Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.
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ABSTRACT:
SUBMITTER: Wu AD
PROVIDER: S-EPMC7780995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC6383505 | biostudies-literature
Project description:BackgroundDopamine signaling is mediated by Gs protein-coupled "D1-like" receptors (D1 and D5) and Gi-coupled "D2-like" receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling.MethodsThe mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.ResultsMessenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.ConclusionsThese results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.
| S-EPMC3847358 | biostudies-literature
Project description:Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) channels and metabotropic (GABA(B)) receptors. GABA(A) channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although GABA(A) channels and GABA(B) receptors have been functionally identified on peripheral nerves in the lung, GABA(A) channels have never been identified on ASM itself. We detected the mRNA encoding of the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, delta-, gamma(1-3)-, pi-, and theta-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, and gamma(2)-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The GABA(A) beta(3)-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific GABA(A) channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the GABA(A)-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional GABA(A) channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease.
| S-EPMC3647697 | biostudies-literature
Project description:Background and purposePrevious studies indicate that prostaglandin EP2 receptors selectively couple to AC2 in non-lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP-dependent responses. The goal of the present study was to identify the cellular microdomains where EP2 receptors stimulate cAMP production.Experimental approachFRET-based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2-camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2-MyrPalm and Epac2-CAAX were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane respectively. Epac2-NLS was used to monitor responses at the nucleus.Key resultsActivation of AC with forskolin or β2 -adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 receptors with butaprost produced cAMP responses that were most readily detected by the non-raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains.Conclusions and implicationsEP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while β2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.
| S-EPMC5522994 | biostudies-literature
Project description:Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.
| S-EPMC4131261 | biostudies-literature
Project description: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.
| S-EPMC3567364 | biostudies-literature
Project description:The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting ?-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca(2+)](i)) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100-300 ?M), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 ?M), blunted subsequent Ca(2+) responses to bradykinin (10 ?M) and S-(-)-Bay K 8644 (10 ?M). In A/J mice, the nebulization of [8]-gingerol (100 ?M), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca(2+)](i) regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including ?(2)-agonists, in airway diseases such as asthma.
| S-EPMC3604064 | biostudies-literature
Project description:Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-β1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-β1 affects the ability of HASM cells to relax in response to β2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-β1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-β1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-β1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-β1 decreases HASM cell β2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying β2-agonist hyporesponsiveness in asthma, and suggest TGF-β1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.
| S-EPMC6670035 | biostudies-literature
Project description:Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.
| S-EPMC7686140 | biostudies-literature
Project description:Obesity is one of the major risk factors for asthma. Previous studies have demonstrated that free fatty acid levels are elevated in the plasma of obese individuals. Medium- and long-chain free fatty acids act as endogenous ligands for the free fatty acid receptors FFAR1/GPR40 and FFAR4/GPR120, which couple to Gq proteins. We investigated whether FFAR1 and FFAR4 are expressed on airway smooth muscle and whether they activate Gq-coupled signaling and modulate airway smooth muscle tone. We detected the protein expression of FFAR1 and FFAR4 in freshly dissected native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by immunoblotting and immunohistochemistry. The long-chain free fatty acids (oleic acid and linoleic acid) and GW9508 (FFAR1/FFAR4 dual agonist) dose-dependently stimulated transient intracellular Ca(2+) concentration ([Ca(2+)]i) increases and inositol phosphate synthesis in HASM cells. Downregulation of FFAR1 or FFAR4 in HASM cells by small interfering RNA led to a significant inhibition of the long-chain free fatty acids-induced transient [Ca(2+)]i increases. Oleic acid, linoleic acid, or GW9508 stimulated stress fiber formation in HASM cells, potentiated acetylcholine-contracted guinea pig tracheal rings, and attenuated the relaxant effect of isoproterenol after an acetylcholine-induced contraction. In contrast, TUG-891 (FFAR4 agonist) did not induce the stress fiber formation or potentiate acetylcholine-induced contraction. These results suggest that FFAR1 is the functionally dominant free fatty acid receptor in both human and guinea pig airway smooth muscle. The free fatty acid sensors expressed on airway smooth muscle could be an important modulator of airway smooth muscle tone.
| S-EPMC4628981 | biostudies-literature