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

Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (K(Ca)) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated K(Ca) currents were blocked by iberiotoxin (IbTX, a large conductance K(Ca) blocker). The H2O2-stimulating effect on large-conductance K(Ca) (BK(Ca)) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BK(Ca) currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BK(Ca) currents. Using RT-PCR and western blot analysis, three subtypes of K(Ca) channels were detected in HCFs: BK(Ca) channels, small-conductance K(Ca) (SK(Ca)) channels, and intermediate-conductance K(Ca) (IK(Ca)) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BK(Ca) currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BK(Ca) channels.
Apoptosis ; Blotting, Western ; Cyclic AMP-Dependent Protein Kinases ; Cyclic GMP-Dependent Protein Kinases ; Fibroblasts* ; Guanosine ; Guanylate Cyclase ; Humans* ; Hydrogen Peroxide* ; Hydrogen* ; Oxidative Stress ; Phosphotransferases ; Potassium Channels, Calcium-Activated ; Protein Kinases*

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of Ca2+-activated potassium channel (KCa channel) and is involved in the activation of KCa channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1, 50 micrometer) and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP, 100 micrometer), a membrane-permeable analogue of cGMP, increased the KCa channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP (100 micrometer), cGMP (100 micrometer), ATP+cGMP (100 micrometer each), PKG (5 U/ microliter), ATP (100 micrometer)+PKG (5 U/ microliter), or cGMP (100 micrometer)+PKG (5 U/ microliter) did not increase the channel activity. ATP (100 micrometer)+cGMP (100 micrometer)+PKG (5 U/ microliter) added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP, 100 micrometer), a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The KCa channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of KCa channels, or some associated proteins in the membrane patch increase the activity of the KCa channel, and the activation may be associated with the vasodilating action.
Adenosine Triphosphate ; Cyclic GMP-Dependent Protein Kinases ; Membranes ; Molsidomine ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Okadaic Acid ; Patch-Clamp Techniques ; Phosphorylation ; Potassium Channels* ; Potassium* ; Protein Phosphatase 2 ; Signal Transduction*

The serine/threonine kinase Akt has been shown to play a role of multiple cellular signaling pathways and act as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI3K). It has been reported that phosphorylated Akt activates eNOS resulting in the production of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). It has been also reported that PKG activates PI3K/Akt signaling. Therefore, it is possible that PI3K, Akt, eNOS, sGC, and PKG form a loop to exert enhanced and sustained activation of Akt. However, the existence of this loop in eNOS-expressing cells, such as endothelial cells or astrocytes, has not been reported. Thus, we examined a possibility that Akt phosphorylation might be enhanced via eNOS/sGC/PKG/PI3K pathway in astrocytes in vivo and in vitro. Phosphorylation of Akt was detected in astrocytes after KA treatment and was maintained up to 72 h in mouse hippocampus. 2 weeks after KA treatment, astrocytic Akt phosphorylation was normalized to control. The inhibition of eNOS, sGC, and PKG significantly decreased Akt and eNOS phosphorylation induced by KA in astrocytes. In contrast, the decreased phosphorylation of Akt and eNOS by eNOS inhibition was significantly reversed with PKG activation. The above findings in mouse hippocampus were also observed in primary astrocytes. These data suggest that Akt/eNOS/sGC/PKG/PI3K pathway may constitute a loop, resulting in enhanced and sustained Akt activation in astrocytes.
Animals ; Astrocytes ; Cyclic GMP-Dependent Protein Kinases ; Endothelial Cells ; Guanylate Cyclase ; Hippocampus ; Kainic Acid ; Mice ; Nitric Oxide ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Phosphotransferases ; Receptors, Growth Factor ; Transducers

PURPOSE: The purpose of this study was to investigate the effect of exogenous nitric oxide (NO) on the proliferation of gastric carcinoma cells and the signaling pathways that regulate these responses. METHODS: MKN-28 cells were obtained from the Korean Cell Line Bank (KCLB) and maintained in DMEM culture media. The effect of sodium nitroprusside (SNP), a NO donor, on the proliferation of a serum-starved gastric carcinoma cell line, MKN-28, was examined by [3H]thymidine incorporation. Western blot was performed to analyze the translocation of protein kinase C (PKC)-deltafrom the cytosol to the plasma membrane of the MKN-28 cells. RESULTS: The proliferation of MKN-28 cells was significantly increased by SNP. It was also found that the proliferation was significantly inhibited by the protein kinase A (PKA) inhibitor, KT5720, and the protein kinase G inhibitor (PKG), KT5823, in SNP-treated cells. The SNP-induced proliferation was also inhibited by the PKC-deltaspecific inhibitor, rottlerin (1mu), but was increased by the PKC-beta inhibitor, Go6976 (1muM). The amount of translocated PKC-deltaprotein in the plasma membrane from the cytosol increased time-dependently after treating the cells with SNP, suggesting that NO activates PKC-delta Anti-inflammatory drugs, including dexamethasone, aspirin, indomethacin, mephenamic acid, and acetaminophen inhibited the SNP-induced proliferation of the cells and blocked of PKC-deltaactivation. CONCLUSION: NO stimulates the proliferation of serum- starved gastric cancer cells. The NO-induced proliferation may be mediated by PKC-delta The inhibitory effect of anti-inflammatory drugs on cell proliferation may be related to the inhibition of PKC-deltaactivity.
Acetaminophen ; Aspirin ; Blotting, Western ; Cell Line ; Cell Membrane ; Cell Proliferation ; Culture Media ; Cyclic AMP-Dependent Protein Kinases ; Cyclic GMP-Dependent Protein Kinases ; Cytosol ; Dexamethasone ; Humans ; Indomethacin ; Nitric Oxide ; Nitroprusside ; Protein Kinase C ; Protein Kinase C-delta* ; Protein Kinases* ; Stomach Neoplasms* ; Tissue Donors

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents (Icap). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on Icap. Membrane-permeable cGMP analogs, 8-bromoguanosine 3', 5'-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on Icap. The PKG inhibitor KT5823 prevented the inhibition of Icap by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.
Animals ; Benzoates ; Carbazoles ; Cyclic GMP-Dependent Protein Kinases ; Ganglia, Spinal ; Guanosine ; Guanylate Cyclase ; Hemoglobins ; Humans ; Imidazoles ; Neurons ; Nitric Oxide ; Nitroprusside ; Penicillamine ; Phosphotransferases ; Proteins ; Rats ; Receptors, Cytoplasmic and Nuclear ; Sensory Receptor Cells ; Spinal Nerve Roots ; Tissue Donors

The relaxation induced by stimulation of the inhibitory non-adrenergic, non-cholinergic (iNANC) nerve is mediated by the release of iNANC neurotransmitters such as nitric oxide (NO), vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP). The mechanisms of NO, VIP or ATP-induced relaxation have been partly determined in previous studies, but the detailed mechanism remains unknown. We tried to identify the nature of iNANC neurotransmitters in the smooth muscle of guinea pig ileum and to determine the mechanism of the inhibitory effect of nitric oxide. We measured the effect of NO-donors, VIP and ATP on the intracellular Ca2+ concentration((Ca2+)i), by means of a fluorescence dye (fura 2) and tension simultaneously in the isolated guinea pig ileal smooth muscle. Following are the results obtained. 1. Sodium nitropnisside (SNP: 10(-5) M) or S-nitro-N-acetyl-penicillamine (SNAP: 10(-5) M) decreased resting (Ca2+)i and tension of muscle. SNP or SNAP also inhibited rhythmic oscillation of (Ca2+)i and tension. In 40mM K+ solution or carbachol (CCh:10(-6) M)-induced precontracted muscle, SNP decreased muscle tension. VIP did not change (Ca2+)i and tension in the resting or precontracted muscle, but ATP increased resting (Ca2+)i and tension in the resting muscle. 2. 1H-(1,2,4)oxadiazol(4,3-a)quinoxalin-1-one (ODQ:1 muM), a specific inhibitor of soluble guanylate cyclase, limited the inhibitory effect of SNP. 3. Glibenclamide (10 muM), a blocker of KATP channel, and 4-aminopyridine (4-AP:5 mM), a blocker of delayed rectifier K channel, apamin (0.1 muM), a blocker of small conductance KCa. channel had no effect on the inhibitory effect of SNP. Iberiotoxin (0.1 muM), a blocker of large conductance KCa channel, significantly increased the resting (Ca2+)i, and tension, and limited the inhibitory effect of SNP. 4. Nifedipine (1 muM) or elimination of external Ca2+ decreased not only resting (Ca2+)i and tension but also oscillation of (Ca2+)i and tension. Ryanodine (5 muM) and cyclopiazonic acid (10 muM) decreased oscillation of (Ca2+)i and tension. 5. SNP decreased Ca2+ sensitivity of contractile protein. In conclusion, these results suggest that 1) NO is an inhibitory neurotransmitter in the guinea pig ileum, 2) the inhibitory effect of SNP on the (Ca2+)i and tension of the muscle is due to a decrease in (Ca2+)i by activation of the large conductance KCa channel and a decrease in the sensitivity of contractile elements to Ca2+ through activation of G-kinase.
4-Aminopyridine ; Adenosine Triphosphate ; Animals ; Apamin ; Carbachol ; Cyclic GMP-Dependent Protein Kinases ; Fluorescence ; Glyburide ; Guanylate Cyclase ; Guinea Pigs* ; Guinea* ; Ileum* ; Muscle Tonus ; Muscle, Smooth* ; Neurotransmitter Agents* ; Nifedipine ; Nitric Oxide ; Relaxation ; Ryanodine ; Sodium ; Vasoactive Intestinal Peptide

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

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