Nox2 oxidase activity accounts for the oxidative stress and vasomotor dysfunction in mouse cerebral arteries following ischemic stroke

Background and Purpose

Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase.

Methods

Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2^-/-) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N ^ω-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting.

Results

Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2^-/- mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2^-/- mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2^-/- mice. Vascular CD45 levels were unchanged by ischemia-reperfusion.

Conclusions

Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.     

Exogenous nitric oxide negatively regulates c-Jun N-terminal kinase activation via inhibiting endogenous NO-induced S-nitrosylation during cerebral ischemia and reperfusion in rat hippocampus

Nitric oxide (NO), synthesized from l -arginine by NO synthases, is a small endogenous free radical with multiple functions. The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in cerebral ischemia and reperfusion. In this study, we found that the NO donor sodium nitroprusside (SNP) can decrease the damage of hippocampal neurons induced by cerebral ischemia and reperfusion. Our current study demonstrates that SNP can suppress the phosphorylation of JNK3 by suppressing the increased S-nitrosylation of JNK3 induced by cerebral ischemia and reperfusion. In contrast, dithiothreitol reversed the effect of SNP on S-nitrosylation of JNK3. Furthermore, the inhibitor of nNOS (7-NI) and the inhibitor of iNOS (AMT) can decrease JNK3 phosphorylation through decreasing S-nitrosylation of JNK3. Our data suggest that endogenous NO synthesized by NO synthases can increase JNK3 phosphorylation by means of S-nitrosylation during global ischemia/reperfusion in rat hippocampus. However, the exogenous NO (SNP) can reverse the effect of endogenous NO by inhibiting S-nitrosylation of JNK3. Together, these results suggest that the exogenous NO may provide a new clue for stroke therapy.     

Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition

Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a powerful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg i.v. each) 24 h before 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size was significantly reduced in DETA/NO-treated mice (30 +/- 2% of risk region in treated mice vs. 50 +/- 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg i.v.), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 +/- 1 vs. 30 +/- 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca(2+)-induced swelling by 20 micromol/l CaCl(2) or by the higher concentration of 200 micromol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 micromol/l CaCl(2), thereby confirming that Ca(2+) induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca(2+)-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator.     

Effect of sodium nitroprusside on hemoglobin oxygen binding properties of the blood during hepatic ischemia-reperfusion in rabbits

The parameters of blood oxygen transport were determined in rabbits during the hepatic ischemia/reperfusion (HIR) with or without sodium nitroprusside (SNP) administration. Hepatic ischemia was induced for 30 min by a. hepatica propria clamping, reperfusion lasted 120 min. Indices of blood oxygen transport (hemoglobin-oxygen affinity index (p50), pO2, pH, pCO2, HCO3-, TCO2, ABE, etc.) and nitrite/nitrate (NO(x)) amounts were measured in blood during HIR. Animals were subdivided into two groups: 1st group--HIR; 2nd--HIR plus SNP infusion (SNP, Sigma, i.v. 10 mcmol/kg). The experiments had shown that HIR led to significant acidic changes in the acid-base balance and high blood p50. The SNP infusion in the 2nd group led to less changing in the p50 values during HIR which were accompanied with high NO(x) levels. We conclude that oxyhemoglobin dissociation curve shift leftwards after SNP administration promotes the maintenance liver during ischemia-reperfusion.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning

Nitric oxide (NO) donors mimic the early phase of ischemic preconditioning (IPC). The effects of nitroxyl (HNO/NO(-)), the one-electron reduction product of NO, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated whether HNO/NO(-), produced by decomposition of Angeli's salt (AS; Na(2)N(2)O(3)), has a cardioprotective effect in isolated perfused rat hearts. Effects were examined after intracoronary perfusion (19 min) of either AS (1 microM), the NO donor diethylamine/NO (DEA/NO, 0.5 microM), vehicle (100 nM NaOH) or buffer, followed by global ischemia (30 min) and reperfusion (30 min or 120 min in a subset of hearts). IPC was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each intervention was assessed by changes in myocardial contractility as well as lactate dehydrogenase (LDH) release and infarct size. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly improved with IPC and pre-exposure to AS, as opposed to control or DEA/NO-treated hearts. Infarct size and LDH release were also significantly reduced in IPC and AS groups, whereas DEA/NO was less effective in limiting necrosis. Co-infusion in the triggering phase of AS and the nitroxyl scavenger, N-acetyl-L-cysteine (4 mM) completely reversed the beneficial effects of AS, both at 30 and 120 min reperfusion. Our data show that HNO/NO(-) affords myocardial protection to a degree similar to IPC and greater than NO, suggesting that reactive nitrogen oxide species are not only necessary but also sufficient to trigger myocardial protection against reperfusion through species-dependent, pro-oxidative, and/or nitrosative stress-related mechanisms.     

Nitric oxide mediates protein kinase C isoform translocation in rat heart during postischemic reperfusion

It is controversial whether nitric oxide (NO) is protective or deleterious against ischemia-reperfusion injury. We examined the effect of NO on PKC isoform translocation and protection against ischemia-reperfusion injury in perfused heart. An NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester, 3.0 microM), administered only during reperfusion but not during ischemia, inhibited the translocation of PKC-alpha, -delta and -epsilon isoforms to the nucleus-myofibril fraction and the translocation of PKC-alpha to the membrane fraction after ischemia (20 min) and reperfusion (10 min) in the perfused rat heart. NO donors, 3-morpholinosydnonimine (SIN-1) or S-nitroso-N-acetylpenicillamine (SNAP) activated purified PKC in vitro. SIN-1 also induced PKC isoform translocation in perfused heart. On the other hand, PKC selective inhibitor, calphostin C (0.2 microM) or chelerythrine (1.0 microM), aggravated the contractile dysfunction of ischemic heart during reperfusion, when they were perfused during reperfusion. These data suggest that NO generated during reperfusion following ischemia activates PKC isoforms and may protect the heart against contractile dysfunction in the perfused rat heart.     

Augmentation of Nitric Oxide,Superoxide, and Peroxynitrite Production During Cerebral Ischemia and Reperfusion in the Rat

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, N-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.     

Cardioprotective effect induced by brief exposure to nitric oxide before myocardial ischemia-reperfusion in vivo.

Administration of nitric oxide (NO) donors during ischemia and reperfusion protects from myocardial injury. However, whether administration of an NO donor during a brief period prior to ischemia protects the myocardium and the endothelium against ischemia-reperfusion injury in vivo is unknown. To study this possibility anesthetized pigs were subjected to 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4h of reperfusion. In initial dose-finding experiments, vehicle or three different doses of the NO donor S-nitroso-N-acetyl-D,L-penicillamin (SNAP; 0.1; 0.5; 2.5 micromol) were infused into the LAD for 3 min starting 13 min during ischemia. Only the 0.5 micromol dose of SNAP reduced infarct size (from 85+/-3% of the area at risk in the vehicle group to 63+/-3% in the SNAP-treated group; p<0.01). There were no significant differences in hemodynamics in the vehicle and SNAP groups during ischemia-reperfusion. Endothelium-dependent dilatation of coronary microvasculature induced by substance P was larger in the SNAP group than in the vehicle group. Myeloperoxidase activity was lower in the ischemic/reperfused myocardial area of pigs given SNAP (4.97+/-0.61 U/g) than in vehicle-treated pigs (8.45+/-0.25 U/g; p<0.05). It is concluded that intracoronary administration of the NO donor SNAP for a brief period before ischemia reduces infarct size, attenuates neutrophil accumulation, and improves endothelial function. These results suggest that NO exerts a classic preconditioning-like protection against ischemia-reperfusion injury in vivo in a narrow concentration range.     

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [¹⁴C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A₂ activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PEpl ethanolamine plasmalogen - PI phosphatidylinositol - PS phosphatidylserine - poly-PI polyphosphoinsoitide - DG diacylglycerol - TG triacylglycerol - FFA free fatty acids - PUFA polyunsaturated fatty acids - MCA middle cerebral artery - CCAs common carotid arteries - HPTLC high performance thin layer chromatography - GLC gas-liquid chromatography - PLA₂ phospholipase A₂ Special issue dedicated to Dr. Leon S. Wolfe.     

Nitric oxide: a trigger for classic preconditioning?

To determine whether nitric oxide (NO) is involved in classic preconditioning (PC), the effect of NO donors as well as inhibition of the L-arginine-NO-cGMP pathway were evaluated on 1) the functional recovery during reperfusion of ischemic rat hearts and 2) cyclic nucleotides during both the PC protocol and sustained ischemia. Tissue cyclic nucleotides were manipulated with NO donors [S-nitroso-N-penicillamine (SNAP), sodium nitroprusside (SNP), or L-arginine] and inhibitors of nitric oxide synthase (N(omega)-nitro-L-arginine methyl ester or N-nitro-L-arginine) or guanylyl cyclase (1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one). Pharmacological elevation in tissue cGMP levels by SNAP or SNP before sustained ischemia elicited functional improvement during reperfusion comparable to that by PC. Administration of inhibitors before and during the PC protocol partially attenuated functional recovery, whereas they had no effect when given after the ischemic PC protocol and before sustained ischemia only, indicating a role for NO as a trigger but not as a mediator. Ischemic PC, SNAP, or SNP caused a significant increase in cGMP and a reduction in cAMP levels after 25 min of sustained ischemia that may contribute to the protection obtained. The results obtained suggest a role for NO (and cGMP) as a trigger in classic PC.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O₂ tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O₂-limited decrease stage; (2) O₂-limited steady stage; (3) neuronal NOS activation stage; (4) acute O₂-recovery elevation stage; and (5) O₂-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.     

Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke.

The efficacy of nitric oxide (NO) treatment in ischemic stroke, though well recognized, is yet to be tested in clinic. NO donors used to treat ischemic injury are structurally diverse compounds. We have shown that treatment of S-nitrosoglutathione (GSNO) protects the brain against injury and inflammation in rats after experimental stroke [M. Khan, B. Sekhon, S. Giri, M. Jatana, A. G. Gilg, K. Ayasolla, C. Elango, A. K. Singh, I. Singh, S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke, J. Cereb. Blood Flow Metab. 25 (2005) 177-192.]. In this study, we tested structurally different NO donors including GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), methylamine hexamethylene methylamine NONOate (MAHMA), propylamine propylamine NONOate (PAPA), 3-morpholinosydnonimine (SIN-1) and compared their neuroprotective efficacy and antioxidant property in rats after ischemia/reperfusion (I/R). GSNO, in addition to neuroprotection, decreased nitrotyrosine formation and lipid peroxidation in blood and increased the ratio of reduced versus oxidized glutathione (GSH/GSSG) in brain as compared to untreated animals. GSNO also prevented the I/R-induced increase in mRNA expression of ICAM-1 and E-Selectin. SNAP and SNP extended limited neuroprotection, reduced nitrotyrosine formation in blood and blocked increase in mRNA expression of ICAM-1 and E-Selectin in brain tissue. PAPA, MAHMA, and SIN-1 neither protected the brain nor reduced oxidative stress. We conclude that neuroprotective action of NO donors in experimental stroke depends on their ability to reduce oxidative stress both in brain and blood.     

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 2009^{1, 2}. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area³. In the early 1990s, endothelin-1 (ET-1) ⁴ was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified ⁵ by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans^5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume⁸. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used⁶. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion^{9, 10}. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.     

Effects of estrous synchronization on response to nitric oxide donors, nitric oxide synthase inhibitors, and endothelin-1 in vitro

Two experiments were conducted to determine the effects of nitric oxide (NO) donors, endothelin-(ET-1), and NO synthase (NOS) inhibitors on bovine luteal function in vitro. In experiment 1, estrus in Brahman cows was synchronized with Synchro-Mate-B (SMB) and day-13-14 corpora luteal slices were weighed, diced and incubated in vitro. Treatments (100 ng/ml) were: vehicle, N[see symbol in text]-nitro-L-arginine-L-methyl ester (L-NAME), N(G)-monomethyl-L-arginine acetate (L-NMMA), diethylenetriamine (DETA), DETA-NONOate, sodium nitroprusside (SNP), or ET-1. In experiment 2, estrus was synchronized with Lutalyse, a Controlled Intravaginal Progesterone Releasing Device (CIDR), or cows were not synchronized. Corpora lutea were collected, weighed, and luteal slices were weighed, diced and incubated in vitro with treatments. Treatments (100ng/ml) were: vehicle, L- NAME, L-NMMA, DETA, DETA-NONOate, sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or endothelin-1. Tissues were incubated in M- 199 for 1 h without treatments and for 4 and 8 h in both experiments with treatments in both experiments. Media were analyzed for progesterone, prostaglandins E2 and F2alpha (PGE2, PGF2alpha) by radioimmunoassay (RIA). Hormone data in experiments 1 and 2 were analyzed by 2 x 7 and 3 x 2 x 8 factorial design for analysis of variance (ANOVA), respectively. Luteal weights in experiment 2 were analyzed by a one-way ANOVA. Concentrations of progesterone in media were similar (P > or = 0.05) among treatments within experiments. Concentrations of PGE2 in media in experiment 1 were undetectable in 90 and 57% of the samples at 4 and 8 h, respectively. PGF2alpha increased (P < or = 0.05) with time, but did not differ (P > or = 0.05) among treatments. Secretion of PGF2alpha was not affected by treatments (P > or = 0.05). In experiment 2, luteal weights of the induced estrous cycle were decreased (P < or = 0.05) by Lutalyse. Concentrations of PGE2 and PGF2alpha increased (P < or = 0.05) with time in control of all three synchronization regimens. DETA-NONOate, SNAP, sodium nitroprusside (NO donors) and ET-1 increased (P < or = 0.05) PGE2 except in the CIDR synchronized group (P > or = 0.05). No treatment increased (P > or = 0.05) PGF2alpha in any synchronization regimen. It is concluded that either SMB containing norgestomet or a CIDR containing progesterone alters luteal secretion of PGE2, Lutalyse lowers luteal weights in the induced estrous cycle, and NO or ET-1 given alone are not luteolytic agents. It is suggested that NO and ET-1 could have indirect antiluteolytic/luteotropic effects via increasing PGE2 secretion by luteal tissue rather than being luteolytic.     

盐酸戊乙奎醚（长托宁）抑制脑缺血／再灌注时谷氨酸的释放及受体机制研究

目的：观察盐酸戊乙奎醚对全脑缺血／再灌注大鼠易损区海马谷氨酸（Glu）及其受体（NMDAR1）的影响,探讨盐酸戊乙奎醚脑保护作用机制。方法：雄性Wistar大鼠60只,随机分为3组（n=20）：假手术组（A组）;缺血再灌注对照组（B组）;盐酸戊乙奎醚干预组,采用Pulsinelli-Brierley四血管阻断法制备全脑缺血模型,实验分两部分进行,各组随机选择10只大鼠于全脑缺血15min再灌注1h、3h、6h,采用脑微透析技术结合高效液相色谱（HPLC）检测大鼠海马细胞外Glu水平的变化,其余10只大鼠于再灌注3h后灌流固定断头取脑,采用免疫组织化学方法,检测海马CA1区NMDAR1蛋白表达。结果：与缺血／再灌注组各对应时点相比较,盐酸戊乙奎醚干预组大鼠海马细胞外Glu含量明显降低,统计结果差异均有显著性（P〈0．05或P〈0．01）;海马CA1区NMDAR1表达明显受抑制,积分光密度、阳性细胞面积、平均灰度值均存在显著性差异（P〈0．05或P〈0．01）。结论：脑缺血／再灌注早期应用盐酸戊乙奎醚不仅减少兴奋性氨基酸释放,还能抑制NMDAR1的高表达而产生脑保护作用。     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.