The Anesthesiology Physician-Scientist Pipeline: Current Status and Recommendations for Future Growth-An Initiative of the Anesthesia Research Council.

The limited number and diversity of resident physicians pursuing careers as physician-scientists in medicine has been a concern for many decades. The Anesthesia Research Council aimed to address the status of the anesthesiology physician-scientist pipeline, benchmarked against other medical specialties, and to develop strategic recommendations to sustain and expand the number and diversity of anesthesiology physician-scientists. The working group analyzed data from the Association of American Medical Colleges and the National Resident Matching Program to characterize the diversity and number of research-oriented residents from US and international allopathic medical schools entering 11 medical specialties from 2009 to 2019. Two surveys were developed to assess the research culture of anesthesiology departments. National Institutes of Health (NIH) funding information awarded to anesthesiology physician-scientists and departments was collected from NIH RePORTER and the Blue Ridge Medical Institute. Anesthesiology ranked eighth to tenth place of 11 medical specialties in the percent of "research-oriented" entering residents, defined as those with advanced degrees (Master's or PhDs) in addition to the MD degree or having published at least 3 research publications before residency. Anesthesiology ranked eighth of 11 specialties in the percent of entering residents who were women but ranked fourth of 11 specialties in the percent of entering residents who self-identified as belonging to an underrepresented group in medicine. There has been a 72% increase in both the total NIH funding awarded to anesthesiology departments and the number of NIH K-series mentored training grants (eg, K08 and K23) awarded to anesthesiology physician-scientists between 2015 and 2020. Recommendations for expanding the size and diversity of the anesthesiology physician-scientist pipeline included (1) developing strategies to increase the number of research intensive anesthesiology departments; (2) unifying the diverse programs among academic anesthesiology foundations and societies that seek to grow research in the specialty; (3) adjusting American Society of Anesthesiologists metrics of success to include the number of anesthesiology physician-scientists with extramural research support; (4) increasing the number of mentored awards from Foundation of Anesthesia Education and Research (FAER) and International Anesthesia Research Society (IARS); (5) supporting an organized and concerted effort to inform research-oriented medical students of the diverse research opportunities within anesthesiology should include the specialty being represented at the annual meetings of Medical Scientist Training Program (MSTP) students and the American Physician Scientist Association, as well as in institutional MSTP programs. The medical specialty of anesthesiology is defined by new discoveries and contributions to perioperative medicine which will only be sustained by a robust pipeline of anesthesiology physician-scientists.

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1ß3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63CreERT2 and KRT5-CreERT2-labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2-labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.

Pharmacokinetics of Gingerols, Shogaols, and Their Metabolites in Asthma Patients.

6-Gingerol and 6-shogaol are the most abundant gingerols and shogaols in ginger root and have been shown to reduce the asthmatic phenotype in murine models of asthma. Several studies have described the pharmacokinetics of gingerols and shogaols in humans following the oral ingestion of ginger, while little was known about the metabolism of these components in humans, particularly in patients with asthma. In this study, a dietary supplement of 1.0 g of ginger root extract was administered to asthma patients twice daily for 56 days and serum samples were drawn at 0.5-8 h on days 0, 28, and 56. The metabolic profiles of gingerols and shogaols in human plasma and the kinetic changes of gingerols, shogaols, and their metabolites in asthma patients collected on the three different visits were analyzed using liquid chromatography-mass spectrometry (LC-MS). Ketone reduction was the major metabolic pathway of both gingerols and shogaols. Gingerdiols were identified as the major metabolites of 6-, 8-, and 10-gingerols. M11 and M9 were identified as the double-bond reduction and both the double-bond and ketone reduction metabolites of 6-shogaol, respectively. Cysteine conjugation was another major metabolic pathway of 6-shogaol in asthma patients, and two cysteine-conjugated 6-shogaol, M1 and M2, were identified as the major metabolites of 6-shogaol. Furthermore, gingerols, shogaols, and their metabolites were quantitated in the human serum collected at different time points during each of the three visits using a very sensitive high-resolution LC-MS method. The results showed that one-third of 6-gingerol was metabolized to produce its reduction metabolites, 6-gingerdiols, and more than 90% of 6-shogaol was metabolized to its phase I and cysteine-conjugated metabolites, suggesting the importance of considering the contribution of these metabolites to the bioavailability and health beneficial effects of gingerols and shogaols. All gingerols, shogaols, and their metabolites reached their peak concentrations in less than 2 h, and their half-lives (t1/2) were from 0.6 to 2.4 h. Furthermore, long-term treatment of ginger supplements, especially after 56 days of treatment, increases the absorption of ginger compounds and their metabolites in asthma patients.

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction in vivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Development of Inhaled GABAA Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders.

We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting ß2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1ß3γ2 GABAAR structural data, suggests binding of the diazepine core between the α1+/γ2- interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 µM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 µM, with rapid onset (15 min) at 25 µM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

Melatonin MT2 receptor is expressed and potentiates contraction in human airway smooth muscle.

Nocturnal asthma is characterized by heightened bronchial reactivity at night, and plasma melatonin concentrations are higher in patients with nocturnal asthma symptoms. Numerous physiological effects of melatonin are mediated via its specific G protein-coupled receptors (GPCRs) named the MT1 receptor, which couples to both Gq and Gi proteins, and the MT2 receptor, which couples to Gi. We investigated whether melatonin receptors are expressed on airway smooth muscle; whether they regulate intracellular cyclic AMP (cAMP) and calcium concentrations ([Ca2+]i), which modulate airway smooth muscle tone; and whether they promote airway smooth muscle cell proliferation. We detected the mRNA and protein expression of the melatonin MT2 but not the MT1 receptor in native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by RT-PCR, immunoblotting, and immunohistochemistry. Activation of melatonin MT2 receptors with either pharmacological concentrations of melatonin (10-100 µM) or the nonselective MT1/MT2 agonist ramelteon (10 µM) significantly inhibited forskolin-stimulated cAMP accumulation in HASM cells, which was reversed by the Gαi protein inhibitor pertussis toxin or knockdown of the MT2 receptor by its specific siRNA. Although melatonin by itself did not induce an initial [Ca2+]i increase and airway contraction, melatonin significantly potentiated acetylcholine-stimulated [Ca2+]i increases, stress fiber formation through the MT2 receptor in HASM cells, and attenuated the relaxant effect of isoproterenol in guinea pig trachea. These findings suggest that the melatonin MT2 receptor is expressed in ASM, and modulates airway smooth muscle tone via reduced cAMP production and increased [Ca2+]i.

Ginger metabolites and metabolite-inspired synthetic products modulate intracellular calcium and relax airway smooth muscle.

Asthma affects millions of people worldwide and its prevalence is increasing. It is characterized by chronic airway inflammation, airway remodeling, and pathologic bronchoconstriction, and it poses a continuous treatment challenge with very few new therapeutics available. Thus, many asthmatics turn to plant-based complementary products, including ginger, for better symptom control, indicating an unmet need for novel therapies. Previously, we demonstrated that 6-shogaol (6S), the primary bioactive component of ginger, relaxes human airway smooth muscle (hASM) likely by inhibition of phosphodiesterases (PDEs) in the ß-adrenergic (cyclic nucleotide PDEs), and muscarinic (phospholipase C, PLC) receptor pathways. However, oral 6S is extensively metabolized and it is unknown if the resulting metabolites remain bioactive. Here, we screened all the known human metabolites of 6S and several metabolite-based synthetic derivatives to better understand their mechanism of action and structure-function relationships. We demonstrate that several metabolites and metabolite-based synthetic derivatives are able to prevent Gq-coupled stimulation of intracellular calcium [Ca2+]i and inositol trisphosphate (IP3) synthesis by inhibiting PLC, similar to the parent compound 6S. We also show that these compounds prevent recontraction of ASM after ß-agonist relaxation likely by inhibiting PDEs. Furthermore, they potentiate isoproterenol-induced relaxation. Importantly, moving beyond cell-based assays, metabolites also retain the functional ability to relax Gq-coupled-contractions in upper (human) and lower (murine) airways. The current study indicates that, although oral ginger may be metabolized rapidly, it retains physiological activity through its metabolites. Moreover, we are able to use naturally occurring metabolites as inspiration to develop novel therapeutics for brochoconstrictive diseases.

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.

Nebulized MIDD0301 Reduces Airway Hyperresponsiveness in Moderate and Severe Murine Asthma Models.

We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABAAR) modulator. The therapeutic efficacy of nebulized MIDD0301 in reducing airway resistance was investigated in spontaneous breathing mice using a whole-body plethysmograph and in unconscious mice using a forced oscillation technique. Prophylactic nebulized MIDD0301 reduced subsequent methacholine-induced bronchoconstriction in ovalbumin and house dust mite allergic asthma models and in normal mice. Nebulized MIDD0301 exhibited comparable or better therapeutic potency compared to nebulized albuterol and oral montelukast. Prophylactic nebulized MIDD0301 was also effective in reducing bronchoconstriction, comparable to nebulized albuterol or fluticasone, in a steroid resistant asthma mouse model induced by intratracheal installation of lipopolysaccharide and interferon-gamma. Oral dexamethasone was ineffective in this model. Nebulized MIDD0301 was also effective in reversing bronchospasm when dosed after methacholine challenge comparable to albuterol. Pharmacokinetic studies showed that about 0.06% of nebulized MIDD0301 entered the mouse lung when using a whole body plethysmograph and therapeutic levels were sustained in the lung for at least 25 min. Consistent with previous reports on orally dosed MIDD0301, high doses of nebulized MIDD0301 resulted in minimal brain exposure and thus no observable adverse sensorimotor or respiratory depression effects occurred. In addition, no adverse cardiovascular effects were observed following 100 mg/kg i.p. dosing. These results further demonstrate that charged imidazodiazepine MIDD0301 can selectively target lung GABAAR without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation.

Reduced allergic lung inflammation and airway responsiveness in mice lacking the cytoskeletal protein gelsolin.

Airway smooth muscle hyperresponsiveness associated with chronic airway inflammation leads to the typical symptoms of asthma including bronchoconstriction and wheezing. Asthma severity is associated with airway inflammation; therefore, reducing airway inflammation is an important therapeutic target. Gelsolin is an actin capping and severing protein that has been reported to be involved in modulation of the inflammatory response. Using mice genetically lacking gelsolin, we evaluated the role of gelsolin in the establishment of house dust mite (HDM) antigen-induced allergic lung inflammation. The genetic absence of gelsolin was found to be protective against HDM sensitization, resulting in reduced lung inflammation, inflammatory cytokines, and Muc5AC protein in bronchoalveolar lavage (BAL) fluid. The number of eosinophils, lymphocytes, and interstitial macrophages in the BAL were increased after HDM sensitization in wild-type mice but were attenuated in gelsolin-null mice. The observed attenuation of inflammation may be partly due to delayed migration of immune cells, because the reduced eosinophils in the BALs from gelsolin-null mice compared with controls occurred despite similar amounts of the chemoattractant eotaxin. Splenic T cells demonstrated similar proliferation rates, but ex vivo alveolar macrophage migration was delayed in gelsolin-null mice. In vivo, the reduced lung inflammation after HDM sensitization in gelsolin-null mice was associated with significantly diminished airway resistance to inhaled methacholine compared with HDM-treated wild-type mice. Our results suggest that modulation of gelsolin expression or function in selective inflammatory cell types that modulate allergic lung inflammation could be a therapeutic approach for asthma.

Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.

Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.

The short-chain free fatty acid receptor FFAR3 is expressed and potentiates contraction in human airway smooth muscle.

Emerging evidence suggests that gut microbiota-derived short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) are important modulators of the inflammatory state in diseases such as asthma. However, the functional expression of the Gi protein-coupled free fatty acid receptors (FFAR2/GPR43 and FFAR3/GPR41) has not been identified on airway smooth muscle (ASM). Classically, acute activation of Gi-coupled receptors inhibits cyclic AMP (cAMP) synthesis, which impairs ASM relaxation and can also induce crosstalk between Gi- and Gq-signaling pathways, potentiating increases in intracellular Ca2+ concentration ([Ca2+]i), favoring ASM contraction. In contrast, chronic activation of Gi-coupled receptors can sensitize adenylyl cyclase resulting in increased cAMP synthesis favoring relaxation. We questioned whether the Gi-coupled FFAR2 or FFAR3 is expressed in human ASM, whether they modulate cAMP and [Ca2+]i, and whether SCFAs modulate human ASM tone. We detected the protein expression of FFAR3 but not FFAR2 in native human ASM and primary cultured human airway smooth muscle (HASM) cells. In HASM cells, acute activation of FFAR3 with SCFAs inhibited forskolin-stimulated cAMP accumulation, but chronic activation did not sensitize cAMP synthesis. SCFAs induced [Ca2+]i increases that were attenuated by pertussis toxin, gallein, U73122, or xestospongin C. Acute treatment with SCFAs potentiated acetylcholine-stimulated [Ca2+]i increases and stress fiber formation in cells and contraction of ex vivo human airway tissues. In contrast, chronic pretreatment of human ASM with propionate did not potentiate airway relaxation. Together, these findings demonstrate that FFAR3 is expressed in human ASM and contributes to ASM contraction via reduced cAMP and increased [Ca2+]i.

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.

Bile acids inhibit cholinergic constriction in proximal and peripheral airways from humans and rodents.

Duodenogastroesophageal reflux (DGER) is associated with chronic lung disease. Bile acids (BAs) are established markers of DGER aspiration and are important risk factors for reduced post-transplant lung allograft survival by disrupting the organ-specific innate immunity, facilitating airway infection and allograft failure. However, it is unknown whether BAs also affect airway reactivity. We investigated the acute effects of 13 BAs detected in post-lung-transplant surveillance bronchial washings (BW) on airway contraction. We exposed precision-cut slices from human and mouse lungs to BAs and monitored dynamic changes in the cross-sectional luminal area of peripheral airways using video phase-contrast microscopy. We also used guinea pig tracheal rings in organ baths to study BA effects in proximal airway contraction induced by electrical field stimulation. We found that most secondary BAs at low micromolar concentrations strongly and reversibly relaxed smooth muscle and inhibited peripheral airway constriction induced by acetylcholine but not by noncholinergic bronchoconstrictors. Similarly, secondary BAs strongly inhibited cholinergic constrictions in tracheal rings. In contrast, TC-G 1005, a specific agonist of the BA receptor Takeda G protein-coupled receptor 5 (TGR5), did not cause airway relaxation, and Tgr5 deletion in knockout mice did not affect BA-induced relaxation, suggesting that this receptor is not involved. BAs inhibited acetylcholine-induced inositol phosphate synthesis in human airway smooth muscle cells overexpressing the muscarinic M3 receptor. Our results demonstrate that select BAs found in BW of patients with lung transplantation can affect airway reactivity by inhibiting the cholinergic contractile responses of the proximal and peripheral airways, possibly by acting as antagonists of M3 muscarinic receptors.

Ginger and its bioactive component 6-shogaol mitigate lung inflammation in a murine asthma model.

Asthma, a common disorder associated with airway inflammation and hyperresponsiveness, remains a significant clinical burden in need of novel therapeutic strategies. Patients are increasingly seeking complementary and alternative medicine approaches to control their symptoms, including the use of natural products. Ginger, a natural product that we previously demonstrated acutely relaxes airway smooth muscle (ASM), has long been reported to possess anti-inflammatory properties, although a precise mechanistic understanding is lacking. In these studies, we demonstrate that chronic administration of whole ginger extract or 6-shogaol, a bioactive component of ginger, mitigates in vivo house dust mite antigen-mediated lung inflammation in mice. We further show that this decrease in inflammation is associated with reduced in vivo airway responsiveness. Utilizing in vitro studies, we demonstrate that 6-shogaol augments cAMP concentrations in CD4 cells, consistent with phosphodiesterase inhibition, and limits the induction of nuclear factor-κB signaling and the production of proinflammatory cytokines in activated CD4 cells. Sustained elevations in cAMP concentration are well known to inhibit effector T cell function. Interestingly, regulatory T cells (Tregs) utilize cAMP as a mediator of their immunosuppressive effects, and we demonstrate here that 6-shogaol augments the Treg polarization of naïve CD4 cells in vitro. Taken together with previous reports, these studies suggest that ginger and 6-shogaol have the potential to combat asthma via two mechanisms: acute ASM relaxation and chronic inhibition of inflammation.

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.