Stability and Detection Limit of Avian Influenza, Newcastle Disease Virus, and African Horse Sickness Virus on Flinders Technology Associates Card by Conventional Polymerase Chain Reaction.

The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens.

Plasma biomarkers associated with survival and thrombosis in hospitalized COVID-19 patients.

Severe coronavirus disease-19 (COVID-19) has been associated with fibrin-mediated hypercoagulability and thromboembolic complications. To evaluate potential biomarkers of coagulopathy and disease severity in COVID-19, we measured plasma levels of eight biomarkers potentially associated with coagulation, fibrinolysis, and platelet function in 43 controls and 63 COVID-19 patients, including 47 patients admitted to the intensive care unit (ICU) and 16 non-ICU patients. COVID-19 patients showed significantly elevated levels of fibrinogen, tissue plasminogen activator (t-PA), and its inhibitor plasminogen activation inhibitor 1 (PAI-1), as well as ST2 (the receptor for interleukin-33) and von Willebrand factor (vWF) compared to the control group. We found that higher levels of t-PA, ST2, and vWF at the time of admission were associated with lower survival rates, and that thrombotic events were more frequent in patients with initial higher levels of vWF. These results support a predictive role of specific biomarkers such as t-PA and vWF in the pathophysiology of COVID-19. The data provide support for the case that hypercoagulability in COVID-19 is fibrin-mediated, but also highlights the important role that vWF may play in the genesis of thromboses in the pathophysiology of COVID-19. Interventions designed to enhance fibrinolysis might prove to be useful adjuncts in the treatment of coagulopathy in a subset of COVID-19 patients.

Diminished androgen levels are linked to irritable bowel syndrome and cause bowel dysfunction in mice.

Functional gastrointestinal disorders (FGIDs) have prominent sex differences in incidence, symptoms, and treatment response that are not well understood. Androgens are steroid hormones present at much higher levels in males than females and could be involved in these differences. In adults with irritable bowel syndrome (IBS), a FGID that affects 5% to 10% of the population worldwide, we found that free testosterone levels were lower than those in healthy controls and inversely correlated with symptom severity. To determine how this diminished androgen signaling could contribute to bowel dysfunction, we depleted gonadal androgens in adult mice and found that this caused a profound deficit in gastrointestinal transit. Restoring a single androgen hormone was sufficient to rescue this deficit, suggesting that circulating androgens are essential for normal bowel motility in vivo. To determine the site of action, we probed androgen receptor expression in the intestine and discovered, unexpectedly, that a large subset of enteric neurons became androgen-responsive upon puberty. Androgen signaling to these neurons was required for normal colonic motility in adult mice. Taken together, these observations establish a role for gonadal androgens in the neural regulation of bowel function and link altered androgen levels with a common digestive disorder.

Anesthetic Management for Endovascular Treatment of Stroke in Patients With Coronavirus Disease 2019: A Case Series.

A significant number of patients with coronavirus disease 2019 develop strokes with large vessel obstructions that may require endovascular treatment for revascularization. Our series focuses on periprocedural issues and the anesthetic management of these patients. We analyzed medical records of 5 patients with positive reverse transcription polymerase chain reaction tests for severe acute respiratory syndrome coronavirus 2 during their hospitalization who underwent endovascular treatment at our hospital between March and mid-June 2020. We found that our patients were different from the typical patients with ischemic stroke in that they had signs of hypercoagulability, hypoxia, and a lack of hypertension at presentation.

Carotid approach to anterior circulation thromboembolectomy in an adult with failing fontan physiology: a case report.

BACKGROUND: Anesthetic management of an adult with failing Fontan physiology is complicated given inherent anatomical and physiological alterations. Neurosurgical interventions including thromboembolectomy may be particularly challenging given importance of blood pressure control and cerebral perfusion. CASE PRESENTATION: We describe a 29 year old patient born with double outlet right ventricle (DORV) with mitral valve atresia who after multi-staged surgeries earlier in life, presented with failing Fontan physiology. She was admitted to the hospital almost 29 years after her initial surgeries to undergo workup for a dual heart and liver transplant in the context of a failing Fontan with elevated end diastolic pressures, NYHA III heart failure symptoms, and liver cirrhosis from congestive hepatopathy. During the workup in the context of holding anticoagulation for invasive procedures, she developed a middle cerebral artery (MCA) stroke requiring a thromboembolectomy via left carotid artery approach. DISCUSSION AND CONCLUSIONS: This case posed many challenges to the anesthesiologist including airway control, hemodynamic and cardiopulmonary monitoring, evaluation of perfusion, vascular access, and management of anticoagulation in an adult patient in heart and liver failure with Fontan physiology undergoing thromboembolectomy for MCA embolic stroke.

Anoctamin 1 antagonism potentiates conventional tocolytic-mediated relaxation of pregnant human uterine smooth muscle.

BACKGROUND: Currently available tocolytic agents are not effective treatment for preterm labor beyond 48 h. A major reason is the development of maternal side effects which preclude the maintenance of an effective steady-state drug concentration. One strategy that can mitigate these side effects is utilizing synergistic drug combinations to reduce the drug concentrations necessary to elicit a clinical effect. We have previously shown that three anoctamin 1 (ANO1) antagonists mediate potent relaxation of precontracted human uterine smooth muscle (USM). In this study, we aimed to determine whether a combination of sub-relaxatory doses of tocolytic drugs in current clinical use [the L-type voltage-gated calcium channel (VGCC) blocker, nifedipine (NIF); and the ß2-adrenergic (ß2AR) agonist, terbutaline (TRB)] will potentiate USM relaxation with two ANO1 antagonists [benzbromarone (BB) and MONNA (MN)]. OBJECTIVE: This study sought to examine the synergistic potency and mechanistic basis of two ANO1 antagonists with currently available tocolytic drugs. Functional endpoints assessed included relaxation of pre-contracting pregnant human USM tissue, inhibition of intracellular calcium release, and reduction of spontaneous transient inward current (STIC) recordings in human uterine smooth muscle cells. METHODS: Human myometrial strips and primary human USM cells were used in organ bath and calcium flux experiments with different combinations of sub-threshold doses of ANO1 antagonists and terbutaline or nifedipine to determine if ANO1 antagonists potentiate tocolytic drugs. RESULTS: The combination of sub-threshold doses of two ANO1 antagonists and current tocolytic drugs demonstrate a significant degree of synergy to relax human pregnant USM compared to the effects achieved when these drugs are administered individually. CONCLUSION: A combination of sub-threshold doses of VGCC blocker and ß2AR agonist with ANO1 antagonists potentiates relaxation of oxytocin-induced contractility and calcium flux in human USM ex vivo. Our findings may serve as a foundation for novel tocolytic drug combinations.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

Light-Mediated Inhibition of Colonic Smooth Muscle Constriction and Colonic Motility via Opsin 3.

Opsin photoreceptors outside of the central nervous system have been shown to mediate smooth muscle photorelaxation in several organs. We hypothesized that opsin receptor activation in the colon would have a similar effect and influence colonic motility. We detected Opsin 3 (OPN3) protein expression in the colonic wall and demonstrated that OPN3 was present in enteric neurons in the muscularis propria of the murine colon. Precontracted murine colon segments demonstrated blue light (BL) -mediated relaxation ex vivo. This photorelaxation was wavelength specific and was increased with the administration of the chromophore 9-cis retinal and a G protein receptor kinase 2 (GRK2) inhibitor. Light-mediated relaxation of the colon was not inhibited by L-NAME or tetrodotoxin (TTX). Furthermore, BL exposure in the presence of 9-cis retinal decreased the frequency of colonic migrating motor complexes (CMMC) in spontaneously contracting mouse colons ex vivo. These results demonstrate for the first time a receptor-mediated photorelaxation of colonic smooth muscle and implicate opsins as possible new targets in the treatment of spasmodic gastrointestinal dysmotility.

An isolated retrograde-perfused newborn mouse heart preparation.

The Langendorff-perfused model is a powerful tool to study biological responses in the isolated heart in the absence of confounders. The model has been adapted recently to enable study of the isolated mouse heart and the effects of genetic manipulation. Unfortunately, the small size and fragility of the mouse heart pose significant challenges, limiting application of the Langendorff model to the study of adult mice. Cardiac development is a complex and dynamic process that is incompletely understood. Thus, establishing an isolated-perfused heart model in the newborn mouse would be an important and necessary advance. Here we present a method to successfully cannulate and perfuse the isolated newborn murine heart. We describe the basic and fundamental physiological characteristics of the ex-vivo retrograde-perfused beating neonatal heart in wild-type C57Bl/6 male mice. Our approach will enable future study of the physiological and pharmacological responses of the isolated immature murine heart to enhance knowledge of how developmental cardiac biology impacts health and disease.•The Langendorff model is a powerful tool to study the heart without confounders.•An isolated-perfused newborn murine heart model has yet to be established.•We demonstrate the first successful isolated neonatal murine heart preparation.

Correction to: Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

This article was updated to correct Joy Y. Vink's name in the author listing.

Stroke and mechanical thrombectomy in patients with COVID-19: technical observations and patient characteristics.

BACKGROUND: COVID-19 infections have been shown to be associated with a range of thromboembolic disease. OBJECTIVE: To describe our endovascular experience in a consecutive series of patients with COVID-19 who presented with large vessel occlusions, and to describe unique findings in this population. METHODS: Mechanical thrombectomy was performed on five consecutive patients with COVID-19 with large vessel occlusions. A retrospective study of these patients was performed. Patient demographics, laboratory values, mechanical thrombectomy technique, and clinical and angiographic outcomes were reviewed. RESULTS: Four patients with COVID-19 presented with anterior circulation occlusions and one patient with COVID-19 presented with both anterior and posterior circulation occlusions. All patients had coagulation abnormalities. Mean patient age was 52.8 years. Three patients presented with an intracranial internal carotid artery occlusion. Two patients presented with an intracranial occlusion and a tandem thrombus in the carotid bulb. One patient presented with an occlusion in both the internal carotid and basilar arteries. Clot fragmentation and distal emboli to a new vascular territory were seen in two of five (40%) patients, and downstream emboli were seen in all five (100%) patients. Patient clinical outcome was generally poor in this series of patients with COVID-19 large vessel occlusion. CONCLUSION: Our series of patients with COVID-19 demonstrated coagulation abnormalities, and compared with our previous experience with mechanical thrombectomy in large vessel occlusion, this group of patients were younger, had tandem or multiple territory occlusions, a large clot burden, and a propensity for clot fragmentation. These patients present unique challenges that make successful revascularization difficult.

Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

Spontaneous preterm birth (sPTB) remains a worldwide healthcare challenge. Preterm labor (PTL) is thought to be the largest reversible cause of sPTB, but current tocolytic therapies are ineffective and associated with systemic side effects from chronic use. Therefore, identifying novel mechanisms that promote human uterine smooth muscle (hUSM) relaxation is essential to improving clinical management of PTL. Here, we aimed to determine if an extraocular opsin receptor (OPN 3,4,5) system is expressed in pregnant hUSM and to characterize how photo-mediated relaxation of pre-contracting hUSM may be facilitated by external application of light. Translational studies were performed with hUSM from healthy late gestation patients (n = 8) and non-pregnant, similarly aged patients undergoing hysterectomy (n = 4). First, RT-PCR screened for mRNA coding for components of the classical extraocular light receptors (OPN 3,4,5). We found a restricted repertoire of opsin receptors (OPN3) expressed in pregnant hUSM tissue. Immunohistochemistry was performed to confirm protein expression. Pre-contracting late gestation hUSM strips were studied in functional organ bath studies to determine if photo-mediated relaxation is intensity or wavelength dependent. Functional organ bath studies revealed acute photo-mediated relaxation occurring in an intensity- and wavelength-dependent manner. Finally, coimmunoprecipitation of OPN3 with Gs following light activation suggests that a component of photo-relaxation occurs via G protein-coupled receptor machinery. This is the first report of light-mediated relaxation of pre-contracted human myometrium. Activation of endogenous light receptors on human myometrium may become a novel, non-invasive tocolytic strategy.

Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone.

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Airway smooth muscle photorelaxation via opsin receptor activation.

Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.

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

INTRODUCTION: γ-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 ß-agonists would affect GABA release from airway epithelial cells through activation of PKA. METHODS: C57/BL6 mice received a pretreatment of a ß-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 (3)H-GABA. (3)H-GABA released was measured during activation and inhibition of PKA and tyrosine kinase signaling pathways. RESULTS: ß-agonist pretreatment prior to methacholine challenge attenuated in vivo GABA release in mouse BAL and (3)H-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. CONCLUSION: ß-agonists decrease GABA release from airway epithelium through the activation of cAMP and PKA. This has important therapeutic implications as ß-agonists and GABA are important mediators of both mucus production and airway smooth muscle tone.

Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling.

The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2'F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca(2+)]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2'F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca(2+)]i, store-operated Ca(2+) entry, and methacholine-induced Ca(2+) oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

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

Severe bronchospasm refractory to ß-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 ß-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 ß-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.

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.

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone.

Chronic obstructive pulmonary disease and asthma are characterized by hyperreactive airway responses that predispose patients to episodes of acute airway constriction. Recent studies suggest a complex paradigm of GABAergic signaling in airways that involves GABA-mediated relaxation of airway smooth muscle. However, the cellular source of airway GABA and mechanisms regulating its release remain unknown. We questioned whether epithelium is a major source of GABA in the airway and whether the absence of epithelium-derived GABA contributes to greater airway smooth muscle force. Messenger RNA encoding glutamic acid decarboxylase (GAD) 65/67 was quantitatively measured in human airway epithelium and smooth muscle. HPLC quantified GABA levels in guinea pig tracheal ring segments under basal or stimulated conditions with or without epithelium. The role of endogenous GABA in the maintenance of an acetylcholine contraction in human airway and guinea pig airway smooth muscle was assessed in organ baths. A 37.5-fold greater amount of mRNA encoding GAD 67 was detected in human epithelium vs. airway smooth muscle cells. HPLC confirmed that guinea pig airways with intact epithelium have a higher constitutive elution of GABA under basal or KCl-depolarized conditions compared with epithelium-denuded airway rings. Inhibition of GABA transporters significantly suppressed KCl-mediated release of GABA from epithelium-intact airways, but tetrodotoxin was without effect. The presence of intact epithelium had a significant GABAergic-mediated prorelaxant effect on the maintenance of contractile tone. Airway epithelium is a predominant cellular source of endogenous GABA in the airway and contributes significant prorelaxant GABA effects on airway smooth muscle force.

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.