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Introduction: Air pollution from diesel combustion is linked in part to the generation of diesel exhaust particles (DEP). DEP exposure induces various processes, including inflammation and oxidative stress, which ultimately contribute to a decline in lung function. Cyclic AMP (cAMP) signaling is critical for lung homeostasis. The impact of DEP on cAMP signaling is largely unknown. Methods: We exposed human bronchial epithelial (BEAS-2B) cells to DEP for 24-72 h and evaluated mitochondrial bioenergetics, markers of oxidative stress and inflammation and the components of cAMP signaling. Mitochondrial bioenergetics was measured at 72 h to capture the potential and accumulative effects of prolonged DEP exposure on mitochondrial function. Results: DEP profoundly altered mitochondrial morphology and network integrity, reduced both basal and ATP-linked respiration as well as the glycolytic capacity of mitochondria. DEP exposure increased gene expression of oxidative stress and inflammation markers such as interleukin-8 and interleukin-6. DEP significantly affected mRNA levels of exchange protein directly activated by cAMP-1 and -2 (Epac1, Epac2), appeared to increase Epac1 protein, but left phospho-PKA levels unhanged. DEP exposure increased A-kinase anchoring protein 1, β2-adrenoceptor and prostanoid E receptor subtype 4 mRNA levels. Interestingly, DEP decreased mRNA levels of adenylyl cyclase 9 and reduced cAMP levels stimulated by forskolin (AC activator), fenoterol (β2-AR agonist) or PGE2 (EPR agonist). Discussion: Our findings suggest that DEP induces mitochondrial dysfunction, a process accompanied by oxidative stress and inflammation, and broadly dampens cAMP signaling. These epithelial responses may contribute to lung dysfunction induced by air pollution exposure. [ABSTRACT FROM AUTHOR]

Background and Purpose: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor‐mediated, compartmentalized responses. Experimental Approach: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA–AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down‐regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. Key Results: Cyclic AMP movement was significantly slower than that of free‐diffusion in hASM cells, and disrupting PKA–AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D‐AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor‐mediated responses. Conclusion and Implications: Our results confirm that AKAP‐anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction--magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively--to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 x 10-7 vs. 2.2 x 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes. [ABSTRACT FROM AUTHOR]

Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments. [ABSTRACT FROM AUTHOR]

Adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. Mammals possess nine isoforms of transmembrane ACs, dubbed AC1-9, that serve as major effector enzymes of G protein-coupled receptors (GPCRs). The transmembrane ACs display varying expression patterns across tissues, giving the potential for them to have a wide array of physiological roles. Cells express multiple AC isoforms, implying that ACs have redundant functions. Furthermore, all transmembrane ACs are activated by Gαs, so it was long assumed that all ACs are activated by Gαs-coupled GPCRs. AC isoforms partition to different microdomains of the plasma membrane and form prearranged signaling complexes with specific GPCRs that contribute to cAMP signaling compartments. This compartmentation allows for a diversity of cellular and physiological responses by enabling unique signaling events to be triggered by different pools of cAMP. Isoform-specific pharmacological activators or inhibitors are lacking for most ACs, making knockdown and overexpression the primary tools for examining the physiological roles of a given isoform. Much progress has been made in understanding the physiological effects mediated through individual transmembrane ACs. GPCR-AC-cAMP signaling pathways play significant roles in regulating functions of every cell and tissue, so understanding each AC isoform's role holds potential for uncovering new approaches for treating a vast array of pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Nicotinic ACh receptors containing the α7 sub-unit (α7-nAChRs) suppress inflammation through a wide range of pathways in immune cells. These receptors are thus potentially involved in a number of inflammatory diseases. However, the detailed mechanisms underlying the anti-inflammatory effects of α7-nAChRs remain to be described.Experimental Approach: Anti-inflammatory effects of α7-nAChR agonists were assessed in both murine macrophages (RAW 264.7) and bone marrow-derived macrophages (BMDM), stimulated with LPS, using immunoblotting, RT-PCR and luciferase reporter assays. The role of adenylyl cyclase-6 in the degradation of Toll-like receptor 4 (TLR4) following endocytosis, was explored via overexpression and knockdown. A mouse model of chronic obstructive pulmonary disease (COPD) induced by porcine pancreatic elastase was used to confirm key findings.Results: Anti-inflammatory effects of α7-nAChRs were largely dependent on adenylyl cyclase-6 activation, as knockdown of adenylyl cyclase-6 considerably reduced the effects of α7-nAChR agonists while adenylyl cyclase-6 overexpression promoted them. We found that α7-nAChRs and adenylyl cyclase-6 are co-localized in lipid rafts of macrophages and directly interact. Activation of adenylyl cyclase-6 led to increased degradation of TLR4. Administration of the α7-nAChR agonist PNU-282987 attenuated pathological and inflammatory end points in a mouse model of COPD.Conclusion and Implications: The α7-nAChRs inhibit inflammation through activating adenylyl cyclase-6 and promoting degradation of TLR4. The use of α7-nAChR agonists may represent a novel therapeutic approach for treating COPD and possibly other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: The ongoing COVID-19 outbreak has caused devastating mortality and posed a significant threat to public health worldwide. Despite the severity of this illness and 2.3 million worldwide deaths, the disease mechanism is mostly unknown. Previous studies that characterized differential gene expression due to SARS-CoV-2 infection lacked robust validation. Although vaccines are now available, effective treatment options are still out of reach. Results: To characterize the transcriptional activity of SARS-CoV-2 infection, a gene signature consisting of 25 genes was generated using a publicly available RNA-Sequencing (RNA-Seq) dataset of cultured cells infected with SARS-CoV-2. The signature estimated infection level accurately in bronchoalveolar lavage fluid (BALF) cells and peripheral blood mononuclear cells (PBMCs) from healthy and infected patients (mean 0.001 vs. 0.958; P < 0.0001). These signature genes were investigated in their ability to distinguish the severity of SARS-CoV-2 infection in a single-cell RNA-Sequencing dataset. TNFAIP3, PPP1R15A, NFKBIA, and IFIT2 had shown bimodal gene expression in various immune cells from severely infected patients compared to healthy or moderate infection cases. Finally, this signature was assessed using the publicly available ConnectivityMap database to identify potential disease mechanisms and drug repurposing candidates. Pharmacological classes of tricyclic antidepressants, SRC-inhibitors, HDAC inhibitors, MEK inhibitors, and drugs such as atorvastatin, ibuprofen, and ketoconazole showed strong negative associations (connectivity score < − 90), highlighting the need for further evaluation of these candidates for their efficacy in treating SARS-CoV-2 infection. Conclusions: Thus, using the 25-gene SARS-CoV-2 infection signature, the SARS-CoV-2 infection status was captured in BALF cells, PBMCs and postmortem lung biopsies. In addition, candidate SARS-CoV-2 therapies with known safety profiles were identified. The signature genes could potentially also be used to characterize the COVID-19 disease severity in patients' expression profiles of BALF cells. [ABSTRACT FROM AUTHOR]

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 E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-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 PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family. [ABSTRACT FROM AUTHOR]

The nongenomic mechanisms by which glucocorticoids modulate β2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on β2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and β2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-β to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4−/− mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-β-stimulated human OA synoviocytes. TGF-β1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 μM) increased intracellular cAMP levels and reduced TGF-β1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-β1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 μM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4−/− synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+ synovium. Prg4−/− synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+ synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis. [ABSTRACT FROM AUTHOR]

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder. [ABSTRACT FROM PUBLISHER]

Background and Purpose: Previous 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 Approach: FRET-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 Results: Activation 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 Implications: EP2 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. [ABSTRACT FROM AUTHOR]

Pulmonary fibroblasts regulate extracellular matrix production and degradation and are critical in maintenance of lung structure, function, and repair, but they also play a central role in lung fibrosis. cAMP-elevating agents inhibit cytokine- and growth factor-stimulated myofibroblast differentiation and collagen synthesis in pulmonary fibroblasts. In the present study, we overexpressed adenylyl cyclase 6 (AC6) in pulmonary fibroblasts and measured cAMP production and collagen synthesis. AC6 overexpression enhanced cAMP production and the inhibition of collagen synthesis mediated by isoproterenol and beraprost, but not the responses to butaprost or PGE2. To examine if increased AC6 expression would impact the development of fibrosis in an animal model, we generated transgenic mice that overexpress AC6 under a fibroblast-specific promoter, FTS1. Lung fibrosis was induced in FTS1-AC6+/− mice and littermate controls by intratracheal instillation of saline or bleomycin. Wild-type mice treated with bleomycin showed extensive peribronchial and interstitial fibrosis and collagen deposition. By contrast, FTS1-AC6+/− mice displayed decreased fibrotic development, lymphocyte infiltration (as determined by pathological scoring), and lung collagen content. Thus, AC6 overexpression inhibits fibrogenesis in the lung by reducing pulmonary fibroblast-mediated collagen synthesis and myofibroblast differentiation. Because AC6 overexpression does not lead to enhanced basal or PGE2-stimulated levels of cAMP, we conclude that endogenous catecholamines or prostacyclin is produced during bleomycin-induced lung fibrosis and that these signals have antifibrotic potential. [ABSTRACT FROM AUTHOR]

Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol. [ABSTRACT FROM AUTHOR]

A growing body of data indicates that multiple signal transduction events in the heart occur via plasma membrane receptors located in signaling microdomains. Lipid rafts, enriched in cholesterol and sphingolipids, form one such microdomain along with a subset of lipid rafts, caveolae, enriched in the protein caveolin. In the heart, a key caveolin is caveolin-3, whose scaffolding domain is thought to serve as an anchor for other proteins. In spite of the original morphologic definition of caveolae ("little caves"), most work related to their role in compartmenting signal transduction molecules has involved subcellular fractionation or immunoprecipitation with anti-caveolin antibodies. Use of such approaches has documented that several G protein-coupled receptors (GPCR), and their cognate heterotrimeric G proteins and effectors, localize to lipid rafts/caveolae in neonatal cardiac myocytes. Our recent findings support the view that adult cardiac myocytes appear to have different patterns of localization of such components compared to neonatal myocytes and cardiac fibroblasts. Such results imply the existence of multiple subcellular microdomains for GPCR-mediated signal transduction in cardiac myocytes, in particular adult myocytes, and raise a major unanswered question: what are the precise mechanism(s) that determine co-localization of GPCR and post-receptor components with lipid rafts/caveolae in cardiac myocytes and other cell types? [ABSTRACT FROM AUTHOR]

The many components of G-protein-coupled receptor (GPCR) signal transduction provide cells with numerous combinations with which to customize their responses to hormones, neurotransmitters, and pharmacologic agonists. GPCRs function as guanine nucleotide exchange factors for heterotrimeric (α, β, γ) G proteins, thereby promoting exchange of GTP for GDP and, in turn, the activation of 'downstream' signaling components. Recent data indicate that individual cells express mRNA for perhaps over 100 different GPCRs (out of a total of nearly a thousand GPCR genes), several different combinations of G-protein subunits, multiple regulators of G-protein signaling proteins (which function as GTPase activating proteins), and various isoforms of downstream effector molecules. The differential expression of such protein combinations allows for modulation of signals that are customized for a specific cell type, perhaps at different states of maturation or differentiation. In addition, in the linear arrangement of molecular interactions involved in a given GPCR-G-protein- effector pathway, one needs to consider the localization of receptors and post-receptor components in subcellular compartments, microdomains, and molecular complexes, and to understand the movement of proteins between these compartments. Co-localization of signaling components, many of which are expressed at low overall concentrations, allows cells to tailor their responses by arranging, or spatially organizing in unique and kinetically favorable ways, the molecules involved in GPCR signal transduction. This review focuses on the role of lipid rafts and a subpopulation of such rafts, caveolae, as a key spatial compartment enriched in components of GPCR signal transduction. Recent data suggest cell-specific patterns for expression of those components in lipid rafts and caveolae. Such domains likely define functionally important, cell-specific regions of signaling by GPCRs and drugs active at those GPCRs. [ABSTRACT FROM AUTHOR]

Pulmonary fibroblasts are recruited to sites of lung injury, where they are activated to produce extracellular matrix proteins and to facilitate repair. However, these cells become dysregulated in pulmonary fibrosis, producing excess collagen at sites of injury and forming fibrotic loci that impair lung function. In this study, we used WI-38 human lung fibroblasts and evaluated the ability of G protein-coupled receptor agonists to increase cAMP production and regulate cell proliferation and collagen synthesis. WI-38 cells increase cAMP in response to the β-adrenergic agonist isoproterenol (Iso), prostaglandin E2 (PGE2), certain prostanoid receptor-selective agonists (beraprost, butaprost), an adenosine receptor agonist, and the direct adenylyl cyclase (AC) activator forskolin (Fsk). Responses to Iso, PGE2, and Fsk were studied in more detail. Each induced a dose-dependent inhibition of serum-stimulated cell proliferation (as measured by [3H]thymidine incorporation) and collagen synthesis (as measured by [3H]proline incorporation, collagenase-sensitive [3H]proline incorporation, or levels of procollagen type 1 C-peptide). Quantitative RT-PCR analyses indicated that elevation in cellular cAMP levels decreases expression of collagen types lα(II) and 5α(I) and increases expression and activity of matrix metalloproteinase 2 (MMP-2). Overexpression of AC type 6 or inhibition of cyclic nucleotide phosphodiesterases also increased cellular cAMP levels and decreased cell proliferation and collagen synthesis. Thus multiple approaches that increase cAMP signaling reduce proliferation and differentiated function in human pulmonary fibroblasts. These results suggest that therapies that raise cAMP levels may prove useful in the treatment of pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Examines the expression and functional role of isoforms cyclooxygenase 1 (COX-1) and 2 (COX-2) in the generation of products involved in basal signal regulation in Madin-Darby canine kidney cell (MDCK). Inhibition of MDCK release of adenosine triphosphate; Induction of COX-2 expression; Isozyme roles and cell functions; COX-2 role in renal functioning.

Focuses on purinoceptors of the Madin-Darby canine kidney (MDCK) cells. Description of the MDCK cells; Evidence of expression from receptor subtypes in MDCK-D1 cells; Regulation of epithelial cells by extracellular nucleotides.

The article reports on the use of orphan G protein-coupled receptors (GPCRs) as the biggest and mst successful aims for pharmacotherapy in cardiovascular areas. It discusses that the orphan receptors have no found endogenous ligand. It infers that the receptors are at the forefront of currently discovered drug efforts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

The article discusses the potential therapeutic value of restraining the Wnt/ β-catenin signaling in pulmonary fibrosis and the diverse aspects of lung injury. It states the effectiveness of pulmonary fibrosis treatment in multiple components of the reparative process fibroblast activation including immune system response, stem cell recruitment and epithelial regeneration. It also mentions the significant of inhibition of Wnt/β-catenin signaling in fibrotic diseases treatment.

The author discusses the study "Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention," by M. Mastri and colleagues, which shows the reduction of fibrosis during the systemic administration of a secreted Frizzled-related protein 2 (sFPR2) antibody to cardiomyopathic hamsters. The author discusses the use of a genetic model of cardiovascular disease in the study and demonstrates the alteration of cardia fibrosis using systemic therapy.

We investigated whether activation of the M2 muscarinic receptor inhibits the relaxant effect of isoproterenol on contraction elicited by other receptors. The muscarinic agonist oxotremorine-M caused a weak contraction in urinary bladder from M3 knockout (KO) mice. When measured in the same tissue in the presence of isoproterenol and α,β-methylene-ATP, oxotremorine-M elicited a robust contractile response, but not in bladder from M2/M3 double KO mice. Similar results were observed when PGF2α was used as the non-muscarinic contractile agent, but not α-methyl-serotonin. The combination of isoproterenol and contractile agent (i.e. α,β-methylene-ATP, PGF2α or α-methyl-serotonin) had little effect because of their opposing actions on contraction. To address whether neuronally released acetylcholine acts on the M2 receptor to inhibit relaxation by isoproterenol, we investigated electrical field stimulation of mouse urinary bladder (0.2 Hz, 2 msec duration, 100 V) in the presence of physostigmine (70 nM). Isoproterenol was approximately 12-fold more potent at inhibiting field-stimulated contractions of urinary bladder from M2 KO mice compared to wild type. Our results show that neuronally released or exogenously applied muscarinic agonist can act on the M2 muscarinic receptor to oppose β-adrenoceptor-mediated relaxation of the detrusor. [ABSTRACT FROM AUTHOR]

We examined the expression and localization of adenylyl cyclase (AC) isoforms and G protein-coupled receptors in guinea pig ileum longitudinal smooth muscle (SM). Using non-detergent cell fractionation and sucrose gradient centrifugation, we isolated caveolae/lipid rafts from ileal SM from Hartley guinea pigs. Buoyant fractions expressed both caveolin-I and caveolin-2 but excluded α-SM actin. Immunoblotting for AC isoforms demonstrated that ileal smooth muscle express AC3, AC5/6 and AC8. Each of these AC isoforms was detected exclusively in buoyant, caveolin-rich fractions, consistent with findings from other cells and tissues. β1-adrenergic receptor (βAR) immunoreactivity was detected in both the buoyant and heavy fractions but prostanoid EP2 receptors were detected only in heavy fractions. Overexpression of AC6 led to prominent AC5/6 immunoreactivity in buoyant fractions and enhanced forskolin- and isoproterenol-induced cAMP accumulation but did not effect PGE2-stimulated responses. AC6 overexpression in ileum altered histamine-induced contractions, reducing the maximal percent contraction relative to KCl without altering histamine potency. Thus, ileal SM expresses predominantly caveolar/lipid raft localized isoforms of AC that are highly co-localized with and functionally coupled to βAR. Prostanoid EP2 receptors do not regulate the activity of these raft-localized AC isoforms. Overexpression of AC6 in ileal SM appears to selectively enhance β-adrenergic receptors responses. [ABSTRACT FROM AUTHOR]

Lipid rafts, rich in cholesterol and sphingolipids, display a high concentration of signaling proteins such as receptors, G-proteins, and effectors molecules including certain isoforms of adenylyl cyclases (AC). The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown, but previous work indicates a role for the C1 domain. In this study, we constructed recombinant proteins that comprise the four segments of the C1 domain of human AC6. The proteins correspond to amino acids 306-387, 388-490, 491-589 and 590-692 of human AC6. We transfected each protein in cos-7 cells, which express caveolin-1, and assessed their localization via a fused GFP. Lipid rafts and caveolae were isolated from plasma membrane using a detergent-based fractionation method and fractions were analyzed by immunoblotting. We also used co-immunoprecipitation and immunoflurescence microscopy to identify the subcellular targeting to each expressed C1 domain fragment. We found that only proteins 306 and 388 were associated with lipid rafts, suggesting that these portions of AC6 play a role in its localization. Thus, certain structural elements responsible for AC6 localization in lipid rafts and caveolac appear to be contained in the proximal portion of the C1 domain. [ABSTRACT FROM AUTHOR]