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1.
Nitric oxide (NO) and hydrogen peroxide (H 2O 2) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H 2O 2 (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H 2O 2 alone. In both cell types, the addition of the NO donor increased H 2O 2 steady-state levels, although with different kinetics: in hepatocytes, the increase in H 2O 2 levels was already evident at early time points while in liver endothelial cells it became evident after 2 h of incubation. NO exposure inhibited H 2O 2 degradation, assessed after addition of 50 µM, 200 µM, or 4 mM authentic H 2O 2, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H 2O 2 degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NO x). Early inhibition of H 2O 2 degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H 2O 2 is due to inhibition of H 2O 2 degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NO x (prevailing in endothelial cells). nitrogen monoxide; catalase; glutathione peroxidase 相似文献
2.
H 2O 2-mediated elevation inendothelial solute permeability is associated with pathological eventssuch as ischemia-reperfusion and inflammation. To understand howH 2O 2 mediates increased permeability, weinvestigated the effects of H 2O 2 administrationon vascular endothelial barrier properties and tight junctionorganization and function. We report that H 2O 2exposure caused an increase in endothelial solute permeability in atime-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H 2O 2 exposurecaused the tight junctional protein occludin to be rearranged fromendothelial cell-cell junctions. Occludin rearrangement involvedredistribution of occludin on the cell surface and dissociation ofoccludin from ZO-1. Occludin also was heavily phosphorylated onserine residues upon H 2O 2 administration. H 2O 2 mediates changes in ERK1/ERK2phosphorylation, increases endothelial solute permeability, and altersoccludin localization and phosphorylation were all blocked by PD-98059,a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest thatH 2O 2-mediated increased endothelial solutepermeability involves the loss of endothelial tight junction integritythrough increased ERK1/ERK2 activation. 相似文献
3.
We correlated theadenine nucleotide (AN) levels and energy charge (EC) at the end of atransient oxidative exposure to the delayed death of neuronal cells.When wild-type (WT) or Bcl-2-overexpressing (BCL-2) human neuroblastomacells (Paju) were exposed to 250 µM H 2O 2for 60 min, the EC of WT cells was unchanged, but that of BCL-2 cellsdecreased from 0.91 ± 0.03 to 0.67 ± 0.02. Depletion of ANs wassignificantly greater in BCL-2 (66.7 ± 2%) than in WT (38.8 ± 2%) cells. Proliferation of both lines decreased, averaging 63 ± 17% of control by 48 h. Exposure to 5 mMH 2O 2caused no further change in ANs in BCL-2 cells but in WT cellsdecreased the EC to 0.45 ± 0.08 and depleted ANs to 41 ± 9% ofcontrol; after 24 h, WT cells became pyknotic and showed DNAfragmentation but no chromatin condensation, whereas BCL-2 cells diedby delayed necrosis. After 10 mMH 2O 2,EC dropped to 0.15 ± 0.1, and both lines were acutely killed. TheEC after an oxidative insult correlated well with further growth ofboth cell lines. A significant decline in EC led to delayed death.Bcl-2 did not protect against the fall in EC or AN depletion, but,although survival was not improved, the mechanism of death appeared tobe different. 相似文献
4.
Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O 2uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18, P < 0.05) and to 1.2 ± 0.1 µM O 2 · min 1 · gtissue 1 (10 µM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H 2O 2) inthe effluent ( r = 0.99, P < 0.01). NO markedlydecreased the O 2 uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O 2uptake and H 2O 2 release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O 2uptake and superoxide anion production (reflected byH 2O 2), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation. 相似文献
5.
We previously reportedthat exposure of endothelial cells to H 2O 2results in a loss of cell-cell apposition and increased endothelialsolute permeability. The purpose of this study was to determine howtyrosine phosphorylation and tyrosine phosphatases contribute tooxidant-mediated disorganization of endothelial cell junctions. Wefound that H 2O 2 caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues.H 2O 2 exposure also results in increasedendothelial monolayer permeability, which was attenuated by pp60, aninhibitor of src kinase. Inhibition of protein tyrosinephosphatase activity by phenylarsine oxide (PAO) demonstrated a similarpermeability profile compared with H 2O 2,suggesting that tyrosine phosphatase activity is important inmaintaining a normal endothelial solute barrier. Immunofluorescence shows that H 2O 2 exposure caused a loss ofpan-reactive cadherin and -catenin from cell junctions that was notblocked by the src kinase inhibitor PP1.H 2O 2 also caused -catenin to dissociate fromthe endothelial cytoskeleton, which was not prevented by PP1. Finally,we determined that PP1 did not prevent cadherin internalization. Thesedata suggest that oxidants like H 2O 2 produce biological effects through protein phosphotyrosine modifications bydecreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability. 相似文献
6.
The effect of oxidants onK +-Cl cotransport (KCC) wasinvestigated in equine red blood cells. Carbon monoxidemimicked O 2. The substituted benzaldehyde, 12C79 (5 mM),markedly increased O 2 affinity. In N 2, however,O 2 saturation was low (<10%) but KCC remained active.Nitrite (NO 2) oxidized heme to methemoglobin (metHb).High concentrations of NO 2 (1 and 5 mM vs. 0.5 mM)increased KCC activity above control levels; it became O 2independent but remained sensitive to other stimuli.1-Chloro-2,4-dinitrobenzene (1-3 mM) depleted reduced glutathione(GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O 2-independent KCCactivity; short exposures and low concentrations (30 min, 0.5 or 1 mM)did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over thelevel found in oxygenated control cells required both accumulation ofmetHb and depletion of GSH. Findings are relevant to understanding thephysiology and pathology of regulation of KCC. 相似文献
7.
We examined theeffect of low concentrations of H 2O 2 on theCa 2+-release channel/ryanodine receptor (RyR) to determineif H 2O 2 plays a physiological role in skeletalmuscle function. Sarcoplasmic reticulum vesicles from frog skeletalmuscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle wereincorporated into lipid bilayers. Channel activity of the frog RyR wasnot affected by application of 4.4 mM (0.02%) ethanol. Openprobability ( Po) of such ethanol-treated RyRchannels was markedly increased on subsequent addition of 10 µMH 2O 2. Increase of H 2O 2to 100 µM caused a further increase in channel activity. Applicationof 4.4 mM ethanol to 10 µM H 2O 2-treated RyRsactivated channel activity. Exposure to 10 or 100 µMH 2O 2 alone, however, failed to increase Po. Synergistic action of ethanol andH 2O 2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12).H 2O 2 at 100-500 µM had no effect onpurified channel activity. Application of FKBP12 to the purified RyR1drastically decreased channel activity but did not alter the effects ofethanol and H 2O 2. These results suggest thatH 2O 2 may play a pathophysiological, butprobably not a physiological, role by directly acting on skeletalmuscle RyRs in the presence of ethanol. 相似文献
8.
Reactive oxygenspecies contribute to diaphragm dysfunction in certainpathophysiological conditions (i.e., sepsis and fatigue). However, the precise alterations induced by reactive oxygen species orthe specific species that are responsible for the derangements inskeletal muscle function are incompletely understood. In this study, weevaluated the effect of the superoxide anion radical (O 2·), hydroxyl radical (·OH), and hydrogenperoxide (H 2O 2) on maximum calcium-activatedforce (F max) and calcium sensitivity of the contractileapparatus in chemically skinned (Triton X-100) single rat diaphragmfibers. O 2· was generated using thexanthine/xanthine oxidase system; ·OH was generated using 1 mMFeCl 2, 1 mM ascorbate, and 1 mMH 2O 2; and H 2O 2 wasadded directly to the bathing medium. Exposure to O 2· or ·OH significantly decreasedF max by 14.5% ( P < 0.05) and 43.9%( P < 0.005), respectively. ·OH had no effect onCa 2+ sensitivity. Neither 10 nor 1,000 µMH 2O 2 significantly altered F max orCa 2+ sensitivity. We conclude that the diaphragm issusceptible to alterations induced by a direct effect of ·OH andO 2·, but not H 2O 2, on thecontractile proteins, which could, in part, be responsible forprolonged depression in contractility associated with respiratorymuscle dysfunction in certain pathophysiological conditions. 相似文献
9.
The mechanism underlying H 2O 2-inducedactivation of frog skeletal muscle ryanodine receptors was studiedusing skinned fibers and by measuring single Ca 2+-releasechannel current. Exposure of skinned fibers to 3-10 mM H 2O 2 elicited spontaneous contractures.H 2O 2 at 1 mM potentiated caffeine contracture.When the Ca 2+-release channels were incorporated into lipidbilayers, open probability ( Po) and open timeconstants were increased on intraluminal addition ofH 2O 2 in the presence of cis catalase,but unitary conductance and reversal potential were not affected.Exposure to cis H 2O 2 at 1.5 mM failedto activate the channel in the presence of trans catalase.Application of 1.5 mM H 2O 2 to the transside of a channel that had been oxidized by cisp-chloromercuriphenylsulfonic acid (pCMPS; 50 µM) still led to anincrease in Po, comparable to that elicited by trans 1.5 mM H 2O 2 without pCMPS.Addition of cis pCMPS to channels that had been treated with orwithout trans H 2O 2 rapidly resulted inhigh Po followed by closure of the channel. Theseresults suggest that oxidation of luminal sulfhydryls in theCa 2+-release channel may contribute toH 2O 2-induced channel activation and musclecontracture. 相似文献
10.
Bursts in reactive oxygen species productionare important mediators of contractile dysfunction duringischemia-reperfusion injury. Cellular mechanisms that mediatereactive oxygen species-induced changes in cardiac myocyte functionhave not been fully characterized. In the present study,H 2O 2 (50 µM) decreased contractility of adultrat ventricular myocytes. H 2O 2 caused aconcentration- and time-dependent activation of extracellularsignal-regulated kinases 1 and 2 (ERK1/2), p38, and c-JunNH 2-terminal kinase (JNK) mitogen-activated protein (MAP)kinases in adult rat ventricular myocytes. H 2O 2 (50 µM) caused transient activation of ERK1/2 and p38 MAP kinase thatwas detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned tobaseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. Theprotein kinase C inhibitor chelerythrine completely blocked JNKactivation and reduced ERK1/2 and p38 activation. The tyrosine kinaseinhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38,activation. H 2O 2-inducedNa +/H + exchanger phosphorylation was blocked bythe MAP kinase kinase inhibitor U-0126 (5 µM). These resultsdemonstrate that H 2O 2-induced activation of MAPkinases may contribute to cardiac myocyte dysfunction duringischemia-reperfusion. 相似文献
11.
The effects of nitric oxide (NO) produced by cardiac inducibleNO synthase (iNOS) on myocardial injury after oxidative stress wereexamined. Interleukin-1 induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,L-arginine enhanced NOproduction in a concentration-dependent manner. Glutathione peroxidase(GPX) activity in myocytes was attenuated by elevated iNOS activity andby an NO donor, S-nitroso- N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition ofH 2O 2(0.1 mM, 1 h). Inhibition of iNOS withN -nitro-L-argininemethyl ester ameliorated the effects of NO-enhancing treatments onmyocardial injury and GPX activity. SNAP augmented the myocardialinjury induced byH 2O 2.Inhibition of GPX activity with antisense oligodeoxyribonucleotide forGPX mRNA increased myocardial injury byH 2O 2.Results suggest that the induction of cardiac iNOS promotes myocardialinjury due to oxidative stress via inactivation of the intrinsicantioxidant enzyme, GPX. 相似文献
12.
Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H 2O 2) toxicity and protect cells against H 2O 2 toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H 2O 2 toxicity in cultured liver endothelial cells over a wide range of NO and H 2O 2 concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H 2O 2 was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H 2O 2-induced cell death. SpNO concentrations >0.1 mM were injurious with low H 2O 2 concentrations, but protective at high H 2O 2 concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H 2O 2 steady-state levels in line with inhibition of H 2O 2 degradation. Thus, the overall effect of NO on H 2O 2 toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H 2O 2 levels and enhancement being predominant with high NO and low H 2O 2 levels. 相似文献
13.
We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to 125I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10 ?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial 125I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial 125I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H 2O 2), did not alter the transendothelial 125I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H 2O 2 generated during PMN activation, did not alter the increase in transendothelial 125I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial 125I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H 2O 2, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc. 相似文献
14.
Mesophyll cells of Vicia faba contain kaempferol and quercetinglycosides. When isolated mesophyll cells were treated with0.1 mM H 2O 2 for 2 h, the levels of these flavonols increasedby 1070% of the control values (mean values, 19.6% and34.4% for kaempferol and quercetin glycosides, respectively).Such increases in levels of flavonols were also observed inisolated vacuoles of mesophyll cells. However, when mesophyllcells and vacuoles were treated with 10 mM H 2O 2)degradationof flavonols was observed. These data suggest that H 2O 2 hastwo effects on the metabolism of flavonols: induction of theirsynthesis and stimulation of their oxidation. (Received March 6, 1989; Accepted July 10, 1989) 相似文献
15.
We examined the effects ofH 2O 2on Cl secretion acrosshuman colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, ameasure of Cl secretion,was inhibited in a concentration-dependent fashion when monolayers werepretreated withH 2O 2for 30 min (30-100% inhibition between 500 µM and 5 mM).Moreover,H 2O 2inhibited 76% of the Cl current across monolayers when the basolateral membranes were permeabilized with nystatin (200 µg/ml). When the apical membrane waspermeabilized with amphotericin B,H 2O 2inhibited the Na + current (ameasure ofNa +-K +-ATPaseactivity) by 68% but increased theK + current more than threefold. Inaddition to its effects on ion transport pathways,H 2O 2also decreased intracellular ATP levels by 43%. We conclude that theprincipal effect ofH 2O 2on colonic Cl secretion isinhibitory. This may be due to a decrease in ATP levels followingH 2O 2treatment, which subsequently results in an inhibition of the apicalmembrane Cl conductance andbasolateral membraneNa +-K +-ATPaseactivity. Alternatively,H 2O 2may alter Cl secretion bydirect action on the transporters or alterations in signal transductionpathways. 相似文献
16.
Salinity impairs plant growth and development, thereby leading to low yield and inferior quality of crops. Nitric oxide (NO) has emerged as an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. In this study, tomato seedlings of Lycopersicum esculentum L. “Micro-Tom” treated with 150 mM sodium chloride (NaCl) conducted decreased plant height, total root length, and leaf area by 25.43%, 24.87%, and 33.67%, respectively. While nitrosoglutathione (GSNO) pretreatment ameliorated salt toxicity in a dose-dependent manner and 10 µM GSNO exhibited the most significant mitigation effect. It increased the plant height, total root length, and leaf area of tomato seedlings, which was 31.44%, 20.56%, and 51.21% higher than NaCl treatment alone, respectively. However, NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) treatment reversed the positive effect of NO under salt stress, implying that NO is essential for the enhancement of salt tolerance. Additionally, NaCl?+?GSNO treatment effectively decreased O2? production and H2O2 content, increased the levels of soluble sugar, glycinebetaine, proline, and chlorophyll, and enhanced the activities of antioxidant enzymes and the content of antioxidants in tomato seedlings in comparison with NaCl treatment, whereas NaCl?+?cPTIO treatment significantly reversed the effect of NO under salt stress. Moreover, we found that GSNO treatment increased endogenous NO content, S-nitrosoglutathione reductase (GSNOR) activity, GSNOR expression and total S-nitrosylated level, and decreased S-nitrosothiol (SNO) content under salt stress, implicating that S-nitrosylation might be involved in NO-enhanced salt tolerance in tomatoes. Altogether, these results suggest that NO confers salt tolerance in tomato seedlings probably by the promotion of photosynthesis and osmotic balance, the enhancement of antioxidant capability and the increase of protein S-nitrosylation levels. 相似文献
17.
The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal
(MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated
for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased
significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the
GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate
reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show
significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly
II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the
H 2O 2 and lipid peroxidation levels. Exogenous NO pre-treatment of the seedlings had little influence on the non-enzymatic and
enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment
had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as
the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results
suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing
their antioxidant defense and MG detoxification systems. 相似文献
18.
Inwardlyrectifying K + current( IKir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, IKir randown completely in <10 min [half time( t1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained IKir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used, IKir ran down by~71%. Mg 2+ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg 2+( t1/2 = 1.8 min). IKir also randown when the pipette solution contained 4 mMMg 2+ + 4 mM5'-adenylylimidodiphosphate( t1/2 = 2.7 min)or 4 mM adenosine 5'- O-(3-thiotriphosphate)( t1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity of IKirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis. 相似文献
19.
We studied the influence ofnitric oxide (NO) endogenously produced by adipocytes in lipolysisregulation. Diphenyliodonium (DPI), a nitric oxide synthase (NOS)inhibitor, was found to completely suppress NO synthesis in intactadipocytes and was thus used in lipolysis experiments. DPI was found todecrease both basal and dibutyryl cAMP (DBcAMP)-stimulatedlipolysis. Inhibition of DBcAMP-stimulated lipolysis by DPI wasprevented by S-nitroso- N-acetyl-penicillamine (SNAP), a NO donor. This antilipolytic effect of DPI was also preventedby two antioxidants, ascorbate or diethyldithiocarbamic acid (DDC).Preincubation of isolated adipocytes with DPI (30 min) before exposureto DBcAMP almost completely abolished the stimulated lipolysis.Addition of SNAP or antioxidant during DPI preincubation restored thelipolytic response to DBcAMP, whereas no preventive effects wereobserved when these compounds were added simultaneously to DBcAMP.Exposure of isolated adipocytes to an extracellular generating systemof oxygen species (xanthine/xanthine oxidase) or toH 2O 2 also resulted in an inhibition of thelipolytic response to DBcAMP. H 2O 2 or DPIdecreased cAMP-dependent protein kinase (PKA) activation. The DPIeffect on PKA activity was prevented by SNAP, ascorbate, or DDC. Theseresults provide clear evidence that 1) the DPI antilipolyticeffect is related to adipocyte NOS inhibition leading to PKAalterations, and 2) endogenous NO is required for the cAMPlipolytic process through antioxidant-related effect. 相似文献
20.
In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability ( Kf), effects that are overcome using an amphipathic quinone (CoQ 1) [ Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψ m) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O 2 consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ 1 was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O 2 consumption (1.5±0.1 nmol/min/mg protein), Δψ m (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ 1 on rotenone-induced changes in ATP, O 2 consumption rate, Δψ m, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [ Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ 1 were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM. 相似文献
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