Marian Wielosz

l-Cysteine sulphinate, endogenous sulphur-containing amino acid, inhibits rat brain kynurenic acid production via selective interference with kynurenine aminotransferase II

In the present study the effect of endogenous sulphur-containing amino acids, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate and L-homocysteate, on the production of glutamate receptor antagonist, kynurenic acid (KYNA), was evaluated. The experiments comprised the measurements of (a). KYNA synthesis in rat cortical slices and (b). the activity of KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs). All studied compounds reduced KYNA production and inhibited the activity of KAT I and/or KAT II, thus acting most probably intracellularly. L-Cysteine sulphinate in very low, micromolar concentrations selectively affected the activity of KAT II, the enzyme catalyzing approximately 75% of KYNA synthesis in the brain. L-Cysteine sulphinate potency was higher than other studied sulphur-containing amino acids, than L-aspartate, L-glutamate, or any other known KAT II inhibitor. Thus, L-cysteine sulphinate might act as a modulator of KYNA formation in the brain.

Bupropion-induced convulsions: Preclinical evaluation of antiepileptic drugs

Bupropion, a unique, non-nicotine smoking cessation aid and an effective antidepressant, is well known to produce seizures following overdosing in humans. However, the experimental background for the usefulness of antiepileptic drugs in the protection against bupropion-induced convulsions has not been established yet. Therefore, we tested if the antiepileptic drugs were able to protect mice against clonic convulsions induced by intraperitoneally (i.p.) administered bupropion in the CD97 dose (139.5 mg/kg). Among 13 tested drugs, clonazepam showed the greatest potency (dose-dependent full protection; ED50 = 0.06 mg/kg, i.p.). No signs of locomotor impairment were observed in the rotarod test after anticonvulsive doses of clonazepam, resulting in a broad therapeutic window and favorable protective index (PI) (33.3). Gabapentin produced dose-dependent protection against convulsions at nontoxic doses (up to 1000 mg/kg), having PI>29. Diazepam in a very high dose showed full protection but its PI (1.7) was much less favorable than that of clonazepam. The PI values for ethosuximide, phenobarbital and valproate were slightly higher than unity and lower than 2, and for topiramate and felbamate were lower than unity. Phenytoin, carbamazepine, and lamotrigine as well as tiagabine failed to block the convulsant effects of bupropion even at doses that caused severe motor impairment. Our results encourage clinical testing of clonazepam against seizures developing after bupropion overdose.

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 ≥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.

Endothelium-dependent production and liberation of kynurenic acid by rat aortic rings exposed to l-kynurenine

Rat aortic slices produced and liberated the endogenous antagonist of glutamate receptors, kynurenic acid, upon exposure to L-kynurenine. Endothelium-denuded slices did not synthesize any measurable amount of kynurenic acid, indicating its endothelial origin. Aortic kynurenic acid production was diminished by modification of the ionic milieu, hypoxia and hypoglycemia, as well as by L-glutamate and L-aspartate, endogenous glutamate receptor agonists, and aminooxyacetic acid, a non-selective inhibitor of aminotransferases and mitochondrial respiration. These data pave the way for future research aimed to clarify the role of kynurenic acid in the physiology and pathology of the endothelium and vasculature.

Propranolol and metoprolol enhance the anticonvulsant action of valproate and diazepam against maximal electroshock

The anticonvulsive potential of classical antiepileptics co-administered with beta-adrenergic receptor antagonists against generalized tonic-clonic seizures was evaluated in the model of maximal electroshock (MES)-induced convulsions. Propranolol, acebutolol, metoprolol and atenolol were tested in the doses not affecting the electroconvulsive threshold. Propranolol and metoprolol lowered the ED(50) of valproate and diazepam. Acebutolol reduced valproates but not diazepams ED(50) value. In contrast, hydrophilic atenolol, not penetrating via blood-brain barrier, affected neither the action of valproate nor diazepam. None of the studied drugs changed the protective activity of carbamazepine and phenytoin against MES. beta-blockers per se did not alter the motor performance of mice. Moreover, propranolol and metoprolol did not influence diazepam-evoked impairment of locomotor activity. The free plasma and brain levels of antiepileptic drugs were not affected by beta-blockers. In conclusion, the use of certain beta-adrenoceptor antagonists, such as propranolol and metoprolol, might improve the antiepileptic potential of valproate and diazepam.

Β-adrenergic enhancement of brain kynurenic acid production mediated via cAMP-related protein kinase A signaling

The central levels of endogenous tryptophan metabolite kynurenic acid (KYNA), an antagonist of N-methyl-d-aspartate (NMDA) and alpha7-nicotinic receptors, affect glutamatergic and dopaminergic neurotransmission. Here, we demonstrate that selective agonists of beta(1)-receptors (xamoterol and denopamine), beta(2)-receptors (formoterol and albuterol), alpha- and beta-receptors (epinephrine), 8pCPT-cAMP and 8-Br-cAMP (analogues of cAMP) increase the production of KYNA in rat brain cortical slices and in mixed glial cultures. Neither betaxolol, beta(1)-adrenergic antagonist, nor timolol, a non-selective beta(1,2)-adrenergic antagonist has influenced synthesis of KYNA in both paradigms. In contrast, KT5720, a selective inhibitor of protein kinase A (PKA), strongly reduced KYNA formation in cortical slices (2-10 microM) and in glial cultures (100 nM). beta-adrenergic antagonists and KT5720 prevented the beta-adrenoceptor agonists-induced increases of KYNA synthesis. In vivo, beta-adrenergic agonist clenbuterol (0.1-1.0 mg/kg) increased the cortical endogenous level of KYNA; the effect was blocked with propranolol (10 mg/kg). beta-adrenoceptors agonists, cAMP analogues and KT5720 did not affect directly the activity of KAT I or KAT II measured in partially purified cortical homogenate. In contrast, the exposure of intact cultured glial cells to pCPT-cAMP, 8-Br-cAMP and formoterol has lead to an enhanced action of KATs. These findings demonstrate that beta-adrenoceptor-mediated enhancement of KYNA production is a cAMP- and PKA-dependent event. PKA activity appears to be an essential signal affecting KYNA formation. Described here novel mechanism regulating KYNA availability may be of a potential importance, considering that various stimuli, among them clinically used drugs, activate cAMP/PKA pathway, and thus could counteract the central deficits of KYNA.

Adenosine A1 receptors and the anticonvulsant potential of drugs effective in the model of 3-nitropropionic acid-induced seizures in mice.

The role of adenosine A1 receptors in the activity of drugs and substances protecting against seizures evoked by mitochondrial toxin, 3-nitropropionic acid (3-NPA) was studied in mice. Non-selective A1/A2 adenosine receptor antagonist, aminophylline and selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) diminished the anticonvulsive effects of diazepam, phenobarbital, valproate and gabapentin. In contrast, A1/A2 adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (8pSPT) not penetrating via blood-brain barrier was ineffective. Aminophylline and DPCPX but not 8pSPT also reversed the protective action of A1/A2 adenosine receptor agonist, 2-chloroadenosine (2-CADO) and selective A1 adenosine receptor agonist, R-N6-phenylisopropyloadenosine (R-PIA), against 3-NPA-evoked convulsions. Obtained results suggest that the central adenosine A1 receptor stimulation may play a role in the anticonvulsive potential of diazepam, phenobarbital, valproate and gabapentin in a novel model of 3-NPA-evoked seizures. Moreover, concomitant application of aminophylline with these drugs may reduce their clinical antiepileptic efficacy, especially among patients suffering from seizures related to the disturbances of mitochondrial respiratory chain.

Memantine increases brain production of kynurenic acid via protein kinase A-dependent mechanism.

We describe a novel aspect of action of memantine ex vivo, in the brain cortical slices and in vitro, in mixed glial cultures. The drug potently increased the production of kynurenic acid, an endogenous tryptophan metabolite blocking N-methyl-D-aspartate (NMDA) and nicotinic alpha7 receptors. In cortical slices memantine, an open-channel NMDA blocker (100-150 microM), but not the competitive NMDA receptor antagonist, LY235959 increased the production of kynurenic acid. Neither SCH23390, D1 receptor antagonist (50 microM) nor raclopride, D2 receptor antagonist (10 microM) changed the memantine-induced effects. Propranolol (100 microM) has partially reduced its action. Selective cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (1 microM), but not selective protein kinase C (PKC) inhibitor, NPC15437 (30 microM) totally reversed the action of memantine. In mixed glial cultures, 2-24 h incubation with memantine (2-50 microM) enhanced the production of kynurenic acid. Memantine (up to 0.5 mM) has not affected the activity of kynurenic acid biosynthetic enzymes. The obtained data suggest that memantine enhances the production of kynurenic acid in PKA-mediated way. This effect may partially contribute to the therapeutic actions of memantine and be of a potential clinical importance.

Nitric oxide and convulsions in 4-aminopyridine-treated mice.

We studied whether N(G)-nitro-L-arginine (NNA), an inhibitor of nitric oxide (NO) synthase as well as L-arginine and molsidomine, two agents elevating NO, influenced convulsions caused by 4-aminopyridine, a K+ channel blocker in mice. NNA, in a dose known to decrease level of NO (40 mg x kg(-1)), enhanced the seizure susceptibility to intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) 4-aminopyridine. L-arginine (500 mg x kg(-1)) and molsidomine (20 mg x kg(-1)) alone did not influence 4-aminopyridine-induced seizure activity. Surprisingly, the proconvulsant effect of NNA upon clonic and tonic seizures was potentiated by molsidomine (20 mg x kg(-1)). No influence of L-arginine on the proconvulsant effect of NNA was found. Taking into account the proconvulsant effect of NNA, an involvement of NO-mediated events in the mechanism of convulsive activity of 4-aminopyridine might be postulated. However, the ineffectiveness of L-arginine and molsidomine to suppress the convulsive activity of 4-aminopyridine as well as a paradoxical potentiation of the proconvulsant effect of NNA by molsidomine seem to exclude the impact of NO pathway on 4-aminopyridine-induced convulsions in mice. Our data suggest that the proconvulsant effect of NNA in this seizure model is caused by other, not related to NO, mechanisms.

Influence of antazoline and ketotifen on the anticonvulsant activity of conventional antiepileptics against maximal electroshock in mice

Experimental studies have indicated that the central histaminergic system plays an important role in the inhibition of seizures through the stimulation of histamine H1 receptors. H1 receptor antagonists, including classical antiallergic drugs, occasionally may induce convulsions in healthy children and patients with epilepsy. The purpose of this study was to investigate the effects of antazoline and ketotifen (two H1 receptor antagonists) on the anticonvulsant activity of antiepileptic drugs against maximal electroshock (MES)-induced convulsions in mice. The following antiepileptic drugs were used: valproate, carbamazepine, diphenylhydantoin and phenobarbital. In addition, the effects of antiepileptic drugs alone or in combination with antazoline or ketotifen were studied on long-term memory (tested in the passive avoidance task) and motor performance (evaluated in the chimney test), acutely and after 7-day treatment with these H1 receptor antagonists. The influence of antazoline and ketotifen on the free plasma and brain levels of the antiepileptics was also evaluated. Antazoline (at 0.5 mg/kg), given acutely and after 7-day treatment, significantly diminished the electroconvulsive threshold. Similarly, ketotifen, after acute and chronic doses of 8 mg/kg markedly reduced the threshold for electroconvulsions. In both cases, antazoline and ketotifen were without effect upon this parameter at lower doses. Antazoline (0.25 mg/kg) significantly raised the ED50 value of carbamazepine against MES (both, acutely and after 7-day treatment). Furthermore antazoline (0.25 mg/kg) also reduced the anticonvulsant activity of diphenylhydantoin, but only after repeated administration, without modifying the brain and free plasma level of this drug. Moreover, valproate and phenobarbital did not change their protective activity when combined with antazoline. Ketotifen (4 mg/kg) possessed a biphasic action, acutely it enhanced the anticonvulsant action of carbamazepine and phenobarbital while, following 7-day treatment, reduced the antiseizure activity of carbamazepine. Ketotifen did not affect the free plasma or brain levels of antiepileptics tested. Only acute antazoline (0.25 mg/kg) applied with valproate impaired the performance of mice evaluated in the chimney test. Ketotifen (4 mg/kg) co-administered with conventional antiepileptic drugs impaired motor coordination in mice treated with valproate, phenobarbital or diphenylhydantoin. Acute and chronic antazoline (0.25 mg/kg) alone or in combination with antiepileptic drugs did not disturb long-term memory, tested in the passive avoidance task. Similarly, ketotifen (4 mg/kg) did not impair long-term memory, acutely and after 7-day treatment. However, valproate alone or in combination with chronic ketotifen (4 mg/kg) worsened long-term memory. The results of this study indicate that H1 receptor antagonists, crossing the blood brain barrier, should be used with caution in epileptic patients. This is because antazoline reduced the protective potential of diphenylhydantoin and carbamazepine. Also, ketotifen reduced the protection offered by carbamazepine and elevated the adverse activity of diphenylhydantoin, phenobarbital and valproate.