Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methacholine (MCh) interacted with M(3) muscarinic receptors in rat parotid tissue slices and induced amylase secretion. MCh- and calcimycin-induced exocytosis was completely inhibited by N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide, N(G)-nitro-L-arginine methylester (L-NAME), 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxaline-1-one, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, suggesting that activations of calmodulin (CaM) kinase II, nitric oxide synthase (NOS), and cGMP-dependent protein kinase (PKG) were coupled with the exocytosis. These suggestions were supported by the results that exposure of the slices to MCh induced a rapid increase in these enzyme activities. Western blot analysis showed that neuronal NOS (nNOS) was expressed in isolated parotid acinar cells of rats. To measure nitric oxide (NO) production in response to the stimulation with MCh in real time, the isolated parotid acinar cells had been preloaded with 4,5-diaminofluorescein diacetate and incubated with the agonist. MCh (1 microM) induced a fast increase in 4,5-diaminofluorescein fluorescence, corresponding to an increase in the NO synthesis in the presence of extracellular Ca(2+) but not in the absence of it. When the isolated parotid acinar cells preloaded with L-NAME or 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) were treated simultaneously with MCh, the increase in the fluorescence also was not observed. The MCh-induced increase in the fluorescence was not observed in the cells incubated in the absence of extracellular calcium, showing the importance of Ca(2+) entry from extracellular sites for MCh-induced NOS activation. These results indicate that nNOS is endogenously present in rat parotid acinar cells and that the rapid activation of this enzyme together with those of CaM kinase II and PKG contributes to MCh-induced amylase secretion.
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PMID:Activation of endogenous nitric oxide synthase coupled with methacholine-induced exocytosis in rat parotid acinar cells. 1190 93

Human and simian immunodeficiency virus (HIV and SIV, respectively) infections are characterized by gradual depletion of CD4+ T cells. The underlying mechanisms of CD4+ T-cell depletion and HIV and SIV persistence are not fully determined. The Nef protein is expressed early in infection and is necessary for pathogenesis. Nef can cause T-cell activation and downmodulates cell surface signaling molecules. However, the effect of Nef on the cell cycle has not been well characterized. To determine the role of Nef in the cell cycle, we investigated whether the SIV Nef protein can modulate cell proliferation and apoptosis in CD4+ Jurkat T cells. We developed a CD4+ Jurkat T-cell line that stably expresses SIV Nef under the control of an inducible promoter. Alterations in cell proliferation were determined by flow cytometry using stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester and bromodeoxyuridine incorporation. Apoptotic cell death was measured by annexin V and propidium iodide staining. Our results demonstrated that SIV Nef inhibited Fas-induced apoptosis in these cells and that the mechanism involved upregulation of the Bcl-2 protein. SIV Nef suppressed CD4+ T-cell proliferation by inhibiting the progression of cells into S phase of the cell cycle. Suppression involved an upregulation of cyclin-dependent kinase inhibitors p21 and p27 and the downregulation of cyclin D1 and cyclin A. In summary, inhibition of apoptosis by Nef can lead to persistence of infected cells and can support viral replication. In addition, a Nef-mediated delay in cell cycle progression may contribute to CD4+ T-cell anergy/depletion seen in HIV and SIV disease.
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PMID:Simian immunodeficiency virus Nef protein delays the progression of CD4+ T cells through G1/S phase of the cell cycle. 1190 98

The present study investigated the role of nitric oxide (NO)/cGMP signal transduction in the M(3) muscarinic acetylcholine receptor (mAChR)-stimulated increase in aquaporin-5 (AQP5) levels in the apical plasma membrane (APM) of rat parotid glands. Pretreatment of rat parotid tissue with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium inhibited both acetylcholine (ACh)- and pilocarpine-induced increases in AQP5 in the APM. NO donors [3-morpholinosydnonimine (SIN-1) and (S)-nitroso-N-acetylpenicillamine (SNAP)] mimicked the effects of mAChR agonists. A selective protein kinase G inhibitor [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg-3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823)] and an NO synthase inhibitor (N(6)-imminoethyl-L-lysine) blocked SIN-1- and SNAP-induced increases in AQP5 in the APM. A calmodulin kinase II inhibitor [(8)-5-isoquinolinesulfonic acid, 4-[2-(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (KN-62)] decreased the pilocarpine-induced increase of AQP5 in the APM. Using diaminofluorescinein-2 diacetate, enhanced NO synthase activity was detected in isolated parotid acinar cells after ACh-treatment. Treatment with dibutyryl cGMP, but not dibutyryl cAMP, induced an increase in AQP5 levels in the APM. BAPTA-AM inhibited the cGMP-induced increase in AQP5 in the APM. Pretreatment of the tissues with a myosin light chain kinase inhibitor [(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9)] inhibited a mAChR-stimulated increase in AQP5 levels in the APM. Although there was a significant ACh-induced increase in AQP5 in the APM in the absence of extracellular Ca(2+), the maximal effect of ACh on the AQP5 levels in the APM occurred in the presence of extracellular Ca(2+). These results suggest that NO/cGMP signal transduction has a crucial role in Ca(2+) homeostasis in the mAChR-stimulated increase in AQP5 levels in the APM of rat parotid glands.
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PMID:The muscarinic acetylcholine receptor-stimulated increase in aquaporin-5 levels in the apical plasma membrane in rat parotid acinar cells is coupled with activation of nitric oxide/cGMP signal transduction. 1202 4

In this study, we identify and investigate the role of protein kinase G (PKG) in cells cultured from human prostatic stroma. Cells were used for immunocytochemistry, contractility or K(+) fluorescent imaging studies. All cultured prostatic stromal cells showed PKG immunostaining. Phorbol 12,13 diacetate (PDA, 1 microM) elicited contractions from human-cultured prostatic stromal cells that could be blocked by both the L-type Ca(2+) channel blocker, nifedipine (3 microM), and the protein kinase C inhibitor, bisindolylmaleimide (1 microM). The nitric oxide donor, sodium nitroprusside (SNP, molar pIC(50) 5.16+/-0.17) and the cGMP-phosphodiesterase inhibitor, zaprinast (50 microM), inhibited PDA (1 microM)-induced contractions. The PKG activator beta-phenyl-1, N(2)-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP, molar pIC(50) 6.96 +/- 0.25) also inhibited PDA (1 microM)-induced contractions. Glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not iberiotoxin (100 nM) or Rp-cAMP (5 microM), reversed this inhibition. In human-cultured prostatic stromal cells loaded with the K(+) fluorescent indicator, 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI), PET-cGMP (300 nM) caused a reduction in intracellular K(+) that was blocked by glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not by iberiotoxin (100 nM). These data are consistent with the hypothesis that, in human-cultured prostatic stromal cells, PKG inhibits contractility through the activation of K(ATP) channels.
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PMID:Protein kinase G activation of K(ATP) channels in human-cultured prostatic stromal cells. 1235 8

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A(2A) adenosine receptor (A(2A)AR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A(2A)AR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha, PKCepsilon, and PKCzeta, and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A(2A)AR occurs following deactivation of PKCzeta.
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PMID:Distinct protein kinase C isoforms mediate regulation of vascular endothelial growth factor expression by A2A adenosine receptor activation and phorbol esters in pheochromocytoma PC12 cells. 1259 Jan 38

The effect of (+/-)cis-2-methylspilo(1,3-oxathiolane-5,3')quinuclidine (SNI-2011) on the secretory pathway of amylase in parotid tissues was investigated. SNI-2011-induced exocytosis was inhibited by a cell-permeable Ca(2+) chelator or inhibitors of calmodulin kinase II, neuronal nitric oxide synthase (nNOS), soluble guanyl cyclase, cyclic GMP-dependent protein kinase (PKG), and myosin light chain kinase, suggesting that these enzymes were coupled with the exocytosis. Stimulation with SNI-2011 of isolated rat parotid acinar cells loaded with 4,5-diaminofluorescein/diacetate (DAF-2/DA) induced a fast increase in DAF fluorescence corresponding to an increase in the NO production. SNI-2011-induced amylase secretion from parotid tissues in nNOS knockout mice has not been observed yet in spite of the expression of muscarinic M(3) receptors and the maintenance of secretory response to isoproterenol in the tissues. These results indicate the implication of the activation of Ca(2+)- and calmodulin-dependent enzymes and NOS-PKG signaling pathway in SNI-2011-induced amylase secretion from parotid acinar cells.
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PMID:Effect of SNI-2011 on amylase secretion from parotid tissue in rats and in neuronal nitric oxide synthase knockout mice. 1262 May 14

Cannabinoids protect neurons from excitotoxic injury. We investigated the mechanisms involved by studying N-methyl-D-aspartate (NMDA) toxicity in cultured murine cerebrocortical neurons in vitro and mouse cerebral cortex in vivo. The cannabinoid agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)-methanone mesylate [R(+)-Win 55212] reduced neuronal death in murine cortical cultures treated with 20 microM NMDA, and its protective effect was attenuated by the CB1 cannabinoid receptor (CB1R) antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-cichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A). Cultures from CB1R-knockout mice were more sensitive to NMDA toxicity than were cultures from wild-type mice. The in vitro protective effect of R(+)-Win 55212 was reduced by pertussis toxin, consistent with signaling through CB1R-coupled G-proteins. The nitric-oxide synthase (NOS) inhibitors 7-nitroindazole (7-NI) and N-omega-nitro-L-arginine methyl ester also reduced NMDA toxicity. In addition, CB1R and neuronal NOS were coexpressed in cultured cortical neurons, suggesting that cannabinoids might reduce NMDA toxicity by interfering with the generation of NO. NOS activity in cerebral cortex was higher in CB1R-knockouts than in wildtype mice, and 7-NI reduced NMDA lesion size. R(+)-Win 55212 inhibited NO production after NMDA treatment of wild-type cortical neuron cultures, measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, and this effect was reversed by SR141716A. In contrast, R(+)-Win 55212 failed to inhibit NO production in cultures from CB1R knockouts. Dibutyryl-cAMP blocked the protective effect of R(+)-Win 55212, and this was reversed by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H89). Cannabinoids seem to protect neurons against NMDA toxicity at least in part by activation of CB1R and downstream inhibition of PKA signaling and NO generation.
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PMID:Molecular mechanisms of cannabinoid protection from neuronal excitotoxicity. 1629 67

Tetrabromobisphenol-A (TBBPA) is one of the worlds most widely used brominated flame retardant. The present study reports effects of TBBPA on primary cultures of cerebellar granule cells (CGC). Using the trypan blue exclusion assay, we show that TBBPA induces death of CGC at low micro molar concentrations. Cell death was reduced by the NMDA receptor antagonist MK-801 (3 microM), the antioxidant vitamin E (50 microM), and in calcium-free buffer. We further demonstrate that TBBPA's toxicity was accompanied by apoptosis-like nuclear shrinkage, chromatin condensation, and DNA fragmentation. Other hallmarks of apoptosis such as caspase activity were, however, absent, indicating an atypical form of apoptosis. TBBPA increased intracellular free calcium in a concentration-dependent manner. TBBPA also induced an increase in extracellular glutamate in a time-dependent manner. TBBPA gave a concentration-dependent increase information reactive oxygen species (ROS) of measured with 2,7-dichlorofluorescein diacetate. The ROS formation was inhibited by the extracellular signal-regulated protein kinase (ERK) inhibitor U0126 (10 microM), the tyrosine kinase inhibitor erbstatin-A (25 microM), eliminating calcium from the buffer and by the superoxide dismutase inhibitor diethyldithio-carbamic acid (DDC, 100 microM). Further analysis with Western blot confirmed phosphorylation of ERK1/2 after exposure to TBBPA. We found that TBBPA induces ROS formation, increases intracellular calcium, extracellular glutamate, and death of CGC in vitro at concentrations comparable to those of polychlorinated biphenyl. These findings implicate TBBPA as a predicted environmental toxin and bring out the importance of awareness of its hazardous effects.
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PMID:In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate. 1720 76

We have recently demonstrated that the rheumatoid arthritis (RA) shared epitope (SE) acts as a ligand that triggers nitric oxide (NO) signaling in opposite cells. Given the known pro-oxidative effect of NO and the proposed role of oxidative stress in the pathogenesis of RA, this study explores whether SE-triggered signaling can increase cellular oxidative stress. cAMP levels, adenylyl cyclase activity, and protein kinase A activity were measured using commercial kits. Generation of reactive oxygen species (ROS) was quantified using the fluorochrome dichlorofluorescein diacetate. Oxidative DNA damage was quantified using the single-cell electrophoresis technique. Here, we report that cells exposed to cell surface SE-positive HLA-DR (human leukocyte antigen-DR) molecules, to cell-free recombinant proteins genetically engineered to express the SE motif, or to SE-positive synthetic peptide showed diminished cAMP-dependent signaling, increased ROS levels, and higher vulnerability to oxidative DNA damage. Introduction of single amino acid substitutions into SE-positive peptides revealed a consensus five-amino acid sequence motif of Q/R-K/R-X-X-A that is necessary and sufficient for SE-triggered signaling. The pro-oxidative effect of the SE could be reversed by inhibiting NO production. We conclude that the SE acts as a signaling ligand that activates an NO-mediated pro-oxidative pathway. The potential contribution of this signaling aberration to RA pathogenesis is discussed.
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PMID:The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway. 1725 42

We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels (Am J Physiol Lung Cell Mol Physiol 279: L611-L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH(2)-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-Ialpha to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved.
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PMID:Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins. 1761 49


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