Cell Biology Group, Woolcock Institute of Medical Research, School of Medical Sciences, The Univ. of Sydney, Sydney, NSW, Australia.
Am J Physiol Lung Cell Mol Physiol. 2012 Feb 1;302(3):L334-42. doi: 10.1152/ajplung.00163.2011. Epub 2011 Nov 18.
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.
β(2)-肾上腺素能受体（β2AR）激动剂通过 cAMP 诱导气道松弛。磷酸二酯酶（PDE）降解并调节 cAMP，而在气道平滑肌（ASM）细胞中，PDE4D 降解 cAMP。长效β(2)-激动剂现在被禁止作为哮喘的单一疗法，有人推测 PDE4D 的增加对此现象有贡献。在这项研究中，我们研究了 PDE4D 在哮喘和非哮喘 ASM 细胞中的表达及其被福莫特罗和布地奈德的调节。来自有或没有哮喘的人的原代 ASM 细胞用转化生长因子（TGF）-β(1)、福莫特罗和/或布地奈德刺激。通过实时 PCR 或 PCR 评估 PDE4D mRNA，以评估剪接变体的产生。通过 Western 印迹评估 PDE4D 蛋白，并研究福莫特罗对 cAMP 产生和 PDE 活性的影响。通过 ELISA 评估白细胞介素（IL）-6。福莫特罗呈剂量依赖性地上调 PDE4D mRNA，存在单个剪接变体 PDE4D5。在 3 至 72 小时之间的时间点，福莫特罗未诱导 PDE4D 蛋白，但诱导并增加了 IL-6 分泌。我们用放线菌素 D 和蛋白酶体抑制剂 MG132 预处理细胞，未发现 PDE4D mRNA、蛋白表达或降解的改变。最后，福莫特罗未改变 PDE 活性。这项研究首次表明，PDE4D5 主要在有和没有哮喘的人的 ASM 细胞中表达，并且福莫特罗不会上调 PDE4D 蛋白的产生。这使我们推测，持续的β2AR 激动剂治疗不太可能引起 PDE4 介导的脱敏。
