Ligustrazine Inhibits Lung Phosphodiesterase Activity in a Rat Model of Allergic Asthma

Objectives. This study sought to examine whether ligustrazine was capable of inhibiting phosphodiesterase (PDE) activity and improving lung function in a rat model of asthma. Methods. Rats were initially sensitized using ovalbumin (OVA) and then were challenged daily with aerosolized OVA beginning 14 days later (30 min/day) to generate a rat model of asthma. Changes in airway function following methacholine (MCh) injection were evaluated by monitoring lung resistance ( R L ) and dynamic lung compliance ( C dyn ) values using an AniRes2005 analytic system. In addition, serum IgE was measured via ELISA, while PDE expression was evaluated via qPCR and western blotting. Key Findings. Ligustrazine significantly impaired allergen-induced lung hyperresponsivity and inflammation in this asthma model system. Ligustrazine treatment was also associated with reduced expression of PDEs including PDE4 in the lungs of these rats. Conclusions. Ligustrazine suppresses airway inflammation and bronchial hyperresponsivity in this rat model system, and these changes are associated with decreased PDE expression at the protein and mRNA levels.

Characterization of Resveratrol, Oxyresveratrol, Piceatannol and Roflumilast as Modulators of Phosphodiesterase Activity. Study of Yeast Lifespan

Our desire to live longer has led to an ever-increasing number of novel antiaging products. However, few molecules have any real effect and new ones need to be studied before they can be used commercially. In this contribution, activation of the caloric restriction (CR) pathway was studied using different three (resveratrol, oxyresveratrol and piceatannol)—a family with demonstrated bioactivity on phosphodiesterase activity. The high-affinity phosphodiesterase type 2 (PDE2) of Saccharomyces cerevisiae was expressed in Escherichia coli, purified and characterized. The activity and the inhibitory activity of each stilbene was studied, and the findings were compared in vitro and in silico with those obtained with roflumilast—a human PDE4 inhibitor widely used in chronic obstructive pulmonary diseases. Finally, an in vivo chronological lifespan assay using WT S. cerevisiae and ΔPDE2 S. cerevisiae strains was carried out. It was demonstrated that stilbenes can modulate yPDE2 activity, increasing the lifespan of the yeast by 18% over a control (in combination with other pathways). In addition, roflumilast increased the lifespan in the WT strain. The findings as a whole would increase the range of lifespan products available and suggest novel uses for approved drugs.

Mechanistic and In silico Characterization of Metal ion Requirements of Escherichia coli Zinc Phosphodiesterase Activity

Zinc phosphodiesterase (ZiPD) participates in the maturation of tRNA precursors. The roles of metal ions in promoting phosphoryl transfer reaction on zinc phosphodiesterase (ZiPD) activity have not been fully characterized. Therefore, this study investigated the effects of some metal ions on phosphodiesterase activity of Escherichia coli ZiPD as well as the binding site and binding affinity of the metal ions. ZiPD activity was measured by monitoring the rate of hydrolysis of bis-para-nitrophenyl phosphate (bis-pNPP) in the presence of some selected divalent metal ions (Mn2+, Co2+, Mg2+ and Zn2+). The results obtained revealed that Mn2+ at 1 mM activated ZiPD activity by 4-fold with binding affinity score of 1.795. Co2+ at 0.5 mM activated ZiPD activity by 2-fold with binding affinity score of 1.773. Mg2+ at 0.5 mM enhanced the binding affinity of ZiPD for bis-pNPP but did not increase the turnover rate of ZiPD. Zn2+ at 1.5 mM activated ZiPD activity by 2-fold via increased affinity of ZiPD for bis-pNPP. In conclusion, the findings from this study showed that Mn2+ and Zn2+ are the most effective stimulatory ions of ZiPD for bis-pNPP while Zn2+ exerted the highest binding affinity of ZiPD for bis-pNPP.

Live-cell assays for cell stress responses reveal new patterns of cell signaling caused by mutations in rhodopsin, α-synuclein and TDP-43

ABSTRACTMany neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. Genetically-encoded fluorescent biosensors are effective tools to examine neuronal activity and signaling in living cells. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling we created live-cell assays for ER-mediated cell stress and second messenger signaling. Analysis of the rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa, revealed increased cell stress levels compared to wild type rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress dependent manner. Analysis of single cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing wild type or P23H mutants, further supporting the notion that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in other neurodegenerative diseases we examined how various mutants of α-synuclein and TDP-43 affected ER-mediated cell stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson’s Disease and ALS demonstrated increases in ER-mediated cell stress. This increased cell stress was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors can provide nuanced, new views of neurodegenerative disease processes.