A1 adenosine receptor-mediated Ins(1,4,5)P3 generation in allergic rabbit airway smooth muscle

Worku Abebe and S. Jamal Mustafa

Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina 27858

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The signal transduction pathway for A1 adenosine receptor in airway smooth muscle from allergic rabbits was studied by investigating the effect of the selective A1 adenosine-receptor agonist N6-cyclopentyladenosine (CPA) on tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] measured by protein binding assay. CPA caused a rapid, transient, and concentration-dependent elevation of Ins(1,4,5)P3 in airways from allergic rabbits. The agonist also produced a concentration-dependent contraction of the airway preparations from these animals. Both the Ins(1,4,5)P3 and contractile responses generated by CPA were attenuated by the phospholipase C (PLC) inhibitor U-73122, indicating the coupling of these responses to PLC. The CPA-induced Ins(1,4,5)P3 production observed in the allergic rabbit tissues was also inhibited by the adenosine-receptor antagonist 8-( p-sulfophenyl)-theophylline, suggesting that the effect was mediated by A1 adenosine receptors. On the other hand, the A2 adenosine-receptor agonist CGS-21680 was ineffective in altering the tissue concentration of Ins(1,4,5)P3, indicating that A2 adenosine receptors may not be involved in the activation of PLC in the allergic rabbit airway smooth muscle. In this preparation, the Gi-Go inhibitor pertussis toxin (PTX) attenuated the CPA-induced Ins(1,4,5)P3 accumulation, providing evidence that the generation of Ins(1,4,5)P3 by A1 adenosine-receptor stimulation is coupled to a PTX-sensitive G protein(s). The results suggest that activation of A1 adenosine receptors in allergic rabbit airway smooth muscle causes the production of Ins(1,4,5)P3 via a PTX-sensitive G protein-coupled PLC, and this signaling mechanism may be involved, at least in part, in the generation of contractile responses. It is hypothesized that this process may contribute to adenosine-induced bronchoconstriction in allergic asthma.

asthma; N6-cyclopentyladenosine; phospholipase C; G protein; airway responsiveness; inositol 1,4,5-trisphosphate

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (Cdyn) 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 Cdyn 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 (10-7 to 10-4 M) in the absence or presence of PLC inhibitors (10-5 M) neomycin and 1-[6-((17beta -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 [10-5 M) 8-( p-sulfophenyl)-theophylline (8-SPT); 20-min preincubation], and a Gi-Go 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).

Contractility studies. Primary, secondary, and tertiary airway tissues isolated from allergic rabbits and prepared as epithelium-denuded rings as described in Ins(1,4,5)P3 assay were mounted in isolated tissue baths containing Krebs-Henseleit solution (95% O2-5% CO2, pH 7.4, 37°C) for measurement of isometric tension (3, 6). The rings were placed under a resting tension of 2 g for primary rings and 1 g for secondary and tertiary rings. After equilibrating for 1 h, the tissues were challenged with KCl (30 mM) until reproducible contractile responses were obtained. After an additional equilibration period of 40 min, cumulative concentration-response curves to CPA (10-8 to 10-4 M) were obtained in all the primary, secondary, and tertiary airway rings. For determination of the involvement of PLC in CPA-induced contractile responses, the concentration-response curves for the agonist were repeated in the presence of neomycin (10-5 M) or U-73122 (10-5 M). Tissues were washed for 1 h between CPA concentration-response curves and incubated in the presence of the inhibitors for 20 min before CPA was added. In preliminary experiments, it was confirmed that no changes in the contractile responses of the airways to CPA occurred with time. At the end of each experiment, tissues were blotted dry and weighed.

Materials. House dust mite extract was purchased from Berkeley Biologicals (Berkeley, CA). Ins(1,4,5)P3 kits were procured from Amersham Radiochemical (St. Louis, MO). CPA, CGS-21680, and 8-SPT were purchased from RBI (Natick, MA). Neomycin was purchased from Sigma (St. Louis, MO). PTX was obtained from List Biological Laboratories (Campbell, CA). U-73122, 1-[6-((17beta -3-methoxyestra-1,3,5-(10)-trien-17-yl)amino)-hexyl]-2,5-pyrrolidinedione (U-73343), and tricyclodecane-9-yl-xanthogenate (D-609) were from Calbiochem (La Jolla, CA).

Data analysis. The levels of Ins(1,4,5)P3 were calculated as picomoles per milligram of tissue weight. Contractile responses to CPA are expressed as the increase in tension (in g) in response to each concentration of the agonist per milligram of tissue weight. pD2 (-log EC50) values, which were used to assess the sensitivities of the airway smooth muscle to CPA, were determined from the concentration-response curves (3). Results are reported as geometric means ± SE calculated with a STAT-MATE program. Data were compared with the Student's t-test and considered to be significant if P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (10-5 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).


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Fig. 1.   Time course of N6-cyclopentyladenosine (CPA; 10-5 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 (open circle ) and allergic (bullet ) rabbits. Each point is mean ± SE of 4-11 different observations. Significantly different (P < 0.05) from: * corresponding basal value; # corresponding control peak value.

Figure 2 illustrates the CPA concentration-response curves for Ins(1,4,5)P3 assessed in primary, secondary, and tertiary tracheal rings from allergic rabbits at peak Ins(1,4,5)P3 accumulation time (10 s). In all the three preparations, CPA induced concentration-related (10-7 to 10-4 M) increases in Ins(1,4,5)P3 production, with pD2 values of 5.20 ± 0.11, 5.08 ± 0.09, and 5.13± 0.06 for primary, secondary, and tertiary rings, respectively.


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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).

Preincubation of tertiary airway smooth muscle rings from allergic rabbits with the adenosine-receptor antagonist 8-SPT (10-5 M) significantly diminished the accumulation of Ins(1,4,5)P3 caused by CPA (10-5 M; Fig. 3). However, 8-SPT had no significant effect on the basal concentration of Ins(1,4,5)P3 in this tissue.


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Fig. 3.   Effect of 8-(p-sulfophenyl)-theophylline (8-SPT; 10-5 M) on CPA (10-5 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).

In contrast to the responses of the airways to CPA, incubation of tertiary airways with the A2 adenosine-receptor agonist CGS-21680 (10-5 M) for 5-20 s did not produce a significant effect on the accumulation of Ins(1,4,5)P3 in either allergic or nonallergic rabbits (Fig. 4).


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Fig. 4.   Time course of CGS-21680 (10-5 M)-induced Ins(1,4,5)P3 generation in tertiary branch of airway smooth muscle from control (open circle ) and allergic (bullet ) rabbits. Each point represents mean ± SE of 4-6 different observations.

Pretreatment of primary, secondary, and tertiary airway preparations from allergic rabbits with the phospholipase C inhibitor neomycin (10-5 M) did not produce a significant effect on CPA (10-5 M)-induced Ins(1,4,5)P3 generation (Fig. 5). Neomycin also did not markedly alter the basal Ins(1,4,5)P3 concentration of the tissue. On the other hand, U-73122 (10-5 M), a newly synthesized PLC inhibitor, abolished the Ins(1,4,5)P3 production elicited by CPA (10-5 M) in all three types of rings from allergic rabbits (Fig. 6). However, U-73122 also did not have a significant effect on basal levels of Ins(1,4,5)P3.


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Fig. 5.   Effect of neomycin (10-5 M) on CPA (10-5 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).


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Fig. 6.   Effect of U-73122 (10-5 M) on CPA (10-5 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).

Pretreatment of primary, secondary, and tertiary airway rings from allergic rabbits with the Gi-Go inhibitor PTX caused a significant attenuation of Ins(1,4,5)P3 generation evoked by CPA (10-5 M; Fig. 7). PTX, however, did not produce an effect on basal levels of Ins(1,4,5)P3 in any of the tissues.


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Fig. 7.   Effect of pertussis toxin (PTX; 100 ng/ml) on CPA (10-5 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).

CPA-induced contractile responses. As described previously (5, 6), the addition of CPA in a cumulative manner (10-8 to 10-4 M) produced concentration-dependent increases in the contractile responses of primary, secondary, and tertiary rings isolated from allergic rabbits (Figs. 8 and 9) . Incubation of the tissues with 10-5 M neomycin produced no significant effects on their contractile responsiveness to CPA as reflected by the maximum responses and pD2 values (Fig. 8, Table 1). However, as with the Ins(1,4,5)P3 data (Fig. 6), pretreatment of primary, secondary, and tertiary airways from allergic rabbits with U-73122 (10-5 M) caused a significant inhibition of the contractile effect of CPA, although the pD2 values for the agonist were not significantly altered by the inhibitor (Fig. 9, Table 2).


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Fig. 8.   Effect of neomycin (10-5 M) on CPA-induced contraction of airway smooth muscle from allergic rabbits. Tissues were incubated with (bullet ) or without (open circle ) neomycin for 20 min before addition of CPA. Each point represents mean ± SE of 4-8 different observations.


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Fig. 9.   Effect of U-73122 (10-5 M) on CPA-induced contraction of airway smooth muscle from allergic rabbits. Tissues were incubated with (bullet ) or without (open circle ) U-73122 for 20 min before addition of CPA. Each point represents mean ± SE of 4 different observations.

                              
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Table 1.   Effect of neomycin on CPA pD2 values and maximum contractile responses in airway smooth muscle rings from allergic rabbits

                              
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Table 2.   Effect of U-73122 on CPA pD2 values and maximum contractile responses in airway smooth muscle rings from allergic rabbits

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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)P3. 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 Gi and Go 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.

    ACKNOWLEDGEMENTS

We thank Drs. Shahid Ali and Weixi Qin for expert technical assistance. We also thank Pam Wynne for typing the manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-50049; the American Lung Association of North Carolina; and a Minority Investigator Research Supplement Award from the NHLBI to W. Abebe.

Present address of W. Abebe: Dept. of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. J. Mustafa, Dept. of Pharmacology, School of Medicine, East Carolina Univ., Greenville, NC 27858.

Received 9 February 1998; accepted in final form 24 July 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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