We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1α was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.

2011-10-01·Life Sciences3区 · 医学

Sildenafil inhibits calcineurin/NFATc2-mediated cyclin A expression in pulmonary artery smooth muscle cells

3区 · 医学

Article

作者: Jing Xu ; Xinming Xie ; Ping He ; Zongfang Li ; Hongyang Shi ; Manxiang Li ; Jiamei Lu ; Ping Fang ; Mei Xie ; Xiaochuang Wang ; Yuan Liu ; Xiuzhen Sun ; Yonghong Zhang ; Zongjuan Ming ; Dexin Zhang ; Yun Liu

AIMSTo examine whether calcineurin/NFAT signaling pathway leads to proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating cell cycle proteins and whether the phosphodiesterase-5 (PDE5) inhibitor sildenafil affects calcineurin/NFAT-induced cell proliferation.MAIN METHODSA [(3)H]thymidine incorporation assay was used to examine DNA synthesis (cell proliferation); cyclin A and NFATc2 expressions were determined by Western blot. Cyclin-dependent kinase 2 (CDK2) activity was measured with an in vitro kinase activity assay, and calcineurin and NFAT activity were evaluated using a calcineurin assay kit and a luciferase activity assay, respectively. A chemical inhibitor or siRNA transfection was used to inhibit calcineurin/NFAT signaling pathway.KEY FINDINGSSerotonin dose-dependently stimulated cyclin A expression in PASMCs. This effect was accompanied by dose-dependent increases in CDK2 activity and the rate of DNA synthesis. At the same time, PASMCs treated with serotonin showed dose-dependent activation of calcineurin/NFAT signaling pathway. Inhibition of calcineurin activity by cyclosporine A or loss of NFATc2 protein by siRNA transfection abolished serotonin-induced cyclin A expression and consequent CDK2 activation and DNA synthesis. We further found that pretreatment of cells with sildenafil suppressed serotonin-triggered activation of calcineurin/NFATc2 signaling pathway and resultant cyclin A expression, CDK2 activation and cell proliferation, while the presence of DT-3 [a specific protein kinase G (PKG) peptide inhibitor] reversed the effects of sildenafil on PASMCs.SIGNIFICANCEOur study suggests that enhanced PKG activity suppresses calcineurin/NFATc2 cascade-mediated cyclin A expression, CDK2 activation and PASMC proliferation to contribute to the overall effects of sildenafil in the treatment of pulmonary hypertension.

2010-11-01·Vascular Pharmacology3区 · 医学

PKG-I inhibition attenuates vascular endothelial growth factor-stimulated angiogenesis

3区 · 医学

Article

作者: Martina Monti ; Federica Finetti ; Charis Roussos ; Ioannis Vasileiadis ; Vasiliki Koika ; Zongmin Zhou ; Andreas Papapetropoulos ; Lucia Morbidelli

Vascular endothelial growth factor (VEGF) stimulates nitric oxide (NO) production, which mediates many of its angiogenic actions. However, the angiogenic pathways that operate downstream of NO following VEGF treatment are not well characterized. Herein, we used DT-2 and DT-3, two highly selective cGMP-dependent protein kinase I peptide inhibitors to determine the contribution of PKG-I in VEGF-stimulated angiogenesis. Incubation of chicken chorioallantoic membranes (CAM) with PKG-I peptide inhibitors decreased vascular length in a dose-dependent manner, with DT-3 being more effective than DT-2. Moreover, inhibition of PKG-I with DT-3 abolished the angiogenic response elicited by VEGF in the rabbit eye cornea. PKG-I inhibition also blocked VEGF-stimulated vascular leakage. In vitro, treatment of cells with VEGF stimulated phosphorylation of the PKG substrate VASP through VEGFR2 activation; the VEGF-stimulated VASP phosphorylation was reduced by DT-2. Pre-treatment of cells with DT-2 or DT-3 inhibited VEGF-stimulated mitogen-activated protein kinase cascades (ERK1/2 and p38), growth, migration and sprouting of endothelial cells. The above observations taken together identify PKG-I as a downstream effector of VEGFR2 in EC and provide a rational basis for the use of PKG-I inhibitors in disease states characterized by excessive neovascularization.

100 项与 DT-003 相关的药物交易

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