INTRODUCTION

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Mast cell has been implicated to play a crucial role in asthma because mast cell degranulation induced by the cross-linking of high-affinity Fc receptor (Fc RI) releases a wide array of inflammatory mediators such as histamine, leukotrienes (LT), and cytokines which can initiate, coordinate, and sustain the allergic inflammatory responses (1). Cumulative evidence obtained from rat basophilic leukemia cell line (RBL-2H3) and bone marrow-derived mouse mast cells shows that activation of nontransmembrane protein tyrosine kinases (PTKs) is the earliest detectable signaling response to F RI cross-linking (2). Ensuing from this is the activation of downstream signaling molecules including phospholipase C (PLC), phosphatidylinositol-3-kinase (PI3K), and mitogen-activated protein kinase (MAPK), which eventually leads to mast cell degranulation (3). Inhibition of tyrosine kinases by genistein or piceatannol, PI3K by LY294002, or MAPK kinase by PD 098059 has been shown to attenuate antigen-induced mast cell degranulation (5, 7).

The Schultz-Dale reaction (10) has been extensively used to examine mast cell degranulation and anaphylactic contraction of airway tissues such as trachea, bronchi, and lung parenchymal strips in vitro (11, 12). It is believed that histamine and peptidoleukotrienes are the two major mast cell-derived mediators responsible for the anaphylactic contraction of isolated airways from both human and guinea pig (11, 12). In guinea pigs, both IgE and IgG are able to sensitize mast cells to specific antigen (13) and cross-linking of their corresponding Fc RI and FcR leads to mast cell degranulation. FcR (e.g., FcRIII) and Fc RI are structurally and functionally related, and both belong to a family of multisubunit antigen receptors (14). Engagement of these cell surface receptors utilizes the same tyrosine kinase signaling cascade for successful signal propagation and cellular activation (1, 2, 4, 15).

The purpose of the present study was to examine the effects of inhibition of several critical signaling molecules of the tyrosine kinase signaling cascade on antigen challenge of guinea-pig airways in vitro. Our results show that inhibition of PTK by genistein or tyrphostin 47, MAPK kinase by PD 098059, PI3K by LY294002, or both MAPK kinase and PI3K by a combination of PD 098059 and LY294002 suppressed the peak anaphylactic bronchial contraction to various extents and promoted relaxation of the contracted airways. These modulatory effects are likely to be associated with their profound inhibitory effects on ovalbumin (OA)-induced release of peptidoleukotrienes and, to a lesser extent, histamine, as shown in chopped lung preparations.

	METHODS

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Sensitization Procedures

Guinea pigs were passively sensitized by a single intraperitoneal injection of 1 mg/kg rabbit IgG antibody against OA (16). The animals were killed 2 d after injection.

Preparation of Bronchial Rings

Male Hartley guinea pigs (Interfauna, U.K. Ltd., East Yorkshire, UK) weighing 350 to 450 g were killed by CO₂ asphyxiation and subsequent decapitation. After thoracotomy, heart and lung were excised en bloc and perfused with 50 ml of Krebs-bicarbonate solution via the pulmonary artery. Lung lobes were isolated for studies on the release of histamine and peptidoleukotrienes. Bronchial rings (approximately 3 mm in length) were obtained from the hilar bronchi and cleaned of any parenchyma. Ring preparations were then suspended isometrically under an optimal resting load of 2 g in organ baths containing 5 ml of Krebs-bicarbonate solution aerated with 95% O₂ and 5% CO₂ at 37° C and of the following composition (mM): NaCl, 118.2; KCl, 4.6; NaHCO₃, 24.8; CaCl₂ · 2 H₂O, 2.5; KH₂PO₄, 1.2; MgSO₄ · 7 H₂O, 1.2; and dextrose, 10.0. Contractile responses were monitored using force-displacement transducers (Grass FT-03) coupled to a MacLab/8 data-recording system (AD Instruments, Castle Hill, Australia). A 90-min equilibration period was allowed before any experimentation was begun, and during this time the bath fluid was changed every 10 min.

Contractile Studies

After equilibration, bronchial rings were contracted to 60 mM KCl. The contraction was defined as the maximal tissue response, to which all subsequent anaphylactic contractions were compared. For antigen challenge studies, ring preparations were preincubated with indomethacin (4 µM) for 30 min before addition of OA (1 µg/ml). Indomethacin has been shown to reduce the production of relaxant prostanoids (e.g., prostaglandin E₂ [PGE₂] and PGI₂) capable of modulating the anaphylactic contraction (13, 17). To evaluate the role of tyrosine kinase signaling cascade in mediating anaphylactic bronchial smooth muscle contraction, various inhibitors were preincubated with bronchial rings 30 min before addition of OA. The effects of inhibitors were compared with those of control ring preparations in the absence of the inhibitors. To examine the potential direct smooth muscle relaxant effects of these signal transduction inhibitors, we studied bronchial contraction induced by histamine, LTD₄, or KCl in the presence and absence of the inhibitors. In the LTD₄-induced bronchial ring contraction study, 4 µM indomethacin and 5 mM L-cysteine were preincubated with the tissue for 30 min. It has been reported that very minute amounts of relaxant prostanoids (e.g., PGE₂ and PGI₂) were able to mask LTD₄-induced canine bronchial contraction and that the addition of indomethacin restored the LTD₄ effect (17). L-cysteine is an inhibitor of an aminopeptidase that converts LTD₄ to less potent LTE₄. Adding L-cysteine has been shown to enhance the smooth muscle contractile response to LTD₄ (17). To further explore the potential direct smooth muscle relaxant effects, inhibitors were added to the bronchi after they reached their peak anaphylactic contractions. Salbutamol (1 µM), a ₂-adrenoceptor agonist, was used as a positive control for airway smooth muscle relaxation.

Release of Mediators from Chopped Lung Preparations

Lung lobes obtained from sensitized guinea pigs were cut into approximately 1-mm³ pieces using a McIlwain tissue chopper (Brinkmann Instruments, Westbury, NY). Fragmented lung preparations were washed thoroughly with oxygenated Krebs-NaHCO₃ buffer before incubation. Duplicate aliquots of 200-mg lung fragments were weighed and placed in plastic scintillation vials containing 2 ml of oxygenated Krebs solution in the presence of 4 µM indomethacin (for histamine release) plus 5 mM L-cysteine (for release of peptidoleukotrienes). Lung samples were then incubated in a shaker bath at 37° C for 45 min before they were challenged with OA for 3 min (histamine release) or 10 min (release of peptidoleukotrienes) (18). To determine the mast cell-stabilizing effects of these signal transduction inhibitors, each of these inhibitors was preincubated with the lung preparation for 30 min before antigen challenge. To determine if these inhibitors have any direct inhibitory effects on the de novo synthesis of peptidoleukotrienes such as inhibition of 5-lipoxygenase activity, exogenous arachidonic acid (AA) at a final concentration of 70 µM was added to the lung fragments alone or together with OA challenge for 10 min in the presence and absence of these inhibitors (18, 19). Diffusates were then collected and stored at 70° C until assay.

Histamine Radioenzymatic Assay

Histamine release from lung samples in response to OA was determined using a radioenzymatic assay as previously described with minor modifications (18). Briefly, a total incubation volume of 60 µl was prepared by sequential addition of 10 µl of biological samples (or H₂O for the blank), 25 µl of H₂O (or H₂O containing 500 pg of histamine as internal standard), and 25 µl of reaction reagent in 12 × 75-mm polypropylene culture tubes. Reaction reagent contained 21 µl of 0.4 M potassium phosphate/0.1% bovine serum albumin (BSA), pH 7.8, 2 µl of histamine N-methyl transferase (HNMT), and 2 µl of tritiated S-adenosylmethionine. After a 1-h incubation in a shaker bath at 2° C, the enzymatic reaction was terminated by the addition of 75 µl of 2.5 M potassium borate, pH 11; 4 ml of toluene-isoamyl alcohol (3:1, vol/vol) was then added to each tube. After centrifugation for 5 min, 3.6 ml of the organic phase, which contained tritiated N--methylhistamine ([³H]-MHm) formed by the HNMT reaction, was transferred to another set of tubes containing 250 µl of 1 M KH₂PO₄, pH 7.1, for back extraction of the [³H]-MHm into the aqueous phase. Tubes were centrifuged for 5 min, and the organic phase was aspirated and discarded. The aqueous phase was mixed with 1.25 ml of toluene-isoamyl alcohol. After centrifugation and removal of the organic phase, 150 µl of the aqueous phase was transferred to scintillation vials containing 500 µl of 1 M KH₂PO₄, pH 7.1. Subsequently, 8 ml of biodegradable counting scintillant was added to each tube. Radioactivity was quantitated by liquid scintillation spectrometry (Beckman LS 3801; Beckman Instruments, Inc., Fullerton, CA).

LT Assay

The release of peptidoleukotrienes from chopped lung preparations in response to OA was quantitated by either radioimmunoassay (RIA) or enzyme immunoassay (EIA) (Amersham Life Science, Buckinghamshire, UK). For RIA, total incubation volumes of 400 µl were prepared by sequential addition of 100 µl of biological samples or LTC₄ standard, 100 µl of [³H]LTC₄, 100 µl of peptidoleukotriene-specific antiserum (cross-reactivity for LTC₄/D₄/E₄ = 100%/100%/ 41%), and 100 µl of assay buffer (pH 7.4) in polypropylene tubes. Antigen-antibody competition reaction was allowed to take place overnight at 2° to 8° C. Dextran-coated charcoal suspension (250 µl) was then added to each reaction tube to adsorb any unbound LT. After centrifugation, the supernatants were transferred to scintillation vials containing 10 ml of scintillant. Radioactivity was measured using liquid scintillation spectrometry. For EIA, total incubation volumes of 100 µl were prepared by sequential addition of 50 µl of assay buffer (pH 7.5), 50 µl of biological samples or LTC₄, and 50 µl of antiserum in wells of microtiter plate precoated with goat anti-rat IgG. The mixtures were incubated at 4° C for 2 h followed by addition of LTC₄ peroxidase conjugate. The plate was then incubated at 4° C for a further 3 h. After incubation, all the wells were thoroughly washed with wash buffer and 150 µl of enzyme substrate, 3,3',5,5'-tetramethylbenzidine- hydrogen peroxide, was added subsequently to each well before the mixtures were incubated on a microtiter plate shaker for 30 min at room temperature. After incubation, 100 µl of 1 M sulfuric acid was added into each well. The contents were mixed thoroughly and the optical density was determined in a microplate reader (Tecan, Salzburg, Austria) at 450 nm. Samples were assayed in duplicate.

Data Analysis

All data are presented as mean ± SEM. Statistical differences in contractile responses to OA challenge, histamine, LTD₄, or KCl, and in the release of mediators in response to OA, in the presence and absence of inhibitors, were analyzed using analysis of variance (ANOVA) followed by Student-Newman-Keuls test (20). The critical level for significance was set at p < 0.05.

Materials

The following drugs and chemicals were used in this study: genistein, tyrphostin 47 (RG50864), PD 098059, and LY294002 (Research Biochemicals Internationals, Natick, MA), OA (grade V), histamine dihydrochloride, indomethacin, L-cysteine, BSA, AA (Sigma Chemical Co., St. Louis, MO), toluene, isoamyl alcohol, boric acid, potassium phosphate (Merck, Darmstadt, Germany), dimethyl sulfoxide (DMSO) (BDH Laboratory Supplies, Poole, UK), rabbit IgG fraction to chicken egg albumin (OA) (Organon Teknicka Corp., Durham, NC), LTD₄ (Cayman Chemical Co., Ann Arbor, MI), LT C₄/D₄/E₄ RIA kit, LT C₄/D₄/E₄ EIA kit and biodegradable liquid scintillant (Amersham Life Science, Buckinghamshire, UK), tritiated S-adenosyl-L-[methyl- ³H]methionine (60 to 85 Ci/mmol), and HNMT (New England Nuclear, Boston, MA). Rabbit anti-OA IgG antibody was stored in sterile H₂O, and all inhibitors in DMSO at 20° C. Indomethacin was dissolved in 4.2% (g/ml) NaHCO₃ stock solution. OA and histamine were prepared fresh in deionized H₂O. All other reagents were of analytical grade and were dissolved in deionized H₂O.

	RESULTS

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Effects of Inhibitors on Anaphylactic Contraction

Sensitized guinea-pig bronchial rings contracted in response to 60 mM KCl with an active force of contraction that amounted to 1.68 ± 0.1 g (n = 64). To investigate the effects of inhibitors of tyrosine kinase signaling cascade on the Schultz-Dale reaction, genistein (30 µM), tyrphostin 47 (50 µM), PD 098059 (30 µM), LY294002 (10 µM), or a combination of PD 098059 (30 µM) and LY294002 (10 µM) was incubated with the ring preparations for 30 min before OA challenge. The concentrations of inhibitors were chosen based on our previous studies (16, 18) and others (5, 6, 9, 21, 22) demonstrating that, at these concentrations, activities of PTK, MAPK kinase, and PI3K could be inhibited. OA at 1 µg/ml was able to induce a strong and sustaining anaphylactic bronchial contraction reaching 2.0 ± 0.1 g (n = 34). Genistein and tyrphostin 47, two PTK inhibitors (21, 22), significantly suppressed (p < 0.05) OA-induced peak bronchial contraction by 42.9% and 32.0%, respectively (Figure 1A). In contrast, LY294002, a specific PI3K inhibitor (23) and PD 098059, a MAPK kinase inhibitor (24, 25), produced only a slight reduction in OA-induced peak bronchial contraction, although the inhibition produced by the former reached significant level (p < 0.05). However, when PD 098059 and LY294002 were used together, they substantially depressed (p < 0.05) OA-induced peak bronchial contraction by 53.9% (Figure 2A). On the other hand, the anaphylactically contracted bronchi relaxed to a greater extent in the presence of any of these inhibitors alone or in combination compared with the DMSO controls (Figures 1B and 2B). In the first 10 min, after OA challenge, the contracted bronchi relaxed by 43.7%, 16.9%, 28.9%, 11.9%, or 88.5% in the presence of genistein, tyrphostin 47, PD 098059, LY294002, or combined PD 098059 and LY294002, respectively, compared with only 4.6% in DMSO controls. Complete relaxation was achieved in 30 min with the PTK inhibitors or combined PD 098059 and LY294002 treatment, and approximately 70% relaxation was reached in 30 min with either LY294002 or PD 098059, whereas substantial anaphylactic bronchial contraction of 50% still remained in the control preparations after 30 min and of 20% after 60 min.

View larger version (21K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 1.   (A) Effects of inhibitors of tyrosine kinase signaling cascade on OA-induced peak anaphylactic contraction of guinea pig bronchi. Bronchial rings were incubated with the indicated concentrations of inhibitors or same volume of DMSO for 30 min before 1 µg/ml OA challenge. A DMSO control was carried out in parallel with each experiment using an inhibitor. Each point represents the mean ± SEM of 4 to 8 experiments. *Significant difference from DMSO vehicle controls, p < 0.05. (B) Time course of OA-induced anaphylactic bronchial contraction in the presence and absence of inhibitors. Each point represents the mean of 3 to 6 experiments. Gen = genistein; T47 = tyrphostin 47; PD = PD 098059; LY = LY294002.

View larger version (14K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Figure 2.   (A) Effect of dual inhibition of MAPK kinase and PI3K inhibitors on OA-induced peak anaphylactic contraction of guinea-pig bronchi. *Significant difference from DMSO vehicle controls, p < 0.05. (B) Effect of combined MAPK kinase and PI3K inhibition on the time course of OA-induced anaphylactic bronchial contraction. PD/LY, PD 098059 (30 µM) + LY294002 (10 µM). A DMSO control was carried out in parallel with each experiment using the inhibitors. Each point represents the mean ± SEM of 4 to 6 experiments.

Effects of Inhibitors on Mediator-induced Bronchial Contraction

To determine whether the attenuation of the peak anaphylactic contraction and rapid relaxation of the contracted bronchi produced by the inhibitors are mediated by blocking the release of mast cell-derived mediators or by direct relaxant effects on airway smooth muscle, we evaluated the effects of these inhibitors on histamine-, LTD₄-, or KCl-induced bronchial ring contraction. All these inhibitors, when used alone or in combination, failed to inhibit bronchial contraction induced by 30 µM histamine, 0.1 µM LTD₄, or 60 mM KCl (Figures 3A-3C and 4A). Compared with salbutamol, these inhibitors also failed to revert the anaphylactically contracted bronchi to any significant extent (Figures 3D and 4B). These findings indicate that their inhibitory effects on anaphylactic contraction were not a result of any potential nonselective activities such as receptor antagonism or inhibition of voltage-dependent calcium channels. However, PD 098059 markedly potentiated (p < 0.05) bronchial contraction induced by histamine and KCl by 17.6% and 17.8%, respectively (Figures 3A and 3C). Potentiation of histamine-induced bronchial contraction (36.2%) was also observed when PD 098059 and LY294002 were used together (Figure 4A). These observations imply that inhibition of MAPK pathway in airway smooth muscle could lead to enhancement of agonist-induced contraction.

View larger version (16K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   Figure 3.   Effects of inhibitors of tyrosine kinase signaling cascade on (A) 30 µM histamine-, (B) 0.1 µM LTD4-, or (C ) 60 mM KCl-induced bronchial contraction. A DMSO control was carried out in parallel with each experiment using an inhibitor. Each point represents the mean ± SEM of 3 to 6 experiments. *Significant difference from DMSO controls, p < 0.05. (D) Reversal effects of inhibitors of tyrosine kinase signaling cascade on existing anaphylactically contracted bronchi. DMSO and salbutamol (1 µM) were used in parallel with the inhibitors as negative and positive controls respectively. Gen = genistein; T47 = tyrphostin 47; PD = PD 098059; LY = LY294002; Sal = salbutamol.

View larger version (15K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 4.   (A) Effects of combined MAPK and PI3K inhibition on 30 µM histamine-, 0.1 µM LTD4-, or 60 mM KCl-induced bronchial contraction. A DMSO control was carried out in parallel with each experiment using the inhibitors. *Significant difference from DMSO vehicle controls, p < 0.05. (B) Reversal effects of combined MAPK kinase and PI3K inhibition on existing anaphylactically contracted bronchi. DMSO and salbutamol (1 µM) were used in parallel with the inhibitors as negative and positive controls respectively. PD/LY, PD 098059 (30 µM) + LY294002 (10 µM); Sal = salbutamol.

Effects of Inhibitors on the Release of Mediators

There were low levels of histamine (81.9 ± 10.1 ng/g tissue) and peptidoleukotrienes (< 1 ng/g tissue) released from the chopped lung preparations. Upon OA challenge of the lung fragments, the release of histamine increased by 40-fold (3,309.2 ± 686.9 ng/g tissue, n = 10) and that of LT increased by 22-fold (22.4 ± 3.3 ng/g tissue, n = 8). OA-induced histamine release from lung fragments was reduced by genistein by 36.1%, tyrphostin 47 by 18.6%, PD 098059 by 40.8%, LY294002 by 27.1%, and combined PD 098059 and LY294002 by 28.3% (Figures 5A and 6A). In contrast, the OA-induced peptidoleukotriene release from lung fragments was substantially inhibited by genistein by 49.0%, tyrphostin 47 by 84.1%, PD 098059 by 72.9%, LY294002 by 39.0%, and combined PD 098059 and LY294002 by 76.3% (Figures 5B and 6B). Overall, the inhibitors of the tyrosine kinase signaling cascade demonstrated greater inhibition on peptidoleukotriene release than on histamine release from lung fragments. To determine if these inhibitors possess other activity such as inhibition of 5-lipoxygenase, we examined the effects of these inhibitors on OA-induced peptidoleukotriene release in the presence of 70 µM exogenous AA (Figures 5C and 6B). AA alone triggered a minor release of peptidoleukotrienes from lung fragments (31.8%, n = 8) compared with 100% release induced by OA challenge. All the inhibitors failed to block the AA-induced release of peptidoleukotrienes, indicating that their inhibitory effects on OA-induced peptidoleukotriene release were not mediated by inhibition of 5-lipoxygenase. When OA and AA were added together to the lung fragments, there was an additive effect on the release of peptidoleukotrienes (152.0 ± 12.0%, n = 8). Genistein (30 µM), tyrphostin 47 (50 µM), PD 098059 (30 µM), LY294002 (10 µM), or combined PD 098059 and LY294002 markedly lessened (p < 0.05) the component of peptidoleukotriene release induced by OA by 44.1, 83.4, 74.7, 59.7, and 82.6%, respectively.

View larger version (18K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 5.   Effects of inhibitors of tyrosine kinase signaling cascade on the release of histamine (A) or peptidoleukotrienes in the absence (B) and presence (C ) of 70 µM AA from OA-stimulated guinea pig chopped lung preparations. In the studies of peptidoleukotriene release, inhibitors were incubated with the lung fragments in the presence of 4 µM indomethacin and 5 mM L-cysteine for 30 min before OA, AA, or combined OA and AA stimulation. Each point represents the mean ± SEM of four experiments. *Significant difference from DMSO controls, p < 0.05. Gen = genistein; T47 = tyrphostin 47; PD = PD 098059; LY = LY294002.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Genistein inhibits PTK activity of the epidermal growth factor receptor and pp60^v-src through competitive inhibition at the ATP-binding domain of the kinases (21). Tyrphostin 47 (RG50864), a derivative of the dihydroxybenzylidene malononitrile class of PTK inhibitors, acts by competitive inhibition at the substrate site of the kinase (22). Their relative potencies against nontransmembrane PTKs (e.g., Lyn, Syk, Btk, Jak, and Fer) remain to be elucidated. PD 098059 is a selective MAPK kinase inhibitor and is reported to have no significant direct inhibitory effect on other serine/threonine kinases such as p42^MAPK, Jun kinase, and myosin light chain kinase (24, 25). LY294002, a compound synthesized by modification of the broad-spectrum tyrosine kinase inhibitor quercetin, has been identified as a highly specific inhibitor of PI3K. It has been shown to block FcRI-mediated signal transduction in RBL-2H3 cells and the subsequent mast cell degranulation (6, 9, 23).

The present study confirmed and extended our previous findings (16) that genistein (30 µM) and tyrphostin 47 (50 µM) significantly suppressed (p < 0.05) OA-induced anaphylactic contraction of the bronchi (Figure 1A) and the release of histamine and peptidoleukotrienes from chopped lung fragments (Figure 5). Both inhibitors did not alter histamine-, LTD₄-, or KCl-induced bronchial contractions (Figures 3A to 3C). However, at higher concentration (50 µM), genistein has been shown to attenuate LTD₄-induced bronchial contraction in our previous study (16). On the other hand, our data showed that these inhibitors blocked the release of peptidoleukotrienes more efficiently than that of histamine from the lung fragments, which may account for the greater relaxation observed in the anaphylactically contracted bronchi (Figures 1B). The inability of genistein or tyrphostin 47 to completely abolish OA-induced anaphylactic responses of the guinea-pig airways may be due to the fact that these inhibitors are not Lyn- or Syk-specific and their inhibitory potencies on these PTKs remain uncertain (21, 22). Higher concentrations of genistein or tyrphostin 47 have been shown to produce nonselective activities such as inhibition of calcium and sodium channels (26). Alternatively, the possibility of additional signaling molecules other than Lyn and Syk being concurrently activated upon FcRI cross-linking in mast cells cannot be excluded. It would be important to reexamine antigen challenge of airways in vitro when Lyn- or Syk-specific inhibitors become available.

To determine the role of downstream MAPK and PI3K signaling pathways in mediating anaphylactic responses, we examined the inhibitory effect of PD 098059 and LY294002, respectively. Although PD 098059 (30 µM) demonstrated a major inhibition on OA-induced histamine release from chopped lung preparation (Figure 5A), it did not significantly attenuate OA-induced peak bronchial contraction, which is consistent with our previous findings (18). In the study to determine the effects of the inhibitors on direct agonist-induced bronchial contractions, PD 098059 was found to significantly potentiate histamine-induced bronchial contraction (Figure 3A). Similar potentiation was also observed when PD 098059 was used in combination with LY294002 (Figure 4A). Besides, there was also a marginal potentiation observed in KCl-induced bronchial contraction in the presence of PD 098059 (Figure 3C). These findings suggest that inhibition of MAPK kinase might produce a general enhancing effect on airway smooth muscle contraction in response to mediators such as histamine. Therefore, this may explain the phenomenon whereby PD 098059 significantly blocked OA-induced release of histamine but only slightly inhibited OA-induced peak bronchial contraction. On the other hand, the anaphylactically contracted bronchi relaxed more extensively in the presence of PD 098059 compared with the control (Figure 1B). This is likely to be related to its substantial inhibition of the release of peptidoleukotrienes as shown in the chopped lung preparations (Figure 5B).

LY294002 (10 µM) slightly attenuated (p < 0.05) the anaphylactic bronchial contraction (Figure 1A) with relatively weak yet significant inhibitory effects (p < 0.05) on the release of histamine and peptidoleukotrienes from chopped lung preparations as compared with the other inhibitors (Figure 5). At 10 µM, LY294002 did not alter mediator-induced bronchial contractions. At higher concentration (30 µM), LY294002 exhibited significant airway smooth muscle relaxant effects (data not shown). These findings imply that PI3K might not play a major role in mediating antigen-induced mast cell degranulation in vitro.

We have also examined the effect of dual inhibition of both MAPK kinase and PI3K by PD 098059 and LY294002, respectively, on antigen challenge of guinea-pig airways. As expected, combined PD 098059 and LY294002 additively inhibited OA-induced peak bronchial contraction by nearly 60% (Figure 2A) and markedly facilitated relaxation of the anaphylactically contracted bronchi (Figure 2B). Whereas the enhanced relaxation of the contracted bronchi to the combined treatment is likely to be associated with the profound inhibition of the release of peptidoleukotrienes, the potent inhibition on OA-induced bronchial peak contraction was not accompanied by any enhanced inhibition of histamine release (Figure 6). These observations indicate that the peptidoleukotrienes are important not only in sustaining the plateau phase of the anaphylactic contraction, but also in contributing to the initial peak phase of the contractile response. On the other hand, combined treatment did not manifest any direct inhibitory effect on mediator-induced bronchial contractions. These findings raise the possibilities that the PLC pathway, the other pathway that also contributes to FcRI-mediated mast cell degranulation, might play a more dominant role in the release of preformed mediators such as histamine from mast cells, whereas PI3K and MAPK pathways might be more important for the synthesis and release of peptidoleukotrienes.

View larger version (12K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 6.   Effect of combined MAPK kinase and PI3K inhibition on the release of histamine (A) or peptidoleukotrienes in the presence or absence (B) of 70 µM AA from OA-stimulated guinea pig chopped lung preparations. In the studies of peptidoleukotriene release, a cocktail of the two inhibitors was incubated with the lung preparations in the presence of 4 µM indomethacin and 5 mM L-cysteine for 30 min before OA, AA, or combined OA and AA stimulation. PD/LY, PD 098059 (30 µM) + LY294002 (10 µM). Each point represents the mean ± SEM of 4 to 6 experiments. *Significant difference from DMSO vehicle controls, p < 0.05.

Peptidoleukotrienes are synthesized by the action of 5-lipoxygenase (5-LO) on AA which is released from membrane phospholipids by the action of phospholipase A₂ (PLA₂) (29). It has been shown that MAPK can activate PLA₂ (5, 30, 31) and MAPK pathway can be stimulated by PI3K (32), indicating that both MAPK and PI3K may be able to alter the level of peptidoleukotrienes by regulating the activity of PLA₂. As such, it is important to determine whether these signal transduction inhibitors have any inhibitory effects on 5-LO by examining AA-induced release of peptidoleukotrienes from lung fragments. All inhibitors, used alone or in combination, did not show any inhibition on AA-triggered release of peptidoleukotrienes (Figures 5C and 6B). These findings indicate that their inhibitory effects on anaphylactic responses are not mediated by other nonspecific activity such as inhibition of 5-LO.

Mast cell-derived mediators such as histamine and peptidoleukotrienes produce a plethora of pathological effects in the airways, including bronchoconstriction, plasma exudation, mucus secretion, enhanced neural sensitivity, and recruitment and activation of inflammatory cells. In addition, they also contribute to the development of airway hyperresponsiveness and airway smooth muscle hyperplasia (29, 33, 34). The detailed signaling mechanisms mediating FcRI-induced mast cell degranulation have been unraveled and a signal transduction-based approach offers a specific strategy to block mast cell activation. Our present findings show that inhibitors of PTKs, MAPK kinase, and PI3K attenuated the anaphylactic bronchial contraction and facilitated relaxation of constricted airways by preventing the release of inflammatory mediators such as histamine and peptidoleukotrienes. To further explore the therapeutic potential of inhibitors of the tyrosine kinase signaling cascade for the treatment of asthma, examination of this class of inhibitors in an in vivo model of allergic asthma is recommended.