Injury or inflammation affecting sensory neurons in the dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spinal central sensitization and neuropathic pain. Recent studies have indicated that, following chronic compression of DRG (CCD) or acute dissociation of DRG (ADD) treatment, both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by activity in cGMP-PKG signaling pathway. Here, we provide evidence supporting the idea that CCD or ADD treatment activates cGMP-PKA signaling pathway in the DRG neurons. The results showed that CCD or ADD results in increase of levels of cGMP concentration and expression of PKG-I mRNA, as well as PKG-I protein in DRG. CCD or ADD treated-DRG neurons become hyperexcitable and exhibit increased responsiveness to the activators of cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMP. Hyperexcitability of the injured neurons is inhibited by cGMP-PKG pathway inhibitors, ODQ and Rp-8-pCPT-cGMPS. In vivo delivery of Rp-8-pCPT-cGMPS into the compressed ganglion within the intervertebral foramen suppresses CCD-induced thermal hyperalgesia. These findings indicate that the in vivo CCD or in vitro ADD treatment can activate the cGMP-PKG signaling pathway, and that continuing activation of cGMP-PKG pathway is required to maintain DRG neuronal hyperexcitability and/or hyperalgesia after these two dissimilar forms of injury-related stress.
Animals ; Cyclic GMP ; analogs & derivatives ; metabolism ; Cyclic GMP-Dependent Protein Kinases ; metabolism ; Ganglia, Spinal ; physiopathology ; Hyperalgesia ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thionucleotides ; metabolism

OBJECTIVETo study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock.

METHODSConfluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with LPS or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 was used to pretreat the endothelial cells before the administration of LPS or PKG activator 8-Br-cGMP.

RESULTSExposure to LPS for 5, 10, 30 and 60 minutes led to a rapid time-dependent increase in endothelial PKG activity (P < 0.01 compared to the blank) and the polar distribution of intracellular filamentous actin and preincubation with KT5823 abolished these effects. 8-Br-cGMP was similar to LPS.

CONCLUSIONSThe results suggested that LPS can mediate PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably induce the endothelial hyperpermeability after septic shock.

Capillary Permeability ; Cyclic GMP ; analogs & derivatives ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; physiology ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Shock, Septic ; metabolism ; Signal Transduction

OBJECTIVETo investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes.

METHODSH9c2 cells, a permanent cell line derived from embryonic rat cardiac tissue, and then randomly divided into control group [PBS, cells exposed to H2O2 (600 µmol/L) for 20 min to induce mitochondrial oxidant damage], resveratrol group (0.01, 0.1, 1, 5, 10 and 20 µmol/L for 20 min at 20 min before exposing to H2O2), resveratrol plus inhibitor group (1 µmol/L KT5823 for 10 min at 10 min before 5 µmol/L resveratrol treatment) and inhibitor group (1 µmol/L KT5823 for 10 min). Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring the decrease of TMRE fluorescence intensity. Immunofluorescence assay was used to observe GSK-3β phosphorylation. The phosphorylation of GSK-3β and VASP were determined by Western blot. To detect intracellular NO, cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy.

RESULTSCompared to the control group, resveratrol (0.01-5 µmol/L) attenuated H2O2-induced mitochondrial damage reflected by attenuating the H2O2-induced TMRE fluorescence intensity decrease in a dose-dependent manner and the efficacy of 10 and 20 µmol/L resveratrol was significantly lower than that of 5 µmol/L resveratrol. Resveratrol also significantly upregulated the protein expression of VASP and increased GSK-3β Ser(9) phosphorylation, which could lead the inactivation of GSK-3β. These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823, while KT5823 alone did not affect GSK-3β and VASP phosphorylation. Confocal microscopy showed that DAF-FM (specific NO indicator) was similar between resveratrol and control group, suggesting that resveratrol did not produce NO.

CONCLUSIONSResveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3β via cGMP/PKG signaling pathway independent of NO-related mechanism.

Animals ; Carbazoles ; pharmacology ; Cell Line ; Cyclic GMP ; metabolism ; Cyclic GMP-Dependent Protein Kinases ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Hydrogen Peroxide ; metabolism ; Mitochondria, Heart ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; Oxidants ; metabolism ; Rats ; Signal Transduction ; drug effects ; Stilbenes ; pharmacology

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.
Animals ; Apigenin ; pharmacology ; Cell Adhesion Molecules ; drug effects ; Cell Hypoxia ; Coronary Vessels ; drug effects ; Cyclic GMP ; analogs & derivatives ; metabolism ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; Glucuronates ; pharmacology ; Microfilament Proteins ; drug effects ; NG-Nitroarginine Methyl Ester ; metabolism ; pharmacology ; Phosphoproteins ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Signal Transduction ; drug effects ; Thionucleotides ; metabolism ; pharmacology ; Vasodilation ; drug effects ; physiology

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.
Animals ; Aorta/injuries/metabolism/*pathology ; Calcium-Binding Proteins/genetics/metabolism ; Cell Proliferation ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/genetics/*metabolism ; Hyperplasia/metabolism ; Microfilament Proteins/genetics/metabolism ; Muscle, Smooth, Vascular/metabolism/pathology ; Myocytes, Smooth Muscle/drug effects/*metabolism ; Nitric Oxide/metabolism ; PPAR gamma/agonists/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Sp1 Transcription Factor/metabolism ; Thiazolidinediones/pharmacology ; Thrombospondins/genetics/metabolism ; Tunica Intima/metabolism/*pathology ; Vascular System Injuries/*metabolism/pathology

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 microM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), methylene blue (MB; 10(-5) M) and 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one, (ODQ; 10(-6) or 10(-7) M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10(-8) or 10(-9) M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K+ channel inhibitor glibenclamide (Gli; 10(-5) M) and the voltage-dependent K+ channel inhibitor 4-aminopyridine (4-AP; 2 x 10(-4) M). These results suggest that CAL induces vasorelaxation by activating K+ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.
Animals ; Aorta/drug effects/metabolism/physiology ; Atrial Natriuretic Factor/pharmacology ; Cyclic GMP/*metabolism ; Cyclic GMP-Dependent Protein Kinases/*metabolism ; Dipeptides/pharmacology ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Humans ; Male ; Methylene Blue/pharmacology ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase/metabolism ; Oxadiazoles/pharmacology ; Phenylephrine/pharmacology ; Phosphorylation ; Potassium Channel Blockers/pharmacology ; Potassium Channels/*agonists ; Propanols/*pharmacology ; Quinoxalines/pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vasoconstriction/*drug effects ; Vasodilation/drug effects ; rho-Associated Kinases/antagonists & inhibitors/*metabolism

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.
Bucladesine/pharmacology ; Carbazoles/pharmacology ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*metabolism ; Dibutyryl Cyclic GMP/pharmacology ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/pharmacology ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Pyrroles/pharmacology ; Research Support, Non-U.S. Gov't ; rhoA GTP-Binding Protein/*metabolism

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.
Action Potentials/drug effects ; Animals ; Biological Transport/drug effects ; Calcium/metabolism ; Calcium Channels, L-Type/*metabolism ; Carbazoles/pharmacology ; Cells, Cultured ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors ; Elapid Venoms/*metabolism/pharmacology ; Enzyme Activation ; Heart ; Heart Ventricles/drug effects ; Myocytes, Cardiac/drug effects ; Patch-Clamp Techniques ; Peptides/*metabolism/pharmacology ; Phosphorylation/drug effects ; Rabbits