Research the relationship between vasodilator-stimulated-phosphoprotein and risk factors for atherosclero-sis / 医学研究生学报

In the past few decades, accumulated experimental and clinical evidences suggest that cyclic guanosine monophos-phate (cGMP) plays an important role in atheroselerosis. Vasodilator-stimulated phosphoprotein (VASP), a major downstream compo-nent of the cGMP signaling cascade, is an actin-binding protein which regulates cell adhesion, morphology change, cell motility and cell proliferation. It has shown that VASP is associated with many factors influencing atherosclerosis, and it plays an important role in the genesis and development of atherosclerosis. The relationships between the occurrence of atherosclerosis and risk factors for athero-sclerosis such as hypertension, hyperglycaemia and hyperlipidemia are reviewed in this article.

Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery / 中国天然药物

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.

Effect of cGMP-dependent protein kinase G inhibitor (D)-DT-2 on the lipopolysaccharide-induced hyporesponsiveness of rat vascular ring / 中华麻醉学杂志

Objective To investigate the effect of specific cGMP-dependent protein kinase G (PKG) inhibitor (D)-DT-2 on the contractile function of rat vascular rings after being exposed to lipopolysaccharide (LPS).Methods The experiment was performed in 2 parts.Part Ⅰ:The Sprague-Dawley rat thoracic aortic rings were randomly divided into 3 groups (n =5 each):KH group,DT-2 group and (D)-DT-2 group.KH,DT-2 and (D)-DT-2 were added to the aortic ring after being dilated with 8-Br-cGMP 50 μmol/L for 25 min and the changes in tension of vascular rings were measured.Part Ⅱ:The rat thoracic aortic rings were randomly divided into 4 groups (n =5 each):control group,LPS group,LPS-DT2 group and LPS-(D)-DT2 group.After being incubated with LPS for 3 h in vitro.The Emax and EC50 were compared among the 4 groups.Results Part Ⅰ:Both DT-2 and (D)-DT-2 could contract the vascular rings dilated with 8-Br-cGMP and the Emax was significantly higher in (D)-DT-2 group than DT-2 group (P ＜0.05).Part Ⅱ]:Both DT-2 and (D)-DT-2 significantly improved the contractile function of vascular ring after being exposed to LPS.Emax was significantly higher,while EC50 was lower in groups DT-2 and (D)-DT-2 than in LPS group (P ＜0.01).Emax was significantly increased,while EC50 was decreased in LPS-(D)-DT-2 group as compared with LPS-DT-2 group (P ＜ 0.05).Conclusion PKG inhibitor can improve the contractile function of the vascular rings incubated with LPS and the efficacy of (D)-DT-2 is better than DT-2 in recovering the vascular reactivity.

PPARgamma modulates vascular smooth muscle cell phenotype via a protein kinase G-dependent pathway and reduces neointimal hyperplasia after vascular injury

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to vascular injury such as angioplasty. Protein kinase G (PKG) has an important role in the process of VSMC phenotype switching. In this study, we examined whether rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, could modulate VSMC phenotype through the PKG pathway to reduce neointimal hyperplasia after angioplasty. In vitro experiments showed that rosiglitazone inhibited the phenotype change of VSMCs from a contractile to a synthetic form. The platelet-derived growth factor (PDGF)-induced reduction of PKG level was reversed by rosiglitazone treatment, resulting in increased PKG activity. This increased activity of PKG resulted in phosphorylation of vasodilator-stimulated phosphoprotein at serine 239, leading to inhibited proliferation of VSMCs. Interestingly, rosiglitazone did not change the level of nitric oxide (NO) or cyclic guanosine monophosphate (cGMP), which are upstream of PKG, suggesting that rosiglitazone influences PKG itself. Chromatin immunoprecipitation assays for the PKG promoter showed that the activation of PKG by rosiglitazone was mediated by the increased binding of Sp1 on the promoter region of PKG. In vivo experiments showed that rosiglitazone significantly inhibited neointimal formation after balloon injury. Immunohistochemistry staining for calponin and thrombospondin showed that this effect of rosiglitazone was mediated by modulating VSMC phenotype. Our findings demonstrate that rosiglitazone is a potent modulator of VSMC phenotype, which is regulated by PKG. This activation of PKG by rosiglitazone results in reduced neointimal hyperplasia after angioplasty. These results provide important mechanistic insight into the cardiovascular-protective effect of PPARgamma.

PKG II inhibits downstream targets of proliferation-related MAPK/ERK in gastric cancer cell line SGC-7901 / 肿瘤

Objective: To investigate the effect of PKG II (cGMP-dependent protein kinase II) on cell proliferation-related MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) downstream targets RSK1 (ribosomal S6 kinase 1) and proto-oncogenes c -Jun and c -Fos in gastric cancer SGC-7901 cells. Methods: Gastric cancer SGC-7901 cells were infected with Ad-LacZ and Ad- PKG II adenovirus, and then the PKG II was overexpressed in SGC-7901 cells. These cells were treated with PKG II specific activator - 8-pCPT-cGMP [8-(p-chlorophenylthio)-cyclic GMP], and then they were stimulated with EGF (epidermal growth factor). The proliferation of SGC-7901 cells was examined by MTT method, and the mRNAs expression levels of c-Jun and c-Fos and the protein expression levels of p-RSK1, c-Jun and c-Fos were examined by RT-PCR and Western blotting, respectively. The nuclear accumulation of p-RSK1 was observed under an immunofluorescence microscope. Results: EGF-induced increase of cell proliferation and the elevation of expressions of c-Jun and c-Fos mRNAs and proteins as well as p-RSK1 (Ser380-) protein in SGC-7901 cells which were infected with PKG II were significantly inhibited after treatment with 8-pCPT-cGMP. The phosphorylation of RSK1 (Ser380) was decreased, and the accumulation of p-RSK1 (Ser380) in nuclei of SGC-7901 cells was increased after stimulation with EGF, while it was decreased after treatment with 8-pCPT-cGMP. Conclusion: Activated PKG II can inhibits the proliferation of gastric cancer cells, phosphorylation of RSK1 (Ser380), nuclear accumulation of p-RSK1(Ser380) and the expressions of c -Jun and c -Fos genes which were induced by EGF. Copyright © 2012 by TUMOR.

Role of cGMP-dependent protein kinase against ethanol neurotoxicity of cerebellar granule cells / 第三军医大学学报

Objective To analyze the function of cGMP-dependent protein kinase(PKG)in the survival of cultured rat cerebellar granule cells(CGC).Methods CGC culture was incubated for 24 h before addition of ethanol and different reagents,such as Deta-NONOate(a NO donor),Br-cGMP and Rp-8-pCPT-cGMPS(a cGMP-dependent protein kinase inhibitor).After the treatment,samples were incubated for another 24 h,then the cells were collected and counted by using hemocytometer.Results Deta-NONOate and Br-cGMP enhanced the cell survival in the absence of ethanol,indicating their neurotrophic effects.They showed neuroprotective effect on the cultured CGC since they could reduce the ethanol-induced cell death.Inhibiting PKG with Rp-8-pCPT-cGMPS could eliminate both neurotrophic and neuroprotective effects mediated by Deta-NONOate and Br-cGMP.Conclusion cGMP-dependent protein kinase plays a key role in the cultured CGC survival and protects the cells against ethanol neurotoxicity.

Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.