Jennifer Danielsson

Functional Expression of the TMEM16 Family of Calcium Activated Chloride Channels in Airway Smooth Muscle

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

Antagonists of the TMEM16A calcium-activated chloride channel modulate airway smooth muscle tone and intracellular calcium.

Background:Perioperative bronchospasm refractory to &bgr; agonists continues to challenge anesthesiologists and intensivists. The TMEM16A calcium-activated chloride channel modulates airway smooth muscle (ASM) contraction. The authors hypothesized that TMEM16A antagonists would relax ASM contraction by modulating membrane potential and calcium flux. Methods:Human ASM, guinea pig tracheal rings, or mouse peripheral airways were contracted with acetylcholine or leukotriene D4 and then treated with the TMEM16A antagonists: benzbromarone, T16Ainh-A01, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid, or B25. In separate studies, guinea pig tracheal rings were contracted with acetylcholine and then exposed to increasing concentrations of isoproterenol (0.01 nM to 10 &mgr;M) ± benzbromarone. Plasma membrane potential and intracellular calcium concentrations were measured in human ASM cells. Results:Benzbromarone was the most potent TMEM16A antagonist tested for relaxing an acetylcholine -induced contraction in guinea pig tracheal rings (n = 6). Further studies were carried out to investigate the clinical utility of benzbromarone. In human ASM, benzbromarone relaxed either an acetylcholine- or a leukotriene D4–induced contraction (n = 8). Benzbromarone was also effective in relaxing peripheral airways (n = 9) and potentiating relaxation by &bgr; agonists (n = 5 to 10). In cellular mechanistic studies, benzbromarone hyperpolarized human ASM cells (n = 9 to 12) and attenuated intracellular calcium flux from both the plasma membrane and the sarcoplasmic reticulum (n = 6 to 12). Conclusion:TMEM16A antagonists work synergistically with &bgr; agonists and through a novel pathway of interrupting ion flux at both the plasma membrane and sarcoplasmic reticulum to acutely relax human ASM.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by β-agonists.

Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

Airway Epithelial Cell Release of GABA is Regulated by Protein Kinase A

Introductionγ-amino butyric acid (GABA) is not only the major inhibitory neurotransmitter in the central nervous system (CNS), but it also plays an important role in the lung, mediating airway smooth muscle relaxation and mucus production. As kinases such as protein kinase A (PKA) are known to regulate the release and reuptake of GABA in the CNS by GABA transporters, we hypothesized that β-agonists would affect GABA release from airway epithelial cells through activation of PKA.MethodsC57/BL6 mice received a pretreatment of a β-agonist or vehicle (PBS), followed by methacholine or PBS. Bronchoalveolar lavage (BAL) was collected and the amount of GABA was quantified using HPLC mass spectrometry. For in vitro studies, cultured BEAS-2B human airway epithelial cells were loaded with 3H-GABA. 3H-GABA released was measured during activation and inhibition of PKA and tyrosine kinase signaling pathways.Resultsβ-agonist pretreatment prior to methacholine challenge attenuated in vivo GABA release in mouse BAL and 3H-GABA release from depolarized BEAS-2B cells. GABA release was also decreased in BEAS-2B cells by increases in cAMP but not by Epac or tyrosine kinase activation.Conclusionβ-agonists decrease GABA release from airway epithelium through the activation of cAMP and PKA. This has important therapeutic implications as β-agonists and GABA are important mediators of both mucus production and airway smooth muscle tone.

Impaired Relaxation of Airway Smooth Muscle in Mice Lacking the Actin-Binding Protein Gelsolin

&NA; Diverse classes of ligands have recently been discovered that relax airway smooth muscle (ASM) despite a transient increase in intracellular calcium concentrations ([Ca2+]i). However, the cellular mechanisms are not well understood. Gelsolin is a calcium‐activated actin‐severing and ‐capping protein found in many cell types, including ASM cells. Gelsolin also binds to phosphatidylinositol 4,5‐bisphosphate, making this substrate less available for phospholipase C&bgr;‐mediated hydrolysis to inositol triphosphate and diacylglycerol. We hypothesized that gelsolin plays a critical role in ASM relaxation and mechanistically accounts for relaxation by ligands that transiently increase [Ca2+]i. Isolated tracheal rings from gelsolin knockout (KO) mice showed impaired relaxation to both a &bgr;‐agonist and chloroquine, a bitter taste receptor agonist, which relaxes ASM, despite inducing transiently increased [Ca2+]i. A single inhalation of methacholine increased lung resistance to a similar extent in wild‐type and gelsolin KO mice, but the subsequent spontaneous relaxation was less in gelsolin KO mice. In ASM cells derived from gelsolin KO mice, serotonin‐induced Gq‐coupled activation increased both [Ca2+]i and inositol triphosphate synthesis to a greater extent compared to cells from wild‐type mice, possibly due to the absence of gelsolin binding to phosphatidylinositol 4,5‐bisphosphate. Single‐cell analysis showed higher filamentous:globular actin ratio at baseline and slower cytoskeletal remodeling dynamics in gelsolin KO cells. Gelsolin KO ASM cells also showed an attenuated decrease in cell stiffness to chloroquine and flufenamic acid. These findings suggest that gelsolin plays a critical role in ASM relaxation and that activation of gelsolin may contribute to relaxation induced by ligands that relax ASM despite a transient increase in [Ca2+]i.

Anoctamin Channels in Human Myometrium: A Novel Target for Tocolysis

Background: Spontaneous preterm labor leading to preterm birth is a significant obstetric problem leading to neonatal morbidity and mortality. Current tocolytics are not completely effective and novel targets may afford a therapeutic benefit. Objective: To determine whether the anoctamin (ANO) family, including the calcium-activated chloride channel ANO1, is present in pregnant human uterine smooth muscle (USM) and whether pharmacological and genetic modulation of ANO1 modulates USM contraction. Methods: Reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical staining were done to determine which members of the ANO family are expressed in human USM. Uterine smooth muscle strips were studied in an organ bath to determine whether ANO1 antagonists inhibit oxytocin-induced USM contractions. Anoctamin 1 small interfering RNA (siRNA) knockdown was performed to determine its effect on filamentous-/globular (F/G)-actin ratio, a measurement of actin polymerization’s role in promoting smooth muscle contraction. Results: Messenger RNA (mRNA) encoding all members of the ANO family (except ANO7) are expressed in pregnant USM tissue. Anoctamin 1 mRNA expression was decreased 15.2-fold in pregnant USM compared to nonpregnant. Anoctamin 1 protein is expressed in pregnant human USM tissue. Functional organ bath studies with pregnant human USM tissue demonstrated that the ANO1 antagonist benzbromarone attenuates the force and frequency of oxytocin-induced contractions. In human USM cells, siRNA knockdown of ANO1 decreases F-/G-actin ratios. Conclusion: Multiple members of the ANO family, including the calcium-activated chloride channel ANO1, are expressed in human USM. Antagonism of ANO1 by pharmacological inhibition and genetic knockdown leads to an attenuation of contraction in pregnant human USM. Anoctamin 1 is a potentially novel target for tocolysis.

GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0 ) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation (P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice (P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration (P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium

BackgroundDopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gi-coupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression.MethodsWe evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively.ResultsThe dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells.ConclusionsThese results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms.

Functional comparison of anoctamin 1 antagonists on human uterine smooth muscle contractility and excitability

Background: Pre-term birth is a major health care challenge throughout the world, and preterm labor represents a potentially reversible component of this problem. Current tocolytics do not improve preterm labor beyond 48 h. We have previously shown that anoctamin 1 (ANO1) channel blockade results in relaxation of pre-contracted human uterine smooth muscle (USM). Three drug classes with reported medicinal effects in humans also have members with ANO1 antagonism. In this study, we compared the ability of representatives from these 3 classes to reduce human USM contractility and excitability. Objective: This study sought to examine the comparative potency of 3 ANO1 antagonists on pregnant human USM relaxation, contraction frequency reduction, inhibition of intracellular calcium release and membrane hyperpolarization. Methods: Experiments were performed using: 1) Ex vivo organ bath (human pregnant tissue), 2) Oxytocin-induced calcium flux (in vitro human USM cells) and 3) Membrane potential assay (in vitro human USM cells). Results: Benzbromarone (BB) demonstrated the greatest potency among the compounds tested with respect to force, frequency inhibition, reducing calcium elevation and depolarizing membrane potential. Conclusion: While all 3 ANO1 antagonists attenuate pregnant human uterine tissue contractility and excitability, BB is the most potent tocolytic drug. Our findings may serve as a foundation for future structure-function analyses for novel tocolytic drug development.