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The Background Behind VRT752271

In differentiated human bronchial epithelial cells exposed to tobacco smoke for 24?h, the cAMP content (Figure?3D) and in parallel PKA activity (Figure?3E) were both reduced compared with controls, and for cAMP the loss was comparable to that observed after 5?min incubation with CSE (Figure?2C). The additional presence of roflumilast N-oxide at 2?nM almost restored the cAMP content (Figure?3D) and PKA activity (Figure?3E), while both cAMP and PKA activity recovered to higher than baseline with 1??M roflumilast N-oxide; this also occurred with dbcAMP, a cAMP analogue serving Histamine H2 receptor as a positive control (Figure?3D,E). The loss in cAMP content secondary to CSE exposure for 24?h prompted us to analyse the activity and expression of PDE4, well-known as the prominent cAMP hydrolysing PDE in human bronchial epithelial cells. Firstly, exposure of differentiated bronchial epithelial cells to CSE at 10% for 24?h induced about a twofold increase in PDE4 activity (Figure?4A), probably as a result of an up-regulation in the expression of PDE4B. Indeed, of the PDE4 subtypes analysed, mRNA transcripts for PDE4B were most markedly increased by 2.8-fold over control (P < 0.05), while the 1.4-fold and 1.3-fold increment in PDE4A and D transcripts, respectively, remained below significance (Figure?4B). Strikingly, quenching ROS by apocynin (100??M) or N-acetylcysteine (1?mM) reversed the CSE-induced NVP-AUY922 purchase increment VRT752271 cell line in PDE4B transcripts (Figure?4C). As an enhancement in the levels of cAMP may prevent CSE-induced oxidative stress (as exemplified in Figure?5), next the effects of dbcAMP (1?mM) on PDE4B expression were investigated. The cAMP analogue prevented the CSE-induced increment in PDE4B transcripts (Figure?4D). In line with this result, inhibiting PDE4 (roflumilast N-oxide at 1??M) abolished the CSE-induced increment in PDE4B transcripts (Figure?4E) and protein (Figure?4F). Roflumilast N-oxide at 2?nM modestly reduced the increment in PDE4B transcripts secondary to CSE (Figure?4E). At this concentration, the PDE4 inhibitor was not found to significantly reduce PDE4B protein based on the densitometric analyses (Figure?4F). The role of ROS and its possible regulation by roflumilast N-oxide was further addressed. Intracellular ROS was quantified as DCF following oxidation from H2DCF-DA. Exposure of differentiated human bronchial epithelial cells to CSE at 10% for 30?min resulted in an approx. fourfold increase in DCF accumulation over control. Pre-incubation with roflumilast N-oxide concentration-dependently reduced the increment in DCF accumulation by about 53 and 86% at 2?nM and 1??M, respectively (Figure?5). dbcAMP (1?mM) reproduced the reduction in DCF accumulation observed with the PDE4 inhibitor. Finally, NAC (1?mM) serving as a control prevented DCF accumulation triggered by CSE (Figure?5).
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