Exhaled nitric oxide concentration in patients after heart transplantation.

Nitric oxide (NO) is present in exhaled air in humans and its level may decrease in heart diseases. In the present study we prospectively investigated how heart transplantation treated with oral immunosuppresive drugs based on ciclosporine A influences the exhaled NO concentration (exNO). The study was performed in 17 patients after heart transplantation in various time after procedure and 15 nonsmoking healthy volunteers as a control group. Patients after heart transplantation were free of clinical signs of rejection. End-tidal concentration of exNO was measured by the use of a chemiluminescence method. We found no statistically significant differences in the exNO level between patients after heart transplantation and healthy controls (6.81+/-2.70 part per billion (ppb) in the transplant group vs. 6.01+/-3.43 ppb in the control group). We conclude that heart transplantation and immunosuppresive therapy do not influence the exhaled NO concentration.

Dual effect of DL-homocysteine and S-adenosylhomocysteine on brain synthesis of the glutamate receptor antagonist, kynurenic acid.

Increased serum level of homocysteine, a sulfur-containing amino acid, is considered a risk factor in vascular disorders and in dementias. The effect of homocysteine and metabolically related compounds on brain production of kynurenic acid (KYNA), an endogenous antagonist of glutamate ionotropic receptors, was studied. In rat cortical slices, DL-homocysteine enhanced (0.1-0.5 mM) or inhibited (concentration inducing 50% inhibition [IC50]=6.4 [5.5-7.5] mM) KYNA production. In vivo peripheral application of DL-homocysteine (1.3 mmol/kg intraperitoneally) increased KYNA content (pmol/g tissue) from 8.47 +/- 1.57 to 13.04 +/- 2.86 (P <0.01; 15 min) and 11.4 +/- 1.72 (P <0.01; 60 min) in cortex, and from 4.11 +/- 1.54 to 10.02 +/- 3.08 (P <0.01; 15 min) in rat hippocampus. High concentrations of DL-homocysteine (20 mM) applied via microdialysis probe decreased KYNA levels in rabbit hippocampus; this effect was antagonized partially by an antagonist of group I metabotropic glutamate receptors, LY367385. In vitro, S-adenosylhomocysteine acted similar to but more potently than DL-homocysteine, augmenting KYNA production at 0.03-0.08 mM and reducing it at > or =0.5 mM. The stimulatory effect of S-adenosylhomocysteine was abolished in the presence of the L-kynurenine uptake inhibitors L-leucine and L-phenyloalanine. Neither the N-methyl-D-aspartate (NMDA) antagonist CGS 19755 nor L-glycine influenced DL-homocysteine- and S-adenosylhomocysteine-induced changes of KYNA synthesis in vitro. DL-Homocysteine inhibited the activity of both KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, whereas S-adenosylhomocysteine reduced only the activity of KAT II. L-Methionine and L-cysteine, thiol-containing compounds metabolically related to homocysteine, acted only as weak inhibitors, reducing KYNA production in vitro and inhibiting the activity of KAT II (L-cysteine) or KAT I (L-methionine). The present data suggest that DL-homocysteine biphasically modulates KYNA synthesis. This seems to result from conversion of compound to S-adenosylhomocysteine, also acting dually on KYNA formation, and in part from the direct interaction of homocysteine with metabotropic glutamate receptors and KYNA biosynthetic enzymes. It seems probable that hyperhomocystemia-associated brain dysfunction is mediated partially by changes in brain KYNA level.

beta-Adrenoceptor blockade enhances the anticonvulsant effect of glutamate receptor antagonists against maximal electroshock.

In this study, we evaluated whether beta-adrenoceptor antagonists may modify the protective efficacy of dizocilpine (MK-801), a NMDA receptor antagonist, and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), a non-NMDA (AMPA/kainate) receptor antagonist, against maximal electroshock-induced seizures in mice. Propranolol, acebutolol, metoprolol and atenolol were used in doses that did not alter the electroconvulsive threshold. Propranolol potentiated the anticonvulsant activity of MK-801 and GYKI 52466, significantly lowering their ED(50) values from 0.38 and 15.0 to 0.15 (P<0.001) and 8.4 mg/kg (P<0.001), respectively. Similarly, metoprolol lowered the ED(50) of MK-801 and GYKI 52466 from 0.38 and 15.0 to 0.17 (P<0.05) and 11.2 mg/kg (P<0.05). Acebutolol enhanced the protective action of GYKI 52466, lowering its ED(50) value from 15.0 to 12.2 mg/kg (P<0.05), but not that of MK-801. Atenolol, not penetrating the blood-brain barrier, did not affect the anticonvulsive efficacy of MK-801 and GYKI 52466. In conclusion, beta-adrenoceptor antagonists may act synergistically with excitatory amino acid receptor antagonists to inhibit generalised tonic-clonic seizures.

Protective effect of adenosine receptor agonists in a new model of epilepsy--seizures evoked by mitochondrial toxin, 3-nitropropionic acid, in mice.

The role of adenosine receptor agonists in the convulsant activity of mitochondrial toxin, 3-nitropropionic acid (3-NPA), was studied in mice. The occurrence of seizures evoked by peripheral application of 3-NPA was inhibited with the use of A1 adenosine receptor agonist, R-N6-phenylisopropyladenosine and A1/A2 agonist, 2-chloroadenosine. Moreover, both drugs prevented 3-NPA-induced mortality. Similarly, A1/A2 agonist, 5'-N-ethylcarboxamidoadenosine, protected against seizures evoked by the intracerebral administration of 3-NPA, and this effect was reversed by the co-application of adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline. Obtained results suggest that A1 adenosine receptor activation may modulate the chain of events leading to the development of 3-NPA-induced seizures.

Kynurenine aminotransferase I activity in human placenta.

The aim of the study was to detect and characterize placental kynurenine aminotransferase I (KAT I) activity in physiological pregnancy at term. Placental KAT I was inhibited by l -glutamine, l -tryptophan, and l -phenylalanine and reached optimum activity at pH 9.8. When pyruvate was used as a co-factor, the KAT I activity was significantly higher than the activity of this enzyme in the presence of 2-oxoglutarate. In the light of our findings placental KAT I seems to have biochemical characteristics of KAT I detected in human brain.

Effect of adenosine receptor agonists on neurodegenerative and convulsive activity of mitochondrial toxin, 3-nitropropionic acid.

3-Nitropropionic acid (3-NPA) is a mitochondrial toxin inhibiting the activity of succinate dehydrogenase. Its experimental application in rodents causes lesions of the striatum resembling the course of Huntington's disease in humans. Recently, we have shown that 3-NPA is also a potent convulsive and proconvulsive agent. This study investigated the effects of adenosine receptor agonists on neurodegeneration and convulsions induced by 3-NPA. Adenosinergic agonists prevented seizures but not striatal neuronal loss evoked by 3-NPA, what suggests that different mechanisms might contribute to these pathologies associated with application of mitochondrial toxin.

Previous prolonged clonic seizures diminish antiepileptic activity of valproate against pentetrazol-evoked convulsions.

Prolonged seizures may alter the brain function in numerous ways. It is conceivable that they might lead to modifications of seizure susceptibility or anticonvulsive drug efficacy, however, only limited data address this issue. Therefore, we have decided to estimate the antiepileptic activity of drugs interfering with GABA-ergic neurotransmission in mice subjected to prolonged clonic seizures 2 weeks before, using pentetrazol test. The activity of valproate, but not diazepam or phenobarbital, was diminished in animals following repetitive clonic seizures. It might be hypothesized that in humans suffering from epilepsy, prolonged seizures in the past might contribute to the lowered efficacy of valproic acid later on.

N(G)-nitro-L-arginine and its methyl ester inhibit brain synthesis of kynurenic acid possibly via nitric oxide-independent mechanism.

The effect of nitric oxide synthase (NOS) inhibitors on the brain production of endogenous glutamate receptor antagonist, kynurenic acid, was estimated in vitro. Under standard incubation conditions N(G)-nitro-L-arginine, but not N(G)-nitro-L-arginine methyl ester, up to 5 mM, or 7-nitroindazole, up to 100 microM, inhibited de novo synthesis of kynurenic acid in cortical slices. However, during prolonged incubation, N(G)-nitro-L-arginine methyl ester also reduced the production of kynurenic acid. The substrate for NOS, L-arginine (up to 5 mM), did not influence kynurenic acid synthesis and did not reverse the N(G)-nitro-L-arginine-evoked changes, suggesting that the observed effects are not related to disturbed generation of NO. Enzymatic studies revealed that N(G)-nitro-L-arginine and its methyl ester blocked the activity of brain kynurenine aminotransferase (KAT) I. The activity of KAT II was diminished only by N(G)-nitro-L-arginine. Kinetic analyses have shown that N(G)-nitro-L-arginine and its methyl ester reduce Vmax and increase Km of KAT I, whereas N(G)-nitro-L-arginine diminishes Vmax of KAT II. In conclusion, we report that N(G)-nitro-L-arginine and its methyl ester impair brain synthesis of kynurenic acid, probably via NO-independent mechanism, what could contribute, at least partially, to the enhancement of neurotoxicity or seizures observed in some experimental designs based on their use.

Proconvulsive effects of the mitochondrial respiratory chain inhibitor--3-nitropropionic acid.

The role of impaired mitochondrial function in processes leading to the generation of seizures was studied in mice. An inhibitor of mitochondrial complex III, 3-nitropropionic acid, which is known to evoke convulsions per se, and was used here in subthreshold dose, enhanced seizures generated by electric current and application of 4-aminopyridine. In contrast, 3-nitropropionic acid did not affect convulsions induced by gamma-aminobutyric acid (GABA) receptor antagonists - bicuculline, pentylenetetrazol and picrotoxin, glycine antagonist - strychnine, cholinomimetic drug-pilocarpine, and kynurenine aminotransferase inhibitor - aminooxyacetic acid. It is hypothesised that deranged mitochondrial metabolism renders the central nervous system more susceptible to factors inducing seizures via direct depolarization.

Formation of endogenous glutamatergic receptors antagonist kynurenic acid--differences between cortical and spinal cord slices.

Rat spinal cord slices produced kynurenic acid (KYNA) upon exposure to L-kynurenine. Aminooxyacetic acid, non-selective aminotransferase inhibitor, and L-glutamate, but neither N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-metyloisoxazolo-4-propionate (AMPA), nor kainate, diminished synthesis of KYNA. L-Glutamate action was less potent in spinal than in cortical slices. Metabotropic agonists, L-(+)-2-amino-4-phosphonobutyrate (L-AP4) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), used in concentrations inhibiting cortical KYNA synthesis, were ineffective in spinal cord. Spinal KYNA production seems less susceptible to inhibitory modulation.

Seizures evoked by mitochondrial toxin, 3-nitropropionic acid: new mechanism of epileptogenesis?

A number of data concerning the central action of mitochondrial toxins, substances impairing mitochondrial synthesis of ATP and thus compromising cellular energy status, has emerged within last years. 3-Nitropropionic acid (3-NPA) is an irreversible inhibitor of succinate dehydrogenase and mitochondrial complex II. The experimental administration of 3-NPA may lead to selective neuronal loss and chorea-like behavioral alterations but, as was recently shown, it also evokes clonic convulsions in rodents. The gathered data suggest that disturbed mitochondrial energy metabolism might initiate the chain of events culminating in seizure episode and that 3-NPA might become a useful tool in studying "mitochondrial" seizures. It has been hypothesized that the resistance to standard anticonvulsive therapy occurring among high proportion of epilepsy sufferers may result from the impairment of mitochondrial energy status due to either genetic predispositions or environmental influences.

Anticonvulsant action of chlormethiazole is prevented by subconvulsive amounts of strychnine and aminophylline but not by bicuculline and picrotoxin.

The anticonvulsant action of chlormethiazole was evaluated with the use of subthreshold doses of convulsants affecting the purinergic, glycinergic and gamma-aminobutyric acid (GABA)-mediated transmission, i.e. aminophylline, strychnine, bicuculline and picrotoxin in the model of generalized tonic-clonic convulsions. Chlormethiazole protected mice against maximal electroshock-induced seizures with an ED50 of 130.8 mg/kg. Aminophylline (100 mg/kg) and strychnine (0.4 mg/kg) reversed the protective action of chlormethiazole against electroconvulsions raising the ED50 values of this drug to 218.6 and 208.6 mg/kg, respectively. In contrast, GABA antagonists, bicuculline and picrotoxin, neither affected the protection provided by chlormethiazole nor did they alter the protective activity of valproate, phenobarbital, diphenylhydantoin and carbamazepine against electroconvulsions. Our results indicate that (a) the anticonvulsant activity of chlormethiazole might be related to its interaction with strychnine-sensitive glycinergic as well as purinergic neurotransmission, (b) purinergic and strychnine-sensitive glycinergic events contribute more prominently than GABAergic ones to the anticonvulsant activity of the drugs providing protection against maximal electroshock-induced convulsions.

NMDA- but not kainate-mediated events reduce efficacy of some antiepileptic drugs against generalized tonic-clonic seizures in mice.

PURPOSE: The aim of this study was to evaluate the efficacy of conventional antiepileptic drugs (AEDs) against the generalized tonic-clonic seizures in mice subjected to the subconvulsive doses of N-methyl-D-aspartate (NMDA) or kainate. METHODS: Mice were given NMDA and kainate in the doses of 50.0 and 9.0 mg/kg i.p., respectively [i.e., equal to 75% of their CD16 values (convulsive dose in 16% of the animals studied)]. Subsequently the anticonvulsive potential of conventional AEDs against the maximal electroshock-induced seizures was estimated. Where necessary, the plasma levels of AEDs were assessed. RESULTS: NMDA or kainate application did not affect the electroconvulsive threshold. NMDA, but not kainate, diminished the antiepileptic activity of diazepam (DZP) and carbamazepine (CBZ), increasing their 50% effective doses (ED50s) from 14.1 and 8.6 to 19.0 and 12.1 mg/kg i.p., respectively. Neither NMDA nor kainate affected the ED50 for valproate (VPA), phenobarbital (PB), or diphenylhydantoin (DPH) against electroconvulsions. NMDA-evoked effects were reversed with the use of the NMDA antagonist, D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116) and were not accompanied by the alterations in the free plasma levels of AEDs. CONCLUSIONS: The NMDA-mediated events, but not kainate-related ones, seem to be involved in the protective action of DZP and CBZ against maximal electroshock-induced seizures. Moreover, it might be concluded that when subthreshold activation of NMDA receptors adds to other epileptogenic factors, DZP and CBZ are less efficacious. Presented data indicate that in such situations, adding the NMDA receptor antagonist (at very low doses) to the AED may yield beneficial therapeutic effects.

AMPA/kainate-related mechanisms contribute to convulsant and proconvulsant effects of 3-nitropropionic acid.

The role of the glutamatergic system in the convulsant and proconvulsant action of a mitochondrial toxin, 3-nitropropionic acid, was studied in mice. The occurrence of 3-nitropropionic acid-induced seizures was inhibited by the alpha-amino-2,3-dihydro-5-methyl-3-oxo-isoxazole-propionate (AMPA)/kainate receptor antagonists, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione disodium (NBQX) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HCI (GYKI 52466), with ED50 of 14.1 (7.9-25.2) and 7.2 (5.3-9.6) mg/kg, respectively. The N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and 3-(2-carboxypiperazine-4-yl)propenyl-1-phosphonic acid (CPPene), were ineffective. Moreover, 3-nitropropionic acid given in a subthreshold dose potently enhanced seizures generated by intracerebroventricular administration of AMPA and kainate, lowering their CD50 from 0.98 (0.83-1.17) and 0.73 (0.64-0.83) to 0.55 (0.45-0.66) (P<0.001) and 0.58 (0.51-0.65) (P<0.05) nmol, respectively. In contrast, NMDA action was not changed by 3-nitropropionic acid application. We conclude that AMPA/kainate-mediated events are involved in proconvulsive and convulsive effects of 3-nitropropionic acid.

Mitochondrial toxin 3-nitropropionic acid evokes seizures in mice.

3-Nitropropionic acid, a potent inhibitor of succinate dehydrogenase which thus compromises cellular energy metabolism, evoked convulsions in mice in a dose-dependent manner. CD50 for clonic seizures was 158.5 (144.1-174.3) mg/kg. Tonic seizures were not observed. Broad-spectrum anticonvulsants, namely diazepam, phenobarbital and valproate, prevented the occurrence of 3-nitropropionic acid-induced seizures with ED50 of 4.9 (3.1-7.6), 33.1 (17.9-61.0) and 389.7 (351.2-432.3) mg/kg, respectively. Diphenylhydantoin-like drugs (diphenylhydantoin, and carbamazepine), anti-absence drugs (trimethadione and ethosuximide) and acetazolamide were ineffective. The characteristics of 3-nitropropionic acid-induced seizures resembled those of convulsions evoked by another mitochondrial toxin, aminooxyacetic acid.

Chlormethiazole anticonvulsive efficacy diminished by N-methyl-D-aspartate but not kainate in mice.

The aim of this study was to evaluate the effect of N-methyl-D-aspartate (NMDA) and kainate used at nonconvulsive doses upon protective efficacy of chlormethiazole against maximal electroshock-induced seizures. NMDA (50 mg/kg, i.p.) reduced the anticonvulsant potency of chlormethiazole increasing its ED50 value from 126.9 to 155.0 mg/kg. The effect of NMDA was completely reversed by the competitive NMDA receptor antagonist D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116) (0.06 mg/kg i.p.). Kainic acid (9 mg/kg i.p.) did not affect the anticonvulsive properties of chlormethiazole. Our results suggest that NMDA but not kainate receptor-mediated events participate in the anticonvulsant action of chlormethiazole.

Acute ammonia treatment in vitro and in vivo inhibits the synthesis of a neuroprotectant kynurenic acid in rat cerebral cortical slices.

The synthesis of kynurenic acid (KYNA) from kynurenine was measured in the cerebral cortical slices. In vitro, ammonium acetate at the subtoxic to toxic concentration range from 1 mM to 10 mM dose-dependently inhibited KYNA synthesis (IC50=2.99 mM). Ammonia treatment in vivo decreased KYNA synthesis by 30%. These results suggest that impaired neuroprotection exerted by KYNA might be a potential contributor to the glutamate receptor-mediated aspect of acute ammonia neurotoxicity.