Introduction

Abstract Introduction Materials & Methods Results Discussion References

Tachykinin receptors are pharmacologically classified into three subtypes, NK₁, NK₂ and NK₃, according to preferential affinity for SP, NKA and NKB, respectively (Buck and Burcher, 1986; Guard and Watson, 1991). In several mammalian species the receptor subtypes involved in airway smooth muscle contraction to tachykinin have been demonstrated. For example, the presence of both NK₁ and NK₂ subtypes is found in the guinea pig airways (Maggi et al., 1991b). The NK₂ subtypes exist in rabbit bronchus (Maggi et al., 1992) and hamster trachea (Maggi et al., 1989) as well as in human bronchus (Advenier et al., 1992). In the functional, autoradiographic and binding studies two different types of tachykinin receptors are found in the rabbit airways; NK₁ receptors are more numerous in the peripheral than central airways, and NK₂ receptors are distributed throughout the airways (Black et al., 1992).

Although the bronchoconstrictor action of SP is less potent than that of histamine or ACh (Lundberg et al., 1983; Finney et al., 1985), SP (10⁹-10⁶ M) causes a dose-dependent contraction in the isolated rabbit airway smooth muscle (Tanaka and Grunstein, 1986). Regarding the responsiveness of in vitro preparations of the rabbit tracheal muscle to EFS, contractile responses to EFS in the presence of SP are more potent than those in the presence of NKA (Inoue et al., 1992). In the in vivo study with rabbits, stimulation of RARs by intravenous administration of SP (0.3-3 µg/kg i.v.), which does not significantly affect peak tracheal pressure, is inhibited but not completely blocked by prior treatment with CP 96, 345, a specific NK₁ receptor antagonist (Bonham et al., 1996). However, studies to determine the rank order of the RAR stimulus potency among SP, NKA and NKB in relation to bronchoconstriction have not been reported in in vivo experiments in the rabbit.

Both NEP and angiotensin-converting enzyme act as the enzymes in the degradation of tachykinins (Skidgel et al., 1984). Devillier et al. (1988) demonstrated that both NKA and NKB may be resistant to NEP, which can hydrolyze SP (Matas et al., 1983), and the SP-cleaving activity of NEP is more potent than that of angiotensin-converting enzyme (Johnson et al., 1985). On the other hand, L 659, 877 is a selective peptide antagonist for NK₂ receptors (Van Giesbergen et al., 1991). Thus, the degradation of both tachykinin and L 659, 877 by peptidases must be considered. In in vivo studies of rabbit trachea, SP-induced contraction of smooth muscle is reduced by atropine (Tanaka and Grunstein, 1986) and SP causes concentration-dependent augmentation of contractile responses to EFS (Armour et al., 1991). These studies have provided evidence that SP may facilitate a cholinergic neurotransmission in the rabbit. By the use of L 659, 877 or atropine in the presence of a NEP inhibitor, the pathophysiological actions among the three tachykinins in regulating rabbit airways would be differentiable. We therefore investigated the effects of SP, NKA and NKB given in the same concentrations (0.2-2.7 µg/kg) on the responses of RARs and R_L before and after administration of L 659, 877 (2.3 and 7.6 µg/kg) or atropine (2 mg/kg) in thiorphan (2 mg/kg)-treated rabbits. The experiments were performed in anesthetized, artificially ventilated rabbits.

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Animal preparations. Fourteen rabbits of either sex, weighing 2.5 to 3.5 kg, were anesthetized with urethane (1 g/kg i.p.). The trachea was exposed through a middle incision in the neck and cannulated below the larynx. The trachea and esophagus were retracted rostrally to obtain space for paraffin pool. After heparin (500 U/kg) was administered into the ear vein, the femoral artery was cannulated for measurement of BP. A polyethylene catheter was positioned in the right atrium via the external jugular vein for administration of drugs or a 0.9% NaCl solution. A polyethylene catheter was also inserted into the femoral vein. Supplemental doses of urethane (0.1-0.2 g/kg/hr i.v.) were administered as required. Rectal temperature was maintained at approximately 37°C by a heating pad.

Measurements of RAR activity and lung mechanics. The technique for recording RARs was performed as follows: A thin filament containing afferent nerve fibers was obtained from the cut left vagus nerve, but leaving the right vagus nerve alone. Afferent impulses of the RARs were identified, as described in a previous study (Matsumoto et al., 1994). The identification of RARs was made initially by their firing pattern with brief and irregular bursts of impulses. The receptors were further confirmed by their rapid adaptation to lung inflation and their characteristic response to forced lung deflation. The unitary RAR activity was amplified and selected by a window discriminator for counting the number of impulses. The RAR activity and the pulse output from a discriminator were recorded on a polygraph.

Drugs. The drugs used in this study were SP (Sigma Chemical, St. Louis MO), NKA (Sigma), NKB (Sigma), thiorphan (Sigma), atropine (Sigma) and L 659, 877 (Funakoshi, Tokyo, Japan). Before the experiments, all drugs were dissolved in either a 0.9% NaCl or dimethyl sulfoxide solution and diluted with a 0.9% NaCl solution.

	Results

Abstract Introduction Materials & Methods Results Discussion References

Effects of SP, NKA and NKB on RAR activity and R_L. Typical examples of the effects of SP, NKA, and NKB given i.v. on RAR activity, P_T, and BP are shown in figure 1, A to C. Both SP (0.8 µg/kg) and NKA (0.7 µg/kg) at the same concentrations caused an increase in RAR activity but did not significantly alter either P_T or , and the responses were associated with hypotension. However, NKB at the dose of 2.4 µg/kg had no significant effect on the three measured respiratory parameters. Figure 2 summarized the responses of RARs and R_L to i.v. injections of SP, NKA and NKB, ranging from 0.2 to 2.7 µg/kg, in 14 different RAR preparations on 14 rabbits. The basal discharge of RARs before SP, NKA and NKB were 1.4 ± 0.2, 1.3 ± 0.2 and 1.3 ± 0.2 impulses/sec, respectively. After administration of SP at 0.3, 0.8 and 2.7 µg/kg the discharges of receptors were increased to 4.6 ± 0.4, 7.2 ± 0.7 and 10.4 ± 1.0 impulses/sec, respectively. The discharges of RARs were increased after NKA administration at 0.2, 0.7 and 2.3 µg/kg to 3.8 ± 0.4, 4.9 ± 0.5 and 7.2 ± 0.7 impulses/sec, respectively. The mean RAR responses to SP with different doses were significantly larger than those to NKA at their respective doses. However, NKB at any dose did not significantly influence the discharge of RARs. The rank order of three tachykinins for RAR stimulus potency was SP > NKA > NKB. The excitatory responses of RAR activity to SP (2.7 µg/kg) and NKA (2.3 µg/kg) at a higher dose lasted for 57 ± 6 and 28 ± 3 sec, respectively, in 14 rabbits. Base-line R_L was 17.6 ± 2.3 cm H₂O/liter/sec. At the doses of SP, NKA or NKB administered, no significant changes in R_L were obtained, which indicates that the three tachykinins used in these concentrations had no bronchoconstrictor effect.

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Responses of RARs, PT, and BP to i.v. administration () of tachykinins. (A) SP (0.8 µg/kg); (B) NKA (2.3 µg/kg); (C) NKB (2.4 µg/kg). 25 sec, 10 sec and 6 sec indicate an elapse of time.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Changes in RAR activity and RL in responses to tachykinins. (I) the dose ranged from 0.2 to 0.3 µg/kg; (II) the dose ranged from 0.7 to 0.8 µg/kg; (III) the dose ranged from 2.3 to 2.7 µg/kg. , SP; , NKA; , NKB. Vertical bars are means ± S.E.; n = 14. *P < .05 for significant difference from control values;  P < .05 for significant difference from NKA effects.

Effects of L 659, 877 on the responses of RAR activity and R_L to SP, NKA and NKB in thiorphan-treated rabbits. Administration of thiorphan (2 mg/kg) to inhibit the actions of NEP did not significantly influence either basal discharge of RARs or base-line R_L (fig. 3). In eight thiorphan-treated animals, administration of SP caused the increases in RAR activity and R_L in a dose-dependent manner but had no significant effect on the RAR and R_L responses to NKA and NKB. Subsequent administration of L 659, 877 (2.3 and 7.6 µg/kg), a NK₂ receptor antagonist, did not cause any significant changes in RAR activity and R_L. In the presence of a NEP inhibitor, this NK₂ receptor blocker that had no effect on augmentation of the increased RAR activity and R_L after SP administration dose-dependently inhibited NKA-induced RAR stimulation. Furthermore, no significant effect of L 659, 877 on the RAR and R_L responses to NKB was found in thiorphan-treated animals.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Changes in RAR activity and RL in responses to SP (A) and NKA (B) before and after L 659, 877 in thiorphan (2 mg/kg)-treated rabbits. , the dose ranged from 0.2 to 0.3 µg/kg; , the dose ranged from 0.7 to 0.8 µg/kg; , the dose ranged from 2.3 to 2.7 µg/kg; 3, after L 659, 877 (2.3 µg/kg); 10, after L 659, 877 (7.6 µg/kg). Vertical bars are means ± S.E. (n = 8). *P < .05 for significant difference from control values;  P < .05 for significant difference from thiorphan effects;  P < .05 for significant difference from L 659, 877 effects.

Effects of atropine on the responses of RAR activity and R_L to SP, NKA and NKB in thiorphan-treated rabbits. In six rabbits, pretreatment with thiorphan (2 mg/kg) augmented stimulation of RAR activity by SP only and the responses were associated with an increase in R_L. Subsequent administration of atropine (2 mg/kg) in animals pretreated with a NEP inhibitor significantly attenuated augmentation of the SP-induced RAR stimulation and inhibited the bronchoconstriction evoked by SP, although the remaining effect of RAR stimulation after SP administration was still observed in the presence of both thiorphan and atropine (fig. 4A). As illustrated in figure 4B, the RAR and R_L responses to NKA at 0.2 to 2.3 µg/kg were not influenced significantly by the treatment with either thiorphan (2 mg/kg) or atropine (2 mg/kg). In addition, no significant effect of atropine on the RAR and R_L responses to NKB was obtained in animals pretreated with thiorphan.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Changes in RAR activity and RL in responses to SP (A) and NKA (B) before and after atropine in thiorphan (2 mg/kg)-treated rabbits. , the dose ranged from 0.2 to 0.3 µg/kg; , the dose ranged from 0.7 to 0.8 µg/kg; , the dose ranged from m 2.3 to 2.7 µg/kg. Vertical bars are means ± S.E.; (n = 6). *P < .05 for significant difference from control values.  P < .05 for significant difference from thiorphan effects; P < .05 for significant difference from atropine effects.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

Both SP and NKA are present in primary afferent nerves of the guinea pig airways (Lundberg and Saria, 1986), and these sensory nerves are sensitive to capsaicin (Lundberg and Saria, 1986; Saria et al., 1988). During vagal stimulation and after administration of capsaicin, tachykinins released from sensory nerves exert contraction of airway smooth muscle (Hua et al., 1984). This contracting action is mediated by activation of both NK₁ and NK₂ receptors (Devillier et al., 1988). Indeed, a noncholinergic component in the bronchoconstriction evoked by vagal stimulation in atropine-treated guinea pigs is dose-dependently inhibited but not completely blocked by administration of MEN 10, 376, a newly developed NK₂ receptor antagonist (Maggi et al., 1991a). In the rabbit, atropine or ipratropium bromide, a nonselective muscarinic receptor antagonist, completely blocks both vagus nerve- and field-stimulated contractions of airway smooth muscle (Bloom et al., 1988; Inoue et al., 1992; Loenders et al., 1992; Matsumoto et al., 1995, 1996). However, it is possible that noncholinergic mechanisms in rabbit airways are involved in the bronchoconstriction evoked by vagal stimulation. For this reason, the contraction of airway smooth muscle is only an indirect measurement of the amount of ACh release and may be altered by other factors.

The RARs and the pulmonary and bronchial C fibers are responsible for the neural airway defense reflexes involving bronchoconstriction, mucus secretion and changes in ventilatory pattern (Coleridge and Coleridge, 1994; Karlsson et al., 1988; Widdicombe, 1977). The RARs are stimulated by lung inflation and deflation (Widdicombe, 1954), chemical stimulants, for example, cigarette smoke (Ravi et al., 1994; Sellick and Widdicombe, 1971), histamine (Matsumoto, 1989; Sellick and Widdicombe, 1971) and ammonia (Matsumoto, 1989) and environmental toxins including ozone (Coleridge et al., 1993). In addition, there is evidence that SP can stimulate RAR activity in the rabbit (Matsumoto et al., 1994; Prabhakar et al., 1987). In the same species, administration of SP results in dose-dependent increases in RAR activity that are significantly inhibited but not completely abolished by CP 96, 345, a specific NK₁ receptor antagonist (Bonham et al., 1996). In this study, both SP and NKA caused concentration-related increases in RAR activity, whereas NKB at any concentration had no significant effect on the receptor activity. After i.v. administration of three tachykinins (SP, NKA and NKB) in the dose from 0.2 to 2.7 µg/kg, no significant increases in R_L were found. Thus, it seems unlikely that SP- and NKA-induced bronchoconstrictions contribute to the increases in RAR activity. The results agree with the observations that stimulation of RARs by SP at the maximum doses (0.3 to 3 µg/kg) is not responsible for bronchoconstriction (Bonham et al., 1996). In comparison with the excitatory responses of RAR activity to SP and NKA at the same concentrations, the magnitudes of increased RAR activity evoked by SP administration were significantly larger than those after NKA injection. Accordingly, the rank order of the RAR stimulus potency that was found in the present, in vivo study was SP > NKA > NKB.

What mechanisms are involved in the SP- and NKA-induced RAR stimuli without bronchoconstriction? Bonham et al. (1996) found that the RAR activities before and after NK₁ receptor blockade increased by approximately 700% and 50%, respectively, even when the increases in peak tracheal pressure were not statistically significant before and after administration of CP 96, 345 that significantly inhibited the increase of RAR activity induced by mild pulmonary venous congestion (approximately 5 mm Hg increases in left pressure). CP 96, 345 is a potent inhibitor of airway microvascular leak (Lei et al., 1992; Lembeck et al., 1992). Because we learned that the concentration-related increases of RAR activity after SP administration were not associated with any significant increase in R_L, it is more likely that the SP-induced RAR stimulation is mediated by its own of increased microvascular permeability and an increase in fluid flux. However, it is difficult to demonstrate the other possible mechanisms such as SP-induced mucus secretion and direct stimulation on NK₁ receptors on RAR nerve endings. In the autoradiographic study, binding of [¹²⁵I]NKA on the rabbit smooth muscle is sparse in central airways and becomes more dense in both vascular smooth muscle and epithelium in the peripheral airways (Black et al., 1992). Considering the fact that the NKA-induced RAR stimulation is not related to the bronchoconstrictor action in the rabbit, NKA receptors generally may be related to the pulmonary vascular function and/or epithelium function to regulate both electrolyte transport and mucus secretion (Xu et al., 1986).

NEP is localized within the epithelial cells, tracheal smooth muscle and epithelium (Johnson et al., 1985; Sekizawa et al., 1987). In rabbits treated with a NEP inhibitor, SP caused further augmentation of concentration-dependent increases in RAR activity, but the receptor responses to NKA and NKB did not show any significant change, which indicated that SP has a thiorphan-sensitive mechanism as suggested by Maggi et al. (1989) in the guinea pig gallbladder. Furthermore, the results are consistent with evidence that NKA and NKB might be resistant to NEP (Devillier et al., 1988). Indeed, we confirmed the potentiating effect of SP-induced RAR stimulation in thiorphan-treated animals. In the presence of a NEP inhibitor, L 659, 877, a selective peptide antagonist for NK₂ receptors, concentration-dependently inhibited excitatory responses of RARs to NKA but had no significant effect on augmentation of dose-related increases of RAR activity induced by SP administration. Because the ligand of L 659, 877 is specific for NK₂ receptors (Van Giesbergen et al., 1991), the results in this study suggest that excitatory responses of RAR activity to SP and NKA are mediated by activation of NK₁ and NK₂ receptors, respectively, and that these receptors are present in the rabbit airways. However, no significant changes of RAR activity in response to NKB were observed in the NEP inhibitor-treated animals after administration of a NK₂ receptor blockade. This implies that the tachykinin effect on RARs does not involve participation of NK₃ receptors.

In rabbit trachea in vivo, atropine inhibits SP-induced bronchoconstriction (Tanaka and Grunstein, 1986) and SP augments cholinergic nerve-induced contractions via a postganglionic, prejunctional mechanism (Armour et al., 1991). Belvisi et al. (1994) demonstrated that exogenous SP and NKA in the absence of a NEP inhibitor potentiate the contractile responses of smooth muscle to EFS of bronchial rings in rabbits; these responses are completely blocked by tetrodotoxin or atropine, which suggests that both SP and NKA may play a significant role in regulating cholinergic neurotransmission. In this study, administration of two tachykinins ranging from 0.2 to 2.7 µg/kg did not show any bronchoconstrictor action. In the case treated with a NEP inhibitor, SP-induced bronchoconstriction (measured as an increase in R_L) occurred, and this effect was significantly inhibited by atropine. Because the contractile responses of rabbit airway smooth muscle to methacholine are not significantly altered by SP (Tanaka and Grunstein, 1986), SP-induced bronchoconstriction in thiorphan-treated animals appears partly as a result of the increased prejunctional release of ACh and, as a result, causes further excitation of SP-induced RAR stimulation. The increases of RAR activity and R_L induced by SP administration were still observed in both thiorphan- and atropine-treated animals, which suggests that the effect of increased SP effective concentration caused by a NEP inhibitor would cause bronchoconstriction. However, NKA at any dose in thiorphan-untreated and -treated rabbits did not cause facilitation of ACh release from the airways. Inoue et al. (1992) also found that atropine did not significantly alter the NKA concentration-response curves in the isolated tracheal smooth muscle preparation in the rabbit. The discrepancy with the observations reported by Belvisi (1994) in in vitro preparations may be caused by the difference in experimental conditions, because the apparent presence of NK₂ receptors in the rabbit peripheral airways is not detected by autoradiographic study (Black et al., 1992). Accordingly, the NK₂ receptors in the rabbit airway might reveal an unusual nature. Further experiments are required to clarify the interaction between NK₂ receptors and cholinergic transmission. Activation of NK₃ receptors because of NKB administration had no effect on the R_L responses before and after atropine in thiorphan-treated animals, which suggests that augmentation of cholinergic neurotransmission is independent of the NK₃ receptors.