Mouse Airway Smooth Muscle Cells Research Articles

Commentary: Effect of curcumin nanoparticles on proliferation and migration of mouse airway smooth muscle cells and airway inflammatory infiltration.

Commentary: Effect of curcumin nanoparticles on proliferation and migration of mouse airway smooth muscle cells and airway inflammatory infiltration.

Effect of curcumin nanoparticles on proliferation and migration of mouse airway smooth muscle cells and airway inflammatory infiltration.

Curcumin (CUR) possesses the capability to inhibit various inflammatory factors, exert anti-inflammatory effects, and alleviate asthma attacks; however, its hydrophobicity and instability significantly impede its clinical application. In this study, we synthesized CUR-loaded nanoparticles (CUR-NPs) and evaluated their impact on the proliferation, migration, and inflammatory infiltration of mouse airway smooth muscle cells (ASMCs), while investigating their underlying mechanisms. To achieve this objective, ASMCs were isolated from BALB/c mice and subjected to TGF-β1-induced cell proliferation and migration. Our findings demonstrate that CUR-NPs effectively regulate the release of CUR within cells with superior intracellular uptake compared to free CUR. The CCK-8 assay results indicate that the blank carrier does not exhibit any cytotoxic effects on cells, thus rendering the impact of the carrier itself negligible. The TGF-β1 group exhibited a significant increase in cell proliferation, whereas treatment with CUR-NPs significantly suppressed TGF-β1-induced cell proliferation. The findings from both the cell scratch assay and transwell assay demonstrated that TGF-β1 substantially enhanced cell migration, while CUR-NPs treatment effectively attenuated TGF-β1-induced cell migration. The Western blot analysis demonstrated a substantial increase in the expression levels of TGF-β1, p-STAT3, and CTGF in ASMCs following treatment with TGF-β1 when compared to the control group. Nevertheless, this effect was effectively counteracted upon administration of CUR-NPs. Furthermore, an asthma mouse model was successfully established and CUR-NPs were administered through tail vein injection. The serum levels of TGF-β1 and the expression levels of TGF-β1, p-STAT3, and CTGF proteins in the lung tissue of mice in the model group exhibited significant increases compared to those in the control group. However, CUR-NPs treatment effectively attenuated this change. Our research findings suggest that CUR-NPs possess inhibitory effects on ASMC proliferation, migration, and inflammatory infiltration by suppressing activation of the TGF-β1/p-STAT3/CTGF signaling pathway, thereby facilitating inhibition of airway remodeling.

Effect of icariin on the H2O2-induced proliferation of mouse airway smooth muscle cells through miR-138-5p regulating SIRT1/AMPK/PGC-1α axis.

Icariin exerts antioxidative and anti-inflammatory effects and is used in the treatment of bronchial asthma. However, the specific modes of action are uncertain. In this study, we investigated whether icariin could modulate the silencing information regulator 2-related enzyme 1 (SIRT1)/adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) axis by regulating miR-138-5p during H2O2-induced proliferation of mouse airway smooth muscle cells (ASMCs). Primary BALB/c mouse ASMCs were cultured using the tissue block adherence method and were induced with hydrogen peroxide (H2O2; 200μmol/L) to establish a bronchial asthma ASMC proliferation model. With the aid of Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) in H2O2-induced ASMCs, the expression of miR-138-5p, SIRT1, AMPK, PGC-1α, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), collagen I, and collagen III protein and mRNA were investigated. The proliferation rate and activities of superoxide dismutase1 (SOD1), reduced glutathione (GSH), malonaldehyde (MDA), and reactive oxygen species (ROS) in ASMCs were determined. The results suggest Compared with the H2O2-induced group, icariin inhibited the miR-138-5p expression; enhanced SIRT1, p-AMPK, and PGC-1α expression; attenuated MDA activity and ROS level; lowered TGF-β1, collagen I, and collagen III expression levels; and decreased the proliferation of ASMCs induced by H2O2. The dual-luciferase reporter gene assay results showed that SIRT1 is a regulatory target of miR-138-5p.The results suggest that Icariin could improve the H2O2-induced proliferation of ASMCs. The mechanism may be related to the increase of activation of SIRT1/AMPK/PGC-1α axis by suppressing the expression of miR-138-5p. Thus, SIRT1 is the regulatory target of miR-138-5p.

Biomimetic in vitro respiratory system using smooth muscle cells on ECIS chips for anti-asthma TCMs screening

Airway smooth muscle (ASM) contraction is a major pathophysiological characteristic of asthma. Although β2-adrenoceptor (β2-AR) agonists are currently used as bronchodilators, they cause rapid effect and long-term agonist-induced desensitization. Thus, it is necessary to search for more effective and safer relaxant agents for ASM cells. In this work, bitter taste receptors (TAS2Rs) were demonstrated to be expressed in primary mouse ASM cells endogenously, and they were considered as new drug targets for asthma treatment. Traditional Chinese medicines (TCMs) contained a wide range of TAS2R agonists and some of them had the efficacy of relieving cough and asthma with less toxic side effects. Then the electronic cell-substrate impedance sensor (ECIS) was used for the first time to establish a method to detect the contraction/relaxation effects of ASM cells. Therefore, we introduced a biomimetic in vitro respiratory system using ASM cells on ECIS chips to screen for potential TCMs against asthma. Quinine, nobiletin, and picfeltarraenin IA screened in this study could effectively inhibit the ASM contraction in a concentration-dependent manner, showing potential value as novel anti-asthma drugs. Furthermore, the effective screening of anti-asthma drugs was realized based on 3D ASM cell arrays and gel imaging system. Consistent results were found and the reliability of the biomimetic in vitro respiratory system for the screening of TCMs against asthma was further verified. The biomimetic system designed in this study has the advantages of operation simplicity, high throughput, non-invasive, real-time, and high sensitivity, and therefore provides a promising drug screening platform for asthma disease.

MicroRNA-370 carried by M2 macrophage-derived exosomes alleviates asthma progression through inhibiting the FGF1/MAPK/STAT1 axis.

Emerging evidence has suggested the functions of exosomes in allergic diseases including asthma. By using a mouse model with asthma induced by ovalbumin (OVA), we explored the roles of M2 macrophage-derived exosomes (M2Φ-Exos) in asthma progression. M2Φ-Exos significantly alleviated OVA-induced fibrosis and inflammatory responses in mouse lung tissues, as well as inhibited abnormal proliferation, invasion, and fibrosis-related protein production in platelet derived growth factor (PDGF-BB) treated primary mouse airway smooth muscle cells (ASMCs). The OVA administration in mice or the PDGF-BB treatment in ASMCs reduced the expression of miR-370, which was detected in M2Φ-Exos by miRNA sequencing. However, treating the mice or ASMCs with M2Φ-Exos reversed the inhibitory effect of OVA or PDGF-BB on miR-370 expression. We identified that the target of miR-370 was fibroblast growth factor 1 (FGF1). Downregulation of miR-370 by Lv-miR-370 inhibitor or overexpression of FGF1 by Lv-FGF1 blocked the protective roles of M2Φ-Exos in asthma-like mouse and cell models. M2Φ-Exos were found to inactivate the MAPK signaling pathway, which was recovered by miR-370 inhibition or FGF1 overexpression. Collectively, we conclude that M2Φ-Exos carry miR-370 to alleviate asthma progression through downregulating FGF1 expression and the MAPK/STAT1 signaling pathway. Our study may offer a novel insight into asthma treatment.

MicroRNA-221 Modulates Airway Remodeling via the PI3K/AKT Pathway in OVA-Induced Chronic Murine Asthma.

BackgroundAirway remodeling is one of the most important pathological features of chronic asthma. This study aimed to determine whether microRNA-221 (hereafter, miR-221) can affect airway remodeling in a mouse model of ovalbumin (OVA)-induced chronic asthma.MethodsAdeno-associated viruses (AAVs) “Bearing miR-221 sponges” were used to downregulate miR-221 in asthmatic mice. Staining with hematoxylin and eosin (H&E), Masson trichrome, and periodic acid–Schiff reagent was used to assess histological changes. The affected signaling pathway in mouse airway smooth muscle cells (ASMCs) was also identified by gene chip technology. A PI3K/AKT-inhibitor (LY294002) was used to confirm the role of the pathway in ASMCs.ResultsThe inhibition of miR-221 in a murine asthma model was found to reduce airway hyper-responsiveness, mucus metaplasia, airway inflammation, and airway remodeling (p < 0.05). Furthermore, miR-221 was found to regulate collagen deposition in the extracellular matrix (ECM) of ASMCs. Bioinformatics analysis and western blot analysis confirmed that the PI3K-AKT pathway was involved in ECM deposition in ASMCs.ConclusionmiR-221 might play a crucial role in the mechanism of remodeling via the PI3K/AKT pathway in chronic asthma and it could be considered as a potential target for developing therapeutic strategies.

TRPV4 Complexes With the Na+/Ca2+ Exchanger and IP3 Receptor 1 to Regulate Local Intracellular Calcium and Tracheal Tension in Mice

Intracellular Ca2+ is critical for regulating airway smooth muscle (ASM) tension. A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels (BKCa, IK, and SK3, respectively). The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.

Exchange protein directly activated by cAMP (Epac) protects against airway inflammation and airway remodeling in asthmatic mice

Backgroundβ2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear.MethodsWe established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement.ResultsWe found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 μM, and ESI-09 promoted SOCE of ASMCs at 10 μM and 100 μM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365.ConclusionsOur results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.

Lentivirus‐ and shRNA‐based smooth muscle‐specific TRPC3 channel knockdown as an emerging therapeutic for asthma

A major pathological feature of asthma is airway smooth muscle (ASM) hyperresponsiveness, which is the rationale for using bronchodilators as the first‐line therapy for asthma. The molecular mechanisms for airway hyperresponsiveness remain incompletely understood. The transcription factor nuclear factor‐κB (NFκB) is increasing known to be important for asthma. Using the TFSEARCH program, we have found that NFκB has specific binding sites on the promoter region of canonical transient receptor potential‐3 (TRPC3) channel, a crucial player for numerous pathological cellular responses in a variety of cells. Our investigations have revealed that tumor necrosis factor‐α (TNFα), an important asthmatic mediator, could significantly augment the luciferase intensity in mouse ASM cells (ASMCs) transfected with TRPC3 channel promoter/PGL3 luciferase reporter vectors, indicating an increase in the channel promoter activity. The TNFα‐induced increase in TRPC3 channel promoter activity was blocked by specific NFκB inhibition. Similarly, NFκB‐dependent increased TRPC3 channel promoter activity was observed in ASMCs from mice with allergen‐evoked asthma. We have also demonstrated that among all 7 TRPC channel members, only TRPC3 channel showed a predominant functional activity in ASMCs. TRPC3 channel gene and protein expression levels were much higher in asthmatic mouse ASMCs. More importantly, intravenous or intranasal delivery of lentiviruses encoding the SMC promoter SM22α‐driven mouse TRPC3 channel shRNAs in mice caused a sufficient knockdown of the channel expression and function in ASMCs. The TRPC3 channel knockdown mice had no asthmatic airway hyperresponsiveness. Moreover, lentiviral shRNA‐based SMC‐specific TRPC3 channel knockdown blocked ASM remodeling (known as another major asthmatic pathological feature) in asthmatic mice. Taken together, we conclude that TRPC3 channel serves as an essential mediator of the functional role of the transcription factor NF‐κB in asthma; as such, NF‐κB underlies the asthmatic increase in TRPC3 channel promoter activity, expression and function in ASMCs, leading to airway hyperresponsiveness and remodeling. Evidently, lentivirus‐ and shRNA‐based SMC‐specific TRPC3 channel knockdown is an emerging therapeutic for asthma.Support or Funding InformationHL122865This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

17β-Estradiol Promotes Apoptosis in Airway Smooth Muscle Cells Through CD38/SIRT1/p53 Pathway.

17β-Estradiol (E2) is the major estrogen secreted by the premenopausal ovary and shows dual effects on cell apoptosis under pathological conditions. E2 was previously shown to increase CD38 mRNA and protein expression in myometrial smooth muscle, but its function and mechanism remain largely unknown. Here we investigated the role of E2 in hypoxia-induced apoptosis in mouse airway smooth muscle cells (ASMCs) and explored the underlying mechanisms. Results showed that E2 significantly increased CD38 expression at both mRNA and protein levels, accompanied with decreased SIRT1 levels in ASMCs. By using primary ASMCs from the wild type (WT) and the smooth muscle-specific CD38 knockout (CD38 KO) mice, we found that the down-regulation of SIRT1 induced by E2 was abolished in CD38 KO AMSCs. E2 promoted the acetylation of p53 in WT cells, and this effect was also diminished in the absence of CD38. In addition, E2 further activated CD38/SIRT1/p53 signal pathway and promoted cell apoptosis during hypoxia. However, these effects were reversed in CD38 KO ASMCs and by the specific SIRT1 activator Resveratrol. We also found that E2 enhanced CD38 expression through estrogen receptor. The data suggested that CD38 is a direct target for E2 which promotes hypoxia-induced AMSC apoptosis through SIRT1/p53 signal pathway.

Inhibitory effects of openers of large-conductance Ca2+-activated K+ channels on agonist-induced phasic contractions in rabbit and mouse bronchial smooth muscle.

Airway smooth muscle expresses abundant BKCa channels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCa channel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 μM GoSlo-SR5-130 to activate BKCa channels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2 by -88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2 by 12-83 mV over a concentration range of 1-30 μM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCa channel openers, shifted V1/2 by 20-79 mV over a concentration range of 0.3-3 μM. In rabbit bronchial rings, exposure to histamine (1 μM) induced phasic contractions after a delay of ~35 min. These were abolished by GoSlo-SR5-130 (30 μM). Nifedipine (100 nM) and CaCCinhA01 (10 μM), a TMEM16A blocker, also abolished histamine-induced phasic contractions. In mouse bronchi, similar phasic contractions were evoked by exposure to U46619 (100 nM) and carbachol (100 nM). In each case, these were inhibited by concentrations of GoSlo-SR5-130 and compound X that shifted the activation V1/2 of BKCa channels in the order of -80 mV. In conclusion, membrane potential-dependent regulation of L-type Ca2+ channels appears to be important for histamine-, U46619-, and carbachol-induced phasic contractions in airway smooth muscle. Contractions can be abolished by BKCa channel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.

SIRT7 regulates the TGF-β1-induced proliferation and migration of mouse airway smooth muscle cells by modulating the expression of TGF-β receptor I

Accumulating evidence shows that sirtuin 7 (SIRT7), a key mediator of many cellular activities, plays an important role in the pathogenesis of various diseases; however, little is known about the role of SIRT7 in asthma, which is characterized by airway remodeling. This study investigated the potential role of SIRT7 in regulating the proliferation and migration of airway smooth muscle (ASM) cells, which are critical events during airway remodeling in asthmatic conditions. The results demonstrated that SIRT7 expression was significantly upregulated in ASM cells treated with transforming growth factor-beta 1 (TGF-β1). Knockdown of SIRT7 inhibited the proliferation, promoted the apoptosis, and suppressed the migration of TGF-β1-treated ASM cells, while overexpression of SIRT7 had the opposite effect. Moreover, knockdown of SIRT7 inhibited protein expression of the TGF-β receptor I (TβRI), whilst overexpression of SIRT7 promoted the expression of TβRI. Importantly, knockdown of TβRI partially reversed the stimulatory effect of SIRT7 overexpression on the TGF-β1-induced proliferation and migration of ASM cells. Taken together, these results demonstrate that SIRT7 is involved in regulating TGF-β1-induced ASM cell proliferation and migration by regulating the expression of TβRI, thus indicating an important role of SIRT7 during airway remodeling in asthma.

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)-induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca2+ that was further enhanced by 5% NaCl, indicating that the HS-induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca2+ . The Rho-associated kinase (ROCK) inhibitor Y-27632 relaxed ACh-induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca2+ sensitization, not by decreasing intracellular Ca2+ .

Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine (n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 (n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS (n = 4-7, P = nonsignificant), it did inhibit calcium oscillations (n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.

Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

Targeting Phosphoinositide 3-Kinase γ in Airway Smooth Muscle Cells to Suppress Interleukin-13-Induced Mouse Airway Hyperresponsiveness

We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca(2+) oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca(2+) transient and increased Ca(2+) oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca(2+) transient by 20 to 30% but markedly attenuated Ca(2+) oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma.

Targeting phosphoinositide 3‐kinase γ in airway smooth muscle cells to suppress interleukin‐13‐induced mouse airway hyperresponsiveness

Excessive activation of different bronchoconstrictor G‐protein coupled receptors (GPCRs) in airway smooth muscle (ASM) contributes to airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. We previously reported that phosphoinositide 3‐kinase γ (PI3Kγ), a down‐stream effector of multiple types of GPCRs, directly regulates ASM contraction by modulating Ca2+ oscillations. The aim of this study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR induced by interleukin‐13 (IL‐13) in a murine model of asthma. We found that intranasal administration of IL‐13 into mice induced AHR after 48 h, as assessed by invasive tracheostomy. Acute treatment with a PI3Kγ‐specific inhibitor attenuated IL‐13 effects in vivo and IL‐13‐augmented airway contractility of precision‐cut mouse lung slices in vitro. In addition, silencing PI3Kγ expression in isolated mouse ASM cells markedly attenuated IL‐13‐augmented Ca2+ oscillations and contractility of ASM cells induced by different bronchoconstrictor GPCRs. Our studies demonstrate that PI3Kγ in ASM cells is important for AHR and acute treatment with a PI3Kγ inhibitor can ameliorate AHR by a broader‐based suppression of unwanted GPCR signaling in a murine model of asthma. This work was supported by American Asthma Foundation Early Excellence Award and State of Nebraska Research Funds.

Gβγ‐activated phosphoinositide 3‐kinase γ regulates airway smooth muscle contraction by modulating calcium oscillations

Airway smooth muscle (ASM) contractile hyper‐responsiveness plays a critical role in asthma. Muscarinic receptors in ASM convey bronchoconstrictor signals by dissociating G‐proteins into active Gα and Gβγ subunits. We found that expression of a Gβγ scavenger peptide suppressed acetylcholine (ACh)‐stimulated mouse ASM cell contraction. Gβγ plays prominent roles in signal transduction through its downstream effectors including PI3Kγ. Immunohistochemistry staining and Western blot indicated expression of PI3Kγ in ASM cells of mouse trachea, lung and cultured ASM cells. Inhibitors of PI3Kγ, but not PI3Kα, β, or δ, attenuated ACh‐stimulated airway contraction of mouse lung slices. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitors still exhibited an ACh‐induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, PI3Kγ inhibitors or silence of endogenous PI3Kγ by siRNA had a small inhibitory effect on the ACh‐stimulated initial Ca2+ transient in mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. Thus, Gβγ/PI3Kγ signaling pathway controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Targeting this pathway may directly impact airway hyper‐responsiveness associated with asthma. Supported by Nebraska State LB595 and 692.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

A Mathematical Analysis of Agonist- and KCl-Induced Ca2+ Oscillations in Mouse Airway Smooth Muscle Cells

Airway hyperresponsiveness is a major characteristic of asthma and is generally ascribed to excessive airway narrowing associated with the contraction of airway smooth muscle cells (ASMCs). ASMC contraction is initiated by a rise in intracellular calcium concentration ([Ca2+]i), observed as oscillatory Ca2+ waves that can be induced by either agonist or high extracellular K+ (KCl). In this work, we present a model of oscillatory Ca2+ waves based on experimental data that incorporate both the inositol trisphosphate receptor and the ryanodine receptor. We then combined this Ca2+ model and our modified actin-myosin cross-bridge model to investigate the role and contribution of oscillatory Ca2+ waves to contractile force generation in mouse ASMCs. The model predicts that: 1), the difference in behavior of agonist- and KCl-induced Ca2+ waves results principally from the fact that the sarcoplasmic reticulum is depleted during agonist-induced oscillations, but is overfilled during KCl-induced oscillations; 2), regardless of the order in which agonist and KCl are added into the cell, the resulting [Ca2+]i oscillations will always be the short-period, agonist-induced-like oscillations; and 3), both the inositol trisphosphate receptor and the ryanodine receptor densities are higher toward one end of the cell. In addition, our results indicate that oscillatory Ca2+ waves generate less contraction than whole-cell Ca2+ oscillations induced by the same agonist concentration. This is due to the spatial inhomogeneity of the receptor distributions.