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1.
Neuroscience Bulletin ; (6): 135-148, 2022.
Article in English | WPRIM | ID: wpr-922667

ABSTRACT

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.


Subject(s)
Action Potentials , HEK293 Cells , Humans , Protein Kinase C/metabolism , Pyramidal Cells/enzymology , Shab Potassium Channels/genetics
2.
Acta cir. bras ; 33(7): 556-564, July 2018. tab, graf
Article in English | LILACS | ID: biblio-949368

ABSTRACT

Abstract Purpose: To investigate the effects of baicalin on inflammatory reaction, oxidative stress and protein kinase D1 (PKD1) and nuclear factor-kappa B (NF-κB) protein expressions in severe acute pancreatitis (SAP) rats. Methods: Sixty rats were divided into sham operation, model, and low-, medium- and high-dose baicalin group. SAP model was established in later 4 groups. The later 3 groups were injected with 0.1, 0.2 and 0.4 ml/100 g 5% baicalin injection, respectively. At 12 h, the serum SAP related indexes and inflammatory factors, peripheral blood CD3 and γδT cell percentages, wet/dry ratio and pancreas ascites volume, oxidative stress indexes and PKD1 and NF-κB protein expressions in pancreatic tissue were determined. Results: Compared with model group, in high-dose baicalin group the wet/dry ratio and ascites volume, serum amylase level, phospholipase A2 activity, TNF-α, IL-1 and IL-6 levels, and pancreatic malondialdehyde level and PKD1 and NF-κB protein expression were significantly decreased (P < 0.05), and peripheral blood CD3 and γδT cell percentages and pancreatic superoxide dismutase and glutathione peroxidase levels were significantly increased (P < 0.05). Conclusion: Baicalin can resist the inflammatory reaction and oxidative stress, and down-regulate protein kinase D1 and nuclear factor-kappa B protein expressions, thus exerting the protective effects on severe acute pancreatitis in rats.


Subject(s)
Animals , Pancreatitis/drug therapy , Flavonoids/pharmacology , Protein Kinase C/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , NF-kappa B/metabolism , Oxidative Stress/drug effects , Pancreatitis/metabolism , Superoxide Dismutase/drug effects , Protein Kinase C/drug effects , Random Allocation , Down-Regulation/drug effects , Reproducibility of Results , NF-kappa B/drug effects , Interleukin-6/blood , Interleukin-1/blood , Tumor Necrosis Factor-alpha/blood , Treatment Outcome , Rats, Sprague-Dawley , CD3 Complex/drug effects , CD3 Complex/blood , Glutathione Peroxidase/drug effects , Glutathione Peroxidase/metabolism , Amylases/drug effects , Amylases/blood , Malondialdehyde/metabolism
3.
Article in English | WPRIM | ID: wpr-30206

ABSTRACT

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-theta (PKCtheta) and protein kinase C-beta (PKCbeta) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCtheta and PKCbeta stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox-/- mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.


Subject(s)
Animals , Aorta/cytology , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Lipoproteins, LDL/metabolism , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , NADPH Oxidases/metabolism , Phosphorylation , Protein Kinase C/metabolism , Signal Transduction , Superoxides/metabolism
4.
J. bras. nefrol ; 36(4): 542-544, Oct-Dec/2014. graf
Article in English | LILACS | ID: lil-731142

ABSTRACT

Introduction: When faced with violet, purple or purplish-blue urine, clinicians should consider urinary tract infection in their differential diagnosis. Case report: A 60-year-old woman with end-stage kidney disease and non-adherence to renal replacement therapy was admitted to our hospital for placement of hemodialysis catheter. During her hospitalization she had purple urine, and purple urine bag syndrome (PUBS) was diagnosed. She was effectively treated with antibiotics and her urine returned to a dark yellow color. Discussion: Although this condition is often easily treated, diagnosing PUBS in chronic renal patients probably means an increased serum concentration of indoxyl sulfate, metabolite that is involved in the progression of both CKD and cardiovascular disease. Conclusion: Hence, in the context of our renal patients, perhaps PUBS is not as benign as supposed. .


Subject(s)
Animals , Rats , Isoenzymes/metabolism , Membrane Glycoproteins/metabolism , Protein Kinase C/metabolism , Proteoglycans/metabolism , Amino Acid Sequence , Molecular Sequence Data , Phosphorylation , Protein Kinase C-alpha , Protein Kinase C-delta
5.
Braz. j. med. biol. res ; 47(9): 789-798, 09/2014. graf
Article in English | LILACS | ID: lil-719317

ABSTRACT

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.


Subject(s)
Animals , Male , Aorta, Thoracic/physiopathology , Aortic Coarctation/physiopathology , Calcium/metabolism , Endothelium, Vascular/physiology , Muscle, Smooth, Vascular/physiopathology , Protein Kinase C/antagonists & inhibitors , Vasoconstriction/physiology , Angiotensin I/analysis , Angiotensin II/analysis , Aorta, Thoracic/injuries , Aorta, Thoracic/surgery , Blotting, Western , Blood Pressure/physiology , Chromatography, High Pressure Liquid , Endothelium, Vascular/injuries , Muscle, Smooth, Vascular/metabolism , Neuromuscular Depolarizing Agents/pharmacology , Phenylephrine/pharmacology , Potassium/pharmacology , Protein Kinase C/metabolism , Radioimmunoassay , Rats, Wistar , /metabolism , Vasoconstriction/drug effects
6.
Article in English | WPRIM | ID: wpr-70517

ABSTRACT

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.


Subject(s)
Apoptosis , Caspases/metabolism , Entamoeba histolytica/enzymology , Humans , Hydrolysis , Jurkat Cells , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , T-Lymphocytes/parasitology
7.
São Paulo; s.n; s.n; 2014. 181 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847078

ABSTRACT

As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCßI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCßI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCßI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCßI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCßI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCßI foi capaz de fosforilar a TIF1ß e inibindo-se a PKCßI por RNAi vimos uma diminuição na expressão da TIF1ß e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1ß. Além disso vimos que inibindo-se a PKCßI com o peptídeo inibidor da PKCßI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCßI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCßI o que sugere que a PKCßI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCßI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1ß um possível alvo direto da PKCßI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCßI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCßI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas


The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCßI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCßI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCßI targets in undifferentiated ESC. We indeed saw that inhibiting PKCßI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCßI we found the adaptor protein, TIF1ßI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCßI phosphorylated TIF1ß and inhibiting PKCßI with RNAi decreased the expression of TIF1ß and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1ß. We also saw that inhibiting PKCßI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCßI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCßI suggesting that PKCßI activates c-Myc repressor proteins. Our studies sugest that PKCßI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1ß a possibly a direct target of PKCßI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCßI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCßI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation


Subject(s)
Embryonic Stem Cells/cytology , Protein Kinase C/metabolism , Cell Differentiation , Chromatin/genetics , Mass Spectrometry/methods , Phosphorylation , Protein Kinase C beta/analysis , Proteomics/instrumentation , Repressor Proteins/genetics , Substrates for Biological Treatment/classification
8.
Braz. j. med. biol. res ; 46(9): 746-751, 19/set. 2013. graf
Article in English | LILACS | ID: lil-686569

ABSTRACT

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.


Subject(s)
Animals , Mice , Hydrogen Peroxide/pharmacology , Intramolecular Oxidoreductases/drug effects , Macrophage Migration-Inhibitory Factors/drug effects , Myocytes, Cardiac/metabolism , Oxidants/pharmacology , Protein Kinase C/metabolism , src-Family Kinases/metabolism , Angiotensin II/metabolism , Blotting, Western , Cell Line , Immunohistochemistry , Intramolecular Oxidoreductases/genetics , Microscopy, Confocal , Macrophage Migration-Inhibitory Factors/genetics , Oxidative Stress/physiology , Protein Kinase Inhibitors/pharmacology , Real-Time Polymerase Chain Reaction , Renin-Angiotensin System/physiology
9.
Article in English | IMSEAR | ID: sea-138997

ABSTRACT

Background & objectives: Chapekar established a model of ovarian tumourigenesis in mice by splenic transplantation of ovaries, resulting in sustained luteinizing hormone (LH) levels because of absence of feedback inhibition. There is increasing evidence of the differential response to LH or hCG under various experimental conditions. The effect of sustained hormonal stimulation in long term cultures is sparsely investigated. The study is aimed to determine the role of hCG and LH stress on caprine ovarian granulosa cells and their downstream signaling in short and long term cultures. Methods: To study the response of hCG and LH stress and downstream signaling, short term cultures were set up by exposing goat ovarian granulosa cells in primary cultures to hCG and LH stress (levels beyond their physiological doses) for 5 days (P0). Cells were sub-cultured at sixth day and subjected to prolonged LH/ hCG stress for two weeks in passage 1(P1) (long term cultures). Downstream cell signaling molecules were assessed. Intracellular cAMP was estimated by ELISA. For PKA and PKC, activity assays were performed. pERK protein expressions in short term cultures were assessed by Western blot and flowcytometry; in long term cultures, pERK expression was analyzed by flowcytometry. Results: Differential effects on cell proliferation were observed in long term cultures, where the untreated and hCG exposed cells showed markedly reduced cell proliferation after second week of exposure while LH treated cells continued to proliferate. Different levels of cAMP, PKA, PKC and phosphorylated ERK1/2 were observed on short term and long term LH stimulation. On sustained hormonal stimulation, cAMP levels were significantly (P<0.05) higher in hCG treated cultures as compared to controls and LH treated cultures. LH led to maximal elevation of ERK in long term cultures. Interpretation & Conclusions: As pERK1/2 promotes cellular proliferation, activation of ERK1/2 in LH treated cultures may be responsible for sustained growth. Prolonged LH treatment promoted growth and proliferation in caprine ovarian granulosa cells whereas prolonged exposure to hCG led to elevated levels of cAMP and decreased the rate of proliferation. Defining the signals and second messengers that act as survival or apoptotic mediators may help in elucidation of the mechanisms controlling proliferation or programmed cell death in granulosa cells.


Subject(s)
Animals , Cell Culture Techniques , Cell Proliferation/drug effects , Chorionic Gonadotropin/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Goats , Granulosa Cells/drug effects , Luteinizing Hormone/pharmacology , Mitogen-Activated Protein Kinase 3/metabolism , Primary Cell Culture , Protein Kinase C/metabolism , Signal Transduction/drug effects
10.
Braz. j. med. biol. res ; 44(3): 193-199, Mar. 2011. ilus
Article in English | LILACS | ID: lil-576067

ABSTRACT

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67 percent, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.


Subject(s)
Humans , Angiotensin II/pharmacology , Inflammation/enzymology , Matrix Metalloproteinase 9/metabolism , Monocytes/drug effects , NF-kappa B/metabolism , Protein-Serine-Threonine Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Monocytes/metabolism , Protein Kinase C/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , /metabolism , rho-Associated Kinases/metabolism
11.
Article in English | WPRIM | ID: wpr-58123

ABSTRACT

Scutellarin is a flavonoid extracted from a traditional Chinese herb, Erigeron breviscapus. The present study investigated the effect of scutellarin on MUC5AC mucin production and the possible mechanism. Human bronchial epithelial 16 (HBE16) cells were pretreated with scutellarin for 60 min, and then exposed to human neutrophil elastase (HNE) or interleukin (IL)-13 for 12 hr. RT-PCR and ELISA were performed to measure the amount of MUC5AC mucin production. The results showed that scutellarin inhibited MUC5AC expression both in mRNA and protein level induced by HNE in a concentration-dependent manner. However, scutellarin failed to inhibit MUC5AC mucin production induced by IL-13. To investigate the intracellular mechanisms associated with the effect of scutellarin on MUC5AC mucin production, western blotting was carried out to examine the phosphorylation of protein kinase C (PKC), signal transducer and activator of transcription 6 (STAT6) and extracellular signal-regulated kinase 1/2 (ERK1/2). The phosphorylation of PKC and ERK1/2 was attenuated after treatment with scutellarin, whereas STAT6 was not significantly affected. Therefore, it is suggested that scutellarin down-regulates MUC5AC mucin production on HBE16 cells via ERK-dependent and PKC-dependent pathways.


Subject(s)
Apigenin/chemistry , Cells, Cultured , Dose-Response Relationship, Drug , Down-Regulation , Epithelial Cells/drug effects , Erigeron/chemistry , Glucuronates/chemistry , Humans , Interleukin-13/pharmacology , Leukocyte Elastase/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mucin 5AC/genetics , Phosphorylation , Protein Kinase C/metabolism , Respiratory Mucosa/drug effects , STAT6 Transcription Factor/metabolism , Signal Transduction
12.
Article in English | WPRIM | ID: wpr-203590

ABSTRACT

The action mode of 4,4'-diaminodiphenylsulfone (DDS) is still under debate, although it has long been used in treatment of several dermatologic diseases including Hansen's disease. In this study, we tested the effect of DDS as an antioxidant on paraquat-induced oxidative stress in non-phagocytic human diploid fibroblasts (HDFs). Overall, preincubation of HDFs with DDS prevented the oxidative stress and the resulting cytotoxic damages caused by paraquat in these cells. The specific effects of DDS in paraquat-treated HDFs are summarized as follows: a) reducing the expression of NADPH oxidase 4 (NOX4) by inhibiting paraquat-induced activation of PKC; b) inhibiting paraquat-induced decreases in mitochondrial complex protein levels as well as in membrane potentials; c) consequently, inhibiting the generation of cytosolic and mitochondrial superoxide anions. Taken together, these findings suggest that DDS would suppress the radical generation in non-phagocytic HDFs during oxidative stress, and that DDS might have the extended potential to be used further in prevention of other oxidative stress-related pathologies.


Subject(s)
Biphenyl Compounds/metabolism , Cell Death/drug effects , Cell Survival/drug effects , Dapsone/pharmacology , Diploidy , Enzyme Activation/drug effects , Fibroblasts/cytology , Free Radical Scavengers/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Male , Mitochondria/drug effects , NADPH Oxidases/genetics , Paraquat/toxicity , Phagocytosis/drug effects , Picrates/metabolism , Protein Kinase C/metabolism , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , Superoxides/metabolism
13.
Article in English | WPRIM | ID: wpr-196698

ABSTRACT

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.


Subject(s)
Phosphatidylinositol 3-Kinase/metabolism , Angiotensin II/metabolism , Animals , Cell Movement , Cells, Cultured , Guanine Nucleotide Exchange Factors/genetics , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , NADPH Oxidases/metabolism , Protein Kinase C/metabolism , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/metabolism , src-Family Kinases/metabolism
14.
Indian J Exp Biol ; 2008 Nov; 46(11): 764-9
Article in English | IMSEAR | ID: sea-61661

ABSTRACT

In the present in vitro study, the involvement of cAMP dependent-protein kinase A (PKA) and calcium-dependent protein kinase C (PKC) in the regulation of forebrain (telencephalon and hypothalamus) tyrosine hydroxylase (TH) activity was demonstrated during the reproductive seasons of the female catfish H. fossilis. In the concentration studies conducted in prespawning phase, cAMP (0.05 nM, 0.5 nM, 1 mM and 2.0 mM) or the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX-0.5-2.0 mM) stimulated enzyme activity. Likewise, the incubation of the enzyme preparations with the cAMP dependent-protein kinase A inhibitor H-89 (1 and 10 microM) and PKC inhibitor calphostin C (cal C; 1 and 10 microM) inhibited enzyme activity in a concentration-dependent manner. In seasonal studies, the incubation of the enzyme preparations with cAMP (1 mM), IBMX (1 mM), H-89 (10 microM) and cal-C (10 microM) produced season-dependent effects on enzyme activity. The stimulatory effect of cAMP and IBMX and the inhibitory effect of H-89 and cal C were greater in the resting and spawning phases. The results suggest the involvement of both signal transduction pathways in TH activation vis-à-vis catecholaminergic activity with a more dominant role by the cAMP-PKA pathway.


Subject(s)
1-Methyl-3-isobutylxanthine/pharmacology , Animals , Brain/enzymology , Calcium/metabolism , Catfishes , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Fossils , Isoquinolines/pharmacology , Naphthalenes/pharmacology , Protein Kinase C/metabolism , Seasons , Signal Transduction , Sulfonamides/pharmacology , Tyrosine 3-Monooxygenase/chemistry
15.
Indian J Biochem Biophys ; 2007 Jun; 44(3): 183-5
Article in English | IMSEAR | ID: sea-26644

ABSTRACT

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.


Subject(s)
Animals , Blood Glucose/metabolism , Carbohydrate Metabolism , Cucumis sativus/metabolism , Cytosol/metabolism , Glycogen/metabolism , Glycogen Phosphorylase/metabolism , Glycogen Synthase/metabolism , Liver/metabolism , Male , Pectins/metabolism , Phosphorylases/metabolism , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley
16.
J Biosci ; 2007 Mar; 32(2): 405-13
Article in English | IMSEAR | ID: sea-110705

ABSTRACT

Free fatty acids are known to play a key role in promoting loss of insulin sensitivity,thereby causing insulin resistance and type 2 diabetes.However,the underlying mechanism involved is still unclear.In searching for the cause of the mechanism,it has been found that palmitate inhibits insulin receptor (IR)gene expression,leading to a reduced amount of IR protein in insulin target cells. PDK1-independent phosphorylation of PKC(eta) causes this reduction in insulin receptor gene expression.One of the pathways through which fatty acid can induce insulin resistance in insulin target cells is suggested by these studies.We provide an overview of this important area,emphasizing the current status.


Subject(s)
Fatty Acids, Nonesterified/metabolism , Gene Expression Regulation/physiology , Humans , Insulin/metabolism , Insulin Resistance/physiology , Models, Biological , Phosphorylation , Protein Kinase C/metabolism , Receptor, Insulin/metabolism , Signal Transduction/physiology
17.
Article in English | WPRIM | ID: wpr-53147

ABSTRACT

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.


Subject(s)
Tumor Suppressor Protein p53/metabolism , Protein Kinase C/metabolism , Phosphorylation , JNK Mitogen-Activated Protein Kinases/physiology , Humans , HeLa Cells , Diterpenes/pharmacology , DNA Fragmentation/drug effects , Cell Death/drug effects , Anthracenes/pharmacology
18.
Article in English | WPRIM | ID: wpr-15699

ABSTRACT

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation.


Subject(s)
Up-Regulation , Receptors, Cell Surface/metabolism , Rats , Protein Kinase C/metabolism , Middle Aged , Microscopy, Electron , Humans , Caveolin 1/metabolism , Caveolae/metabolism , Brain/metabolism , Animals , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Aging/metabolism , Aged, 80 and over , Aged
19.
Biol. Res ; 39(3): 531-539, 2006. ilus
Article in English | LILACS | ID: lil-437385

ABSTRACT

Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50 percent. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.


Subject(s)
Animals , Mice , Glucose/metabolism , Insulin , Islets of Langerhans , Phorbol Esters/pharmacology , Protein Kinase C/drug effects , Serotonin/metabolism , Calcium Signaling/drug effects , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Electrochemistry , Fluorometry , Islets of Langerhans/drug effects , Protein Kinase C/metabolism , Pulsatile Flow/drug effects
20.
Gac. méd. Méx ; 141(4): 259-266, jul.-ago. 2005. graf
Article in Spanish | LILACS | ID: lil-632075

ABSTRACT

En este estudio se investigaron los sitios probables de la acción inhibitoria de prolactina (Prl) sobre la esteroidogénesis ovárica inducida por la hormona folículo estimulante (FSH). Para esta finalidad se estudió la capacidad de cultivos primarios de células de la granulosa de la rata de sintetizar estradiol y AMPc bajo la estimulación con FSH o de activadores de la vía dependiente de AMPc en presencia de Prl humana. La participación de otros sistemas de transducción de señal como los dependientes de PKC y proteínas Gi en los mecanismos de acción inhibitoria de la Prl fue también investigada utilizando inhibidores de estos sistemas como la calfostina C y la toxina pertusis. Los resultados demostraron la habilidad de la Prl de alterar la esteroidogénesis previa y posterior a la generación de AMPc, muy probablemente por mecanismos que involucran la activación de la subunidad catalítica de la adenilato ciclasa, así como a través de interactuar con sistemas de transducción de señal dependientes de PKC y proteínas sensibles a la toxina pertusis. Nuestros resultados sugieren un mecanismo de interacción entre receptores acoplados a proteínas G con aquéllos acoplados a cinasas de tirosinas mediado muy probablemente por vías de señalización dependientes de proteínas Gi.


We studied the sites of prolactin inhibition upon FSH induced ovarian steroidogenesis and the ability of prolactin (Prl) to inhibit the synthesis of estradiol and cAMP accumulation under the stimulation of FSH or cAMP dependent activators. The participation of other signal pathways such as PKC and Gi proteins on the inhibitory actions of Prl was also investigated using calfostine C andpertusis toxin as inhibitors. Results showed a dose dependent prolactin decrease in FSH-induced estradiol and cAMP production prior and after the generation of the cyclic nucleotide by a mechanism involving the catalytic subunit of adenyl cyclase and/or through activation of PKC or by the interaction with pertusin toxin sensitive G proteins. Our results suggest a mechanism by which G protein coupled receptors are linked with those coupled with tyrosine kinase through the involvement of a Gi protein mediated mechanism.


Subject(s)
Animals , Female , Rats , Estradiol/biosynthesis , Granulosa Cells/metabolism , Prolactin/pharmacology , Analysis of Variance , Adenylyl Cyclases/metabolism , Catalysis , Cells, Cultured , Cyclic AMP/metabolism , Enzyme Activation , Follicle Stimulating Hormone/pharmacology , GTP-Binding Proteins , Granulosa Cells/drug effects , Naphthalenes/pharmacology , Pertussis Toxin/pharmacology , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Rats, Wistar , Receptors, FSH/metabolism , Signal Transduction , Stimulation, Chemical
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