Airway Smooth Muscle Contraction Research Articles

Diacylglycerol Kinase is a Keystone Regulator of Signaling Relevant to the Pathophysiology of Asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGK) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA through a series of biochemical reactions is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of a and z isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex, and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma such as contraction and proliferation of airway smooth muscle (ASM) cells, and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

Chamaecyparis lawsoniana and Its Active Compound Quercetin as Ca2+ Inhibitors in the Contraction of Airway Smooth Muscle.

The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.

Superoxide Anions Inhibit Intracellular Calcium Response in Porcine Airway Smooth Muscle Cells.

Background Superoxide anions (O 2 - ) have multiple effects on pulmonary parenchyma altering cell proliferation, cellular metabolism, and airway smooth muscle (ASM) contraction. Intracellular calcium ([Ca 2+ ] i ) concentration plays a significant role in the regulation of ASM contraction, relaxation, proliferation, and gene expression. Objective We investigated the effects of O 2 - on agonist-stimulated changes in [Ca 2+ ] i in ASM cells. Design/Methods Fura-2 AM-loaded, freshly isolated porcine ASM (PASM) cells were used to examine [Ca 2+ ] i release in response to acetylcholine (ACh), histamine, endothelin, caffeine, and thapsigargin (TPG) in the presence or absence of extracellular Ca 2+ . Results Exposure of PASM cells to xanthine and xanthine oxidase (X + XO) resulted in a time-dependent generation of O 2 - , inhibited by superoxide dismutase (SOD). Preincubating PASM cells with X + XO for 15- or 45-minute inhibited net [Ca 2+ ] i responses to ACh, histamine, caffeine, and TPG compared with control cells. Pretreating PASM cells with SOD for 30 minutes mitigated the inhibitory effect of X + XO treatment on ACh-induced Ca 2+ elevation suggesting role of O 2 - . X + XO treatment also inhibited caffeine- and TPG-induced Ca 2+ elevation suggesting effect of O 2 - on [Ca 2+ ] i release and reuptake mechanisms. Conclusion Superoxide attenuates [Ca 2+ ] i release, reuptake, and may interfere with physiological functions of ASM cells.

RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms

Regulators of G protein signaling (RGS proteins) constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase activating (GAP) activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle (ASM) contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and ASM contraction whereas RGS4 knockout (KO) mice unexpectedly have decreased AHR due to increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GAP activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared to either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3 kinase (PI3K) and inhibited PI3K-dependent PGE2 secretion elicited by TGFβ in airway epithelial cells. Together these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx.

Airway smooth muscle (ASM) contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i) caused by its release from the sarcoplasmic reticulum (SR) or by extracellular Ca2+ influx. Major channels involved in Ca2+ influx in ASM cells are L-type voltage-dependent Ca2+ channels (L-VDCCs) and nonselective cation channels (NSCCs). Transient receptor potential vanilloid 4 (TRPV4) is an NSCC recently studied in ASM. Mechanical stimuli, such as contraction, can activate TRPV4. We investigated the possible activation of TRPV4 by histamine (His)- or carbachol (CCh)-induced contraction in guinea pig ASM. In single myocytes, the TRPV4 agonist (GSK101) evoked an increase in [Ca2+]i, characterized by a slow onset and a plateau phase. The TRPV4 antagonist (GSK219) decreased channel activity by 94%, whereas the Ca2+-free medium abolished the Ca2+ response induced by GSK101. Moreover, GSK101 caused Na+ influx in tracheal myocytes. GSK219 reduced the Ca2+ peak and the Ca2+ plateau triggered by His or CCh. TRPV4 blockade shifted the concentration-response curve relating to His and CCh to the right in tracheal rings and reduced the maximal contraction. Finally, the activation of TRPV4 in single myocytes increased the Ca2+ refilling of the SR. We conclude that contraction of ASM cells after stimulation with His or CCh promotes TRPV4 activation, the subsequent influx of Ca2+ and Na+, and the opening of L-VDCCs. The entry of Ca2+ into ASM cells via TRPV4 and L-VDCCs contributes to optimal smooth muscle contraction.

B-308 Focal Adhesion Kinase Activation is Involved in Tension Development During Contractile Stimulation of Mouse Detrusor Smooth Muscle to Modulate Bladder Dysfunction-Related Lower Urinary Tract Symptoms

Abstract Background The function of the bladder is to store and expel urine. Bladder dysfunction-related lower urinary tract symptoms (LUTS) is a common urological problem and negatively affects health-related quality of life. Investigation of detrusor muscle contraction mechanisms is necessary to understand its underlying pathophysiology. Voiding occurs through bladder muscle contraction, which is mediated primarily by the increased intracellular Ca2+ concentration-induced myosin regulatory light chain (MLC) phosphorylation. Recent studies showed that focal adhesion kinase (FAK)-regulated focal adhesion assembly and actin polymerization are involved in vascular and airway smooth muscle contraction. However, the role of FAK activation in visceral smooth muscle contraction is not well documented. This study aims to determine if FAK activation is involved in mouse detrusor smooth muscle contraction. Methods Normal adult male and female mouse bladders were harvested. The trigone and dome areas were cut, and the bladder body was used. The mucosa was carefully removed. Detrusor muscle strips (1.5–2 × 8–10 mm) were prepared and then suspended in a 20 mL tissue bath filled with physiological Krebs solution and bubbled with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The isometric contraction was recorded. After establishing a stable baseline, the strips were stimulated with 120 mM potassium chloride (KCl), which was used to normalize the contractility induced by subsequent stimuli. One of the following specific inhibitors or the same volume of vehicle (DMSO) was applied and incubated for 30 min: PF-573228 (2 µM, a FAK inhibitor) or latrunculin B (1 µM, an inhibitor of actin polymerization). Then, the contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5–10−4.5 M ) were measured. In a separate experiment, the detrusor strips were stimulated with CCh (10 µM) after incubation with PF-573228 (2 µM) or vehicle (DMSO) for 30 min. Once the peak contraction was reached, the strips were snap-frozen for immunoblotting analysis of the phosphorylated FAK (p-FAK) and MLC (p-MLC). Statistical analysis was performed (GraphPad Prism 6). The contractile responses to KCl and EFS between vehicle and inhibitor groups were compared using a paired t-test. Nonlinear regression was used to compare CCh-induced dose-response curves. For the expression of phosphorylated proteins, one-way ANOVA, followed by Tukey’s posthoc test, was used for analysis. Results KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle group (P < 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P < 0.05) or latrunculin B at 16 and 32 Hz (P < 0.01). Following the application of PF-573228 or latrunculin B, CCh-induced dose-response contractions were significantly lower than those in the corresponding vehicle-treated strips (P = 0.0021 and 0.0003, respectively). Immunoblotting analysis demonstrated that CCh stimulation caused increased expression of p-FAK and p-MLC, whereas preincubation with PF-573228 led to decreased expression of p-FAK but not p-MLC Conclusion FAK activation is involved in tension development induced by contractile stimulation in mouse detrusor muscle, probably through enhancing actin polymerization, but not MLC phosphorylation.

Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models

BackgroundRhinovirus (RV) infection of airway epithelial cells triggers asthma exacerbations, during which airway smooth muscle (ASM) excessively contracts. Due to ASM contraction, airway epithelial cells become mechanically compressed. We previously reported that compressed human bronchial epithelial (HBE) cells are a source of endothelin-1 (ET-1) that causes ASM contraction. Here, we hypothesized that epithelial sensing of RV by TLR3 and epithelial compression induce ET-1 secretion through a TGF-β receptor (TGFβR)-dependent mechanism.MethodsTo test this, we used primary HBE cells well-differentiated in air–liquid interface culture and two mouse models (ovalbumin and house dust mite) of allergic airway disease (AAD). HBE cells were infected with RV-A16, treated with a TLR3 agonist (poly(I:C)), or exposed to compression. Thereafter, EDN1 (ET-1 protein-encoding gene) mRNA expression and secreted ET-1 protein were measured. We examined the role of TGFβR in ET-1 secretion using either a pharmacologic inhibitor of TGFβR or recombinant TGF-β1 protein. In the AAD mouse models, allergen-sensitized and allergen-challenged mice were subsequently infected with RV. We then measured ET-1 in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR) following methacholine challenge.ResultsOur data reveal that RV infection induced EDN1 expression and ET-1 secretion in HBE cells, potentially mediated by TLR3. TGFβR activation was partially required for ET-1 secretion, which was induced by RV, poly(I:C), or compression. TGFβR activation alone was sufficient to increase ET-1 secretion. In AAD mouse models, RV induced ET-1 secretion in BALF, which positively correlated with AHR.ConclusionsOur data provide evidence that RV infection increased epithelial-cell ET-1 secretion through a TGFβR-dependent mechanism, which contributes to bronchoconstriction during RV-induced asthma exacerbations.

Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle.

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

Sphingosine-1-phosphate induces airway smooth muscle hyperresponsiveness and proliferation

The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signalling in the regulation of airway smooth muscle contraction, airway hyperresponsiveness and airway remodelling but evidence from human studies is lacking. We have compared the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo and in murine airways dissected from healthy and house dust mite (HDM) sensitised animals. Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilisation assays were used to study human airway smooth muscle cell responses. S1P did not induce contraction of airways isolated from healthy and HDM exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, 30 minutes exposure to S1P induced a significant, concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways respectively. HDM-sensitised mice demonstrated a significant increase in methacholine responsiveness which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human airway smooth muscle cells, an effect mediated through the S1P type 2 receptor, as shown by selective antagonism and S1PR2 siRNA knockdown. Our data suggest that S1P released locally into the airways may be involved in the regulation of airway smooth muscle hyperresponsiveness and hyperplasia, defining a novel target for future therapies.

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.

A dynamical model of TGF-β activation in asthmatic airways.

Excessive activation of the regulatory cytokine transforming growth factor $\beta $ (TGF-$\beta $) via contraction of airway smooth muscle (ASM) is associated with the development of asthma. In this study, we develop an ordinary differential equation model that describes the change in density of the key airway wall constituents, ASM and extracellular matrix (ECM), and their interplay with subcellular signalling pathways leading to the activation of TGF-$\beta $. We identify bistable parameter regimes where there are two positive steady states, corresponding to either reduced or elevated TGF-$\beta $ concentration, with the latter leading additionally to increased ASM and ECM density. We associate the former with a healthy homeostatic state and the latter with a diseased (asthmatic) state. We demonstrate that external stimuli, inducing TGF-$\beta $ activation via ASM contraction (mimicking an asthmatic exacerbation), can perturb the system irreversibly from the healthy state to the diseased one. We show that the properties of the stimuli, such as their frequency or strength, and the clearance of surplus active TGF-$\beta $, are important in determining the long-term dynamics and the development of disease. Finally, we demonstrate the utility of this model in investigating temporal responses to bronchial thermoplasty, a therapeutic intervention in which ASM is ablated by applying thermal energy to the airway wall. The model predicts the parameter-dependent threshold damage required to obtain irreversible reduction in ASM content, suggesting that certain asthma phenotypes are more likely to benefit from this intervention.

Inherent differences of small airway contraction and Ca2+ oscillations in airway smooth muscle cells between BALB/c and C57BL/6 mouse strains.

BALB/c and C57BL/6 mouse strains are widely used as animal model in studies of respiratory diseases, such as asthma. Asthma is characterized by airway hyperresponsiveness, which is eventually resulted from the excessive airway smooth muscle (ASM) contraction mediated by Ca2+ oscillations in ASM cells. It is reported that BALB/c mice have inherently higher airway responsiveness, but show no different contractive response of tracheal ring as compared to C57BL/6 mice. However, whether the different airway responsiveness is due to the different extents of small airway contraction, and what's underlying mechanism remains unknown. Here, we assess agonist-induced small airway contraction and Ca2+ oscillations in ASM cells between BALB/c and C57BL/6 mice by using precision-cut lung slices (PCLS). We found that BALB/c mice showed an intrinsically stronger extent of small airway narrowing and faster Ca2+ oscillations in ASM cells in response to agonists. These differences were associated with a higher magnitude of Ca2+ influx via store-operated Ca2+ entry (SOCE), as a result of increased expression of SOCE components (STIM1, Orai1) in the ASM cells of small airway of BALB/c mice. An established mathematical model and experimental results suggested that the increased SOC current could result in increased agonist-induced Ca2+ oscillations. Therefore, the inherently higher SOC underlies the increased Ca2+ oscillation frequency in ASM cells and stronger small airway contraction in BALB/c mice, thus higher airway responsiveness in BALB/c than C57BL/6 mouse strain.

Inhibition of airway smooth muscle contraction and proliferation by LIM kinase inhibitor, LIMKi3

PurposeCurrent medical treatment for asthma aims to inhibit airway smooth muscle (ASM) contraction and proliferation, however, the efficacy of available treatment options is unsatisfactory. Therefore, we explored the effect of LIM domain kinase (LIMK) inhibitor - LIMKi3, on ASM to improve the understanding of ASM contraction and proliferation mechanisms, and to investigate new therapeutic targets. Materials and methodsAsthma model was induced in rats by intraperitoneal injection of ovalbumin. Using phospho-specific antibodies, we examined LIMK, phosphorylated LIMK, cofilin and phosphorylated cofilin. ASM contraction was studied in organ bath experiments. ASM cells proliferation was studied with cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. ResultsImmunofluorescence indicated that LIMKs are expressed in ASM tissues. Western blot revealed that LIMK1 and phospho-cofilin were significantly elevated in asthma ASM tissues. The LIMK inhibitor, LIMKi3 (1 ​μM) could reduce cofilin phosphorylation and therefore inhibit contraction of ASM tissues, and induce actin filament breakdown as well as cell proliferation reduction in cultured human ASM cells. ConclusionsASM contraction and proliferation in asthma may underlie the effects of LIMKs. Small molecule LIMK inhibitor, LIMKi3, might be a potential therapeutic strategy for asthma.

The Umami taste receptor TAS1R1/TAS1R3 is expressed and potentiates contraction of airway smooth muscle

Introduction: In inflamed tissues, local glutamate concentrations increase due to increased vascular permeability and glutamate release from immune cells. Hence, local concentrations of glutamate in the airways could be increased in asthmatics and may modulate asthmatic symptoms. Classically, glutamate activates both ionotropic and metabolic glutamate receptors. In addition, the umami taste receptor TAS1R1/TAS1R3 which is coupled to Gi and Gs protein also acts as a sensor of glutamate. Although the functional expression of taste receptors outside the oral cavity has been identified, the expression of TAS1R1/TAS1R3 in the airway has never been described. Here, we questioned whether TAS1R1/TAS1R3 is expressed in airway smooth muscle (ASM) and modulates ASM tone. Methods: We evaluated the expression of TAS1R1 and TAS1R3 on human ASM (HASM) cells using RNA-seq, RT-PCR, and immunoblots. The effects of monosodium glutamate (MSG) on cAMP production were evaluated in the presence or absence of Gi protein inhibitor pertussis toxin (PTX; 50 ng/ml), Gs protein inhibitor NF449, TAS1R1/TAS1R3 allosteric modulator (IMP; 150 μM) or the cells were transfected with siRNA targeting TAS1R1/TAS1R3. To evaluate whether the activation of TAS1R1/TAS1R3 modulates acetylcholine (ACh; EC50)-induced ASM contraction, mouse tracheal rings suspended under isometric tension in organ baths were treated with MSG (100 μM) ± IMP, followed by ACh. The contribution of TAS1R1/TAS1R3 to ASM relaxation induced by cumulatively increasing concentration of isoproterenol was also evaluated. Results: We detected the expression of TAS1R1 and TAS1R3 in HASM cells. MSG significantly inhibited forskolin-stimulated cAMP production in HASM cells (84.1 ± 3.0% of forskolin alone; n = 7, P &lt; 0.01), which was potentiated by IMP (72.0 ± 4.9% of forskolin alone; n = 7, P &lt; 0.01) or NF449 (83.8 ± 1.9% of forskolin alone; n = 5, P &lt; 0.01). In contrast, the inhibitory effect of MSG on cAMP production was reversed by pretreatment of the cells with PTX or knockdown of TAS1R1 and TAS1R3. Although MSG alone did not affect basal or ACh-induced contractile ASM tone, MSG potentiated ACh-induced ASM contraction in the presence of IMP (112.4 ± 1.6% of previous ACh-induced ASM contraction; n = 11). Furthermore, cotreatment with MSG and isoproterenol resulted in a leftward shift in the isoproterenol relaxation concentration-response curve compared with treatment with isoproterenol alone (EC50 = 16.3 nM vs. 31.0 nM, respectively), which was attenuated in the presence of IMP (EC50 = 33.6 nM) (n = 6). Conclusion: Functional TAS1R1/TAS1R3 are expressed on ASM, and Gi-signaling is greater than Gs-signaling for cAMP production. Activation of TAS1R1/TAS1R3 potentiates ACh-induced ASM contraction. These findings suggest that activation of the TAS1R1/TAS1R3 and Gi leading to crosstalk potentiation of Gq-coupled receptors could be an important regulator of airway contractile responses. Grant-in-Aid for JSPS Fellows (20J20813) (to Sasaki H.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Local targeting of TSLP: feat or defeat

The physiological role of airway smooth muscle remains highly elusive given the poor overall understanding of the regulatory elements and their influence in healthy humans [1]. Paradoxically, the pathophysiological contribution of airway smooth muscle contraction to asthma is better understood than the physiological role of this contraction in non-asthmatics: early and late asthmatic responses highlight immediate-immune cell-free and delayed-immune cell-dependent bronchoconstriction, respectively [2]. Reducing the late asthmatic response with the inhaled anti-TSLP antibody ecleralimab raises more questions than providing answers <https://bit.ly/3vOkEIN> We thank David Manley for revising the English in this editorial.

Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice

Obesity increases the severity of airway hyperresponsiveness (AHR) in individuals with asthma, but the mechanism is not well elucidated. G-protein coupled receptor 40 (GPR40) has been found to induce airway smooth muscle contraction after activated by long-chain fatty acids (LC-FFAs), suggesting a close correlation between GPR40 and AHR in obese. In this study, C57BL/6 mice were fed a high-fat diet (HFD) to induce obesity with or without ovalbumin (OVA) sensitization, the regulatory effects of GPR40 on AHR, inflammatory cells infiltration, and the expression of Th1/Th2 cytokines were evaluated by using a small-molecule antagonist of GPR40, DC260126. We found that the free fatty acids (FFAs) level and GPR40 expression were greatly elevated in the pulmonary tissues of obese asthmatic mice. DC260126 greatly reduced methacholine-induced AHR, ameliorated pulmonary pathological changes and decreased inflammatory cell infiltration in the airways in obese asthma. In addition, DC260126 could down-regulate the levels of Th2 cytokines (IL-4, IL-5, and IL-13) and pro-inflammatory cytokines (IL-1β, TNF-α), but elevated Th1 cytokine (IFN-γ) expression. In vitro, DC260126 could remarkedly reduce oleic acid (OA)-induced cell proliferation and migration in HASM cells. Mechanistically, the effects that DC260126 alleviated obese asthma was correlated with the down-regulation of GTP-RhoA and Rho-associated coiled-coil-forming protein kinase 1 (ROCK1). Herein, we proved that targeting of GPR40 with its antagonist helped to mitigate multiple parameters of obese asthma effectively.

β-Adrenergic Receptor Antagonism of Cholinergic Stimulation of Airway Smooth Muscle Contraction: An Old Receptor Requires a Fresh Look.

β-Adrenergic Receptor Antagonism of Cholinergic Stimulation of Airway Smooth Muscle Contraction: An Old Receptor Requires a Fresh Look.

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

Exercise Reduces Airway Smooth Muscle Contraction in Asthmatic Rats via Inhibition of IL-4 Secretion and Store-Operated Ca2+ Entry Pathway.

Increased evidence has shown that aerobic exercise reduces airway hyperresponsiveness in asthmatic individuals. However, the underlying mechanisms of action remain elusive. This study aimed to investigate the effect of exercise on airway smooth muscle (ASM) contractile function in asthmatic rats, and uncover the possible involvement of interleukin 4 (IL-4) and the store-operated Ca2+ entry (SOCE) pathway. In this study, chicken ovalbumin was used to induce asthma in male Sprague-Dawley rats. The exercise group received moderate-intensity aerobic exercise training for 4 weeks. IL-4 concentrations in bronchoalveolar lavage fluid (BALF) samples were evaluated by enzyme linked immunosorbent assay. The contractile function of the ASM was investigated using tracheal ring tension experiments and intracellular Ca2+ imaging techniques. Western blot analysis was used to evaluate expression levels of calcium-release activated calcium (CRAC) channel protein (Orai) and stromal interaction molecule 1 (STIM1) in ASM. Our data showed that the carbachol-stimulated, SOCE-mediated contraction of rat ASM was significantly increased in asthmatic rats, which could be abolished by exercise. Pharmacological studies revealed that GSK5498A and BTP-2, selective blockers of CRAC channels significantly inhibited SOCE-induced ASM contraction. In addition, exercise inhibited the up-regulation of IL-4 in BALF as well as STIM1 and Orai expression in the ASM of asthmatic rats. In line with these observations, we demonstrated that pretreatment of the ASM with IL-4 up-regulated the expression level of STIM1, Orai1 and Orai2, thereby promoting SOCE-mediated ASM contraction. The data in this study reveal that aerobic exercise may improve the ASM contractile function in asthmatic rats by inhibiting IL-4 secretion and by down-regulating the expression of STIM1, Orai1 and Orai2, thus decreasing excessive SOCE-mediated ASM contraction in asthmatic rats.