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 distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-) > Br(-)--NO3(-) > F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.
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PMID:A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel. 1266 33

Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 microg/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 microg/kg per hour for 28 days). Ca2+-activated K+ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKIalpha versus cGKIbeta, since cGKIbeta is less sensitive to cGMP activators than cGKIalpha. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKIalpha-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKIbeta protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKIbeta mRNA and a decrease in cGKIalpha mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKIbeta was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKIbeta in VSMCs previously ascribed exclusively to cGKIalpha. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.
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PMID:Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension: novel mechanism for nitrate tolerance in vascular smooth muscle. 1464 36

Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches: (1) EPR spin trapping using colloid Fe(DETC)2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (>80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 micromol/L, and 1 micromol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (>10 micromol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. The full text of this article is available online at http://www.circresaha.org.
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PMID:Does nitric oxide mediate the vasodilator activity of nitroglycerin? 1455 Dec 41

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.
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PMID:Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect. 1465 76

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.
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PMID:Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 1487 7

Erythropoietin is protective against cardiac ischemia, but the underlying mechanisms are unknown. We determined whether erythropoietin (0.5 - 10.0 U/ml) confers acute cardioprotection in infant rabbit hearts and the contribution of protein kinases, nitric oxide synthase and potassium channels to the underlying mechanism. Hearts from normoxic infant New Zealand White rabbits (n=8/group) were isolated and perfused in the Langendorff mode. Biventricular function was recorded under steady-state conditions prior to 30 min global no-flow ischemia and 35 min reperfusion. Administration of erythropoietin for 15 min immediately prior to ischemia resulted in a concentration-dependent increase in recovery of left and right ventricular developed pressure in rabbit hearts following myocardial ischemia and reperfusion. The optimal concentration of erythropoietin that afforded maximum recovery of developed pressure was manifest at 1.0 U/ml. Erythropoietin (1.0 U/ml) treatment resulted in phosphorylation of PKC, p38 MAP kinase and p42/44 MAP kinase. The cardioprotective effects of erythropoietin were abolished by the protein kinase inhibitors SB203580 (p38 MAP kinase), PD98059 (p42/44 MAP kinase) and chelerythrine (PKC) as well as the potassium channel blockers glibenclamide, HMR 1098, 5-HD and Paxilline. Nitrite and nitrate release from hearts before (2.3 +/- 0.9 nmol/min/g) and after (2.4 +/- 1.9 nmol/min/g) 15 min treatment with erythropoietin (1.0 U/ml) were not different. L-NAME and L-NMA did not block the cardioprotective effect of erythropoietin. We conclude the rapid activation of potassium channels and protein kinases by erythropoietin represents an important new mechanism for increasing cardioprotection.
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PMID:Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels. 2751 2

BACKGROUND: Under pathological conditions, microglia produce proinflammatory mediators which contribute to neurologic damage, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress microglial activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOS2) and the possible role that IL-1beta plays in that response. METHODS: Rat cortical microglia were stimulated with bacterial lipopolysaccharide (LPS) to induce NOS2 expression (assessed by nitrite and nitrate accumulation, NO production, and NOS2 mRNA levels) and IL-1beta release (assessed by ELISA). Effects of NE were examined by co-incubating cells with different concentrations of NE, adrenergic receptor agonists and antagonists, cAMP analogs, and protein kinase (PK) A and adenylate cyclase (AC) inhibitors. Effects on the NFkappaB:IkappaB pathway were examined by using selective a NFkappaB inhibitor and measuring IkappaBalpha protein levels by western blots. A role for IL-1beta in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells. RESULTS: LPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NFkappaB inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of beta2-adrenergic receptors (beta2-ARs), and reduced loss of inhibitory IkBalpha protein. NE effects were replicated by dibutyryl-cyclic AMP. However, co-incubation with either PKA or AC inhibitors did not reverse suppressive effects of NE, but instead reduced nitrite production. A role for IL-1beta was suggested since NE potently blocked microglial IL-1beta production. However, incubation with a caspase-1 inhibitor, which reduced IL-1beta levels, had no effect on NO production; incubation with IL-receptor antagonist had biphasic effects on nitrite production; and NE inhibited nitrite production in caspase-1 deficient microglia. CONCLUSIONS: NE reduces microglial NOS2 expression and IL-1beta production, however IL-1beta does not play a critical role in NOS2 induction nor in mediating NE suppressive effects. Changes in magnitude or kinetics of cAMP may modulate NOS2 induction as well as suppression by NE. These results suggest that dysregulation of the central cathecolaminergic system may contribute to detrimental inflammatory responses and brain damage in neurological disease or trauma.
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PMID:Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1beta production. 1528 93

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN- and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations >1 microM. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.
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PMID:Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tetranitrate with other organic nitrates. 1533 69

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.
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PMID:Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas. 1566 51

S-Nitrosating nitric oxide (NO) donors like S-nitrosoglutathione (GSNO) induce a persistent inhibition of vascular tone, through the formation of releasable NO stores. In this study, we investigate whether GSNO also induces NO stores-related effects in vessels exhibiting tolerance to glyceryl-trinitrate. Rat aortic rings treated with glyceryl-trinitrate (100 microM for 1 h) exhibited increased level of superoxide and a decrease in NO elevation and relaxation induced by subsequent addition of glyceryl-trinitrate. In glyceryl-trinitrate-treated rings as in controls, pre-exposure to GSNO (1 microM for 30 min) induced a persistent hyporesponsiveness to noradrenaline and a relaxant response to N-acetylcysteine (a low molecular weight thiol which can displace NO from NO stores), both of which being inhibited by guanylyl-cyclase or cyclic GMP-dependent protein kinase inhibitors. These data indicate that GSNO can promote the formation of releasable NO stores in arteries exhibiting increased superoxide level and tolerance to glyceryl-trinitrate. Formation of releasable NO stores is of potential interest to restore the protective effect of NO in organic nitrate-tolerant blood vessels.
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PMID:Formation of releasable NO stores by S-nitrosoglutathione in arteries exhibiting tolerance to glyceryl-trinitrate. 1587 16


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