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)

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced beta-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2'-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38alpha-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCdelta (protein kinase Cdelta), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1beta and interferon-gamma, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.
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PMID:Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells. 1609 52

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).
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PMID:Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle. 1615 88

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.
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PMID:Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness. 1632 56

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.
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PMID:Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines. 1662 25

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) may act as a critical enzyme for nitric-oxide-induced vasodilation. In this study, the role of PKG in regulation of basal tension and in relaxation induced by nitrovasodilators in coronary arteries was determined. Under basal conditions, Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, evoked a significant contraction of isolated porcine coronary arteries, which was prevented by nitro-L: -arginine or the removal of the endothelium. Relaxation to nitroglycerin and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) in vessels preconstricted with U46619 was largely abolished by 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one (ODQ) and inhibited by 48 to 79% by Rp-8-Br-PET-cGMPS. Relaxation of the vessels to 8-Br-cGMP was inhibited by 56% by Rp-8-Br-PET-cGMPS. The basal activity of PKG but not that of cyclic adenosine monophosphate-dependent protein kinase (PKA) was inhibited by nitro-L: -arginine, ODQ, or Rp-8-Br-PET-cGMPS. The activity of PKG but not that of PKA was increased by nitroglycerin and DETA NONOate in intact vessels and increased by cGMP in the tissue homogenates. These effects were abolished by Rp-8-Br-PET-cGMPS but not by myristoylated PKI, a specific inhibitor of PKA. These results suggest that in porcine coronary arteries, PKG is involved in the regulation of basal tension and plays a primary role in relaxation induced by nitrovasodilators, whereas PKA may play a minor role.
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PMID:cGMP-dependent protein kinase in regulation of basal tone and in nitroglycerin- and nitric-oxide-induced relaxation in porcine coronary artery. 1737 6

Smooth muscle expresses the Ialpha and the Ibeta isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at approximately 6 weeks. We reconstituted mice with the cGKIalpha or -Ibeta isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKIalpha or the -Ibeta coding sequences were placed under the control of the SM22alpha promoter to express either isoform selectively in smooth muscle cells (SM-Ialpha or SM-Ibeta transgene). To generate smooth muscle-specific cGKIalpha or cGKIbeta rescue mice, the SM-Ialpha or SM-Ibeta transgenes were crossed on a cGKI-/- genetic background. The levels of cGKIalpha or -Ibeta expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKIalpha or -Ibeta rescue mice, expression of the isozymes was not detectable in non-smooth muscle tissues and cells. Median survival time of the Ialpha and Ibeta rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP-induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone- or K+-induced [Ca2+]i levels. The cGKIalpha and cGKIbeta rescue mice did not show a significant difference in intestinal passage time of BaSO4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions.
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PMID:Rescue of cGMP kinase I knockout mice by smooth muscle specific expression of either isozyme. 1804 24

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10(-6) M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 +/- 0.12, 0.42 +/- 0.08, and 0.11 +/- 0.01 amol/mug, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 +/- 33, 272 +/- 20, and 238 +/- 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 +/- 84, 815 +/- 81, and 549 +/- 78 pmol P(i).min(-1).mg soluble protein(-1)), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 +/- 0.22, 2.64 +/- 0.25, and 2.85 +/- 0.28 pmol P(i).min(-1).ng cGKI(-1), respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.
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PMID:Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity. 1895 58

Alcohol damages the developing brain and can lead to fetal alcohol syndrome. One of alcohol's most important neuropathologic effects is neuronal death. As neurons mature, they become less vulnerable to alcohol-induced death because they acquire a protective signaling pathway, mediated by nitric oxide (NO). This pathway is the NO-cGMP-cyclic GMP-dependent protein kinase G (NO-cGMP-PKG) pathway. The goal of the present studies was to determine whether nuclear factor kappa B (NF-kappaB) is the downstream effector through which the NO-cGMP-PKG pathway signals its neuroprotective effects against alcohol. An activator of NF-kappaB, tumor necrosis factor-alpha (TNF-alpha), protected immature cerebellar granule neuron cultures against alcohol-induced cell death in a dose-dependent fashion. The protective effect of TNF-alpha was similar in magnitude to the protective effects of NMDA and DETA-NONOate, both of which are NO-cGMP-PKG pathway activators. Blockade of the pathway at its first step with NAME, second step with LY83583, or third step with PKG inhibitor increased alcohol-induced cell death and the vulnerability of mature neurons to alcohol toxicity. TNF-alpha protected the neurons, even when the NO-cGMP-PKG pathway was blocked at upstream sites. NF-kappaB activation inhibitor (NFi) worsened alcohol-induced cell death and blocked the protective effects of NO-cGMP-PKG pathway activators and TNF-alpha. TNF-alpha reduced the alcohol vulnerability of immature neurons, while NFi increased the vulnerability of mature neurons. Both NMDA and TNF-alpha led to the phosphorylation and degradation of IkappaBalpha, demonstrating that both agents can activate NF-kappaB in cerebellar granule cells. Thus, NF-kappaB plays a critical role in the acquisition of alcohol resistance by maturing neurons and is a key downstream effector through which the NO-cGMP-PKG pathway signals its neuroprotective effects against alcohol.
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PMID:Nitric oxide utilizes NF-kappaB to signal its neuroprotective effect against alcohol toxicity. 1913 70

We examined early and late alterations in gene expression patterns and phosphorylation levels of key regulators of selected signaling pathways in U937 cells exposed to various (*)NO fluxes. cDNA microarray analysis and real-time quantitative PCR identified 45 NO-sensitive genes (>or=2-fold change), among which KLF2, KLF6, TSC22D3, DDIT4, MKP-5 (up-regulated), KIF23, histone H4, ARL6IP2, CLNS1A, SLC7A6, CDKN3, SRP19, and BCL11A (down-regulated) have not been reported before. For two selected genes, KLF2 and DDIT4, the sensitivity to (.)NO was also proven at the protein level. Among the examined genes, only KLF2 had a higher sensitivity to slow release of NO (DETA-NO) than to high-dose, short-duration exposure (DPTA-NO), reaching an about 50-fold increase in mRNA level. Our study revealed that fast and slow NO donors activate similar signaling pathways and induce phosphorylation of MAP kinases and downstream transcription factors ATF2 and c-Jun. Inhibitory analysis of major signaling pathways showed that activity of p38 MAPK and tyrosine kinases is indispensable for gene induction in cells exposed to DPTA-NO, whereas G-protein Rho suppression caused superinduction of KLF2 in (*)NO-stimulated cells. Finally, we showed that both (*)NO donors caused a marked decrease in phosphorylation of p70S6K, an mTOR substrate and regulator of mRNA translation, and protein kinase Akt, an upstream positive regulator of mTOR.
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PMID:Variation in gene expression profiles of human monocytic U937 cells exposed to various fluxes of nitric oxide. 1989 11

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPT1 but not that of PP1cdelta. The extent of reduction in total MYPT1 protein level was comparable to that of MYPT1 (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8-bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation.
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PMID:Increased degradation of MYPT1 contributes to the development of tolerance to nitric oxide in porcine pulmonary artery. 2041 85


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