Inhibition of nitric oxide synthesis aggravates reperfusion injury after hepatic ischemia and endotoxemia

The potential role of nitric oxide (NO) was investigated in the pathophysiology of liver injury after priming with 20 min hepatic ischemia-reperfusion and administration of .5 mg/kg Salmonella enteritidis endotoxin. Liver injury during the early reperfusion phase of 4 h was characterized by severe vascular oxidant stress, lipid peroxidation (LPO), neutrophil infiltration, and a 33% reduction of the microvascular blood flow in the liver. Inhibition of NO synthesis with N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) aggravated liver injury by 90%, reduced LPO, and did not affect liver neutrophils but further impaired microvascular blood flow. Treatment with the NO-donor spermine-NONOate or L-arginine did not affect these parameters in postischemic animals, however, treatment did restore all values of L-NAME-treated animals back to disease control levels. These data suggest that endogenous NO formation is sufficient to limit ischemic liver injury during reperfusion but inhibition of NO synthesis will result in additional ischemic damage. NO may also be involved in scavenging of superoxide in the vasculature and in inducing LPO.     

NMDA receptor-mediated refinement of a transient retinotectal projection during development requires nitric oxide

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

Lack of nitric oxide contributes to vasospasm during ischemia/reperfusion injury

Vasospasm can be a complication after free tissue transfer and replant operations. Recent studies suggest that vasospasm may be due to endothelium dysfunction, resulting in impairment of nitric oxide production. The present experiment was designed to investigate acute responses of the microcirculation of skeletal muscle to local interarterial infusion of sodium nitroprusside (a direct donor of nitric oxide and thus an endothelium-independent vasodilator) or acetylcholine chloride (which stimulates endothelium release of endogenous nitric oxide) during reperfusion after 4 hours of warm ischemia. Male Sprague-Dawley rats, each weighing 100 to 120 gm, were anesthetized with sodium pentobarbitone and were surgically prepared with vascular isolated and denervated cremaster muscles that were subjected to 4 hours warm ischemia and 2 hours of reperfusion. Sodium nitroprusside (10(-3) M), acetylcholine chloride (10(-4) M), or normal saline (eight rats for each group) were administered by local infusion (0.1 ml/hour) through the femoral artery into the natural blood flow of the cremaster. The arterial tree in the cremaster was observed and arteriole diameters (A1-A4) were measured using intravital microscopy. The number of arteriole branches having temporary stoppage of flow were counted in each cremaster. The results from this study show that local infusion of sodium nitroprusside, but not acetylcholine chloride, prevents ischemia/reperfusion vasoconstriction in A3 and A4 arterioles and thus improves microvascular blood flow. Generalized vasoconstriction caused by topically applied norepinephrine (10(-6) M) to sham ischemia cremasters could be completely reversed by the local infusion of 10(-4) M acetylcholine chloride. These results indicate that vasospasm after ischemia/reperfusion may be related to temporary endothelial cell dysfunction, resulting in the inability to produce sufficient nitric oxide during early reperfusion. Vascular smooth muscle, however, is responsive to locally administered sodium nitroprusside infusion (which is thought to provide exogenous nitric oxide).     

Stimulation of beta2-adrenoceptors inhibits apoptosis in rat brain after transient forebrain ischemia

We have previously demonstrated that the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo was most likely mediated by an increased nerve growth factor (NGF) expression. In the present study, we examined whether clenbuterol was capable of inhibiting apoptosis caused by ischemia. Transient forebrain ischemia was performed in male Wistar rats (300 to 350 g) by clamping both common carotid arteries and reducing the blood pressure to 40 mm Hg for 10 minutes. Clenbuterol (0.1, 0.5, and 1.0 mg/kg intraperitoneally) was administered 3 hours before ischemia or immediately after ischemia. The brains were removed for histologic evaluation 7 days after ischemia. The time course of DNA fragmentation was determined 1, 2, 3 and 4 days after ischemia. Staining with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was used for further analysis of DNA fragments in situ 3 days after ischemia. The NGF protein was assayed by enzyme-linked immunosorbent assay. Ten-minute forebrain ischemia damaged 80% to 90% of the neurons in the hippocampal CA1 region evaluated 7 days after ischemia. Pretreatment with clenbuterol (0.5 and 1.0 mg/kg) reduced the neuronal damage by 18.1% (P < 0.01) and 13.1% (P < 0.05), respectively. The neuroprotective effect also was found when clenbuterol (0.5 mg/kg) was administered immediately after ischemia (P < 0.05). The DNA laddering appeared in striatum 1 day and in hippocampus 2 days after ischemia and peaked on the third day in both regions. The DNA laddering was nearly abolished in the hippocampus and partially blocked in striatum and cortex by 0.5 mg/kg clenbuterol. These results were confirmed by TUNEL staining. Clenbuterol (0.5 mg/kg intraperitoneally) elevated the NGF protein level by 33% (P < 0.05) in the hippocampus and 41% (P < 0.05) in the cortex 6 hours after ischemia. Three days after ischemia, the NGF levels in these regions were no longer different between the clenbuterol-treated and control groups. This study clearly demonstrates that clenbuterol possesses a neuroprotective activity and a marked capacity to inhibit DNA degradation after global ischemia. The results suggest that clenbuterol increases NGF expression during the first hours after global ischemia and thereby protects neurons against apoptotic damage.     

Brainstem auditory evoked potentials during brainstem ischemia and reperfusion in gerbils

To evaluate the reversibility of neural function in the brainstem following ischemia, we investigated the effect of transient brainstem ischemia on the brainstem auditory evoked potential in gerbils. Brainstem ischemia was produced by bilateral extracranial occlusion of vertebral arteries. Local cerebral blood flow was measured by quantitative autoradiography after 5 min of ischemia and was reduced to less than 3 ml/100 g per min in the pons and lower midbrain, indicating severe and reproducible brainstem ischemia. During brainstem ischemia, brainstem auditory evoked potential waveforms disappeared completely. After a brief ischemic insult (5 min), all four brainstem auditory evoked potential components recovered to normal. After longer ischemic insults (10-30 min), brainstem auditory evoked potential components never recovered to normal. Microtubule-associated protein 2 immunoreactivity revealed differential vulnerability of the acoustic relay nuclei in the brainstem. Neurons in the lateral lemniscus were most vulnerable, followed in order by neurons in the trapezoid body, the superior olive and the cochlear nucleus. We also demonstrated a close relationship between the reversibility of ischemia-induced changes on brainstem auditory evoked potential and ischemic lesions of these relay nuclei. These data may be useful for evaluating the therapeutic window of thrombolytic therapy during acute vertebrobasilar occlusion.     

Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats

During reperfusion after ischemia, deleterious biochemical processes can be triggered that may antagonize the beneficial effects of reperfusion. Research into the understanding and treatment of reperfusion injury (RI) is an important objective in the new era of reperfusion therapy for stroke. To investigate RI, permanent and reversible unilateral middle cerebral artery/common carotid artery (MCA/CCA) occlusion (monitored by laser Doppler) of variable duration in Long-Evans (LE) and spontaneously hypertensive (SH) rats and unilateral MCA and bilateral CCA occlusion in selected LE rats was induced. In LE rats, infarct volume after 24 hours of permanent unilateral MCA/CCA occlusion was 31.1 +/- 34.6 mm3 and was only 28% of the infarct volume after 120 to 300 minutes of reversible occlusion plus 24 hours of reperfusion, indicating that 72% of the damage of ischemia/reperfusion is produced by RI. When reversible ischemia was prolonged to 480 and 1080 minutes, infarct volume was 39.6 mm3 and 16.6 mm3, respectively, being indistinguishable from the damage produced by permanent ischemia and significantly smaller than damage after 120 to 300 minutes of ischemia. Reperfusion injury was not seen in SH rats or with bilateral CCA occlusion in LE rats, in which perfusion is reduced more profoundly. Reperfusion injury was ameliorated by the protein synthesis inhibitor cycloheximide or spin-trap agent N-tert-butyl-alpha-phenylnitrone pretreatment.     

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.     

Blockade of tetrahydrobiopterin synthesis protects neurons after transient forebrain ischemia in rat: a novel role for the cofactor

The generation of nitric oxide (NO) aggravates neuronal injury. (6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) is an essential cofactor in the synthesis of NO by nitric oxide synthase (NOS). We attempted to attenuate neuron degeneration by blocking the synthesis of the cofactor BH4 using N-acetyl-3-O-methyldopamine (NAMDA). In vitro data demonstrate that NAMDA inhibited GTP cyclohydrolase I, the rate-limiting enzyme for BH4 biosynthesis, and reduced nitrite accumulation, an oxidative metabolite of NO, without directly inhibiting NOS activity. Animals exposed to transient forebrain ischemia and treated with NAMDA demonstrated marked reductions in ischemia-induced BH4 levels, NADPH-diaphorase activity, and caspase-3 gene expression in the CA1 hippocampus. Moreover, delayed neuronal injury in the CA1 hippocampal region was significantly attenuated by NAMDA. For the first time, these data demonstrate that a cofactor, BH4, plays a significant role in the generation of ischemic neuronal death, and that blockade of BH4 biosynthesis may provide novel strategies for neuroprotection.     

Effect of endogenous nitric oxide on energy metabolism of rat heart mitochondria during ischemia and reperfusion

The effects of ischemia and postischemic reperfusion on the functions of the heart and its mitochondria were studied with special attention to the effect of nitric oxide (NO) by treatment of rat hearts with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or its noninhibitory isomer N(G)-nitro-D-arginine methyl ester (D-NAME). NO generated during reperfusion caused increase in coronary flow (CF), but had no effect on the left ventricular pressure (LVP) or heart rate (HR). The ATP level of the heart decreased during ischemia and was not completely restored by introduction of oxygen during reperfusion due to damage of complexes I and II of the respiratory chain of mitochondria by NO. Inhibition of the respiratory chain resulted in generation of hydrogen peroxide, and NO and NO-derived species generated after production of NO caused further damage of various proteins in mitochondria, such as complexes I and II of the respiratory chain and pyruvate dehydrogenase (PDH). These results suggested that NO generated on reperfusion was the primary cause of mitochondrial dysfunction by damage of complexes I and II of the respiratory chain, with consequent increase of CF in the heart.     

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Late-onset lipid peroxidation and neuronal cell death following transient forebrain ischemia in rat brain

The receptor encoded by the W (c-kit) locus is expressed on the membrane of mouse primordial germ cells, whereas its ligand termed stem cell factor (SCF), encoded by the Sl locus, is expressed on the membrane of somatic cells associated with both the primordial germ cell migratory pathways and homing sites. Using an in vitro short time assay which allows a quantitative measure of adhesion between cells, in the present paper we show that SCF/c-kit interaction can modulate primordial germ cell adhesion to somatic cells. We report that the adhesiveness of 11.5 dpc primordial germ cells to four types of somatic cells in culture (TM4 cells, STO fibroblasts, bone marrow stromal cells and gonadal somatic cells) is significantly reduced by antibodies directed against c-kit receptor or SCF, as well by soluble SCF. This SCF/c-kit mediated adhesion seems independent of SCF-induced tyrosine autophosphorylation of c-kit receptor. Moreover, primordial germ cells showed a poor ability to adhere to a bone marrow stromal cell line carrying the Sl(d) mutation (unable to synthesize membrane-bound SCF). This adhesiveness was not further impaired by anti-c-kit antibody. These results demonstrate that SCF/c-kit interaction contributes to the adhesion of primordial germ cells to somatic cells in culture and suggest that the role played by SCF in promoting survival, proliferation and migration of these cells in vitro and in vivo, demonstrated by several studies, might depend on the ability of the membrane-bound form of this cytokine to directly mediate primordial germ cell adhesion to the surrounding somatic cells.     

Sleep and nitric oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.     

Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat

The immunosuppressant drug cyclosporin A (CsA) is considered to be inherently protective in conditions of ischemia, e.g. in hepatic and cardiac tissue. However, investigations of effects of CsA on neuronal tissue have been contradictory, probably because the blood-brain barrier (BBB) is virtually impermeable to CsA. In the present study, we exploited the finding that the insertion of a syringe needle into brain parenchyma obviously disrupts the BBB and allows influx of CsA, and explored whether CsA, given as intraperitoneal injections daily for 1 week before and 1 week after forebrain ischemia of 7 or 10 min duration, ameliorates the damage incurred to the hippocampal CA 1 sector. In other experiments, the needle insertion and the first i.p. injection of CsA were made 30 min after the start of recirculation, with continued daily administration of CsA during the postinsult week. In animals which were injected with CsA in daily doses of 10 mg kg-1, but in which no needle was inserted, the drug failed to ameliorate CA1 damage, whether the ischemia had a duration of 7 or 10 min. Likewise, needle insertion had no effect on CA1 damage if CsA was not administered. In contrast, when CsA was given to animals with a needle insertion, CA1 damage was dramatically ameliorated, whether treatment was initiated 1 week before ischemia, or 30 min after the start of recirculation. The effect of CsA seemed larger than that of any other drug proposed to have an anti-ischemic effect in forebrain/global ischemia. Injection of tritiated CsA in one animal with BBB disruption lead to detectable radioactivity throughout the ventricular system, suggesting a generalised increase of the entry of CsA across the BBB. The results demonstrate that immunosuppressants of the type represented by CsA markedly ameliorate delayed neuronal damage after transient forebrain ischemia, provided that they can pass the BBB. It is discussed whether the effect of the drug is one involving calcineurin, a protein phosphatase, or if CsA counteracts a permeability transition of the inner mitochondrial membrane, assumed to occur in response to adverse conditions, e.g. gradual accumulation of Ca2+ in the mitochondria in the postischemic period.     

Alterations in [3H]L-N(G)-nitroarginine binding in brain after transient global or transient focal ischemia in gerbils and rats

Adverse physiological effects can often interfere with the use of nitric oxide (NO) as a therapeutic agent, especially when it is systemically generated from prodrugs. NO which is generated and delivered site-specifically by transdermal donors may be useful in the treatment of parasitic, bacterial or viral skin infections without causing systemic side effects. Three diazeniumdiolates (formerly "NONOate"), including two water soluble compounds, (Z)-1-[N-2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1- ium-1,2-diolate (DETA-NO) and (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate (DPTA-NO), and one insoluble compound, DPTA-NO grafted to dextran microspheres (DPTA-NO-g-dextran) were used to transdermally deliver NO to rats. Dextran microspheres were obtained by simultaneously grafting DPTA-NO to dextran and cross-linking dextran with CNBr in an oil-water emulsion. Suspended in hydrogel, DETA-NO, DPTA-NO, and DPTA-NO-g-dextran were applied three times to depilated rats at 4 day intervals. Results show that metabolic urinary nitrate levels increase with time (24-48 h), reach a maximum, and return to baseline by the fourth day. DPTA-NO applications produced an average maximum nitrate level of 94.2 mumol/day +/- 34.2 mumol S.D. compared to the average maximum nitrate level of 39.8 mumol/day +/- 8.6 mumol S.D. obtained from treatment with DETA-NO. These results suggest that DPTA-NO delivered NO more efficiently than DETA-NO. When DPTA-NO-g-dextran microspheres were used as the NO donor, results comparable to DPTA-NO were observed with an average maximum nitrate level of 14.9 mumol/day +/- 3.0 mumol S.D. These and other conclusive data indicate that, via these diazeniumdiolates, NO can be delivered effectively through rat skin.     

Azulenyl nitrones: colorimetric detection of oxyradical end products and neuroprotection in the gerbil transient forebrain ischemia/reperfusion model

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.     

Involvement of nitric oxide and cGMP in the protective effect of neurotensin on hepatocytes

STUDY OBJECTIVE: Tetanus antibody levels have been shown to be inadequate in 50% of patients older than 65 years. Although immunization recommendations have been made for this age group, the efficacy of this intervention has not been well documented. We sought to determine the difference in tetanus antibody levels after the administration of one tetanus toxoid immunization to geriatric patients without adequate titers. METHODS: Thirty-five patients older than 65 years at a large urban comprehensive care geriatric center who were documented to have inadequate tetanus antibody titers were each given one tetanus toxoid immunization. Repeat titers were obtained at least 2 months after the immunization with the use of enzyme-linked immunosorbent assay (Bindazyme kit; the Binding Site Corporation, Birmingham, England). We considered tetanus antibody levels greater than .17 IU/mL protective. RESULTS: The mean age was 79.4 years; 30 of 35 (86%) were female. Repeat tetanus antibody titers were obtained an average of 123 days (range, 63 to 204 days) after immunization with tetanus toxoid. The mean preimmunization antibody titer was .1 IU/mL (range, .04 to .16 IU/mL). After immunization, antibody titers increased a mean of .61 IU/mL (range, -.01 to 2.23 IU/mL; 95% confidence interval, .35 to .87 IU/mL). Thirty of the 35 patients who received a single injection of tetanus toxoid (86%) developed protective titers. We found no relationship between seroconversion and age, sex, or medical history; nor did we find a relationship between antibody level and time elapsed since immunization when repeat titers were obtained. CONCLUSION: Administration of one tetanus toxoid injection affords protective immunity in many geriatric patients.     

Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.     

Interaction of nitric oxide and endothelin-1 in ischemia/reperfusion injury of rat heart

Although several studies have demonstrated that nitric oxide appears to be cardioprotective and endothelin-1 (ET-1) deleterious in myocardial ischemia/reperfusion injury, their interactions in the intact heart are unknown. Therefore, coronary effluent and interstitial fluid ("transudate") levels of ET-1 and cyclic GMP, an indirect measure of nitric oxide production, were determined simultaneously in normoxic and reperfused hearts and compared with myocardial and coronary function. Rat hearts were buffer-perfused at 9 ml/min/g heart wet weight for 45 min (baseline), followed either by another 45 min of perfusion (normoxia), or 15 min of total global ischemia and 30 min reperfusion. Hearts received, from 42-90 min, either vehicle, the inhibitor of nitric oxide formation NG-nitro-L-arginine (L-NNA; 200 micromol/l), the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 micromol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Both mediators were released preferentially into the vascular lumen which resulted in similar luminal and interstitial concentrations of cyclic GMP, but three-fold higher levels of ET-1 in tissue because of the higher effluent than transudate flow rate. L-NNA increased the release of ET-1 and worsened coronary function, whereas SNAP had opposite effects. On reperfusion, considerable functional impairment was observed, although levels of cyclic GMP both in the vascular and tissue compartment were not reduced, but even increased. Reperfusion functional impairment was aggravated after inhibiting the synthesis of nitric oxide, whereas SNAP restored cardiac and coronary function close to pre-ischemic level. Deterioration of function corresponded with an increased level, and improvement with a decreased level of intersitial ET-1 at the onset of reperfusion. PD 142893 was similarly cardioprotective as SNAP both in normoxia and reperfusion. These results suggest that in reperfusion, cardiac function is depressed, despite increased rather than decreased endogenous nitric oxide production, largely due to the prevalence of the deleterious effects of ET-1 which are overcome by antagonism of ET receptors or exogenous nitric oxide supplied by SNAP.