Human Bronchial Smooth Muscle Research Articles

Effect of dexamethasone on voltage-gated Na + channel in cultured human bronchial smooth muscle cells

Voltage-gated Na + channel ( I Na) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I Na, by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10 − 6 M) did not affect I Na. However, the percentage of the cells with I Na was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I Na adjusted by cell capacitance in cells with I Na was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that α and β subunits mRNA of I Na mainly consisted of SCN9A and SCN1β, respectively. Treatment with dexamethasone for 24–48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I Na encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor.

Inhibition of Glycogen Synthase Kinase-3β Is Sufficient for Airway Smooth Muscle Hypertrophy

We examined the role of glycogen synthase kinase-3beta (GSK-3beta) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3beta, each of which inhibit GSK-3beta activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-beta, a proasthmatic cytokine. GSK-3beta inhibition increased mRNA expression of alpha-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon) attenuated LiCl- and SB216763-induced protein synthesis and expression of alpha-actin and SM22, indicating that eIF2B is required for GSK-3beta-mediated airway smooth muscle hypertrophy. eIF2Bepsilon siRNA also blocked CT-1- but not TGF-beta-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3beta-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3beta, blocked protein synthesis and alpha-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-beta. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased alpha-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3beta phosphorylation. These data suggest that inhibition of the GSK-3beta/eIF2Bepsilon translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-beta-induced hypertrophy does not depend on GSK-3beta/eIF2B signaling.

Mechanisms by Which S-Albuterol Induces Human Bronchial Smooth Muscle Cell Proliferation

Background: Racemic albuterol is a 50:50 mixture of the R-isomer, levalbuterol, and the S-isomer, S-albuterol. S-Albuterol increases airway hyperresponsiveness to spasmogens, exacerbates asthmatic conditions and stimulates cell growth, whereas levalbuterol attenuates cell growth in culture. The mechanisms of S-albuterol-induced cell proliferation are not well understood. We studied the role of albuterol isomers and intracellular cell cycle regulators on proliferation of human bronchial smooth muscle cells. Methods: Serum-starved cells (72 h) were fed test agents for 24 h and cell proliferation was measured. The expression of nuclear factor-ĸB inhibitory protein IĸBα, nuclear factor-ĸB, cyclin-dependent kinases 2 and 4, interleukin (IL)-6, and retinoblastoma and platelet-activating factor (PAF) receptor protein were measured by Western blotting. Results:S-Albuterol, PAF and platelet-derived growth factor stimulated cell proliferation, but levalbuterol and the racemic mixture inhibited cell proliferation compared with the effect of 5% fetal bovine serum alone. The proliferative effect of platelet-derived growth factor on S-albuterol was not additive, suggesting that the 2 mediators act by different mechanisms. S-Albuterol induced greater expression of all the measured proteins than either levalbuterol, the racemic mixture or 5% fetal bovine serum. S-Albuterol stimulated IL-6 secretion and abolished the ability of levalbuterol to inhibit IL-6 secretion. Conclusion: Our data show that S-albuterol stimulates cell proliferation by activating expression and phosphorylation of several intracellular mitogenic proteins and may exacerbate asthma by stimulating the release of IL-6. Induction of PAF receptor protein expression by S-albuterol strongly suggests that S-albuterol may exert its adverse effects by binding to a G protein-coupled receptor such as the PAF receptor.

β1‐ and β2‐Adrenoceptor polymorphisms and cardiovascular diseases

Beta(1)- and beta(2)-adrenoceptors (AR) play a pivotal role in regulation of the cardiovascular system. Both beta-AR subtypes are polymorphic. There are two major single nucleotide polymorphisms (SNPs) in the beta(1)-AR gene: the Ser49Gly and Arg389Gly beta(1)-AR polymorphisms. In vitro, in recombinant cell systems Gly49 beta(1)-AR is much more susceptible to agonist-promoted downregulation than Ser49 beta(1)-AR, while Arg389 beta(1)-AR is three to four times more responsive to agonist-evoked stimulation than Gly389 beta(1)-AR. There are three major SNPs in the beta(2)-AR gene: the Arg16Gly, Gln27Glu and Thr164Ile beta(2)-AR polymorphisms (occur in humans only in the heterozygous form). In recombinant cell systems Gly16 beta(2)-AR is much more susceptible to agonist-promoted downregulation while Glu27 beta(2)-AR is rather resistant to agonist-induced downregulation but only in combination with Arg16, that occurs naturally extremely rare. Thr164 beta(2)-AR is three to four times more responsive to agonist-evoked stimulation than Ile164 beta(2)-AR. This review summarizes results from various studies on the possible relationship of these polymorphisms to cardiovascular diseases. At present it appears to be clear that, for cardiovascular diseases such as hypertension, coronary artery disease and chronic heart failure, beta(1)- and beta(2)-AR polymorphisms do not play a role as disease-causing genes; however, they might affect drug responses. Thus, it might be possible, by assessing the beta(1)-AR genotype, to predict responsiveness to beta(1)-AR agonist and -blocker treatment: patients homozygous for the Arg389 beta(1)-AR polymorphism should be good responders while patients homozygous for the Gly389 beta(1)-AR polymorphism should be poor responders or non-responders. Furthermore, subjects heterozygous for the Thr164Ile beta(2)-AR polymorphism exhibit blunted responses to beta(2)-AR stimulation. Finally, the Arg16Gln27 beta(2)-AR haplotype appears to be - at least in human vascular and bronchial smooth muscles - rather susceptible to agonist-induced desensitization (in contrast to the recombinant cell system findings), and might have some predictive value for poor outcome of heart failure. However, future large prospective studies have to replicate these findings in order to substantiate their clinical relevance.

Transcriptional regulation of the human CD97 promoter by Sp1/Sp3 in smooth muscle cells

The EGF-TM7 receptor CD97 shows different features of expression and function in muscle cells compared to hematopoetic and tumor cells. Since the molecular function and regulation of CD97 are poorly understood, this study aimed at defining its basal transcriptional regulation in smooth muscle cells (SMCs). The computational analysis of the CD97 5′-flanking region revealed that the TATA box-lacking promoter possesses several GC-rich regions as putative Sp1/Sp3 binding sites. Transfection studies with serially deleted promoter constructs demonstrated that the minimal promoter fragment resided in the − 218/+ 45 region containing one out of five identified GC-boxes in the leiomyosarcoma cell line SK-LMS-1 and human bronchial smooth muscle cells (HbSMCs). Mutation of the most proximal GC-site in CD97 reporter gene constructs caused a significant decrease in promoter activity. Gel shift assays and chromatin immunoprecipitation revealed that Sp1 and Sp3 bound specifically to the most proximal GC-site. Furthermore, we showed that Sp1 and Sp3 over-expression activates CD97 promoter activity in HEK293 cells. Our data characterize for the first time the activity of the human CD97 promoter which is controlled by Sp1/Sp3 transcription factors in SMCs.

Interleukin-4 and Interleukin-13 Enhance Human Bronchial Smooth Muscle Cell Proliferation

Background: T_H2 inflammation and bronchial smooth muscle cell (BSMC) hyperplasia are characteristic features of asthma, but whether these phenomena are linked remains unknown. This study aims to define the effect of the T_H2 cytokines IL-4 and IL-13 on human BSMC proliferation when administered alone or in combination with the fibroblast growth factor 2 (FGF2) growth factor. In addition, the effects of the proinflammatory mediators TNFα and IL-1β and the involvement of members of the well-known family of platelet-derived growth factor (PDGF) mitogens were tested. Methods: BSMC proliferation was measured by crystal violet staining and PDGF and PDGF receptor (PDGFR) expression were determined by RT-PCR, immunocytochemistry, ELISA, flow cytometry and dot plot analysis. Results: Neither IL-4 nor IL-13 alone induced BSMC proliferation, despite both being potent inducers of PDGF-CC. However, following a pretreatment with FGF2, which increased PDGFR α chain expression, both IL-4 and IL-13 increased FGF2-induced BSMC proliferation in a time- and concentration-dependent manner. TNFα and IL-1β did not affect basal or FGF2-induced BSMC proliferation, but both proinflammatory mediators enhanced the proliferative synergism between FGF2 and the T_H2 cytokines. Conclusions: IL-4 and IL-13 potently induce FGF2-primed BSMC proliferation via an autocrine loop involving PDGFRα and PDGF-CC, and this proliferative synergism is amplified by proinflammatory cytokines.

Transforming growth factor-β promoted vascular endothelial growth factor release by human lung fibroblasts

Purpose : The human lung fibroblast may act as an immunomodulatory cell by providing pro-inflammatory cytokines and chemokines, which are important in airway remodeling. Vascular endothelial growth factor (VEGF) induces mucosal edema and angiogenesis. Thymus and activation regulated chemokine (TARC) induces selective migration of T helper 2 cells. We investigated whether human lung fibroblasts produced VEGF and TARC, and the effects were augmented with the co-culture of fibroblasts and human bronchial smooth muscle cells (HBSMC), and whether dexamethasone can inhibit the proliferation and the release of VEGF in lung fibroblasts. Methods : Human lung fibroblasts were cultured with and without HBSMC, growth-arrested in serum-deprived medium, and pretreated with dexamethasone for 16 hours. After 24-hour stimulation with platelet derived growth factor-BB (PDGF-BB) and/or transforming growth factor- β (TGF- β), culture supernatant was harvested for assays of VEGF and TARC. Cell prolife ration was assayed using BrdU cell proliferation ELISA kit. Results : 1) The release of VEGF was significantly increased after stimulation with TGF- β, and its release was augmented when co-stimulated with PDGF and TGF- β. 2) VEGF release induced by PDGF or TGF- β was inhibited by dexamethasone. 3) There was no synergistic effect on the release of VEGF when human lung fibroblasts were co-cultured with HBSMC. 4) Dexamethasone did not suppress human lung fibroblasts proliferations. 5) Neither TGF- β nor PDGF induced TARC release from lung fibroblasts. Conclusion : Human lung fibroblasts may modulate airway remodeling by release of VEGF, but they have no synergistic effects when co-cultured with HBSMC. Dexamethasone suppresses VEGF release, not proliferation of lung fibroblast. (Korean J Pediatr 2008;51:879-885)

Endothelin-1 in exhaled breath condensate of stable and unstable asthma patients

Endothelins are proinflammatory, profibrotic, broncho- and vasoconstrictive peptides, which play an important role in the development of airway inflammation and remodeling in asthma. The study was undertaken to evaluate the endothelin-1 (ET-1) levels in exhaled breath condensate (EBC) of asthmatics with different degree in asthma severity. EBC was collected from 31 patients with allergic asthma (11 with steroid-naïve mild asthma, 10 with ICS-treated, stable mild-to-moderate asthma, 10 with ICS-treated unstable, severe asthma) and 7 healthy volunteers. In the three groups of asthmatics, ET-1 concentrations in EBC were significantly higher than in healthy volunteers. ET-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. There was a significant correlation between ET-1 levels and FENO in the three groups of asthmatics and between ET-1 and blood eosinophil counts in the group of patients with unstable asthma. Measurements of ET-1 in EBC may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma.

Leukotriene D4‐induced, epithelial cell‐derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Cysteinyl-leukotrienes (cys-LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys-LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. This study aims to investigate the regulatory role of leukotriene (LT)D(4) on TGF-beta1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. HEK293 cells stably transfected with cys-LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD(4) and TGF-beta1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD(4)-treated cells was then assayed for its proliferative effect on primary human BSMC. TGF-beta1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD(4) stimulation. The results demonstrated that LTD(4)-induced TGF-beta1 mRNA production in a time- and concentration-dependent manner in 293LT1. TGF-beta1 secretion was also up-regulated and CM from LTD(4)-treated 293LT1 was shown to increase BSMC proliferation in a TGF-beta1-dependent manner. The increased expression of TGF-beta1 mRNA by LTD(4) also occured in A549 and NHBE cells via a CysLT1-dependent mechanism. In conclusion, elevated expression of cys-LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF-beta1 production by airway epithelial cells.

A Novel Ca2+ Influx Pathway Activated by Mechanical Stretch in Human Airway Smooth Muscle Cells

In response to mechanical stretch, airway smooth muscle exhibits various cellular functions such as contraction, proliferation, and cytoskeletal remodeling, all of which are implicated in the pathophysiology of asthma. We tested the hypothesis that mechanical stretch of airway smooth muscle cells increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by activating stretch-activated (SA) nonselective cation channels. A single uniaxial stretch (3 s) was given to human bronchial smooth muscle cells cultured on an elastic silicone membrane. After the mechanical stretch, a transient increase in [Ca(2+)](i) was observed. The [Ca(2+)](i) increase was significantly dependent on stretch amplitude. The augmented [Ca(2+)](i) due to stretch was completely abolished by removal of extracellular Ca(2+) and was markedly attenuated by an application of Gd(3+), an inhibitor of SA channels, or ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor. In contrast, the stretch-induced rises of [Ca(2+)](i) were not altered by other Ca(2+) channel inhibitors such as nifedipine, BTP-2, and SKF-96365. Moreover, the [Ca(2+)](i) increases were not affected by indomethacin, a cyclooxygenase inhibitor, U-73122, a phospholipase C inhibitor, or xestospongin C, an inhibitor of the inositol-trisphosphate receptor. These findings demonstrate that a novel Ca(2+) influx pathway activated by mechanical stretch, possibly through the Ca(2+)-permeable SA channel activated directly by stretch rather than by indirect mechanisms via intracellular messenger production, is involved in human airway smooth muscle cells. A molecular candidate for the putative SA channel may be one of the members of the TRPV channel family. Thus, abnormal Ca(2+) homeostasis in response to excessive mechanical strain would contribute to the pathogenesis of asthma.

Effect of 1,25‐(OH)2D3 (a vitamin D analogue) on passively sensitized human airway smooth muscle cells

In asthma, airway smooth muscle cell (ASMC) hyperplasia plays an important role in airway remodelling. Increased expression of matrix metalloproteinases-9 (MMP-9), a disintegrin and metalloprotease 33 (ADAM33) in ASMCs are also relevant to asthmatic airway remodelling. 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has potent antiproliferative properties in vitro in various cell types; however, its role in ASMCs is not well understood. This study investigated the effect of 1,25-(OH)(2)D(3) on passively sensitized human bronchial (airway) smooth muscle cell (HASMC) proliferation and MMP-9 and ADAM33 expressions. The effect of 1,25-(OH)(2)D(3) on cell proliferation was examined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium-bromide colorimetry assay; cell cycle analysis by flow cytometry; and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The expression of MMP-9 and ADAM33 in HASMCs was investigated by real-time quantitative PCR and Western Blot analysis. 1,25-(OH)(2)D(3) effectively suppressed passively sensitized HASMC proliferation, proliferating cell nuclear antigen expression and G(1)/S transition in HASMCs passively sensitized with asthmatic serum. Further analysis showed that 1,25-(OH)(2)D(3) significantly down-regulated the expressions of protein for MMP-9 and ADAM33, as well as their mRNA levels in passively sensitized HASMCs. 1,25-(OH)(2)D(3) has direct inhibitory effects on passively sensitized HASMCs in vitro, including inhibition of cell proliferation and expression of MMP-9 and ADAM33, suggesting a possible beneficial role for 1,25-(OH)(2)D(3) in preventing and treating asthmatic airway remodelling.

Effects of neurotrophins on human bronchial smooth muscle cell migration and matrix metalloproteinase-9 secretion

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have been found to be upregulated in inflammatory pulmonary diseases, including asthma. The functional role for the neurotrophins in the airways is still not known, but it has been proposed that neurotrophins induce airway hyperreactivity and tissue remodeling. Bronchial smooth muscle cells have been suggested to be involved in the remodeling process through their capacity to proliferate, migrate, and secrete inflammatory mediators and matrix metalloproteinases (MMPs). Therefore, we studied the effect of NGF, BDNF, and NT-3 on human bronchial smooth muscle cell (HBSMC) migration and MMP-2 and MMP-9 secretion. Immunocytochemistry studies showed that HBSMCs expressed the neurotrophin receptors TrkA, TrkB, and TrkC. BDNF, NT-3, and NGF increased MMP-9, but not MMP-2, secretion as shown by zymography. BDNF and NT-3, but not NGF, stimulated HBSMC migration as evaluated by Boyden chamber. Taken together, our data indicate that the neurotrophins may stimulate events important for airway remodeling.

Volume-regulated Chloride Channel-3 in PDGF-Induced Proliferation of Human Bronchial Smooth Muscle Cells

Volume-regulated Chloride Channel-3 in PDGF-Induced Proliferation of Human Bronchial Smooth Muscle Cells

TLR3-Mediated Synthesis and Release of Eotaxin-1/CCL11 from Human Bronchial Smooth Muscle Cells Stimulated with Double-Stranded RNA

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

Acetylcholine-Induced Asynchronous Calcium Waves in Intact Human Bronchial Muscle Bundle

Calcium (Ca2+) is an important activator of the contractile machinery in airway smooth muscle (ASM). While agonist-induced Ca2+ signals are well characterized in animal ASM, little is known about what occurs in adult human ASM. In this study, we examined the Ca2+ signal elicited by acetylcholine (ACh) in smooth muscle cells of the intact human bronchial muscle strips obtained from fresh surgical specimens in relation to muscle contraction. We found that ACh induces repetitive Ca2+ waves that spread along the longitudinal axis of individual cells in the intact human bronchial smooth muscle strips. These Ca2+ waves display no apparent synchronization between neighboring cells, and their generation precedes force development. Comparison of the ACh concentration dependence of tissue contraction and selected parameters of the asynchronous Ca2+ waves (ACW) reveals that the graded force generation by ACh-stimulated human bronchial muscle strips is achieved by differential recruitment of cells to initiate Ca2+ waves and by enhancement of the frequency of ACW once the cells are recruited. Furthermore, pharmacologic characterization shows that the ACW are produced by repetitive cycles of SR Ca2+ release via ryanodine-sensitive channels followed by SR Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+ ATPase. Extracellular Ca2+ entry involving receptor-operated channels/store-operated channels, reverse-mode Na+/Ca2+ exchange, and to a lesser extent L-type voltage-gated Ca2+ channels is required to maintain the ACW. These findings for the first time demonstrate the occurrence and the role of ACW in excitation-contraction coupling in adult human ASM.

Sphingosine 1-Phosphate Causes Airway Hyper-Reactivity by Rho-Mediated Myosin Phosphatase Inactivation

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca(2+) sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

Endothelin-1 induces hypertrophy and inhibits apoptosis in human airway smooth muscle cells

Endothelin-1 (ET-1), a G protein-coupled receptor-activating peptide, is increased in airway epithelium, plasma, and bronchoalveolar lavage fluid of asthmatic patients. We hypothesized that ET-1 may contribute to the increased airway smooth muscle mass found in severe asthma by inducing hypertrophy and inhibiting apoptosis of smooth muscle cells. To investigate this hypothesis, we determined that treatment of primary human bronchial smooth muscle cells with ET-1 dose dependently [10(-11)-10(-7) M] inhibited the apoptosis induced by serum withdrawal. ET-1 treatment also resulted in a significant increase in total protein synthesis, mediated through both ET(A) and ET(B) receptors, cell size, as well as increased expression of myosin heavy chain, alpha-smooth muscle actin, and calponin. ET-1-induced hypertrophy was accompanied by activation of JAK1/STAT-3 and MAPK1/2 (ERK1/2) cell signaling pathways. Inhibition of JAK1/STAT-3 pathways by piceatannol or ERK1/2 by the MAPK/ERK kinase 1/2 inhibitor U0126 blunted the increase in total protein synthesis. The hypertrophic effect of ET-1 was equivalent to that of the gp130 cytokine oncostatin M and greater than that induced by cardiotrophin-1. ET-1 induced release of IL-6 but not IL-11, leukemia inhibitory factor, oncostatin M, or cardiotrophin-1, although treatment of cells with IL-6 alone did not induce hypertrophy. These results suggest that ET-1 is a candidate mediator for the induction of increased smooth muscle mass in asthma and identify signaling pathways activated by this mediator.

Role of RhoA Inactivation in Reduced Cell Proliferation of Human Airway Smooth Muscle by Simvastatin

Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Simvastatin (0.1-1.0 muM) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.

Adenosine A1 Receptors Mediate Mobilization of Calcium in Human Bronchial Smooth Muscle Cells

Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca(2+)](i)) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca(2+)](i). In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A(2A) receptors and adenosine A(3) receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A(1) receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP(3)) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A(1) receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP(3) signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.

The TH2 Cytokines, IL-4 and IL-13, Induce Human Bronchial Smooth Muscle Proliferation

The TH2 Cytokines, IL-4 and IL-13, Induce Human Bronchial Smooth Muscle Proliferation