INTRODUCTION

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ASTHMA IS A DISEASE characterized by a variety of respiratory conditions, including airway inflammation, hyperresponsiveness, and obstruction. It affects ~10% of the population in industrialized countries, and this figure is anticipated to increase in the next decade (12, 26). Although the cause of asthma remains obscure, a number of stimuli have been recognized to trigger the airway hyperresponsiveness associated with the disease. This has been supported by several lines of evidence from clinical and experimental observations. In humans, adenosine has been shown to induce bronchoconstriction in asthmatic but not in normal subjects (16). Airway smooth muscle isolated from individuals with allergic asthma have also been demonstrated to be hyperreactive to adenosine (9). In addition, the concentration of adenosine in bronchoalveolar lavage fluid of asthmatic subjects has been observed to be elevated (17). In a rabbit model of asthma that simulates the human condition of the disease, adenosine has been shown to cause bronchoconstriction and bronchial hyperresponsiveness (4, 6, 7). Similarly, the nucleoside has been observed to induce contraction of airway smooth muscle isolated from this allergic rabbit model (4, 6). The bronchial lavage concentration of adenosine has also been reported to be elevated in this model (5). On the other hand, all these characteristics associated with the disease have been found to be lacking in age-matched nonallergic rabbits (4-7).

A further study (6) conducted in our laboratory demonstrated that the adenosine-induced bronchoconstriction observed in the allergic rabbit model of asthma was mediated via A₁ adenosine receptors. El-Hashim et al. (18) from another laboratory also reported similar results with this model. These in vivo results were also confirmed by in vitro experiments with the use of isolated airway smooth muscle preparations from allergic rabbits (4, 6). In addition, specific A₁ adenosine-receptor binding sites have been shown in the lung plasma membranes from allergic rabbits (6). Furthermore, A₁ adenosine receptor-mediated contractile responses observed in allergic rabbit airway smooth muscle was found to be linked to increased intracellular levels of calcium (4).

It is generally agreed that calcium-mobilizing agonists elicit contraction of airway smooth muscle by mechanisms that involve activation of phospholipase C (PLC) via receptor-coupled GTP-binding proteins (G proteins) (13). PLC consists of a family of related isozymes with distinct tissue distribution and biochemical requirements (36, 37). Activation of this enzyme induces the breakdown of plasma membrane phosphatidylinositol 4,5-bisphosphate, resulting in the generation of the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and 1,2-diacylglycerol (DAG) (36). Ins(1,4,5)P₃ can mobilize calcium from nonmitochondrial intracellular stores, such as sarcoplasmic reticulum, and this effect has been suggested to contribute to the initiation of contraction. DAG, on the other hand, activates protein kinase C, which is believed to be involved in the maintenance of contractile responses together with calcium entering the cells from extracellular sources. The present study was undertaken to further understand the mechanism of action of adenosine in asthma by investigating the effect of A₁ adenosine-receptor stimulation on PLC-induced generation of Ins(1,4,5)P₃ in airway smooth muscle from the allergic rabbit model. The N⁶-substituted adenosine analog N⁶-cyclopentyladenosine (CPA) was used as an A₁ adenosine-receptor agonist. This compound was previously reported to be a potent bronchoconstrictor in this model of asthma and is considered to be a useful marker of airway hyperresponsiveness (4, 6, 7).

	MATERIALS AND METHODS

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Induction of asthma. Asthma was induced in allergic rabbits according to the established methods used in our laboratory (7). Briefly, New Zealand White rabbit littermates free of Pasteurella were immunized intraperitoneally within 24 h of birth with 312 allergen units of dust mite allergen extract suspended in 10% kaolin once every week for the first month and then biweekly for the following 3 months. Such immunized rabbits preferentially generate allergen-specific IgE antibody and positively respond to aeroallergen challenges with early- and late-phase asthmatic responses as well as hyperresponsiveness (7, 25). This model closely simulates events occurring in human asthmatics (25). Age-matched nonimmunized littermates treated under identical conditions were used as controls, hereafter referred to as nonallergic rabbits. To evaluate airway sensitivity to adenosine, increasing doses of the nucleoside ranging from 0.16 to 20 mg/ml in 0.9% saline were aerosolized in anesthetized rabbits as reported previously (6, 7). Total lung resistance and dynamic compliance (C_dyn) were recorded on an automated pulmonary mechanics respiratory analyzer (model 6, Buxco Electronics, Troy, NY). From this, the concentration of adenosine producing a 50% reduction in C_dyn from baseline (PC₅₀) was determined for each rabbit (6, 7).

Ins(1,4,5)P₃ assay. Airways were removed from allergic and nonallergic rabbits and cleaned of fat and parenchymal tissue down to the tertiary branches. Primary, secondary, and tertiary rings were prepared and denuded of the epithelium as previously described (4, 6). The rings were mounted in isolated tissue baths containing gassed (95% O₂-5% CO₂) Krebs-Henseleit solution with a composition of (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11 glucose, and 2.5 CaCl₂ (pH 7.4, 37°C). After a 1-h equilibration period, the tissues were treated with 30 mM KCl for 5 min. Tissues were then washed and further equilibrated for 40 min. During the equilibration periods, the bathing solution was changed every 15-20 min. The airway rings were then incubated with the A₁ adenosine-receptor agonist CPA for various times (10-30 s) and at different concentrations (10⁷ to 10⁴ M) in the absence or presence of PLC inhibitors (10⁵ M) neomycin and 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122; 20-min preincubation), an adenosine-receptor antagonist [10⁵ M) 8-( p-sulfophenyl)-theophylline (8-SPT); 20-min preincubation], and a G_i-G_o inhibitor [100 ng/ml of pertussis toxin (PTX); 3-h preincubation] (31). For comparison purposes, a few key experiments were also performed in tertiary airway rings with the use of the A₂ adenosine-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680). After incubation with drugs, the rings were frozen in liquid nitrogen and stored at 80°C until assayed for Ins(1,4,5)P₃ levels as described by Abebe and MacLeod (1, 2). Control tissues that were not treated with drugs were also frozen to measure basal Ins(1,4,5)P₃ contents. The frozen rings were weighed, pulverized, and homogenized in 6% TCA, followed by centrifugation. The supernatants were treated four times with four volumes of ether to remove the TCA. Ins(1,4,5)P₃ levels in the aqueous extracts were determined with protein-binding assay kits (1, 2).

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	RESULTS

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As reported previously (6, 7), at the time of death, the majority of dust mite-sensitized rabbits revealed bronchial hyperresponsiveness to adenosine. The PC₅₀ for adenosine in these animals was 2.96 ± 0.45 mg/ml (n = 15). Age-matched nonallergic rabbits demonstrated a much higher PC₅₀ at values > 20 mg/ml.

CPA-induced Ins(1,4,5)P₃ generation. The time course of Ins(1,4,5)P₃ accumulation in response to CPA in primary, secondary, and tertiary tracheal rings from allergic and control rabbits is shown in Fig. 1. In all three preparations from both groups of animals, CPA (10⁵ M) caused a rapid rise in Ins(1,4,5)P₃ contents that reached a peak 10 s after administration of the agonist. The levels of Ins(1,4,5)P₃ then declined with time, returning close to basal levels 20 s after CPA addition, and continued to decline slightly over a period of 30 s. However, in all airways from nonallergic rabbits, the peak Ins(1,4,5)P₃ levels in response to CPA were not significantly greater than the corresponding basal levels. On the other hand, the CPA-induced maximum generation of the polyphosphate in all the primary, secondary, and tertiary rings from allergic rabbits was significantly higher than the corresponding values measured in the absence of the agonist. Basal Ins(1,4,5)P₃ contents in the three types of airways from allergic rabbits were similar to those assessed in corresponding tissues from nonallergic rabbits. However, the maximum Ins(1,4,5)P₃ levels measured in the secondary and tertiary airway preparations from allergic rabbits in response to CPA were significantly greater than the peak Ins(1,4,5)P₃ levels attained in response to the agonist in corresponding nonallergic tissues. The concentrations of Ins(1,4,5)P₃ did not differ between nonallergic and allergic rabbit airways at any other time points of incubation with CPA (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time course of N6-cyclopentyladenosine (CPA; 105 M)-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation in primary, secondary, and tertiary branches of airway smooth muscle from control () and allergic () rabbits. Each point is mean ± SE of 4-11 different observations. Significantly different (P < 0.05) from: * corresponding basal value; # corresponding control peak value.

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View larger version (12K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for CPA-induced Ins(1,4,5)P3 generation in airway smooth muscle from allergic rabbits. [CPA], concentration of CPA. Tissues were incubated with CPA for 10 s before reaction was terminated. b, Basal Ins(1,4,5)P3 contents determined before CPA was added to tissues. Each point is mean ± SE of 4-8 different observations. * Significantly different from corresponding basal value (P < 0.05).

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View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of 8-(p-sulfophenyl)-theophylline (8-SPT; 105 M) on CPA (105 M)-induced Ins(1,4,5)P3 generation in tertiary branch of airway smooth muscle from allergic rabbits. Tissues were pretreated with 8-SPT for 20 min before being incubated with CPA for 10 s. Each bar is mean ± SE of 4 different observations. * Significantly different from all other values (P < 0.05).

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View larger version (11K): [in this window] [in a new window]   Fig. 4.   Time course of CGS-21680 (105 M)-induced Ins(1,4,5)P3 generation in tertiary branch of airway smooth muscle from control () and allergic () rabbits. Each point represents mean ± SE of 4-6 different observations.

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View larger version (41K): [in this window] [in a new window]   Fig. 5.   Effect of neomycin (105 M) on CPA (105 M)-induced Ins(1,4,5)P3 generation in primary, secondary, and tertiary branches of airway smooth muscle from allergic rabbits. Tissues were pretreated with neomycin for 20 min before being incubated with CPA for 10 s. Each bar is mean ± SE of 4-5 different observations. * Significantly different from basal and neomycin alone-treated values within the group (P < 0.05).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Effect of U-73122 (105 M) on CPA (105 M)-induced Ins(1,4,5)P3 generation in airway smooth muscle from allergic rabbits. Tissues were pretreated with U-73122 for 20 min before being incubated with CPA for 10 s. Each bar is mean ± SE of 4-6 different observations. * Significantly different from all other values within the group (P < 0.05).

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View larger version (39K): [in this window] [in a new window]   Fig. 7.   Effect of pertussis toxin (PTX; 100 ng/ml) on CPA (105 M)-induced Ins(1,4,5)P3 generation in airway smooth muscle from allergic rabbits. Tissues were pretreated with PTX for 3 h before being incubated with CPA for 10 s. Each bar is mean ± SE of 4-9 different observations. * Significantly different from all other values within the group (P < 0.05).

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View larger version (12K): [in this window] [in a new window]   Fig. 8.   Effect of neomycin (105 M) on CPA-induced contraction of airway smooth muscle from allergic rabbits. Tissues were incubated with () or without () neomycin for 20 min before addition of CPA. Each point represents mean ± SE of 4-8 different observations.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Effect of U-73122 (105 M) on CPA-induced contraction of airway smooth muscle from allergic rabbits. Tissues were incubated with () or without () U-73122 for 20 min before addition of CPA. Each point represents mean ± SE of 4 different observations.

	DISCUSSION

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In the present study, we have presented evidence that the A₁ adenosine-receptor agonist CPA induces a rapid, transient, and concentration-dependent elevation in Ins(1,4,5)P₃ contents in allergic rabbit airway smooth muscle. However, the agonist was unable to produce such an effect in airways from nonallergic rabbits. These findings, therefore, suggest that activation of A₁ adenosine receptors results in the production of Ins(1,4,5)P₃ in allergic but not in normal rabbit airway smooth muscle. The involvement of adenosine receptors in this process is further supported by our observation that the commonly used adenosine-receptor antagonist 8-SPT (3) could selectively inhibit the CPA-induced Ins(1,4,5)P₃ production in this tissue. Because the A₂ adenosine-receptor agonist CGS-212680 did not elicit Ins(1,4,5)P₃ generation in either allergic or nonallergic rabbits, A₂ receptors may not be involved in this mechanism. Consistent with the Ins(1,4,5)P₃ data, we also found that CPA could cause contraction of airway smooth muscle from allergic but not from nonallergic rabbits. In both the allergic and nonallergic tissues, CGS-21680 was also ineffective in inducing contraction of all three types of airway tissues (data not shown). Although the contractile data presented here are similar to those previously reported (4, 6), the observation of enhanced production of Ins(1,4,5)P₃ in the allergic rabbit airways in response to A₁ adenosine-receptor stimulation is the first of its kind, although activation of this receptor in DDT₁ MF-2 smooth muscle cells by other investigators (19) has also been shown to be associated with increased generation of Ins(1,4,5)P_3. The fact that the PLC inhibitor U-73122 could attenuate both the CPA-induced contraction and Ins(1,4,5)P₃ generation in rabbit airways suggests that both of these responses are mediated, at least in part, via PLC-linked mechanisms. Because the contractile responses generated by CPA could not be inhibited by the phosphatidylcholine-specific PLC inhibitor D-609 (33) (data not shown), it is believed that the CPA-induced responses are mediated via phosphatidylinositol-specific PLC. U-73122 is a novel aminosteroid that has been reported by others to be a selective inhibitor of phosphatidylinositol-specific PLC in platelets (10), neutrophils (35), neuroblastoma (38), and guinea pig airway smooth muscle (32). The current data obtained with allergic rabbit airways provide evidence for the existence of an additional biological system containing at least this PLC, which is susceptible to the inhibitory action of U-73122. On the other hand, the lack of alteration of basal Ins(1,4,5)P₃ contents by U-73122 and the ineffectiveness of its nonactive analog U-73343 (25) on contractile responses (data not shown) confirm the absence of nonspecific action of the inhibitor on the allergic rabbit tissues. The failure of neomycin to attenuate the contractile as well as the Ins(1,4,5)P₃ responses indirectly indicates that both of these events share a common cellular mechanism, in this case, a mechanism that is not susceptible to inhibition by this compound. Neomycin has also been reported to be ineffective in attenuating certain agonist-induced contractions and phosphatidylinositol hydrolysis (including those produced by carbamylcholine and histamine) in guinea pig trachea (14), although many studies (15, 28, 29, 31, 32, 34) have indicated otherwise in a number of biological systems. The reason for these inconsistent observations is unclear, but there is the suggestion that this could be related to differences in the compartmentalization of receptor-coupled phosphatidylinositol 4,5-bisphosphate to a domain that is inaccessible to neomycin (14).

In the present investigation, the rapid increase in formation of Ins(1,4,5)P₃ observed in the allergic airway smooth muscle in response to CPA is in close agreement with the hypothesis that Ins(1,4,5)P₃ may be responsible for the initiation of agonist-induced tension development in this tissue (13). On the other hand, the fact that the production of the second messenger caused by CPA was transient suggests that it may not directly contribute to the maintenance of the contractile response (4, 13, 24). In this respect, the influx of extracellular calcium and protein kinase C activation by DAG, which is also partly generated by hydrolysis of phosphatidylinositol 4,5-bisphosphate, may have a role in the maintenance of contraction (4, 13). It is believed that whereas the augmented formation of Ins(1,4,5)P₃ in response to CPA was due to the activation of PLC, the decrease in the levels of the polyphosphate after an initial increase could have resulted from its metabolism to inositol 1,4-bisphosphate and/or inositol 1,3,4,5-tetrakisphosphate by activated Ins(1,4,5)P₃ 5-phosphatase and/or Ins(1,4,5)P₃ 3-kinase present in the cytosol (8, 21). The decline in Ins(1,4,5)P₃ levels observed does not appear to be related to a desensitization phenomenon because this was not recorded in the CPA concentration-response curves for Ins(1,4,5)P₃ production.

Comparison of the concentration-response curves for CPA indicates that the pD₂ values for Ins(1,4,5)P₃ production in the three types of airway rings were less than the corresponding pD₂ values for contraction. Similar discrepancies between pD₂ values for agonist-induced contractile and biochemical responses have been reported by other investigators for various smooth muscle preparations (22, 39). Several possible explanations can be forwarded for this observation. First, the assay used to measure Ins(1,4,5)P₃ may not be as sensitive as that which detected the contractility changes. Second, there could be amplification between second messenger production and contractile responses, such as agonist-induced enhancement of calcium sensitivity of contractile proteins (22, 39). Last, there may be involvement of PLC-independent mechanisms and/or a greater efficiency of coupling between A₁ adenosine-receptor activation and the influx of extracellular calcium in generating contractile responses to submaximal concentrations of CPA (27).

In an attempt to assess the involvement of G proteins in A₁ adenosine receptor-mediated Ins(1,4,5)P₃ generation in the allergic rabbit airway smooth muscle, we determined the effect of PTX on CPA-induced production of the second messenger. The results reveal that PTX inhibited Ins(1,4,5)P₃ formation elicited by CPA in this smooth muscle. This observation suggests that in the allergic rabbit smooth muscle CPA can evoke, at least in part, the generation of Ins(1,4,5)P₃ through PTX-sensitive pathways. Because PTX is known to ADP-ribosylate G_i and G_o proteins (20, 23), it is likely that these proteins may be involved in the coupling of the A₁ receptor to PLC in this tissue model. In support of this, it has been previously shown that increased intracellular levels of Ins(1,4,5)P₃ caused by the activation of A₁ adenosine receptors by CPA and adenosine in DDT₁ MF-2 smooth muscle cells are mediated via PTX-sensitive G proteins (19).

The contractile data demonstrate that the CPA-induced responses tended to be more pronounced from peripheral to central airways. This observation confirms previous results from our laboratory (6), further indicating that the smooth muscle surrounding smaller airways is more sensitive to A₁ adenosine-receptor stimulation. However, unlike the contractile responses, the corresponding changes in Ins(1,4,5)P₃ levels of the tissues were smaller, although a similar trend of enhancement was detected. The reason for this discrepancy between the biochemical and physiological responses is not clear. However, by analogy with vascular preparations (11), one possible explanation could be that as the size of the airways decreases, the contractile response becomes more and more dependent on extracellular calcium rather than on Ins(1,4,5)P₃-releasable intracellular calcium.

In our study, the contribution of non-smooth muscle to the changes in Ins(1,4,5)P₃ contents was assumed to be minimal. This is because a previous study by Langlands et al. (24) demonstrated that the levels of Ins(1,4,5)P₃ present in cartilage, which is a major component of the airway mass, are below the level of detection for the assay system used here. Furthermore, because the epithelium was removed from the tissues in the present experiments, its contribution to Ins(1,4,5)P₃ contents was minimized. Therefore, the changes seen in Ins(1,4,5)P₃ were the result of the action of CPA on the airway smooth muscle.

The present results could be relevant to the pathophysiology of asthma in several aspects. Endogenous adenosine has been shown to be increased in allergic asthma both in the rabbit model we used and in human asthmatics (5, 17). Whereas a basal value of 2.87 µM was reported in bronchoalveolar lavage fluid of allergic rabbits, the basal concentration of the nucleoside in nonallergic animals was found to be below detection level (5). In humans, the reported basal adenosine contents were 2.55 and 0.72 µM in asthmatic and normal subjects, respectively (17). The demonstration of an increased concentration of adenosine in allergic asthma provides evidence for the role of endogenous adenosine in provoking bronchoconstriction in asthma. This observation is supported by the fact that the adenosine-receptor antagonist theophylline is efficacious in the treatment of asthma, and bronchospasm is one of the potential side effects of the adenosine-uptake inhibitor dipyridamole when used in thallium myocardial perfusion imaging (30). However, the reason(s) for the increased adenosine concentration in allergic asthma and its source(s) are unclear. In its action as a bronchoconstrictor, it is likely that the nucleoside may, at least in part, act directly on the airway smooth muscle adenosine receptors. In support of this, we have documented that in airway preparations from allergic rabbits no inflammatory cells, including mast cells, could be detected histologically and that the mast cell secretagogue compound 48/80 was without effect on the responses of the airways to adenosine-receptor agonists (S. Ali and S. J. Mustafa, unpublished data). On the basis of the CPA data we obtained in the present study, it can therefore be speculated that at least part of the endogenous adenosine-induced airway smooth muscle reactivity under allergic conditions is linked to phosphatidylinositol-specific PLC activity. This implies the possibility that inhibitors of this enzyme can have the potential for developing antiasthma drugs.

In conclusion, our results demonstrate that CPA via A₁ adenosine receptors induces PLC-mediated Ins(1,4,5)P₃ generation and contraction in allergic rabbit airway smooth muscle. This coupling process involves PTX-sensitive G proteins. The results suggest that PLC activation may play an important role in adenosine-induced bronchoconstriction in asthma and such an understanding can be useful for possible therapeutic exploitation of the signaling pathway.