Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

1465-9921-6-85 RRJ Research Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction Schaafsma Dedmer d.schaafsma@rug.nl Gosens Reinoud r.gosens@rug.nl Bos T I Sophie i.s.t.bos@rug.nl Meurs Herman h.meurs@rug.nl Zaagsma Johan j.zaagsma@rug.nl Nelemans S Adriaan s.a.nelemans@rug.nl

Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Respiratory Research 1465-9921 2005 6 1 85 http://respiratory-research.com/content/6/1/85 1604864710.1186/1465-9921-6-85 13 5 2005 27 7 2005 27 7 2005 2005 Schaafsma et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.

Methods

Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F_2α(PGF_2α) and prostaglandin E₂(PGE₂) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP₁-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF_2α-and PGE₂-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.

Results

Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF_2α-and PGE₂-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF_2α-and PGE₂-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP₁-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.

Conclusion

The results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.

Background

Growth factors have been reported to be involved in proliferation and differentiation of smooth muscle cells from a variety of tissues, including vasculature and airways 12. In addition, several growth factors have been shown to induce contraction of vascular smooth muscle 34. The mechanisms by which growth factors induce contraction have only been partly elucidated. Recent evidence has indicated that growth factor-receptors, such as the insulin-like growth factor-1 (IGF-1)-receptor, can activate the Rho/Rho-kinase pathway directly 5 and may be involved in smooth muscle contraction via Rho-kinase 6. Smooth muscle contraction is mainly regulated by the phosphorylation level of the 20 kDa regulatory myosin light chain (MLC) 7. MLC phosphorylation can be initiated by an increase in intracellular Ca²⁺-concentration ([Ca²⁺]_i) followed by the Ca²⁺-calmodulin-dependent activation of myosin light chain kinase (MLCK). The extent of MLC phosphorylation is determined by the ratio of MLCK (MLC-phosphorylation) to myosin light chain phosphatase (MLCP)(MLC-dephosphorylation) activities 8. Activated Rho-kinase mainly exerts its effect through inhibition of MLCP, resulting in an enhanced MLC phosphorylation and thus an increased level of contraction at a fixed [Ca²⁺]_i(Ca²⁺-sensitization) 69.

In bovine airway smooth muscle, it has been demonstrated that prolonged incubation with growth factors modulates the phenotypic state of the muscle 1011. They have also been described to exert acute contractile effects on guinea pig tracheal smooth muscle 1213. Recently, we showed that growth factors are also capable of inducing human bronchial smooth muscle contraction. Thus, angiotensin II as well as IGF-1 induced a sustained contraction, which was completely dependent on Rho-kinase 14.

These observations may be of pathophysiological and pharmacotherapeutical interest, as expression levels both of growth factors (EGF)15 and of receptors of growth factors (EGF15, PDGF1516) have been found elevated in asthmatic airways. Also, increased levels of PDGF have been found in exhaled breath condensate of asthmatic children with severe airflow limitation 17. Moreover, previous studies showed an augmented role of Rho-kinase in acetylcholine induced bronchial smooth muscle contraction after repeated allergen challenge in rats 1819. Furthermore, we have recently demonstrated that the process of active allergic sensitization by itself, without subsequent allergen exposure, is sufficient to induce an enhanced role of Rho-kinase in guinea pig airway smooth muscle contraction ex vivo and airway resistance in vivo 20. Therefore, a better understanding of the mechanisms by which growth factors induce a Rho-kinase dependent contraction is of pathophysiological and pharmacotherapeutical interest.

Epidermal growth factor (EGF) causes contraction of guinea pig tracheal smooth muscle via arachidonic acid metabolism in which presumably a tyrosine kinase and phospholipase A₂are involved 1213. It is well documented that receptor tyrosine kinases can activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)-kinase (MEK)212223. Activation of MAPK by MEK may result in the activation of cytosolic phospholipase A₂(cPLA₂) 24 and subsequent production of arachidonic acid and prostaglandins. Several studies have demonstrated that contractile prostaglandins are dependent on Rho-kinase 2025. Altogether, it can be hypothesized that growth factor-induced contraction is mediated via the MEK-dependent, cPLA₂-mediated production of prostaglandins and subsequent activation of Rho-kinase. Therefore, we investigated the effects of inhibition of Rho-kinase, MEK and cyclooxygenase (COX) on growth factor-induced prostaglandin-production and contraction, using guinea pig tracheal smooth muscle preparations. In addition, we investigated the effects of selective prostaglandin receptor antagonists on growth factor-induced contraction.

Methods

Animals

Outbred specified pathogen-free male Dunkin Hartley guinea pigs (Harlan, Heathfield, U.K.), weighing 500–700 g, were used in this study. All protocols described in this study were approved by the University of Groningen Committee for Animal Experimentation.

Isometric tension measurements

After experimental concussion and rapid exsanguination the trachea was removed and transferred to Krebs-Henseleit (KH) buffer solution (composition in mM: NaCl 117.5, KCl 5.6, MgSO₄1.18, CaCl₂2.5, NaH₂PO₄1.28, NaHCO₃25.00 and D-glucose 5.55; pregassed with 95% O₂and 5% CO₂; pH 7.4) at 37°C. The trachea was carefully prepared free of serosa and connective tissue. In some cases, the airway epithelium was carefully removed by moving a 15-cm woollen thread up and down the trachea twice. Epithelium denudation was confirmed by histological examination after fixating cryostat sections (5 μm) in acetone and staining with hematoxylin eosin. Single open-ring tracheal preparations were prepared and mounted for isometric recording, using Grass FT-03 transducers, in 20 ml water-jacketed organ baths (37°C) containing KH solution. During a 90 min equilibration period, with washouts every 30 min, resting tension was gradually adjusted to 0.5 g. Subsequently, the preparations were precontracted with 20 and 40 mM KCl. Following two wash-outs, maximal relaxation was established by the addition of 0.1 μM isoprenaline and tension was re-adjusted to 0.5 g, immediately followed by two changes of fresh KH-buffer. After another equilibration period of 30 min EGF (0.1, 1, 3, 10 or 30 ng/ml) or PDGF (0.1, 1, 3, 10 or 30 ng/ml) was applied or cumulative concentration response curves (CRCs) were constructed to stepwise increasing concentrations of histamine (1 nM – 100 μM), PGE₂(1 nM – 3μM) or PGF_2α(1 nM – 10 μM). When maximal agonist-induced contraction was obtained, the tracheal rings were washed several times and maximal relaxation was established using isoprenaline. When used, the inhibitors of Rho-kinase (Y-27632, 1 μM), MAPK-ERK-kinase (MEK) (U-0126, 3 μM) or COX (indomethacin, 3 μM) were applied to the organ bath 30 min before agonist addition. This was also the case for the FP-receptor-and EP₁-receptor-antagonists AL-8810 and AH-6809 (10 μM both, applied individually to separate preparations), respectively.

Measurement of prostaglandin F_2αand prostaglandin E₂production

Guinea pig tracheal rings were incubated using a 24-wells plate at 37°C. Each well contained 1 ml KH-buffer and 7 tracheal rings. Twenty-one rings were isolated from every trachea, so three conditions per preparation could be tested. Following a 30 min pre-incubation period, 100 μl of the medium was taken as the first sample. Subsequently, PDGF (10 ng/ml) was applied. To determine the time dependency of prostaglandin (PG)-production, samples were collected at 5, 10, 15, 20 and 30 min after PDGF-addition. Sampling was performed under a 95 % O₂/ 5 % CO₂atmosphere. PGF2α-and PGE₂-production was determined using an ELISA-assay according to the manufacturer's protocol (R&D Systems, U.K.).

Data analysis

All data represent means ± s.e. mean from n separate experiments. Statistical significance of differences was evaluated using either a one way analysis of variance (ANOVA) followed by a Bonferroni post-test or by a paired or unpaired two-tailed Student's t-test when appropriate, and significance was accepted when P<0.05.

Chemicals

Platelet-derived growth factor AB (PDGF-AB, human recombinant) was from Bachem (Bubendorf, Switzerland) and epidermal growth factor (human recombinant), indomethacin, histamine dihydrochloride and (-)-isoprenaline hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). PGF_2αwas obtained from Pharmacia and Upjohn (Puurs, Belgium) and PGE₂was from BIOMOL (U.S.A). 1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene (U-0126), (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) and 6-isopropoxy-9-xanthone-2-carboxylic acid (AH-6809) were obtained from Tocris Cookson Ltd. (Bristol, U.K.). 9α, 15R-dihydroxy-1 1β-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16, 17, 18, 19, 20-pentanor-prosta-5Z, 13E-dien-1-oic acid (AL-8810) was obtained from Cayman Chemical (U.S.A). All other chemicals were of analytical grade.

Results

To investigate the contractile effects of EGF and PDGF on guinea pig tracheal smooth muscle, CRCs of the growth factors were constructed (Fig. 1). Both EGF and PDGF were capable of inducing concentration-dependent contractions, with a potency (EC₅₀) of 6.7 ± 2.3 ng/ml for EGF and 6.4 ± 2.8 ng/ml for PDGF. As shown in Fig. 2, both growth factors induced a slowly developing sustained contraction, which was prevented almost completely in the presence of either Y-27632 (1 μM), U-0126 (3 μM) or indomethacin (3 μM). Also, basal myogenic tone (expressed with respect to maximal relaxation established with isoprenaline) was abolished by these inhibitors (Fig. 2).

Figure 1

EGF (A)-and PDGF (B)-induced contraction of guinea pig open-ring tracheal smooth muscle preparations

EGF (A)-and PDGF (B)-induced contraction of guinea pig open-ring tracheal smooth muscle preparations. Responses shown are corrected for basal myogenic tone, which amounted to 0.21 ± 0.06 g on average (0 % growth factor effect). Maximal effects were reached at concentrations of 30 ng/ml and amounted 0.31 ± 0.04 g (EGF, 100 % effect) and 0.24 ± 0.06 g (PDGF, 100 % effect), corresponding to 19.8 ± 2.8 % and 15.3 ± 4.1 %, respectively, of maximal histamine-induced contraction. Data represent means ± s.e. mean of seven (EGF) or four (PDGF) experiments, each performed in duplicate.

Figure 2

Effects of Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM) on (A) EGF (10 ng/ml)-and (B) PDGF (10 ng/ml)-induced guinea pig trachealsmooth muscle contraction

Effects of Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM) on (A) EGF (10 ng/ml)-and (B) PDGF (10 ng/ml)-induced guinea pig trachealsmooth muscle contraction. Data represent means ± s.e. mean of five (EGF) or six (PDGF) experiments, each performed in duplicate.

Since both MEK-(U-0126) and COX-inhibition (indomethacin) prevented growth factor-induced contraction, we envisaged that growth factor-induced prostaglandin production would be responsible for the observed contractions.

Stimulation of tracheal smooth muscle preparations with PDGF for 30 min greatly enhanced the release of prostaglandin E₂(PGE₂) by 255 ± 78 % (from 963 ± 245 to 2762 ± 138 pg/ml; p < 0.01 at t = 30 min; Fig. 3A) and prostaglandin F_2α(PGF_2α) by 182 ± 38 % (from 1093 ± 204 to 2929 ± 570 pg/ml; p < 0.05 at t = 30 min; Fig. 3B). As shown in Fig. 4, both the release of PGE₂and PGF_2αwere significantly reduced in the presence of U-0126 (3 μM) and indomethacin (3 μM). In contrast to growth factor-induced contraction, no significant effect of treatment with Y-27632 (1 μM) was found on PGE₂(p = 0.23) or PGF_2α(p = 0.08) release. These findings would suggest that prostaglandins produced in response to growth factor stimulation are capable of inducing a Rho-kinase-dependent contraction. Application of PGE₂caused ASM contraction in concentrations up to 0.03 μM (pEC₅₀= 8.22 ± 0.07, E_max= 58.3 ± 11.2 %), but caused relaxation in higher concentrations (Fig. 5A). Indeed, Rho-kinase inhibition resulted in a decreased potency (pEC₅₀= 7.9 ± 0.2; p < 0.05) and maximal contraction (E_max= 11.7 ± 3.5 %; p < 0.05) of PGE₂-induced contraction. PGF_2α-induced contractions (pEC₅₀= 6.8 ± 0.2; E_max= 71.9 ± 8.2 %) were dependent on Rho-kinase as well, as indicated by the significantly decreased potency (pEC₅₀= 6.2 ± 0.2 ; p < 0.05) and maximal contraction (E_max= 41.8 ± 9.3 %; p < 0.05) after treatment with Y-27632 (Fig. 5B).

Figure 3

Growth factor-induced PGE₂(A) and PGF_2α(B) release from guinea pig tracheal smooth muscle preparations

Growth factor-induced PGE₂(A) and PGF_2α(B) release from guinea pig tracheal smooth muscle preparations. Basal release amounted to 963 ± 245 pg/ml (PGE₂) and 1093 ± 204 pg/ml (PGF_2α). Data represent means ± s.e.mean of five experiments.

Figure 4

Effects of U-0126 (3 μM), indomethacin (3 μM) and Y-27632 (1 μM) on growth factor-induced PGE₂(A) and PGF_2α(B) release. Data represent means ± s.e.mean of six (PGE₂) and five (PGF_2α) experiments. *p < 0.05, **p < 0.01 compared to PDGF.

Figure 5

Effects of Rho-kinase inhibition on prostaglandin-induced contraction

Effects of Rho-kinase inhibition on prostaglandin-induced contraction. PGE₂(A)-and PGF_2α(B)-induced contraction in the absence and presence of Y-27632 (1 μM) of guinea pig open-ring tracheal smooth muscle preparations. Data represent means ± s.e.mean of four (PGE₂) and seven (PGF_2α) experiments, each performed in duplicate.

To establish the functional contribution of the contractile PGE₂-sensitive EP₁-receptor and the PGF_2α-sensitive FP-receptor to growth factor-induced contraction, the selective EP₁-receptor antagonist AH-6809 (10 μM) and the selective FP-receptor antagonist AL-8810 (10 μM) were used. Both EGF-and PDGF-induced contractions were significantly reduced after treatment with AL-8810 (46,7 ± 13.0 % and 52.7 ± 13.2 % inhibition, respectively; p < 0.01 both), whereas contractions were almost abolished after treatment with AH-6809 (95.1 ± 3.1 % and 94.4 ± 4.7 % inhibition, respectively; p < 0.001 both)(Fig. 6A,B). To determine whether the epithelium was the source of the prostaglandins involved in growth factor-induced contraction, the effects of AL-8810 and AH-6809 on epithelium-denuded tracheal preparations were studied. Complete denudation was achieved as illustrated in Fig. 7. In these preparations, PDGF induced a slightly higher contraction compared to that in intact preparations, however the difference was not significant. Similar to intact preparations, PDGF-induced contraction was significantly reduced by both AL-8810 (48.8 ± 7.1 % inhibition; p < 0.05; Fig. 6C) and AH-6809 (92.1 ± 3.0 % inhibition; p < 0.01); Fig. 6C). Moreover, the inhibition in denuded preparations was very similar to that in intact preparations, both for AL-8810 and AH-6809, indicating that FP-and EP₁-receptor stimulation involved in growth factor-induced contraction occurs independently of epithelium.

Figure 6

EGF (10 ng/ml, A)-and PDGF (10 ng/ml, B,C)-induced contraction of intact (A,B) and epithelium-denuded (C) guinea pig open-ring tracheal smooth muscle preparations in the absence or presence of AL-8810 (10 μM) or AH-6809 (10 μM). Data represent means ± s.e. mean of five (A,B) and three (C) experiments, each performed in duplicate. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control.

Figure 7

Representative photomicrograph of an intact (A) and epithelium-denuded (B) tracheal preparation

Representative photomicrograph of an intact (A) and epithelium-denuded (B) tracheal preparation. The photographs were taken at 100 × magnification.

Discussion

In this study we demonstrate that the growth factors EGF and PDGF induce contractions of guinea pig tracheal smooth muscle in a concentration dependent fashion. The concentration-effect range of EGF and PDGF (0.1 – 30 ng/ml) represents a pharmacological range very similar to other effects, such as mitogenesis of airway smooth muscle 2610. Since contractile effects of EGF have previously been associated with the production of eicosanoids 13 and contractions induced by of IGF-1 and angiotensin II appeared to be dependent on Rho-kinase 1314, we analyzed whether contractions induced by submaximal concentrations of growth factors are dependent not only on Rho-kinase, but also on COX and MEK. This might be characteristic for growth factor-induced contraction, since potency and maximal contraction of histamine were shown to be independent of Rho-kinase, COX 20 and MEK (Schaafsma et al, unpublished observations) in guinea pig tracheal smooth muscle. Similarly, muscarinic receptor mediated contractions are only partially Rho-kinase-dependent 2728, further illustrating the agonist-dependent role of Rho-kinase mediated calcium sensitization.

The role of Rho-kinase in growth factor-mediated effects could depend on the duration of growth factor stimulation. For instance, phenotypic modulation, as a consequence of 8 days stimulation with growth factors, or growth factor-induced proliferation of bovine tracheal smooth muscle, has been shown to be independent of Rho-kinase. However, in accordance with the effects of Rho-kinase inhibition on growth factor-induced contraction of human isolated bronchus 14, we demonstrate that Y-27632 fully inhibits growth factor-induced contraction of guinea pig tracheal smooth muscle. This indicates that growth factor-induced acute (smooth muscle contraction) and long term (e.g. modulation of smooth muscle phenotype) effects in airway smooth muscle may be differentially dependent on Rho-kinase.

Since MEK and COX inhibition almost abrogated growth factor-induced contraction, it can be suggested that growth factor-induced contraction relies on the production of prostaglandins. In several studies, it has been demonstrated that cytosolic phospholipase A₂(PLA₂) can be activated in response to growth factors in a MAPK-dependent fashion, which results in subsequent arachidonic acid production 293031. In addition, contractile activity of EGF in guinea pig tracheal smooth muscle has been reported to be inhibited by indomethacin and by the phospholipase A₂inhibitor mepacrine 12. As indicated by our results, PGF_2αand PGE₂are being produced in response to PDGF-stimulation in a time-dependent fashion, similar to that of growth factor-induced contraction. Both prostaglandins are contractile agonists for airway smooth muscle 203233. Contractions induced by (exogenous) PGF_2αand PGE₂were found to be largely dependent on Rho-kinase activity, which corresponds to observations in vascular smooth muscle 2534, indicating that Rho-kinase plays an essential role in PGF_2α-and PGE₂-induced contractions. Interestingly, Rho-kinase inhibition had a more pronounced effect on PGE₂-than on PGF_2α-induced contractions. This can be explained, however, by realizing that the EP₂-receptor mediated relaxation 35, as seen with the higher PGE₂-concentrations, is suppressing the contractile phase more effectively when its Rho-kinase-dependent component is being inhibited.

In addition to direct contractile effects on guinea pig airway smooth muscle, PGF_2αhas been shown to augment cholinergic responsiveness of bovine airway smooth muscle 36, indicating an important role for PGF_2αin regulating airway smooth muscle tone. PGF_2αhas been described to exert its contractile effects on smooth muscle through the FP-receptor 3738. Also, PGF_2α-induced Ca²⁺-mobilization in vascular smooth muscle cells was dose-dependently inhibited by the selective FP-receptor antagonist AL-8810 39. In our study, a selective and effective concentration of AL-8810 4039 reduced EGF-and PDGF-induced contractions, indicating that PGF_2αcontributes to growth factor-induced contraction through the FP-receptor.

Smooth muscle contractions induced by PGE₂are predominantly mediated through activation of the EP₁-receptor 4132. In guinea pig airway smooth muscle it has been previously found that PGE₂-induced contractions could be dose-dependently inhibited by the EP₁-receptor antagonist SC-19220 without modulating the relaxant activity (Van Amsterdam, 1991). Also, like PGF_2α, PGE₂enhances cholinergic airway responsiveness of bovine airway smooth muscle 36. In the present study we found that growth factor-induced contraction of guinea pig tracheal smooth muscle is essentially dependent on EP₁-receptor stimulation, since the selective EP₁-receptor antagonist AH-6809 36 abrogated growth factor-induced contractions. Interestingly, these contractions were partially inhibited by FP-receptor blockade as well. From these observations, it may be hypothesized that PGF_2α-mediated contractions partially rely on EP₁-receptor stimulation (possibly by releasing small amounts of PGE₂, selectively activating EP₁-receptors) and that synergistic contractile effects of concomitant EP₁-and FP-receptor stimulation occur.

Several growth factors, including EGF and PDGF, have been implicated in airway inflammation as they can be released from inflammatory cells, such as macrophages and eosinophils. Moreover, they can be derived from extravasated plasma, epithelial cells and the airway smooth muscle itself 242. Growth factors are involved in tissue repair processes, therefore growth factor-induced contraction could protect damaged areas in the airways from the environment during these processes. In the pathophysiology of asthma, the repair process is usually not restricted to a single segment of the airways and growth factors may then contribute to airflow obstruction. Inhibition of such contractions might therefore be relevant under such pathophysiological conditions.

Conclusion

Our overall results indicate that EGF and PDGF induce airway smooth muscle contraction through contractile prostaglandins. These prostaglandins are presumably produced by the consecutive actions of MEK, cytosolic PLA₂and COX and in turn are dependent on Rho-kinase for their contractile effects (Fig. 8). Since growth factor-induced contractions were inhibited by antagonists of contractile prostaglandin receptors both in intact and epithelium-denuded preparations, it can be concluded that the prostaglandins involved in growth factor-induced contraction are not primarily derived from the epithelium. Since both growth factors and increased Rho-kinase activity are associated with pathophysiological conditions and growth factor-induced contraction is fully Rho-kinase dependent, inhibition of Rho-kinase might be of therapeutical interest in the treatment of inflammatory (airway) diseases.

Figure 8

Putative mechanism of growth factor-induced airway smooth muscle contraction

Putative mechanism of growth factor-induced airway smooth muscle contraction. Growth factors, like EGF and PDGF, bind to their receptors with intrinsic tyrosine kinase activity (RTK) and activate MAPK, which may result in increased levels of arachidonic acid (AA) via cytosolic phospholipase A₂(cPLA₂) activation. As a consequence of cyclooxygenase (COX)-mediated conversion of AA, prostaglandins (PGs) are produced. These (contractile) prostaglandins, like PGF_2αand PGE₂, may in turn couple to their receptors and induce an airway smooth muscle contraction which is largely dependent on Rho-kinase. U-0126, indomethacin (indo) and Y-27632 are inhibitors of MAPK, COX and Rho-kinase, respectively.

Abbreviations

AA, arachidonic acid; AHR, airway hyperresponsiveness; ASM, airway smooth muscle; COX, cyclooxygenase; cPLA₂, cytosolic phospholipase A₂; CRC, cumulative concentration response curve; EGF, epidermal growth factor; EP₁-receptor, prostaglandin E₂-receptor type 1; FP-receptor, prostaglandin F_2α-receptor; IGF-1, insulin-like growth factor-1; Indo, indomethacin; KH, Krebs-Henseleit; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase (MEK); pEC₅₀, -log₁₀of the concentration causing 50 % of the effect; PDGF, platelet-derived growth factor; PG, prostaglandin; PGE₂, prostaglandin E₂; PGF_2α, prostaglandin F_2α; RTK, receptors with intrinsic tyrosine kinase activity

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

DS designed and coordinated the study, performed a major part of the experiments, performed the statistical analysis and drafted the manuscript. RG participated in the design of the study, assisted in performing part of the experiments and contributed to the preparation of the manuscript. ISTB substantially assisted in performing the experiments. HM participated in the design of the study and the interpretation of the results. JZ participated in the design of the study, interpretation of results and final revision of the manuscript. SAN supervised the study, participated in its design and in the preparation of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank the Netherlands Asthma Foundation for financial support (grant 01.83).

The insulin-like growth factor axis: A review of atherosclerosis and restenosis Bayes-Genis A Conover CA Schwartz RS Circ Res 2000 86 125 130 10666406 Airway smooth muscle as a target in asthma Hirst SJ Clin Exp Allergy 2000 30 Suppl 1 54 59 10.1046/j.1365-2222.2000.00099.x 10849477 Tyrphostin attenuates platelet-derived growth factor-induced contraction in aortic smooth muscle through inhibition of protein tyrosine kinase(s) Sauro MD Thomas B J Pharmacol Exp Ther 1993 267 1119 1125 8263773 Vasoconstriction: a new activity for platelet-derived growth factor Berk BC Alexander RW Brock TA Gimbrone MAJ Webb RC Science 1986 232 87 90 3485309 Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF Taya S Inagaki N Sengiku H Makino H Iwamatsu A Urakawa I Nagao K Kataoka S Kaibuchi K J Cell Biol 2001 155 809 820 10.1083/jcb.200106139 11724822 Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells Fukata Y Amano M Kaibuchi K Trends Pharmacol Sci 2001 22 32 39 10.1016/S0165-6147(00)01596-0 11165670 Invited review: regulation of myosin phosphorylation in smooth muscle Pfitzer G J Appl Physiol 2001 91 497 503 11408468 Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase SOMLYO ANDREWP SOMLYO AVRILV Physiol Rev 2003 83 1325 1358 14506307 Rho/Rho-kinase mediated signaling in physiology and pathophysiology Wettschureck N Offermanns S J Mol Med 2002 80 629 638 10.1007/s00109-002-0370-2 12395147 Functional characterization of serum- and growth factor-induced phenotypic changes in intact bovine tracheal smooth muscle Gosens R Meurs H Bromhaar MM McKay S Nelemans SA Zaagsma J Br J Pharmacol 2002 137 459 466 10.1038/sj.bjp.0704889 12359627 Insulin induces a hypercontractile airway smooth muscle phenotype Gosens R Nelemans SA Hiemstra M Grootte Bromhaar MM Meurs H Zaagsma J Eur J Pharmacol 2003 481 125 131 10.1016/j.ejphar.2003.08.081 14637184 Contraction of guinea pig trachea by epidermal growth factor--urogastrone Patel P Itoh H Lederis K Hollenberg MD Can J Physiol Pharmacol 1988 66 1308 1312 3149204 Mechanisms of epidermal growth factor-induced contraction of guinea pig airways Nasuhara Y Munakata M Sato A Amishima M Homma Y Kawakami Y Eur J Pharmacol 1996 296 161 168 10.1016/0014-2999(95)00692-3 8838452 Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent Gosens R Schaafsma D Grootte Bromhaar MM Vrugt B Zaagsma J Meurs H Nelemans SA Eur J Pharmacol 2004 494 73 76 10.1016/j.ejphar.2004.04.035 15194453 Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway Amishima M Munakata M Nasuhara Y Sato A Takahashi T Homma Y Kawakami Y Am J Respir Crit Care Med 1998 157 1907 1912 9620926 Airway fibroblasts exhibit a synthetic phenotype in severe asthma Lewis CC Chu HW Westcott JY Tucker A Langmack EL Sutherland ER Kraft M J Allergy Clin Immunol 2005 115 534 540 10.1016/j.jaci.2004.11.051 15753901 Analysis of Growth Factors and Inflammatory Cytokines in Exhaled Breath Condensate from Asthmatic Children Leung TF Wong GW Ko FW Li CY Yung E Lam CW Fok TF Int Arch Allergy Immunol 2005 137 66 72 10.1159/000085106 15832052 Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats Chiba Y Takada Y Miyamoto S MitsuiSaito M Karaki H Misawa M Br J Pharmacol 1999 127 597 600 10.1038/sj.bjp.0702585 10401547 Enhanced Ca2+ sensitization of the bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats Chiba Y Sakai H Suenaga H Kamata K Misawa M Res Commun Mol Pathol Pharmacol 1999 106 77 85 11127810 Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction Schaafsma D Gosens R Bos IS Meurs H Zaagsma J Nelemans SA Br J Pharmacol 2004 143 477 484 10.1038/sj.bjp.0705903 15381630 The EGF receptor as central transducer of heterologous signalling systems Zwick E Hackel PO Prenzel N Ullrich A Trends Pharmacol Sci 1999 20 408 412 10.1016/S0165-6147(99)01373-5 10577253 Signaling from G-protein-coupled receptors to mitogen-activated protein (MAP)-kinase cascades Lopez-Ilasaca M Biochem Pharmacol 1998 56 269 277 10.1016/S0006-2952(98)00059-8 9744561 Beyond Herceptin and Gleevec Fischer OM Streit S Hart S Ullrich A Curr Opin Chem Biol 2003 7 490 495 10.1016/S1367-5931(03)00082-6 12941424 cPLA2 is phosphorylated and activated by MAP kinase Lin LL Wartmann M Lin AY Knopf JL Seth A Davis RJ Cell 1993 72 269 278 10.1016/0092-8674(93)90666-E 8381049 Essential role of rho kinase in the Ca2+ sensitization of prostaglandin F(2alpha)-induced contraction of rabbit aortae Ito K Shimomura E Iwanaga T Shiraishi M Shindo K Nakamura J Nagumo H Seto M Sasaki Y Takuwa Y J Physiol 2003 546 823 836 10.1113/jphysiol.2002.030775 12563007 Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis Kelleher MD Abe MK Chao TS Jain M Green JM Solway J Rosner MR Hershenson MB Am J Physiol 1995 268 L894 L901 7611431 Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype Gosens R Schaafsma D Meurs H Zaagsma J Nelemans SA Eur J Pharmacol 2004 483 71 78 10.1016/j.ejphar.2003.10.027 14709328 Muscarinic excitation-contraction coupling mechanisms in tracheal and bronchial smooth muscles Janssen LJ Wattie J Lu-Chao H Tazzeo T J Appl Physiol 2001 91 1142 1151 11509509 Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells Margolis BL Holub BJ Troyer DA Skorecki KL Biochem J 1988 256 469 474 3146974 The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets Bornfeldt KE Campbell JS Koyama H Argast GM Leslie CC Raines EW Krebs EG Ross R J Clin Invest 1997 100 875 885 508260 9259587 Platelet-derived growth factor stimulates phospholipase C-gamma 1, extracellular signal-regulated kinase, and arachidonic acid release in rat myometrial cells: contribution to cyclic 3',5'-adenosine monophosphate production and effect on cell proliferation Boulven I Palmier B Robin P Vacher M Harbon S Leiber D Biol Reprod 2001 65 496 506 11466218 EP1 receptor blockade attenuates both spontaneous tone and PGE2-elicited contraction in guinea pig trachealis Ndukwu IM White SR Leff AR Mitchell RW Am J Physiol 1997 273 L626 L633 9316498 Beta-adrenoceptor responsiveness in non-allergic and allergic airways - An in-vitro approach Van Amsterdam RG 1991 68 69 Involvement of Rho-kinase in contraction of guinea-pig aorta induced by prostanoid EP3 receptor agonists Shum WW Le GY Jones RL Gurney AM Sasaki Y Br J Pharmacol 2003 139 1449 1461 10.1038/sj.bjp.0705393 12922932 Receptors and pathways mediating the effects of prostaglandin E2 on airway tone Tilley SL Hartney JM Erikson CJ Jania C Nguyen M Stock J McNeisch J Valancius C Panettieri RAJ Penn RB Koller BH Am J Physiol Lung Cell Mol Physiol 2003 284 L599 L606 12618422 Augmentation of bovine airway smooth muscle responsiveness to carbachol, KCl, and histamine by the isoprostane 8-iso-PGE2 Catalli A Janssen LJ Am J Physiol Lung Cell Mol Physiol 2004 287 L1035 L1041 10.1152/ajplung.00138.2004 15257985 International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes Coleman RA Smith WL Narumiya S Pharmacol Rev 1994 46 205 229 7938166 Prostaglandins and leukotrienes: advances in eicosanoid biology Funk CD Science 2001 294 1871 1875 10.1126/science.294.5548.1871 11729303 Real-time intracellular Ca2+ mobilization by travoprost acid, bimatoprost, unoprostone, and other analogs via endogenous mouse, rat, and cloned human FP prostaglandin receptors Kelly CR Williams GW Sharif NA J Pharmacol Exp Ther 2003 304 238 245 10.1124/jpet.102.042556 12490597 AL-8810: a novel prostaglandin F2 alpha analog with selective antagonist effects at the prostaglandin F2 alpha (FP) receptor Griffin BW Klimko P Crider JY Sharif NA J Pharmacol Exp Ther 1999 290 1278 1284 10454504 Characterization of prostanoid receptors mediating actions of the isoprostanes, 8-iso-PGE(2) and 8-iso-PGF(2alpha), in some isolated smooth muscle preparations Sametz W Hennerbichler S Glaser S Wintersteiger R Juan H Br J Pharmacol 2000 130 1903 1910 10.1038/sj.bjp.0703522 10952681 Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle McKay S Sharma HS Respir Res 2002 3 11 64809 11806846 10.1186/rr160