Publications by authors named "Raymond B Penn"

81 Publications

Diacylglycerol Kinase Inhibition Reduces Airway Contraction by Negative Feedback Regulation of Gq-signaling.

Am J Respir Cell Mol Biol 2021 Jul 22. Epub 2021 Jul 22.

Thomas Jefferson University, 6559, Center for Translational Medicine, Philadelphia, Pennsylvania, United States;

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors (GPCRs) causes airway hyperresponsiveness (AHR) in asthma. Activation of Gq-coupled GPCRs leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKα and ζ isoform knockout mice are protected from the development of allergen-induced AHR. Here we aimed at determining the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζwhile pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Further, we employed human precision-cut lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pre-treatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and myosin light chain phosphatase in ASM cells. Further, DGK inhibition decreased Gq agonist-induced calcium elevation, generation of IP3, and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.
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http://dx.doi.org/10.1165/rcmb.2021-0106OCDOI Listing
July 2021

Autocrine regulation of airway smooth muscle contraction by diacylglycerol kinase.

J Cell Physiol 2021 Jul 18. Epub 2021 Jul 18.

Division of Pulmonary, Allergy and Critical Care Medicine, Center for Translational Medicine, Jane & Leonard Korman Respiratory Institute, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Diacylglycerol kinase (DGK), a lipid kinase, catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid, thereby terminating DAG-mediated signaling by Gq-coupled receptors that regulate contraction of airway smooth muscle (ASM). A previous study from our laboratory demonstrated that DGK inhibition or genetic ablation leads to reduced ASM contraction and provides protection for allergen-induced airway hyperresponsiveness. However, the mechanism by which DGK regulates contractile signaling in ASM is not well established. Herein, we investigated the role of prorelaxant cAMP-protein kinase A (PKA) signaling in DGK-mediated regulation of ASM contraction. Pretreatment of human ASM cells with DGK inhibitor I activated PKA as demonstrated by the phosphorylation of PKA substrates, VASP, Hsp20, and CREB, which was abrogated when PKA was inhibited pharmacologically or molecularly using overexpression of the PKA inhibitor peptide, PKI. Furthermore, inhibition of DGK resulted in induction of cyclooxygenase (COX) and generation of prostaglandin E (PGE ) with concomitant activation of Gs-cAMP-PKA signaling in ASM cells in an autocrine/paracrine fashion. Inhibition of protein kinase C (PKC) or extracellular-signal-regulated kinase (ERK) attenuated DGK-mediated production of PGE and activation of cAMP-PKA signaling in human ASM cells, suggesting that inhibition of DGK activates the COX-PGE pathway in a PKC-ERK-dependent manner. Finally, DGK inhibition-mediated attenuation of contractile agonist-induced phosphorylation of myosin light chain 20 (MLC-20), a marker of ASM contraction, involves COX-mediated cAMP production and PKA activation in ASM cells. Collectively these findings establish a novel mechanism by which DGK regulates ASM contraction and further advances DGK as a potential therapeutic target to provide effective bronchoprotection in asthma.
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http://dx.doi.org/10.1002/jcp.30528DOI Listing
July 2021

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

FASEB J 2021 07;35(7):e21674

Department of Medicine, Pulmonary and Critical Care Medicine, Center for Translational Medicine, Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.
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http://dx.doi.org/10.1096/fj.202100154RDOI Listing
July 2021

Can GPCRs Be Targeted to Control Inflammation in Asthma?

Adv Exp Med Biol 2021 ;1304:1-20

Center for Translational Medicine, Division of Pulmonary, Allergy, & Critical Care Medicine Jane & Leonard Korman Respiratory Institute, Sidney Kimmel Medical College Thomas Jefferson University, Philadelphia, PA, USA.

Historically, the drugs used to manage obstructive lung diseases (OLDs), asthma, and chronic obstructive pulmonary disease (COPD) either (1) directly regulate airway contraction by blocking or relaxing airway smooth muscle (ASM) contraction or (2) indirectly regulate ASM contraction by inhibiting the principal cause of ASM contraction/bronchoconstriction and airway inflammation. To date, these tasks have been respectively assigned to two diverse drug types: agonists/antagonists of G protein-coupled receptors (GPCRs) and inhaled or systemic steroids. These two types of drugs "stay in their lane" with respect to their actions and consequently require the addition of the other drug to effectively manage both inflammation and bronchoconstriction in OLDs. Indeed, it has been speculated that safety issues historically associated with beta-agonist use (beta-agonists activate the beta-2-adrenoceptor (βAR) on airway smooth muscle (ASM) to provide bronchoprotection/bronchorelaxation) are a function of pro-inflammatory actions of βAR agonism. Recently, however, previously unappreciated roles of various GPCRs on ASM contractility and on airway inflammation have been elucidated, raising the possibility that novel GPCR ligands targeting these GPCRs can be developed as anti-inflammatory therapeutics. Moreover, we now know that many GPCRs can be "tuned" and not just turned "off" or "on" to specifically activate the beneficial therapeutic signaling a receptor can transduce while avoiding detrimental signaling. Thus, the fledging field of biased agonism pharmacology has the potential to turn the βAR into an anti-inflammatory facilitator in asthma, possibly reducing or eliminating the need for steroids.
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http://dx.doi.org/10.1007/978-3-030-68748-9_1DOI Listing
May 2021

Honing in on the effectors of oxidative stress in the asthmatic lung: oxidised phosphatidylcholines.

Authors:
Raymond B Penn

Eur Respir J 2021 02 17;57(2). Epub 2021 Feb 17.

Center for Translational Medicine, Dept of Medicine, Thomas Jefferson University, Philadelphia, PA, USA

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http://dx.doi.org/10.1183/13993003.03736-2020DOI Listing
February 2021

Enlightening Ways to Relax Airway Smooth Muscle: Opsins.

Am J Respir Cell Mol Biol 2021 01;64(1):4-6

Korman Respiratory Institute Thomas Jefferson University Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1165/rcmb.2020-0468EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781008PMC
January 2021

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

Proc Natl Acad Sci U S A 2020 11 23;117(45):28485-28495. Epub 2020 Oct 23.

Department of Pharmacology, Rutgers-Robert Wood Johnson Medical School, The State University of New Jersey, Piscataway, NJ 08854;

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca]) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca] and relaxation of ASM cells. Of note, OR2W3-evoked [Ca] mobilization and ASM relaxation required Ca flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.
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http://dx.doi.org/10.1073/pnas.2003111117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668088PMC
November 2020

Mast cells in asthma: Here I am, stuck in the middle with you.

Authors:
Raymond B Penn

Eur Respir J 2020 07 2;56(1). Epub 2020 Jul 2.

Dept of Medicine, Division of Pulmonary and Critical Care Medicine, Center for Translational Medicine, Jane and Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA

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http://dx.doi.org/10.1183/13993003.01337-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643049PMC
July 2020

The proton-sensing receptor ovarian cancer G-protein coupled receptor 1 (OGR1) in airway physiology and disease.

Curr Opin Pharmacol 2020 04 29;51:1-10. Epub 2020 Apr 29.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Institute, Thomas Jefferson University, 1020 Locust St., Suite 543G JAH, Philadelphia, PA, 19107, United States. Electronic address:

Numerous G protein-coupled receptors (GPCRs) regulate multiple airway functions and play fundamental roles in normal and aberrant airway and lung physiology. Thus, GPCRs are prime candidates of targeting by disease therapeutics. The intriguing proton-sensing GPCR Ovarian cancer G-protein coupled receptor 1 (OGR1; aka GPR68) has recently been shown capable of regulating airway smooth muscle (ASM) contraction and proliferation. Although the study of OGR1 has been confounded by the fact that the proton is the presumed cognate ligand of OGR1, recent studies have begun to identify novel ligands and modulators capable of regulating the diverse signaling, and functional role of OGR1. Such studies offer hope for OGR1-targeting drugs as therapeutics for obstructive lung diseases such as asthma. Herein, we review the literature to date detailing the receptor biology and pharmacology of OGR1, receptor function in the airway, and describe the potential clinical utility of OGR1-modulating drugs.
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http://dx.doi.org/10.1016/j.coph.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529780PMC
April 2020

A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation.

J Biol Chem 2020 03 21;295(10):3316-3329. Epub 2020 Jan 21.

Center for Translational Medicine and Korman Respiratory Institute, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.
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http://dx.doi.org/10.1074/jbc.RA119.010004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062156PMC
March 2020

Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2.

FASEB J 2020 02 16;34(2):2126-2146. Epub 2019 Dec 16.

Department of Medicine, Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA, USA.

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.
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http://dx.doi.org/10.1096/fj.201901301RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018560PMC
February 2020

Agonist-specific desensitization of PGE-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts.

Naunyn Schmiedebergs Arch Pharmacol 2020 05 28;393(5):843-856. Epub 2019 Dec 28.

Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, 92672, USA.

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3',5'-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E (PGE) can stimulate cAMP production through prostaglandin E (EP) and EP receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE shifted the concentration-response curve to PGE rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP- and EP-selective agonists and antagonists suggested that PGE-stimulated cAMP responses in HFL-1 cells are mediated by EP receptors. EP receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE pretreatment. PGE treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE causes agonist-specific desensitization of EP receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.
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http://dx.doi.org/10.1007/s00210-019-01800-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328663PMC
May 2020

Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors.

Cell 2019 10;179(4):895-908.e21

Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. Electronic address:

The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2019.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838683PMC
October 2019

Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

FASEB J 2019 08 1;33(8):9008-9016. Epub 2019 May 1.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, The Jane and Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Na/H exchanger regulatory factor 1 (NHERF1; also known as ezrin-radixin-moesin-binding phosphoprotein 50) is a PSD-95, disc large, zona occludens-1 adapter that acts as a scaffold for signaling complexes and cytoskeletal-plasma membrane interactions. NHERF1 is crucial to β-2-adrenoceptor (βAR)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized βAR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP-mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho-vasodilator-stimulated phosphoprotein (p-VASP) by isoproterenol (ISO), prostaglandin E (PGE), or forskolin (FSK) as well as the induction of p-heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO-, PGE-, and FSK-induced IL-6 gene expression and cytokine production without affecting cAMP-mediated phosphodiesterase 4D (PDE4D) gene expression, phospho-cAMP response element-binding protein (p-CREB), and cAMP response element (CRE)-Luc, or PDGF-induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO-induced chromatin-binding of the transcription factor CCAAT-enhancer-binding protein-β (c/EBPβ). c/EBPβ knockdown almost completely abrogated the cAMP-mediated IL-6 but not PDE4D gene expression. The differential regulation of cAMP-induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.-Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.
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http://dx.doi.org/10.1096/fj.201900323RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662985PMC
August 2019

Methods to Investigate β-Arrestin-Mediated Regulation of GPCR Function in Human Airway Smooth Muscle.

Methods Mol Biol 2019 ;1957:69-82

Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, Jane and Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA.

Arrestin proteins were originally characterized as regulators of GPCR desensitization, and that function alone was sufficient to promote extreme interest in their study. It is now appreciated that arrestins also function as mediators of GPCR trafficking and G protein-independent signaling. This latter function places them as prominent players in the emerging field of qualitative signaling, which promises to launch a new area of pharmacology that defines ligands with selectivity/bias toward either G protein-dependent or -independent signaling. To meet the demands of research into arrestin function, methodology has evolved accordingly over the last three decades since the discovery of the arrestin family. Herein we describe state-of-the-art approaches for studying the role of arrestins (β-arrestin1 aka arrestin 2, β-arrestin2 aka arrestin 3) in GPCR function in a primary cell type, cultured airway smooth muscle cells.
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http://dx.doi.org/10.1007/978-1-4939-9158-7_4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857537PMC
July 2019

Regulation of ovarian cancer G protein-coupled receptor-1 expression and signaling.

Am J Physiol Lung Cell Mol Physiol 2019 05 6;316(5):L894-L902. Epub 2019 Feb 6.

Department of Medicine, Center for Translational Medicine and Division of Pulmonary, Allergy and Critical Care Medicine; and Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, Pennsylvania.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pH) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pH or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pH and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pH or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.
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http://dx.doi.org/10.1152/ajplung.00426.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589584PMC
May 2019

NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy.

Am J Pathol 2019 04 30;189(4):847-867. Epub 2019 Jan 30.

Department of Medicine, Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania; Division of Urology, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.
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http://dx.doi.org/10.1016/j.ajpath.2018.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458544PMC
April 2019

Cooperativity of E-prostanoid receptor subtypes in regulating signaling and growth inhibition in human airway smooth muscle.

FASEB J 2019 04 2;33(4):4780-4789. Epub 2019 Jan 2.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Prostaglandin E2 (PGE) is produced in the airway during allergic lung inflammation and both promotes and inhibits features of asthma pathology. These mixed effects relate to 4 E-prostanoid (EP) receptor subtypes (EP1, 2, 3 and 4) expressed at different levels on different resident and infiltrating airway cells. Although studies have asserted both EP2 and EP4 expression in human airway smooth muscle (HASM), a recent study asserted EP4 to be the functionally dominant EP subtype in HASM. Herein, we employ recently-developed subtype-selective ligands to investigate singular or combined EP2 and EP4 receptor activation in regulating HASM signaling and proliferation. The subtype specificity of ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) was first demonstrated in human embryonic kidney 293 cells stably expressing different EP receptor subtypes. EP receptor knockdown and subtype-selective antagonists demonstrated EP2 and EP4 receptor responsiveness in HASM cells to the specific ONO compounds, whereas PGE appeared to preferentially signal via the EP4 receptor. Both singular EP2 and EP4 receptor agonists inhibited HASM proliferation, and combined EP2 and EP4 receptor agonism exhibited positive cooperativity in both chronic G-mediated signaling and inhibiting HASM proliferation. These findings suggest both EP2 and EP4 are functionally important in HASM, and their combined targeting optimally inhibits airway smooth muscle proliferation.-Michael, J. V. Gavrila, A., Nayak, A. P., Pera, T., Liberato, J. R., Polischak, S. R., Shah, S. D., Deshpande, D. A., Penn, R. B. Cooperativity of E-prostanoid receptor subtypes in regulating signaling and growth inhibition in human airway smooth muscle.
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http://dx.doi.org/10.1096/fj.201801959RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436661PMC
April 2019

Give Me a Fork: Can Autophagy Research Solve the Riddle of Airway Remodeling in Asthma?

Am J Respir Cell Mol Biol 2019 May;60(5):494-496

1 Jane and Leonard Korman Lung Institute Thomas Jefferson University Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1165/rcmb.2018-0353EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503612PMC
May 2019

New targets for resolution of airway remodeling in obstructive lung diseases.

F1000Res 2018 30;7. Epub 2018 May 30.

Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, USA.

Airway remodeling (AR) is a progressive pathological feature of the obstructive lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). The pathology manifests itself in the form of significant, progressive, and (to date) seemingly irreversible changes to distinct respiratory structural compartments. Consequently, AR correlates with disease severity and the gradual decline in pulmonary function associated with asthma and COPD. Although current asthma/COPD drugs manage airway contraction and inflammation, none of these effectively prevent or reverse features of AR. In this review, we provide a brief overview of the features and putative mechanisms affecting AR. We further discuss recently proposed strategies with promise for deterring or treating AR.
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http://dx.doi.org/10.12688/f1000research.14581.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981194PMC
May 2018

Pepducins as a potential treatment strategy for asthma and COPD.

Curr Opin Pharmacol 2018 06 2;40:120-125. Epub 2018 May 2.

Sidney Kimmel Medical College, Center for Translational Medicine, Jane and Leonard Korman Lung Institute, Thomas Jefferson University, Philadelphia, PA 19107, United States.

Current therapies to treat asthma and other airway diseases primarily include anti-inflammatory agents and bronchodilators. Anti-inflammatory agents target trafficking and resident immunocytes and structural cells, while bronchodilators act to prevent or reverse shortening of airway smooth muscle (ASM), the pivotal tissue regulating bronchomotor tone. Advances in our understanding of the biology of G protein-coupled receptors (GPCRs) and biased agonism offers unique opportunities to modulate GPCR function that include the use of pepducins and allosteric modulators. Recent evidence suggests that small molecule inhibitors of Gα as well as pepducins targeting G-coupled receptors can broadly inhibit contractile agonist-induced ASM function. Given these advances, new therapeutic approaches can be leveraged to diminish the global rise in morbidity and mortality associated with asthma and chronic obstructive pulmonary disease.
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http://dx.doi.org/10.1016/j.coph.2018.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345380PMC
June 2018

Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

FASEB J 2018 02 4;32(2):862-874. Epub 2018 Jan 4.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.
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http://dx.doi.org/10.1096/fj.201700555RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888400PMC
February 2018

Gα facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

Br J Pharmacol 2017 Dec 12;174(23):4383-4395. Epub 2017 Nov 12.

Rutgers Institute for Translational Medicine and Science, Child Health Institute, Rutgers University, New Brunswick, NJ, USA.

Background And Purpose: PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G family proteins activate ROCK pathways in other cell types, their role in M muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined.

Experimental Approach: Gα coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction.

Key Results: Knockdown of M receptors or Gα attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα coimmunoprecipitated with M receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS.

Conclusions And Implications: Gα plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma.
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http://dx.doi.org/10.1111/bph.14040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715591PMC
December 2017

Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone.

Sci Rep 2017 05 3;7(1):1477. Epub 2017 May 3.

Department of Medicine, Division of Gastroenterology & Hepatology, Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, PA, USA.

The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p.
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http://dx.doi.org/10.1038/s41598-017-01550-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431208PMC
May 2017

An Official American Thoracic Society Research Statement: Current Challenges Facing Research and Therapeutic Advances in Airway Remodeling.

Am J Respir Crit Care Med 2017 01;195(2):e4-e19

Background: Airway remodeling (AR) is a prominent feature of asthma and other obstructive lung diseases that is minimally affected by current treatments. The goals of this Official American Thoracic Society (ATS) Research Statement are to discuss the scientific, technological, economic, and regulatory issues that deter progress of AR research and development of therapeutics targeting AR and to propose approaches and solutions to these specific problems. This Statement is not intended to provide clinical practice recommendations on any disease in which AR is observed and/or plays a role.

Methods: An international multidisciplinary group from within academia, industry, and the National Institutes of Health, with expertise in multimodal approaches to the study of airway structure and function, pulmonary research and clinical practice in obstructive lung disease, and drug discovery platforms was invited to participate in one internet-based and one face-to-face meeting to address the above-stated goals. Although the majority of the analysis related to AR was in asthma, AR in other diseases was also discussed and considered in the recommendations. A literature search of PubMed was performed to support conclusions. The search was not a systematic review of the evidence.

Results: Multiple conceptual, logistical, economic, and regulatory deterrents were identified that limit the performance of AR research and impede accelerated, intensive development of AR-focused therapeutics. Complementary solutions that leverage expertise of academia and industry were proposed to address them.

Conclusions: To date, numerous factors related to the intrinsic difficulty in performing AR research, and economic forces that are disincentives for the pursuit of AR treatments, have thwarted the ability to understand AR pathology and mechanisms and to address it clinically. This ATS Research Statement identifies potential solutions for each of these factors and emphasizes the importance of educating the global research community as to the extent of the problem as a critical first step in developing effective strategies for: (1) increasing the extent and impact of AR research and (2) developing, testing, and ultimately improving drugs targeting AR.
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http://dx.doi.org/10.1164/rccm.201611-2248STDOI Listing
January 2017

β Agonists.

Handb Exp Pharmacol 2017 ;237:23-40

Division of Respiratory Medicine, University of Nottingham, Nottingham, NG7 2RD, UK.

History suggests β agonists, the cognate ligand of the β adrenoceptor, have been used as bronchodilators for around 5,000 years, and β agonists remain today the frontline treatment for asthma and chronic obstructive pulmonary disease (COPD). The β agonists used clinically today are the products of significant expenditure and over 100 year's intensive research aimed at minimizing side effects and enhancing therapeutic usefulness. The respiratory physician now has a therapeutic toolbox of long acting β agonists to prophylactically manage bronchoconstriction, and short acting β agonists to relieve acute exacerbations. Despite constituting the cornerstone of asthma and COPD therapy, these drugs are not perfect; significant safety issues have led to a black box warning advising that long acting β agonists should not be used alone in patients with asthma. In addition there are a significant proportion of patients whose asthma remains uncontrolled. In this chapter we discuss the evolution of β agonist use and how the understanding of β agonist actions on their principal target tissue, airway smooth muscle, has led to greater understanding of how these drugs can be further modified and improved in the future. Research into the genetics of the β adrenoceptor will also be discussed, as will the implications of individual DNA profiles on the clinical outcomes of β agonist use (pharmacogenetics). Finally we comment on what the future may hold for the use of β agonists in respiratory disease.
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http://dx.doi.org/10.1007/164_2016_64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480238PMC
September 2017

Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a.

Am J Physiol Gastrointest Liver Physiol 2016 11 15;311(5):G964-G973. Epub 2016 Sep 15.

Department of Medicine, Division of Gastroenterology & Hepatology, Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania.

A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.
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http://dx.doi.org/10.1152/ajpgi.00290.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130548PMC
November 2016

Bronchoprotection and bronchorelaxation in asthma: New targets, and new ways to target the old ones.

Pharmacol Ther 2016 08 23;164:82-96. Epub 2016 Apr 23.

Center for Translational Medicine and Jane and Leonard Korman Lung Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, United States. Electronic address:

Despite over 50years of inhaled beta-agonists and corticosteroids as the default management or rescue drugs for asthma, recent research suggests that new therapeutic options are likely to emerge. This belief stems from both an improved understanding of what causes and regulates airway smooth muscle (ASM) contraction, and the identification of new targets whose inhibition or activation can relax ASM. In this review we discuss the recent findings that provide new insight into ASM contractile regulation, a revolution in pharmacology that identifies new ways to "tune" G protein-coupled receptors to improve therapeutic efficacy, and the discovery of several novel targets/approaches capable of effecting bronchoprotection or bronchodilation.
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http://dx.doi.org/10.1016/j.pharmthera.2016.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942340PMC
August 2016

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by β2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

Am J Respir Cell Mol Biol 2016 08;55(2):234-42

1 Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Texas.

Mice lacking the endogenous β2-adrenoceptor (β2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of β2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various β2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous β2AR agonists on allergic lung inflammation can be explained by qualitative β2AR signaling. The β2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a β-arrestin-dependent pathway. Previous studies suggest that β-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the β2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing β2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of β2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by β-agonists.
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http://dx.doi.org/10.1165/rcmb.2015-0373OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979368PMC
August 2016

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

Nature 2015 Nov 9;527(7579):477-83. Epub 2015 Nov 9.

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7365, USA.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.
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http://dx.doi.org/10.1038/nature15699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4796946PMC
November 2015
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