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.

Agonism of the TMEM16A calcium-activated chloride channel modulates airway smooth muscle tone.

TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.

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.

Targeting the γ-Aminobutyric Acid A Receptor α4 Subunit in Airway Smooth Muscle to Alleviate Bronchoconstriction.

We previously demonstrated that airway smooth muscle (ASM) cells express γ-aminobutyric acid A receptors (GABA(A)Rs), and that GABA(A)R agonists acutely relax ASM. Among the GABA(A)R α subunits, human ASM cells express only α4 and α5, providing the opportunity for selective pharmacologic targeting. Novel GABA(A)R-positive allosteric modulators designed for enhanced α4/α6 subunit selectivity were synthesized using iterative computational analyses (CMD-45 and XHe-III-74). Studies using oocyte heterologous expression systems confirmed that CMD-45 and XHe-III-74 led to significantly greater augmentation of currents induced by a 3% maximal effective concentration (EC3) of GABA [EC3]-induced currents in oocytes expressing α4 or α6 subunits (along with ß3 and γ2) compared with other α subunits. CMD-45 and XHe-III-74 also led to greater ex vivo relaxation of contracted wild-type mouse tracheal rings compared with tracheal rings from GABA(A)R α4 subunit (Gabra4) knockout mice. Furthermore, CMD-45 and XHe-III-74 significantly relaxed precontracted human ASM ex vivo, and, at a low concentration, both ligands led to a significant leftward shift in albuterol-mediated ASM relaxation. In vivo, inhaled XHe-III-74 reduced respiratory system resistance in an asthmatic mouse model. Pretreatment of human ASM cells with CMD-45 and XHe-III-74 inhibited histamine-induced increases in intracellular calcium concentrations in vitro, an effect that was lost when calcium was omitted from the extracellular buffer, suggesting that inhibition of calcium influx due to alterations in plasma membrane potential may play a role in the mechanism of ASM relaxation. Selective targeting of the GABA(A)R α4 subunit with inhaled ligands may be a novel therapeutic pathway to treat bronchoconstriction, while avoiding sedative central nervous system effects, which are largely mediated by α1-3 subunit-containing GABA(A)Rs in the brain.

Attenuation of airway smooth muscle contractility via flavonol-mediated inhibition of phospholipase-Cß.

Enhanced contractility of airway smooth muscle (ASM) is a major pathophysiological characteristic of asthma. Expanding the therapeutic armamentarium beyond ß-agonists that target ASM hypercontractility would substantially improve treatment options. Recent studies have identified naturally occurring phytochemicals as candidates for acute ASM relaxation. Several flavonoids were evaluated for their ability to acutely relax human and murine ASM ex vivo and murine airways in vivo and were evaluated for their ability to inhibit procontractile signaling pathways in human ASM (hASM) cells. Two members of the flavonol subfamily, galangin and fisetin, significantly relaxed acetylcholine-precontracted murine tracheal rings ex vivo (n = 4 and n = 5, respectively, P < 0.001). Galangin and fisetin also relaxed acetylcholine-precontracted hASM strips ex vivo (n = 6-8, P < 0.001). Functional respiratory in vivo murine studies demonstrated that inhaled galangin attenuated the increase in lung resistance induced by inhaled methacholine (n = 6, P < 0.01). Both flavonols, galangin and fisetin, significantly inhibited purified phosphodiesterase-4 (PDE4) (n = 7, P < 0.05; n = 7, P < 0.05, respectively), and PLCß enzymes (n = 6, P < 0.001 and n = 6, P < 0.001, respectively) attenuated procontractile Gq agonists' increase in intracellular calcium (n = 11, P < 0.001), acetylcholine-induced increases in inositol phosphates, and CPI-17 phosphorylation (n = 9, P < 0.01) in hASM cells. The prorelaxant effect retained in these structurally similar flavonols provides a novel pharmacological method for dual inhibition of PLCß and PDE4 and therefore may serve as a potential treatment option for acute ASM constriction.

A new paradigm for the role of smooth muscle cells in the human cervix.

BACKGROUND: Premature cervical remodeling resulting in spontaneous preterm birth may begin with premature failure or relaxation at the internal os (termed "funneling"). To date, we do not understand why the internal os fails or why funneling occurs in some cases of premature cervical remodeling. Although the human cervix is thought to be mostly collagen with minimal cellular content, cervical smooth muscle cells are present in the cervix and can cause cervical tissue contractility. OBJECTIVE: To understand why the internal os relaxes or why funneling occurs in some cases of premature cervical remodeling, we sought to evaluate cervical smooth muscle cell content and distribution throughout human cervix and correlate if cervical smooth muscle organization influences regional cervical tissue contractility. STUDY DESIGN: Using institutional review board-approved protocols, nonpregnant women <50 years old undergoing hysterectomy for benign indications were consented. Cervical tissue from the internal and external os were immunostained for smooth muscle cell markers (α-smooth muscle actin, smooth muscle protein 22 calponin) and contraction-associated proteins (connexin 43, cyclooxygenase-2, oxytocin receptor). To evaluate cervical smooth muscle cell morphology throughout the entire cervix, whole cervical slices were obtained from the internal os, midcervix, and external os and immunostained with smooth muscle actin. To correlate tissue structure with function, whole slices from the internal and external os were stimulated to contract with 1 µmol/L of oxytocin in organ baths. In separate samples, we tested if the cervix responds to a common tocolytic, nifedipine. Cervical slices from the internal os were treated with oxytocin alone or oxytocin + increasing doses of nifedipine to generate a dose response and half maximal inhibitory concentration. Student t test was used where appropriate. RESULTS: Cervical tissue was collected from 41 women. Immunohistochemistry showed cervical smooth muscle cells at the internal and external os expressed mature smooth muscle cell markers and contraction-associated proteins. The cervix exhibited a gradient of cervical smooth muscle cells. The area of the internal os contained 50-60% cervical smooth muscle cells that were circumferentially organized in the periphery of the stroma, which may resemble a sphincter-like pattern. The external os contained approximately 10% cervical smooth muscle cells that were randomly scattered in the tissue. In organ bath studies, oxytocin stimulated the internal os to contract with more than double the force of the external os (1341 ± 693 vs 523 ± 536 integrated grams × seconds, respectively, P = .009). Nifedipine significantly decreased cervical tissue muscle force compared to timed vehicle control (oxytocin alone) at doses of 10(-5) mol/L (vehicle 47% ± 15% vs oxytocin + nifedipine 24% ± 16%, P = .007), 10(-4) mol/L (vehicle 46% ± 16% vs oxytocin + nifedipine -4% ± 20%, P = .003), and 10(-3) mol/L (vehicle 42% ± 14% vs oxytocin + nifedipine -15% ± 18%, P = .0006). The half maximal inhibitory concentration for nifedipine was 1.35 × 10(-5) mol/L. CONCLUSION: Our findings suggest a new paradigm for cervical tissue morphology-one that includes the possibility of a specialized sphincter at the internal os. This new paradigm introduces novel avenues to further investigate potential mechanisms of normal and premature cervical remodeling.

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.

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

BACKGROUND: Perioperative bronchospasm refractory to ß 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 µM) ± 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 ß 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 ß agonists and through a novel pathway of interrupting ion flux at both the plasma membrane and sarcoplasmic reticulum to acutely relax human ASM.

Calcium-activated chloride channels anoctamin 1 and 2 promote murine uterine smooth muscle contractility.

OBJECTIVE: To determine the presence of calcium activated chloride channels anoctamin 1 (ANO1) and 2 (ANO2) in human and murine uterine smooth muscle (MUSM) and evaluate the physiologic role for these ion channels in murine myometrial contractility. STUDY DESIGN: We performed reverse transcription polymerase chain reaction to determine whether ANO1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of ANO1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of ANO1 and 2 in murine uterine tissue was evaluated using electrophysiologic studies, organ bath, and calcium flux experiments. RESULTS: ANO1 and 2 are expressed in human and MUSM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous MUSM contractions. Blockade of ANO1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium. CONCLUSION: The calcium activated chloride channels ANO1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis.

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.

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.

Chloride in airway smooth muscle: the ignored anion no longer?

This Perspectives accompanies an Editorial Focus that summarizes new developments concerning the role of chloride in airway smooth muscle physiology. We provide several observations and mechanistic insights to reconcile recent experimental evidence with existing paradigms concerning chloride channel-mediated effects on airway smooth muscle tone. In addition, we highlight the potentially complex and dynamic nature that chloride currents and membrane potential have on calcium handling and airway smooth muscle contractility.

Targeting the restricted α-subunit repertoire of airway smooth muscle GABAA receptors augments airway smooth muscle relaxation.

The prevalence of asthma has taken on pandemic proportions. Since this disease predisposes patients to severe acute airway constriction, novel mechanisms capable of promoting airway smooth muscle relaxation would be clinically valuable. We have recently demonstrated that activation of endogenous airway smooth muscle GABA(A) receptors potentiates ß-adrenoceptor-mediated relaxation, and molecular analysis of airway smooth muscle reveals that the α-subunit component of these GABA(A) receptors is limited to the α(4)- and α(5)-subunits. We questioned whether ligands with selective affinity for these GABA(A) receptors could promote relaxation of airway smooth muscle. RT-PCR analysis of GABA(A) receptor subunits was performed on RNA isolated by laser capture microdissection from human and guinea pig airway smooth muscle. Membrane potential and chloride-mediated current were measured in response to GABA(A) subunit-selective agonists in cultured human airway smooth muscle cells. Functional relaxation of precontracted guinea pig tracheal rings was assessed in the absence and presence of the α(4)-subunit-selective GABA(A) receptor agonists: gaboxadol, taurine, and a novel 8-methoxy imidazobenzodiazepine (CM-D-45). Only messenger RNA encoding the α(4)- and α(5)-GABA(A) receptor subunits was identified in RNA isolated by laser capture dissection from guinea pig and human airway smooth muscle tissues. Activation of airway smooth muscle GABA(A) receptors with agonists selective for these subunits resulted in appropriate membrane potential changes and chloride currents and promoted relaxation of airway smooth muscle. In conclusion, selective subunit targeting of endogenous airway smooth muscle-specific GABA(A) receptors may represent a novel therapeutic option for patients in severe bronchospasm.

Novel expression of a functional glycine receptor chloride channel that attenuates contraction in airway smooth muscle.

Airway smooth muscle (ASM) contraction is an important component of the pathophysiology of asthma. Taurine, an agonist of glycine receptor chloride (GlyR Cl(-)) channels, was found to relax contracted ASM, which led us to question whether functional GlyR Cl(-) channels are expressed in ASM. Messenger RNA for ß (GLRB), α1 (GLRA1), α2 (GLRA2), and α4 (GLRA4) subunits were found in human (Homo sapiens) and guinea pig (Cavia porcellus) tracheal smooth muscle. Immunoblotting confirmed the protein expression of GLRA1 and GLRB subunits in ASM. Electrical activity of cultured human ASM cells was assessed using a fluorescent potentiometric dye and electrophysiological recordings. Glycine increased current and significantly increased fluorescence in a dose-dependent manner. The GlyR Cl(-) channel antagonist strychnine significantly blocked the effects of glycine on potentiometric fluorescence in ASM cells. Guinea pig airway ring relaxation of ACh-induced contractions by isoproterenol was significantly left-shifted in the presence of glycine. This effect of glycine was blocked by pretreatment with the GlyR Cl(-) channel antagonist strychnine. Glycine treatment during tachykinin- and acetylcholine-induced contractions significantly decreased the maintenance of muscle force compared to control. GlyR Cl(-) channels are expressed on ASM and regulate smooth muscle force and offer a novel target for therapeutic relaxation of ASM.

Functional expression of γ-amino butyric acid transporter 2 in human and guinea pig airway epithelium and smooth muscle.

γ-Amino butyric acid (GABA) is a primary inhibitory neurotransmitter in the central nervous system, and is classically released by fusion of synaptic vesicles with the plasma membrane or by egress via GABA transporters (GATs). Recently, a GABAergic system comprised of GABA(A) and GABA(B) receptors has been identified on airway epithelial and smooth muscle cells that regulate mucus secretion and contractile tone of airway smooth muscle (ASM). In addition, the enzyme that synthesizes GABA, glutamic acid decarboxylase, has been identified in airway epithelial cells; however, the mechanism(s) by which this synthesized GABA is released from epithelial intracellular stores is unknown. We questioned whether any of the four known isoforms of GATs are functionally expressed in ASM or epithelial cells. We detected mRNA and protein expression of GAT2 and -4, and isoforms of glutamic acid decarboxylase in native and cultured human ASM and epithelial cells. In contrast, mRNA encoding vesicular GAT (VGAT), the neuronal GABA transporter, was not detected. Functional inhibition of (3)H-GABA uptake was demonstrated using GAT2 and GAT4/betaine-GABA transporter 1 (BGT1) inhibitors in both human ASM and epithelial cells. These results demonstrate that two isoforms of GATs, but not VGAT, are expressed in both airway epithelial and smooth muscle cells. They also provide a mechanism by which locally synthesized GABA can be released from these cells into the airway to activate GABA(A) channels and GABA(B) receptors, with subsequent autocrine and/or paracrine signaling effects on airway epithelium and ASM.

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.

Extracellular Matrix Rigidity Modulates Human Cervical Smooth Muscle Contractility-New Insights into Premature Cervical Failure and Spontaneous Preterm Birth.

Spontaneous preterm birth (sPTB), a major cause of infant morbidity and mortality, must involve premature cervical softening/dilation for a preterm vaginal delivery to occur. Yet, the mechanism behind premature cervical softening/dilation in humans remains unclear. We previously reported the non-pregnant human cervix contains considerably more cervical smooth muscle cells (CSMC) than historically appreciated and the CSMC organization resembles a sphincter. We hypothesize that premature cervical dilation leading to sPTB may be due to (1) an inherent CSMC contractility defect resulting in sphincter failure and/or (2) altered cervical extracellular matrix (ECM) rigidity which influences CSMC contractility. To test these hypotheses, we utilized immunohistochemistry to confirm this CSMC phenotype persists in the human pregnant cervix and then assessed in vitro arrays of contractility (F:G actin ratios, PDMS pillar arrays) using primary CSMC from pregnant women with and without premature cervical failure (PCF). We show that CSMC from pregnant women with PCF do not have an inherent CSMC contractility defect but that CSMC exhibit decreased contractility when exposed to soft ECM. Given this finding, we used UPLC-ESI-MS/MS to evaluate collagen cross-link profiles in the cervical tissue from non-pregnant women with and without PCF and found that women with PCF have decreased collagen cross-link maturity ratios, which correlates to softer cervical tissue. These findings suggest having soft cervical ECM may lead to decreased CSMC contractile tone and a predisposition to sphincter laxity that contributes to sPTB. Further studies are needed to explore the interaction between cervical ECM properties and CSMC cellular behavior when investigating the pathophysiology of sPTB.