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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]

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing β2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 μM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates β2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein Gαs and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (ADCY5) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.

We explored whether the M2 muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M3 muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M3 selectivity. Then, we used 4-DAMP mustard to inactivate M3 responses in the guinea pig ileum to attempt to reveal direct, M2 receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M2 receptor knockout, and M3 receptor knockout mice characterized by negative log EC50 (pEC 50 ) values ± SEM of 6.75 ± 0.03, 6.26 ± 0.05, and 6.99 ± 0.08, respectively. The corresponding E max values in wild-type and M2 receptor knockout mice were approximately the same, but that in the M3 receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration–response curve of oxotremorine-M in wild-type ileum resembled that of the M3 knockout mouse in terms of its pEC 50 , E max, and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M3 responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M3 knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M3 response. The guinea pig ileum, therefore, lacks a direct, highly potent, M2-contractile component but may have a direct, lower potency M2 component.

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]

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 [PGE.sub.2]. 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.sup.+/-] 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.sup.+/-] 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 [PGE.sub.2]-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. cAMP; lipid rafts doi: 10.1152/ajplung.00429.2009.

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 [3-adrenergic agonist isoproterenol (Iso), prostaglandin [E.sub.2] (PG[E.sub.2]), certain prostanoid receptor-selective agonists (beraprost, butaprost), an adenosine receptor agonist, and the direct adenylyl cyclase (AC) activator forskolin (Fsk). Responses to ISO, PG[E.sub.2], and Fsk were studied in more detail. Each induced a dose-dependent inhibition of serum-stimulated cell proliferation (as measured by [[sup.3]H]thymidine incorporation) and collagen synthesis (as measured by [[sup.3]H]proline incorporation, collagenase-sensitive [[sup.3]H]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 1[alpha](II) and 5[alpha](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. [beta]-adrenergic receptors; prostaglandin; prostanoid receptors; pulmonary fibrosis; extracellular matrix

Heterotrimeric GTP- binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a [Gα.sub.s]-specific GAP. The RGS domain of RGS-PX1 specifically interacted with [Gα.sub.s], accelerated its GTP hydrolysis, and attenuated [Gα.sub.s]-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking., Heterotrimeric G proteins relay extracellular signals initiated by hormones, neurotransmitters, chemokines, and sensory stimuli through G protein-coupled receptors to intracellular effectors and trigger a variety of physiological responses (1, 2). [...]

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]

Madin-Darby canine kidney (MDCK) cells release ATP upon mechanical or biochemical activation, initiating P2Y receptor signaling that regulates basal levels of multiple second messengers, including cAMP (J Biol Chem 275: 11735-11739, 2000). Data shown here document inhibition of cAMP formation by [Gd.sup.3+] and niflumic acid, channel inhibitors that block ATP release. cAMP production is stimulated via [Ca.sup.2+]-dependent activation of cytosolic phospholipase [A.sub.2], release of arachidonic acid (AA), and cyclooxygenase (COX)-dependent production of prostaglandins, which activate prostanoid receptors coupled to [G.sub.s] and adenylyl cyclase. In the current investigation, we assessed the expression and functional role of the two known isoforms of COX, COX-1 and COX-2. Treatment of cells with either a COX-1-selective inhibitor, SC-560, or COX-2-selective inhibitors, SC-58125 or NS-398, inhibited basal and UTP-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulated cAMP formation, implying this response is in part attributable to an action of AA metabolites. These findings imply an important role for the inducible form of COX, COX-2, under basal conditions. Indeed, COX-2 expression was readily detectable by immunoblot, and treatments that induce or reduce COX-2 expression in other cells (interleukin-1[Beta], tumor necrosis factor-[Alpha], phorbol ester, or dexamethasone) had minimal or no effect on the levels of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-[D.sub.1] cells and participates in basal and [P2Y.sub.2]-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function. adenosine 3',5'-cyclic monophosphate; adenosine 5'-triphosphate release; arachidonic acid; prostaglandin [E.sub.2]