β2-Agonists upregulate PDE4 mRNA but not protein or activity in human airway smooth muscle cells from asthmatic and nonasthmatic volunteers

β(2)-Adrenergic receptor (β2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting β(2)-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-β(1), formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with β2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis.     

Bronchial inflammation induced PKCζ over-expression: involvement in mechanical properties of airway smooth muscle

Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of α-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O₂⁻ generated via the oxidase system activated with TS.     

β-Adrenergic receptor population is up-regulated by increased cyclic adenosine monophosphate concentration in chicken skeletal muscle cells in culture

Summary Skeletal muscle hypertrophy is promoted in vivo by administration of β-adrenergic receptor (βAR) agonists. Chicken skeletal muscle cells were treated with 1 μM isoproterenol, a strong βAR agonist, between days 7 and 10 in culture. βAR population increased by approximately 40% during this treatment; however, the ability of the cells to synthesize cyclic adenosine monophosphate (cAMP) was diminished by twofold. Neither the basal concentration of cAMP nor the quantity of myosin heavy chain (MHC) was affected by the 3-d exposure to isoproterenol. To understand further the relationship between intracellular cAMP levels, βAR population, and muscle protein accumulation, intracellular cAMP levels were artificially elevated by treatment with 0–10 μM forskolin for 3 d. The basal concentration of cAMP in forskolintreated cells increased up to sevenfold in a dose-dependent manner. Increasing concentrations of forskolin also led to an increase in βAR population, with a maximum increase of approximately 40–60% at 10 μM forskolin. A maximum increase of 40–50% in the quantity of MHC was observed at 0.2 μM forskolin, but higher concentrations of forskolin reduced the quanity of MHC back to control levels. At 0.2 μM forskolin, intracellular levels of cAMP were higher by approximately 35%, and the βAR population was higher by approximately 30%. Neither the number of muscle nuclei fused into myotubes nor the percentage of nuclei in myotubes was affected by forskolin at any of the concentrations studied.     

TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.     

Integrin α7β1 is a redox-regulated target of hydrogen peroxide in vascular smooth muscle cell adhesion

Upon adhesion to laminin-111, aortic smooth muscle cells initially form membrane protrusions with an average diameter of 2.9μm. We identified these protrusions also as subcellular areas of increased redox potential and protein oxidation by detecting cysteine sulfenic acid groups with dimedone. Hence, we termed these areas oxidative hot spots. They are spatially and temporally transient during an early stage of adhesion and depend on the activity of the H(2)O(2)-generating NADPH oxidase 4. Presumably located on cellular protrusions, integrin α7β1 mediates adhesion and migration of vascular smooth muscle cells to laminins of their surrounding basement membrane. Using protein chemistry and mass spectrometry, two specific oxidation sites within the integrin α7 subunit were identified: one located in its genu region and another within its calf 2 domain. Upon H(2)O(2) treatment, two cysteine residues are oxidized thereby unlocking a disulfide bridge. The genu region is a hinge, around which the integrin domains pivot between a bent/inactive and an upright/active conformation. Also, cysteine oxidation within the calf 2 domain permits conformational changes related to integrin activation. H(2)O(2) treatment of α7β1 integrin in concentrations of up to 100μM increases integrin binding activity to laminin-111, suggesting a physiological redox regulation of α7β1 integrin.     

Paradoxical attenuation of β2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase

The limiting component within the receptor-G protein-effector complex in airway smooth muscle (ASM) for β(2)-adrenergic receptor (β(2)-AR)-mediated relaxation is unknown. In cardiomyocytes, adenylyl cyclase (AC) is considered the "bottleneck" for β-AR signaling, and gene therapy trials are underway to increase inotropy by increasing cardiac AC expression. We hypothesized that increasing AC in ASM would increase relaxation from β-agonists, thereby providing a strategy for asthma therapy. Transgenic (TG) mice were generated with approximately two- to threefold overexpression of type 5 AC (AC5) in ASM. cAMP and airway relaxation in response to direct activation of AC by forskolin were increased in AC5-TG. Counter to our hypothesis, isoproterenol-mediated airway relaxation was significantly attenuated (～50%) in AC5-TG, as was cAMP production, suggesting compensatory regulatory events limiting β(2)-AR signaling when AC expression is increased. In contrast, acetylcholine-mediated contraction was preserved. G(αi) expression and ERK1/2 activation were markedly increased in AC5-TG (5- and 8-fold, respectively), and β-AR expression was decreased by ～40%. Other G proteins, G protein-coupled receptor kinases, and β-arrestins were unaffected. β-agonist-mediated airway relaxation of AC5-TG was normalized to that of nontransgenic mice by pertussis toxin, implicating β(2)-AR coupling to the increased G(i) as a mechanism of depressed agonist-promoted relaxation in these mice. The decrease in β(2)-AR may account for additional relaxation impairment, given that there is no enhancement over nontransgenic after pertussis toxin, despite AC5 overexpression. ERK1/2 inhibition had no effect on the phenotype. Thus perturbing the ratio of β(2)-AR to AC in ASM by increasing AC fails to improve (and actually decreases) β-agonist efficacy due to counterregulatory events.