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Protein Kinase G (protein + kinase_g)
Selected AbstractscGMP-enhancing- and ,1A/,1D -adrenoceptor blockade-derived inhibition of Rho-kinase by KMUP-1 provides optimal prostate relaxation and epithelial cell anti-proliferation efficacyTHE PROSTATE, Issue 13 2007Chi-Ming Liu Abstract Background Soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) and Rho kinase (ROCK2) pathways are important in the regulation of prostate smooth muscle tone. This study is aimed to examine the relaxation activities of a sGC activator and PDE5A/ROCK2 inhibitor KMUP-1 in rat prostate and associated anti-proliferation activity in human prostatic epithelial cells. Methods The action characteristics of KMUP-1 were identified by isometric tension measurement, receptor binding assay, Western blotting and radioimmunoassay in rat prostate. Anti-proliferation activity of KMUP-1 in human prostatic epithelial PZ-HPV-7 cells was identified using flow cytometry and real time QRT-PCR. Results KMUP-1 inhibited phenylephrine-induced contractility in a concentration-dependent manner. KMUP-1 possessed potent ,1A/,1D -adrenoceptor binding inhibition activity, increased cAMP/cGMP levels and increased the expression of sGC, PKG, and PKA protein in rat prostate. Moreover, KMUP-1 inhibited phenylephrine-induced ROCK2 expression. KMUP-1 inhibited cell growth, arrested the cell cycle at G0/G1 phase and increased the expression of p21 in PZ-HPV-7 cells. Conclusions These results broaden our knowledge of sGC/cGMP/PKG and ROCK2 regulation on the relaxation and proliferation of prostate, which may help in the design of benign prostate hyperplasia (BPH) therapies that target these signaling pathways. KMUP-1 possesses the potential benefit in the treatment of BPH by its ,1A/,1D -adrenoceptor blockade, sGC activation, inhibition of PDE5A and ROCK2 and p21 protein enhancement, leading to attenuation of the smooth muscle tone and the proliferation of epithelial PZ-HPV-7 cells. The synergistic contribution of these pathways by KMUP-1 may benefit BPH patients with lower urinary tract symptoms. Prostate 67: 1397,1410, 2007. © 2007 Wiley-Liss, Inc. [source] Protein kinase G is involved in ammonia-induced swelling of astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 2009Agnieszka Konopacka Abstract Ammonia-induced swelling of astrocytes is a primary cause of brain edema associated with acute hepatic encephalopathy. Previous studies have shown that ammonia transiently increases cGMP in brain in vivo and in cultured astrocytes in vitro. We hypothesized that protein kinase G (PKG), an enzyme activated by cGMP and implicated in regulation of cell shape, size, and/or volume in peripheral and CNS cells, may play a role in the ammonia-induced astrocytic volume increase. Treatment of cultured rat cortical astrocytes with 1 or 5 mM NH4Cl (ammonia) for 24 h increased their cell volume by 50% and 80% above control, respectively, as measured by confocal imaging followed by 3D computational analysis. A cGMP analog, 8-(4-chlorophenylthio)-cGMP, increased the cell volume in control cells and potentiated the increase in 1 mM ammonia-treated cells. A soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) abrogated, and a PKG inhibitor [8-(4-chlorophenylthio)-cGMP-thioate, Rp-isomer] dose-dependently reduced the cell volume-increasing effect of 5 mM ammonia. The results suggest that (i) PKG may play a permissive role in ammonia-induced astrocytic swelling and (ii) elevation of brain cGMP associated with acute exposure to ammonia in vivo may aggravate the ensuing brain edema. [source] Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator-induced relaxation of porcine coronary veinsBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2007H Qi Background and purpose: Coronary venous activity is modulated by endogenous and exogenous nitrovasodilators. The present study was to determine the role of protein kinase G (PKG) in the regulation of the basal tension and nitrovasodilator-induced relaxation of coronary veins. Experimental approach: Effects of a PKG inhibitor on the basal tension and responses induced by nitroglycerin, DETA NONOate, and 8-Br-cGMP in isolated porcine coronary veins were determined. Cyclic cGMP was measured with radioimmunoassay. PKG activity was determined by measuring the incorporation of 32P from ,- 32P-ATP into the specific substrate BPDEtide. Key results: Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, increased the basal tension of porcine coronary veins and decreased PKG activity. The increase in tension was 38% of that caused by nitro- L -arginine. Relaxation of the veins induced by nitroglycerin and DETA NONOate was accompanied with increases in cGMP content and PKG activity. These effects were largely eliminated by inhibiting soluble guanylyl cyclase with ODQ. The increase in PKG activity induced by the nitrovasodilators was abolished by Rp-8-Br-PET-cGMPS. The relaxation caused by these dilators and by 8-Br-cGMP at their EC50 was attenuated by the PKG inhibitor by 51,66%. Conclusions and implications: These results suggest that PKG is critically involved in nitric oxide-mediated regulation of the basal tension in porcine coronary veins and that it plays a primary role in relaxation induced by nitrovasodilators. Since nitric oxide plays a key role in modulating coronary venous activity, augmentation of PKG may be a therapeutic target for improving coronary blood flow. British Journal of Pharmacology (2007) 152, 1060,1069; doi:10.1038/sj.bjp.0707479; published online 24 September 2007 [source] Nitric oxide regulates cell survival in purified cultures of avian retinal neurons: involvement of multiple transduction pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 2 2007T. A. Mejía-García Abstract Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S -nitroso-acetyl- d - l -penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5,-phosphate (8Br-cGMP) (GMP) or 3-(5,-hydroxymethyl-2,-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc-aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re-feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)-dependent way, and strengthens the notion that NO plays an important role during CNS development. [source] Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ ATP-sensitive K+ channel pathwayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Gerly A. C. Brito Abstract The aim of the present study was to determine the effect of pertussis toxin (PTX) on inflammatory hypernociception measured by the rat paw pressure test and to elucidate the mechanism involved in this effect. In this test, prostaglandin E2 (PGE2) administered subcutaneously induces hypernociception via a mechanism associated with neuronal cAMP increase. Local intraplantar pre-treatment (30 min before), and post-treatment (5 min after) with PTX (600 ng/paw1, in 100 µL) reduced hypernociception induced by prostaglandin E2 (100 ng/paw, in 100 µL, intraplantar). Furthermore, local intraplantar pre-treatment (30 min before) with PTX (600 ng/paw, in 100 µL) reduced hypernociception induced by DbcAMP, a stable analogue of cAMP (100 µg/paw, in 100 µL, intraplantar), which indicates that PTX may have an effect other than just Gi/G0 inhibition. PTX-induced analgesia was blocked by selective inhibitors of nitric oxide synthase (L-NMMA), guanylyl cyclase (ODQ), protein kinase G (KT5823) and ATP-sensitive K+ channel (Kir6) blockers (glybenclamide and tolbutamide). In addition, PTX was shown to induce nitric oxide (NO) production in cultured neurons of the dorsal root ganglia. In conclusion, this study shows a peripheral antinociceptive effect of pertussis toxin, resulting from the activation of the arginine/NO/cGMP/PKG/ATP-sensitive K+ channel pathway. [source] Enhancement of learning behaviour by a potent nitric oxide-guanylate cyclase activator YC-1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Wei-Lin Chien Abstract Memory is one of the most fundamental mental processes, and various approaches have been used to understand the mechanisms underlying this process. Nitric oxide (NO), cGMP and protein kinase G (PKG) are involved in the modulation of synaptic plasticity in various brain regions. YC-1, which is a benzylindazole derivative, greatly potentiated the response of soluble guanylate cyclase to NO (up to several hundreds fold). We have previously shown that YC-1 markedly enhances long-term potentiation in hippocampal and amygdala slices via NO-cGMP-PKG-dependent pathway. We here further investigated whether YC-1 promotes learning behaviour in Morris water maze and avoidance tests. It was found that YC-1 shortened the escape latency in the task of water maze, increased and decreased the retention scores in passive and active avoidance task, respectively. Administration of YC-1 30 min after foot-shock stimulation did not significantly affect retention scores in response to passive avoidance test. Administration of scopolamine, a muscarinic antagonist, markedly impaired the memory acquisition. Pretreatment of YC-1 inhibited the scopolamine-induced learning deficit. The enhancement of learning behaviour by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor L-NAME and PKG inhibitors of KT5823 and Rp-8-Br-PET-cGMPS, indicating that NO-cGMP-PKG pathway is also involved in the learning enhancement action of YC-1. YC-1 is thus a good drug candidate for the improvement of learning and memory. [source] Protein kinase G is involved in ammonia-induced swelling of astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 2009Agnieszka Konopacka Abstract Ammonia-induced swelling of astrocytes is a primary cause of brain edema associated with acute hepatic encephalopathy. Previous studies have shown that ammonia transiently increases cGMP in brain in vivo and in cultured astrocytes in vitro. We hypothesized that protein kinase G (PKG), an enzyme activated by cGMP and implicated in regulation of cell shape, size, and/or volume in peripheral and CNS cells, may play a role in the ammonia-induced astrocytic volume increase. Treatment of cultured rat cortical astrocytes with 1 or 5 mM NH4Cl (ammonia) for 24 h increased their cell volume by 50% and 80% above control, respectively, as measured by confocal imaging followed by 3D computational analysis. A cGMP analog, 8-(4-chlorophenylthio)-cGMP, increased the cell volume in control cells and potentiated the increase in 1 mM ammonia-treated cells. A soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) abrogated, and a PKG inhibitor [8-(4-chlorophenylthio)-cGMP-thioate, Rp-isomer] dose-dependently reduced the cell volume-increasing effect of 5 mM ammonia. The results suggest that (i) PKG may play a permissive role in ammonia-induced astrocytic swelling and (ii) elevation of brain cGMP associated with acute exposure to ammonia in vivo may aggravate the ensuing brain edema. [source] Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2000B. Sorin Abstract There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx. [source] von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanismJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2006R. RIBA Summary.,Background: The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data. Objectives: To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF. Methods: We used three independent markers of NO signaling, [3H] l -citrulline production, cGMP accrual and immunoblotting of vasodilator,stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF. Results: VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] l -citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin ,IIb,3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent. Conclusions: We demonstrate that VWF activates eNOS through a specific Ca2+ -dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG. [source] A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: In vivo characterization and structure/function relationshipsMASS SPECTROMETRY REVIEWS, Issue 4 2008Arjen Scholten Abstract Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity. © 2008 Wiley Periodicals, Inc. Mass Spec Rev 27: 331,353, 2008 [source] Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator-induced relaxation of porcine coronary veinsBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2007H Qi Background and purpose: Coronary venous activity is modulated by endogenous and exogenous nitrovasodilators. The present study was to determine the role of protein kinase G (PKG) in the regulation of the basal tension and nitrovasodilator-induced relaxation of coronary veins. Experimental approach: Effects of a PKG inhibitor on the basal tension and responses induced by nitroglycerin, DETA NONOate, and 8-Br-cGMP in isolated porcine coronary veins were determined. Cyclic cGMP was measured with radioimmunoassay. PKG activity was determined by measuring the incorporation of 32P from ,- 32P-ATP into the specific substrate BPDEtide. Key results: Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, increased the basal tension of porcine coronary veins and decreased PKG activity. The increase in tension was 38% of that caused by nitro- L -arginine. Relaxation of the veins induced by nitroglycerin and DETA NONOate was accompanied with increases in cGMP content and PKG activity. These effects were largely eliminated by inhibiting soluble guanylyl cyclase with ODQ. The increase in PKG activity induced by the nitrovasodilators was abolished by Rp-8-Br-PET-cGMPS. The relaxation caused by these dilators and by 8-Br-cGMP at their EC50 was attenuated by the PKG inhibitor by 51,66%. Conclusions and implications: These results suggest that PKG is critically involved in nitric oxide-mediated regulation of the basal tension in porcine coronary veins and that it plays a primary role in relaxation induced by nitrovasodilators. Since nitric oxide plays a key role in modulating coronary venous activity, augmentation of PKG may be a therapeutic target for improving coronary blood flow. British Journal of Pharmacology (2007) 152, 1060,1069; doi:10.1038/sj.bjp.0707479; published online 24 September 2007 [source] Cardioprotection: spotlight on PKGBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2007M V Cohen Classical ischaemic preconditioning, delayed or second window preconditioning and postconditioning are forms of cardioprotection that are dependent on cell surface receptors, intracellular signalling molecules and kinases that ultimately block formation of the mitochondrial permeability transition. The latter is presumed to cause myocardial necrosis as well as apoptosis, so prevention of its formation upon resumption of perfusion after a prolonged coronary occlusion should be cardioprotective. In all of these forms of cardioprotection, formation of cGMP and activation of protein kinase G (PKG) are recognized to be key steps in the signal transduction pathway. Burley et al. highlight the roles of cGMP and PKG in their comprehensive review. They describe the basic biology of PKG and emphasize its compartmentalization, which may be responsible for the frustration induced by assays for PKG in whole cell lysates and for the spurious conclusions about the role of PKG in cardioprotection. This review will be useful to both the novice and the seasoned investigator. British Journal of Pharmacology (2007) 152, 833,834; doi:10.1038/sj.bjp.0707453; published online 17 September 2007 [source] YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Ming-Shyan Chang YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-,, -,, -,, -, and -, isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-,, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-,. The MEK inhibitor, PD 98059 (10,50 ,M), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-, activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression. British Journal of Pharmacology (2002) 136, 558,567; doi:10.1038/sj.bjp.0704777 [source] | |||||||||||||