5 min was shown to increase RL in anesthetized and atropinized guinea pigs (10). Bronchoconstriction induced by inhalation of cold air for 10 min was proposed to be mediated entirely by tachykinins released from sensory nerve endings, because it was blocked by pretreatment with the selective antagonist of NK₂-receptors SR 48968 (10). Involvement of kinins was indicated by the observation that a kinin B₂-receptor antagonist, HOE 140, abolished bronchoconstriction induced by cold air (10). The present experiments show that the mechanisms involved in the increase in RL induced by cold-air inhalation for 5 min and unmasked by inhibition of NOS are similar to those shown to be responsible for the bronchoconstriction caused by inhalation of cold air for 10 min (10). They also show that inhibition of cold-air-induced bronchoconstriction by HOE 140 and SR 48968 was complete, suggesting that kinins and tachykinins share a common final pathway, causing the bronchoconstriction induced by cold air when NO release is blocked.

Capsaicin-sensitive primary sensory neurons that contain and release tachykinins are referred to as polymodal nociceptors because they are stimulated by heat, chemical stimuli, and high-threshold mechanical stimuli (19). Although a few reports have suggested that some mechano- and cold-sensitive A-receptors may be activated by capsaicin (20), capsaicin-sensitive sensory neurons do not generally appear to be activated directly by low temperature. This physiologic finding was recently confirmed by neurochemical findings that dorsal-root-ganglion neurons in culture release substance P (SP) after exposure to hypertonic media, but not to a physiologic solution maintained at a low temperature (21). It therefore appears unlikely that cold air alone stimulates airway sensory nerves. In a variety of guinea pig tissues, including the airways, BK augments its own proinflammatory action via release of sensory neuropeptides, namely the tachykinins substance P (SP) and neurokinin A (NKA) (22). In contrast, there is no evidence that inflammation induced by tachykinins is mediated by kinin release. This suggests that bronchoconstriction induced by exposure of guinea pig airways to cold air for 5 min after NOS inhibition is due to the following cascade: cold air, in a first phase of inflammation, causes the release of kinins. Apparently, kinins do not contribute directly to smooth-muscle contraction, but instead excite sensory nerves, thus releasing the proinflammatory tachykinins (23), which cause bronchoconstriction via activation of NK₂-receptors. With regard to the component of bronchoconstriction induced by cold-air inhalation for 10 min that is resistant to BK-receptor blockade (10), it is possible than mediators others than BK are released by cold air, and stimulate sensory nerves to release bronchoconstrictor tachykinins.

The novel mechanism that we have described in this report is the involvement of the L-Arg-NOS pathway in the modulation of bronchoconstriction induced by inhalation of cold air in guinea pigs. Two pieces of evidence point to this conclusion. First, exposure to cold air for 5 min, which by itself did not affect tracheobronchial tone, caused marked bronchoconstriction when guinea pigs were pretreated with two different NOS inhibitors, L-NAME and L-NMMA, but not with their inactive enantiomers. Second, the increase in RL caused by cold-air inhalation in the presence of L-NAME was reversed by L-Arg in a stereospecific manner. Next, we addressed the question of the mechanism by which cold air causes the release of bronchorelaxant NO. Because kinins and tachykinins are released in the airways after exposure to cold air, it is possible that these two classes of mediators are involved in the release of NO. An alternative hypothesis is that cold air on its own stimulates the release of NO. Observations in previous studies (16) and the present study that bronchoconstriction induced by aerosolized capsaicin (an effect completely mediated by tachykinins released from sensory nerves) is unaffected by NOS inhibitors excludes the possibility that tachykinins may release bronchorelaxant NO from guinea pig airways. This conclusion is further supported by the finding that stimulation of NK₂-receptors, which mediate cold-air-induced bronchoconstriction, with the selective agonist [ Ala⁸]NKA (4) failed to increase cGMP levels in strips of guinea pig trachealis muscle in vitro.

This involvement of kinins in the release of NO that follows cold-air inhalation is supported by a large body of evidence: bronchoconstriction induced by aerosolized BK is exaggerated by aerosolized (16) or intravenous NOS inhibitors; epithelium-dependent relaxation induced by BK in tracheal-tube preparations of guinea pigs was changed to contraction by L-NAME or L-NMMA (8); BK, via B₂-receptor stimulation, increased cGMP in strips of guinea pig trachealis in vitro. These indirect pieces of evidence for a role of kinins in the release of NO after cold-air inhalation are strongly supported by the data obtained with receptor antagonists in the present study. Cold-air inhalation for 5 min did not influence baseline bronchoconstriction. Similarly, no change in baseline RL was observed after cold-air inhalation and pretreatment with a kinin B₂-receptor antagonist. However, RL decreased significantly after cold-air inhalation in guinea pigs pretreated with a tachykinin NK₂-receptor antagonist. Assuming that this relaxation is mediated by NO release if tachykinins were involved in the release of NO following exposure to cold air, pretreatment with SR 48968 should have inhibited cold-air-induced relaxation of resting airways. On the other hand, if cold air released NO on its own, cold-air inhalation should have reduced bronchial tone despite kinin B₂-receptor blockade. The observation that blockade of tachykinin NK₂-receptors resulted in a relaxation of non-preconstricted guinea pig airways points to kinins as the mediators that release NO after exposure to cold air. In fact, should kinins release both NO and tachykinins, the relaxant effect of kinins would be unmasked by an NK₂-receptor antagonist, as we in fact observed after pretreatment with SR 48968. To our knowledge, this is the first evidence that bronchorelaxant NO is released by an endogenously released autacoid.

The present data do not give any indication about the source of NO released after cold-air inhalation. However, previous studies have shown that kinins, as well as other mediators, release bronchorelaxant NO from the airway epithelium (3, 8), and kinin B₂-receptors have been described on airway epithelial cells of guinea pigs (24). Thus, it is possible that increased kinin levels in the airways after exposure to cold air cause the release of NO from the epithelium. The pathophysiologic relevance of the findings in the present study is unknown. It is interesting to note that in guinea pigs, the involvement of endogenous kinins in bronchoconstriction (6) and endogenous NO in bronchorelaxation (25) has been demonstrated in models of inflammation such as anaphylaxis. It has recently been reported that bronchoconstriction in response to inhaled BK is markedly increased by inhibition of the L-Arg- NOS pathway in patients with moderate asthma (26). Further studies may elucidate whether NO is involved in the modulation of bronchoconstriction induced in asthmatic subjects by agents that might increase kinin levels in the airways.

Inhalation of cold air worsens symptoms of exercise-induced asthma (27), and asthma-like symptoms are more common in cross-country skiers (breathing large volumes of cold air) than in the general population (28). Dry-gas hyperpnea-induced bronchoconstriction (HIB) in guinea pigs is an effect that phenomenologically resembles hyperpnea-induced bronchoconstriction in human subjects with exercise-induced asthma. Dry-gas HIB in guinea pigs is mediated by tachykinin release from sensory nerves (29), a mechanism similar to that activated by cold air. If tachykinins play a role in HIB in human subjects with exercise-induced asthma, or in the worsening of this condition when these subjects breathe cold air, the recently discovered nonpeptide tachykinin receptor antagonists may be of some benefit in this pathology.