Modulation of extracellular glutamate concentration by nitric oxide synthase inhibitor in rat transient forebrain ischemia

The purpose of the present study was to clarify the effect of topical administration of a nitric oxide synthase inhibitor on extracellular glutamate concentration in transient forebrain ischemia. Two microdialysis probes were inserted into the bilateral striata of Wistar rats. N^G-Nitro-l-arginine (l-NNA) with or withoutl-arginine was topically administered into the unilateral striatum through one of the microdialysis probes, while Ringer's solution was perfused into the contralateral striatum as the control, and 14 minutes of forebrain ischemia was applied. The extracellular glutamate concentration during ischemia and subsequent reperfusion was statistically significantly higher on the 100 μMl-NNA-perfused side than on the control side, but 1 MMl-NNA was ineffective. When 100 μMl-NNA was perfused together with 500 μMl-arginine, the glutamate concentration did not differ from that on the control side. Moreover, administration of 500 μMl-arginine significantly suppressed the glutamate elevation after reperfusion. The fact that the lower dose ofl-NNA increased the accumulation of glutamate during ischemia and reperfusion without altering the blood flow may indicate that nitric oxide affords protection against ischemic neuronal damage. However, since the higher dose ofl-NNA did not affect the glutamate concentration, it appears that the effect of nitric oxide on extracellular glutamate concentration in forebrain ischemia differs, depending on the degree of the inhibition of NOS activity.     

Inhibition of nitric oxide synthase dramatically potentiates seizures induced by kainic acid and pilocarpine in rats

We investigated whether the severity of convulsions evoked by kainic acid and pilocarpine is modified in nitric oxide synthase inhibitor-treated rats. We found that chronic treatment (4 days) withN_w-nitro-l-arginine greatly potentiates seizures induced by both convulsants suggesting a potential role for nitric oxide in mechanisms regulating seizure induction and propagation.     

N-methyl-d-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats

Intracerebral microdialysis technique was utilized to study the effect ofN^G-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor, onN-methyl-d-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mMd-(−)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mMN^G-nitro-l-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA-evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats.     

l-NAME inhibits pentylenetetrazole and strychnine-induced seizures in mice

Nitric oxide (NO) formation has been shown in many neuronal tissues subserves a variety of functions. N-Methyl-d-aspartate (NMDA) receptor stimulation which releases nitric oxide and raises cGMP levels, mediates epileptiform activity induced by various agents. Disinhibition of inhibitory neurotransmitter γ-aminobutyric acid (GABA) and/or activation of NMDA receptor appears to be factors involved in the initiation and generalization of the pentylenetetrazole (PTZ) induced seizures. In the present study, we examined the effects of N^ω-nitro-l-arginine methylester (l-NAME) which inhibits nitric oxide synthase, on PTZ and strychnine induced seizures in mice. l-NAME (100 mg/kg) significantly prolonged the onset time of tonic generalized extension without affecting myoclonic jerks and tonic-clonic convulsions. l-NAME (200 mg/kg) significantly delayed three characteristic behavioral changes including first myoclonic jerk (FMJ), generalized clonic seizure (GCS) and tonic generalized extension (TGE). The effects of l-NAME were reversed by l-arginine (1000 mg/kg). l-NAME (100 and 200 mg/kg) significantly delayed the onset time of strychnine induced TGE. The effects of both doses of l-NAME were reversed by l-arginine. In conclusion, our results demonstrate that NO synthase inhibition suppresses the onset time of PTZ and strychnine induced seizures. Under the light of our current knowledge NO synthase inhibitors seem far away to be considered as a group of antiepileptic drugs. On the other hand there are some strong evidences about the role of NO in central pathophysiological mechanisms. © 1997 Elsevier Science B.V. All rights reserved.     

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

It has previously been suggested that increases of l-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase (P<0.01) in nNOS protein and a concomitant 2.4-fold increase (P<0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.     

Changes in serum prolactin after electroconvulsive and epileptic seizures

Summary Serial serum prolactin (PRL) concentrations were measured after epileptic seizures and seizures following electroconvulsive therapy (ECT). There was a large and rapid rise in PRL after ECT seizures but a much more variable PRL response after spontaneous seizures. Only epileptic seizures of longer duration and of grand mal character resulted in a more substantial rise in PRL. In ECT seizures there was a significant positive correlation between the duration of seizures and the rise in postictal PRL. The postictal PRL curves over 24h were similar in both spontaneous seizures and ECT seizures. Interictally there were no differences in PRL levels between epileptic patients compared to patients with other neurological diseases or healthy volunteers. Chronic treatment with drugs affecting dopamine transmission had a profound effect on PRL secretion, and a dose-dependent significant increase in PRL with neuroleptics was observed.     

A double-blind placebo-controlled trial ofl-threonine in amyotrophic lateral sclerosis

Summary Fifteen patients with the unequivocal diagnosis of amyotrophic lateral sclerosis (ALS) completed a 1-year randomized double-blind placebo-controlled trial ofl-threonine (2g daily). During the study, patients in the placebo group showed a decline in functional status consistent with the natural history of ALS, which was not statistically different from outcome in the patients in thel-threonine group.     

Effects of Central Injection of Kyotorphin and l-Arginine on Oxytocin and Vasopressin Release and Blood Pressure in Conscious Rats

Intracerebroventricular (ICV) administration of an inhibitor of nitric oxide synthase (NOS) increases oxytocin but not vasopressin secretion, in dehydrated rats [38]. Surprisingly, central injection of l-arginine, the substrate for NOS, caused a similar effect. Kyotorphin (l-tyrosyl-l-arginine), a dipeptide formed from l-arginine by kyotorphin synthetase in the brain may mediate this magnocellular response. Therefore, the dose and time responses of hormone release were compared following ICV injection of kyotorphin and l-arginine to conscious rats that were normally hydrated or deprived of water for 24 h. In water-sated rats, both l-arginine and kyotorphin increased blood pressure and plasma glucose levels coincident with elevating circulating levels of oxytocin, but not vasopressin. In dehydrated animals, both l-arginine and kyotorphin increased plasma oxytocin levels with a similar time course but only kyotorphin decreased vasopressin release. d-arginine, like l-arginine, stimulated secretion of oxytocin, indicating a nonstereospecific effect. A kyotorphin receptor antagonist (l-leucyl-l-arginine) given ICV to dehydrated animals elevated plasma oxytocin and prevented the decrease in vasopressin levels after kyotorphin. Thus, kyotorphin, but not l-arginine, appears to attenuate release of vasopressin either directly from magnocellular neurons or indirectly via modulating compensatory reflexes activated by the pressor response. On the other hand, an excess of l-arginine and kyotorphin within the CNS may mimic the stress response by augmenting release of oxytocin and activating the sympathetic nervous system to increase blood pressure and plasma glucose levels.     

Nitric oxide reduces body temperature and sympathetic input to brown adipose tissue during PGE1-hyperthermia

The firing rate of the nerves innervating interscapular brown adipose tissue (IBAT) and IBAT and colonic temperatures (T_IBAT and T_c) were monitored in urethane-anaesthetized male Sprague-Dawley rats. These variables were measured for 40 min before (baseline values) and 40 min after a 4 μmoles l-arginine (l-arg) or 400 nmoles nitroprusside (NP) injection in a lateral cerebral ventricle and an intracerebroventricular administration of 500 ng prostaglandin E₁ (PGE₁). The same variables were monitored in other rats with l-arg or NP or PGE₁ administration alone. No drug was injected in control rats. The results show that l-arg or NP injection reduces the increases in firing rate, T_IBAT, T_c induced by PGE₁ These findings suggest that nitric oxide is important in the control of thermogenic changes during the PGE₁ hyperthermia.     

Nitric oxide and hydroxyl radicals initiate lipid peroxidation by NMDA receptor activation

In this experiment, we used direct electron paramagnetic resonance (EPR) spectra to measure lipid peroxidation by hydroxyl radical (.OH), nitric oxide (.NO) and lipid radical (.L). NMDA-receptor associated lipid peroxidation is thought to act through .OH in induction of neurotoxicity. The origin of .OH generation was found to arise mainly from peroxynitrite anion produced from O(2)(-) and .NO rather than from Fenton's reaction. This study verified that .OH generation from interactive reactions between .NO and O(2)(-) initiates NMDA-induced lipid peroxidation of PC12 cells.     

Spinal neuronal pathology associated with continuous intrathecal infusion ofN-methyl-d-aspartate in the rat

Summary Continuous intrathecal infusion ofN-methyl-d-aspartate (NMDA) at the level of the lumbar enlargement of the spinal cord in middle-aged rats produced dose-dependent toxicity of spinal cord neuronal systems. Toxicity was enhanced by coadministration of glycine, but was significantly reduced when NMDA was coadministered with the competitive inhibitordl-2-amino-5-phosphovaleric acid or the noncompetitive inhibitor MgSO₄. The toxic effects of NMDA were manifest most dramatically and at the lowest concentrations in the neuropil, while neuronal loss was obvious at higher concentrations. The distribution and intensity of reactive astrocytosis was consistent with the known regional and subcellular distribution of NMDA receptors in the spinal cord of rats. The increase in ribosomes and rough endoplasmic reticulum observed in anterior horn cells suggested an increase of cell metabolism reflecting either a nonspecific response to injury or a specific increase in cell metabolism secondary to sustained activation of NMDA receptors. The present studies implicate excitatory amino acid receptors of the NMDA type in producing toxicity to selected neuronal populations of the spinal cord. This model provides a system for studies of the protective effects and rescue of neuronal populations susceptible to the toxic effects of excitatory amino acids.Supported by the ALS Society of Canada     

The nature and time course of neuronal vacuolation induced by the N-methyl-D-aspartate antagonist MK-801

N-Methyl-D-aspartate (NMDA) antagonists cause neuronal vacuolation in the posterior cingulate and retrosplenial cortex of the rat. Because the nature of neuronal pathologic changes due to NMDA antagonists may affect the potential clinical use of this class of drugs, we undertook experiments to define the nature and time course of the vacuolation caused by high-dose (5 mg/kg) MK-801 (dizocilpine, 5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine). Ultrastructural examination revealed the vacuoles to be not a form of hydropic cellular degeneration, but rather a dilatation of several intracellular compartments, chiefly endoplasmic reticulum and mitochondria. Study of the time course of the alterations revealed no light or ultrastructural features of neuronal necrosis in over 1 thousand neurons examined in layers 3 and 4 of the cingulate and retrosplenial cortex, 153 of which were vacuolated. The vacuoles resolved over time by decreasing in magnitude. Oxalate-pyroantimonate methodology revealed no redistribution of cell calcium in either vacuolated or non-vacuolated neurons. At 6 h, when vacuoles were consistently prominent in glutaraldehyde-fixed plastic-embedded tissue, a separate series of experiments was undertaken to vary methods of tissue preparation, and determine conditions under which vacuolation occurs. Frozen sections revealed no vacuoles. Subsequent paraffin embedding of the previously frozen tissue revealed no vacuoles, but vacuoles were seen in paraffin after perfusion fixation. Immersion fixation with brain refrigeration for 12 h prior to fixation revealed no vacuoles. Alcohol fixation also led to no visible vacuoles. We conclude that the vacuolation induced by NMDA antagonists is a reaction to aldehyde fixation of perturbed but living neurons, resulting in artifactual distortion of multiple intracellular compartments.Supported by a grant (MT-9935) from the Medical Research Council of Canada     

Different mechanisms of l-arginine induced dilation of brain arterioles in normotensive and hypertensive rats

We evaluated the response of pial arterioles to l-arginine in anesthetized normotensive rats and spontaneously hypertensive rats equipped with a closed cranial window. Topical application of 10⁻⁶ − 10⁻⁴ mol/l l-arginine, which is known to be the endogenous substrate for the synthesis of nitric oxide, induced dose-dependent arteriolar vasodilation. The response was more pronounced in hypertensive than in normotensive rats (at the concentration of 10⁻⁴ mol/l l-arginine: 18.3 ± 3.3% vs. 6.7 ± 1.7%, respectively, means ± S.E.). The stereoisomer d-arginine had no effect in hypertensive rats. Topical application of the nitric oxide synthase inhibitor N-nitro-l-arginine converted l-arginine-induced dilation to constriction in normotensive and hypertensive rats. The cyclooxygenase inhibitor indomethacin (5 μg/ml cerebrospinal fluid) also blocked the dilation in both strains. Photochemical endothelial injury blocked l-arginine-induced dilation in normotensive rats, but only partly antagonized the response in hypertensive animals. Intravenous or topical pretreatment with the free radical scavenger superoxide dismutase significantly reduced the dilating response to 10⁻⁴ mol/l l-arginine in hypertensive rats. Superoxide dismutase did not significantly change the response to l-arginine in normotensive animals. It is concluded that nitric oxid formation in the endothelium and liberation of cyclooxygenase products cause l-arginine-induced dilation in normotensive rats. While nitric oxide and cyclooxygenase products are also involved in l-arginine-induced dilation in spontaneously hypertensive rats, superoxide radicals contribute to the enhanced response in this strain. This mechanism appears to be specific for the hypertensive animals and is only partly dependent on an intact endothelium.     

Parkinson's tremor,relief by an antiaminic drug (BC 105)

Summary Tremor is among the parkinsonian symptoms one of the most difficult to control. Even the treatment by l-DOPA does not give satisfactory results. According to recent biochemical hypothesis, a loss of functional balance of cerebral serotonin and histamine seems to be responsible for the symptom. The results obtained by the administration of antihistaminic, antiserotoninic, antiaminic and acetylcholinic drugs were quantitatively compared by using a piezoelectric accelerometer. The antihistaminic drug is found to be very active but unfavourable side effects are present, while the antiaminic drug is not less active, with the advantage of being free of such effects.

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Zusammenfassung Der Tremor ist unter den Parkinson-Symptomen mit am schwierigsten zu kontrollieren. Selbst die Behandlung mit l-DOPA gibt keine zufriedenstellenden Ergebnisse. Neuere biochemische Hypothesen lassen es wahrscheinlich erscheinen, daß eine funktionelle Balance zwischen dem cerebralen Serotonin und Histamin verlorengeht und so das Symptom entsteht. Die Ergebnisse der Behandlung mit Antihistaminen, Antiserotoninen, Antiaminen und Acetylcholin werden quantitativ in ihrer Wirkung verglichen, wobei ein piezoelektrischer Accelerometer benutzt wird. Die antihistaminischen Medikamente sind sehr wirksam, haben aber ungünstige Nebenwirkungen, während die antiamine Medikation weniger wirksam ist, aber dafür frei von allen Nebenwirkungen bleibt.

     

Intraventricular infusion of N-methyl-D-aspartate

This study documents the ultrastructural features of acute neuronal injury following N-methyl-D-aspartate (NMDA) receptor activation. NMDA (100 nmol/microliters) or vehicle was infused over a 15-min period into the lateral ventricle of adult rats. After perfusion fixation, specimens demonstrating normal and abnormal patterns of vascular permeability to horseradish peroxidase were sampled for ultrastructural analysis. In NMDA-infused rats, brain regions exhibiting protein extravasation contained swollen dendritic profiles and abnormal neuronal perikarya. Although periventricular regions were most severely affected, parenchymal abnormalities were also detected in the cerebral cortex, septum, striatum, thalamus, hypothalamus and cerebellum. Mildly affected dendrites contained dark compact mitochondria, while in severely swollen dendrites mitochondria were enlarged with ruptured cristae. Focal sites of plasma membrane disruption were also detected within swollen dendrites. Swollen neurons commonly displayed peripheral pallor and increased numbers of cytoplasmic vacuoles. Other neurons appeared dark and shrunken, some containing disrupted mitochondria and pyknotic nuclei. Pretreatment with the NMDA antagonist MK-801 (2 mg/kg) attenuated the neuronal and dendritic alterations. In conditions where cerebrospinal fluid levels of glutamate are abnormally elevated, excessive NMDA receptor activation may lead to early vascular and neuronal complications which could work in concert to promote brain injury.     

Increased nitric oxide caused by the ketogenic diet reduces the onset time of kainic acid-induced seizures in ICR mice

Although the antiepileptic effects of the ketogenic diet (KD) are well documented, the mechanisms underlying this action remain obscure. Nitric oxide (NO) has long been thought to play a role in regulating seizures. However, the effects of the KD on endogenous NO production have not been characterized. Therefore, the present study was designed to examine the effect of the KD on endogenous NO production, as well as the precise role of NO in kainic acid (KA)-induced seizures, in male ICR mice. We first found that preadministration of the KD for 4 weeks increased endogenous NO generation in the hippocampus. We also demonstrated that the increase in NO induced by the KD resulted from increased neuronal NO synthase (nNOS) activity and exerted an antiepileptic effect on KA-induced seizures, based on the results of experiments using NOS-knockout mice and two NOS inhibitors, N-omega-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). These data suggest that the antiepileptic effects of the KD might be mediated, at least in part, by increased NO levels in the hippocampus.     

On the relationship between l-DOPA therapy and CSF monoamine metabolites in parkinson's disease

Summary HVA, the main dopamine catabolite, was shown to be decreased in CSF of parkinsonian patients. A significant relationship was also shown between clinical improvement and HVA concentration increase in CSF. It was, however, recently claimed that such increase was not specific.A study on the CSF concentrations of HVA and 5-HIAA (the main serotonin metabolite) was undertaken on 10 parkinsonian patients. The determinations were made before and after l-DOPA therapy, when a satisfactory clinical status was achieved. The results obtained clearly demonstrate that a large increase of HVA was found in CSF during l-DOPA therapy, but no relationship was found between HVA levels and clinical improvement. A decrease of 5-HIAA in CSF during l-DOPA treatment was found.These results are discussed in the light of a relationship between dopamine and serotonin during l-DOPA therapy.     

Changes in electroretinogram and serum potassium during l-DOPA treatment in parkinsonism

Summary The relationship of l-DOPA plasma level, parameters of ERG and severity of extrapyramidal symptoms after a single dose of l-DOPA was investigated in 11 patients suffering from parkinsonism of idiopathic or arteriosclerotic origin.After a drug-free night, each patient received his/her usual morning dose of l-DOPA. In the subsequent 3 h, the ERG recordings, blood levels and clinical ratings of extrapyramidal symptoms significantly dropped after a delay of 60 min in relation to the occurrence of the peak plasma l-DOPA level.The initial b wave amplitudes as well as initial serum potassium values were abnormally high. There was a statistically significant correlation between the decrease of b wave amplitude (b) and the potassium normalization index (i.e. the ratio between the observed decrease of serum potassium and the pretreatment difference from the middle normal potassium value).A definite interpretation of the data cannot be provided until more knowledge about the origin of b wave of ERG is available. It can be concluded tentatively that dopaminergic processes influence electrophysiological reactivity of the retina.     

Nitric oxide is involved in sustained and delayed cell death of rat retina following transient ischemia

We have investigated the role of nitric oxide (NO) in the rat retina following ischemic injury induced by transient increase of intraocular pressure. The thickness of both the inner plexiform layer and inner nuclear layer decreased during early postischemic stages (up to 1 week). In late postischemic stages (2-4 weeks), the thickness of the outer nuclear layer (ONL) decreased markedly. Thus, mechanisms other than excitotoxic ones may contribute to postischemic retinal cell death. Treatment of rats with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, significantly reduced ischemic damage. Our findings suggest that NO is involved in the mechanism of ischemic injury, and plays a key role in the delayed and sustained cell death in the ONL following transient retinal ischemia.