The immunosuppressor st1959, a 3,5-diaryl-s-triazole derivative, inhibits T cell activation by reducing NFAT nuclear residency.

3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole (ST1959) has shown therapeutic effects in several animal models of autoimmune diseases. In this study the effects of ST1959 were further investigated in a murine model of colitis. The evidence obtained indicates that the beneficial effects exerted by ST1959 rely upon a decreased local immunological response. The cellular effects of ST1959 were additionally investigated on human peripheral blood mononuclear cells and Jurkat T cells by measuring cytokine production, cell proliferation and activation of a set of transcription factors. ST1959 decreases human T cell proliferation and inhibits cytokine expression at the transcriptional level. Moreover, at doses inhibiting cytokine production, ST1959 blocks phorbol 12-myristate 13-acetate (PMA) and ionomycin-induced nuclear factor protein of activated T cell (NFAT1) activity, without impairing AP-1- and NF-kB-dependent transcription. Immunofluorescence data show that ST1959 inhibits the nuclear residency of NFAT1 in both Jurkat and human peripheral blood mononuclear cells activated with PMA/ionomycin. leptomycin B, an inhibitor of CRM1/exportin-1alpha-dependent nuclear export, reverted the inhibitory effect of ST1959 on NFAT1 nuclear localization. This indicates that ST1959 may increase the nuclear export of NFAT1, downregulating NFAT1 activity via a mechanism different from that of cyclosporin A, since it does not affect NFAT phosporylation/dephosphorylation steps. These findings provide new insights into the molecular mechanisms underlying the immunomodulatory activity of ST1959.

Peroxynitrite-dependent activation of src tyrosine kinases lyn and hck in erythrocytes is under mechanistically different pathways of redox control.

Peroxynitrite, the product of superoxide and nitric oxide radicals, is considered one of the major oxidants formed in vivo under intense oxidative stress. We have previously reported the upregulation by peroxynitrite of src kinase activity in red blood cells. In this study, we investigated the mechanisms of peroxynitrite action and we demonstrate that two src kinases (lyn and hck) are activated through different pathways involving cysteine-dependent or -independent oxidations. Activation of hck by peroxynitrite or by hydrogen peroxide could be explained by reversible SH redox changes, whereas lyn was unaffected by hydrogen peroxide and its direct activation by peroxynitrite occurred through a still unknown modification(s) not reverted by SH reduction or inhibited by SH alkylation. Moreover, lyn could be activated also downstream by peroxynitrite-activated hck. The cross talk between lyn and hck was selective, since activated hck did not activate the non-src kinase syk. This study illustrates the complexity of redox-dependent src regulation and suggests that one reason for src heterogeneity may be a peculiar difference in their sensitivity to physiological oxidants. Irrespectively of the activation pathway, the final effect of peroxynitrite is the amplification of tyrosine-dependent signaling, a finding of general interest in nitric oxide-related pathophysiology.

Epileptogenic activity of two peptides derived from diazepam binding inhibitor after intrahippocampal injection in rats.

Peptides DBI 42-50 (DRPGLLDLK) and DBI 43-50 (RPGLLDLK) are synthetic fragments of an 18 amino acid peptide called octadecaneuropeptide (QATVGDVNTDRPGLLDLK), a brain derivative of diazepam-binding inhibitor (DBI). The two peptides were unilaterally injected into the dorsal hippocampus (granule cells of dentate gyrus) of freely moving adult rats. The electroencephalographic (EEG) pattern was continuously recorded from bilateral hippocampal and cortical electrodes, and the animals' behavior was observed throughout the experiment. A dose of 100 nmol peptide 42-50 was required to reliably cause EEG alterations (seizures and spiking). EEG changes, defined as seizures, were characterized by discrete repetitive periods of high-frequency and/or multispike complexes and/or high-voltage synchronized spike or wave activity. EEG seizures were often associated with a frozen appearance of the animal and "wet dog shakes." Tonic-clonic convulsions were not observed. EEG seizures induced by peptide 42-50 were prevented by 90 mg/kg PK 11195, a selective antagonist of a novel GABAA receptor-linked subtype of a benzodiazepine (BDZ) receptor, but were unaffected by flumazenil, an agonist of the "central" type of BDZ receptor and by D(-)2-amino-7-phosphonoheptanoic acid, a selective antagonist of the N-methyl-D-aspartate subtype of excitatory amino acid receptors. Light microscopy showed no neuropathological changes in the injected hippocampus. The data show that these DBI-derived peptide fragments induce a typical pattern of limbic seizures in rats. DBI and/or its natural processing products may play a role in the pathophysiology of epilepsy.

A peptidase-resistant cyclic octapeptide analogue of somatostatin (SMS 201-995) modulates seizures induced by quinolinic and kainic acids differently in the rat hippocampus.

Electroencephalographic (EEG) seizures were measured in rats after intrahippocampal injection of 120 nmol quinolinic acid into the stratum radiatum CA1 or 0.19 nmol kainic acid in the dentate gyrus or in the stratum radiatum. Injection of 5 micrograms SMS 201-995, a peptidase-resistant cyclic octapeptide analogue of somatostatin, into the stratum radiatum, 15 min before quinolinic acid, did not significantly modify the number of seizures and the total time in seizures. Five micrograms SMS 201-995 injected into the stratum radiatum reduced the number of seizures induced by kainic acid in the same area and the total time spent in seizures by 58% and 75%, respectively (Student's t-test; P less than 0.01). In both instances the latency to the first ictal episode was not significantly modified. Lesions of the medial septum, which reduced the activity of choline-o-acetyl-transferase (CAT) in the dorsal hippocampus by greater than 90% after one week did not significantly affect seizures induced by quinolinic acid. In rats lesioned in the medial septum, 5 micrograms SMS 201-995 reduced the total time spent in seizures by 43%, without changing the number of ictal episodes and raised the latency to the first quinolinic acid-induced seizure by 53% (ANOVA 2 x 2, P less than 0.05) but had no effect on these measures in the corresponding sham-operated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of enantiomers of propranolol on desipramine-induced anti-immobility in the forced swimming test in the rat.

The effect of d- and l-enantiomers of propranolol on desipramine-induced anti-immobility effects and on brain desipramine levels was studied in the rat. Intraperitoneal propranolol and desipramine were administered three times, 25, 6, 2 and 24, 5, 1 h respectively, before the test. It was found that l-propranolol but not d-propranolol, at the same doses (2.5 and 5 mg/kg), antagonized 20 mg/kg desipramine without altering desipramine brain levels. It is suggested that blockade of beta-adrenergic receptors rather than membrane-stabilizing or pharmacokinetic effects is responsible for the antagonism of propranolol toward desipramine.

Kynurenic acid synthesis by human glioma.

Biopsy material from human gliomas obtained during neurosurgery was used to investigate whether pathological human brain tissue is capable of producing kynurenic acid (KYNA), a natural brain metabolite which can act as an antagonist at excitatory amino acid receptors. Upon in vitro exposure to 40, 200 or 1000 microM L-kynurenine, the immediate bioprecursor of KYNA, freshly prepared tissue slices in a dose-dependent fashion produced KYNA which was detected in the incubation medium. De novo synthesized KYNA was identified by several chromatographic procedures. Astrocytomas produced significantly more KYNA than glioblastomas.

Seasonal and circadian variations of behavioural response to antidepressants in the forced swimming test in rats.

In order to verify whether the variability reported to occur in the effectiveness of antidepressants in the forced swimming test (FST) was due to seasonal and/or circadian variations, the anti-immobility effect induced by several antidepressants was assessed in rats every month throughout the year or every 4 hours for 2 consecutive days. Amineptine (20 mg/kg), amitriptyline (15 mg/kg), desipramine (20 mg/kg), imipramine (15 mg/kg) and mianserin (15 mg/kg) exerted the maximal effect in March. The effect of nomifensine (5 mg/kg) was constant throughout the year. Unlike imipramine and mianserin, nomifensine induced an anti-immobility effect that was maximal during the light phase of the light/dark cycle and was constant in continuous darkness.

Effect of serotoninergic drugs on stress-induced hyperthermia (SIH) in mice.

8-OH-DPAT (2.5-10 mg/kg) and buspirone (10 mg/kg) but not 5,7-DHT (200 micrograms/mouse), pCPA (75 and 150 mg/kg, three times), ritanserin (0.1 and 0.2 mg/kg), LY 53857 (1.5 and 3 mg/kg), GR 38032 F (0.1-100 micrograms/kg), TFMPP (5 and 20 mg/kg) and mCPP (2.5 and 5 mg/kg) antagonized the rise in body temperature that occurs to the last mice removed from their group housing, which was termed as stress-induced hyperthermia (SIH). Ro 15-1788, at a dose which blocked the effect of diazepam on SIH, did not reverse the anxiolytic effect of buspirione. Instead, when cerebral 5-HT content was reduced to 50% by 5,7-DHT-induced lesion, the effect of buspirone on SIH was decreased. TFMPP 5 mg/kg did not shorten significantly the onset of SIH as could have been expected by an anxiogenic drug, while the dose of 20 mg/kg did not modify the pattern of SIH at all. The lower dose of TFMPP evoked a hyperthermic and the higher a hypothermic response.

Studies on the potential neurotoxic and convulsant effects of increased blood levels of quinolinic acid in rats with altered blood-brain barrier permeability.

Intravenous injection of 450 mg/kg quinolinic acid (Quin), an endogenous kynurenine metabolite with excitotoxic properties, induced only minor electroencephalographic (EEG) modifications and no neurotoxicity in rats with a mature blood-brain barrier (BBB). BBB permeability was altered in rats by focal unilateral irradiation of the cortex (7 mm in diameter and 5 mm in depth) with protons (60 Gy, 9 Gy/min). Three days after irradiation, Evans blue dye staining showed BBB breakdown in the dorsal hippocampus of the irradiated hemisphere. No neurotoxic or convulsant effects were observed as a consequence of the radiation itself. When BBB-lesioned rats were challenged with 225 mg/kg Quin iv, epileptiform activity was observed on EEG analysis. Tonic-clonic seizures were induced by 225-450 mg/kg Quin. Light microscopic analysis showed a dose-related excitotoxic type of lesion restricted to the hippocampus ipsilateral to the irradiated side. Neuro-degeneration was prevented by local injection of 120 nmol D(-)2-amino-7-phosphonoheptanoic acid, a selective N-methyl-D-aspartate receptor antagonist. No lesions or EEG or behavioral modifications occurred after 450 mg/kg nicotinic acid, an inactive analog of Quin. The potential neurotoxic and convulsant effects of increased blood levels of Quin under conditions of altered BBB permeability are discussed.

Kinetics of MK-801 and its effect on quinolinic acid-induced seizures and neurotoxicity in rats.

MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate], a noncompetitive antagonist of the N-methyl-D-aspartate-type of excitatory aminoacid receptors, was measured in plasma and brain tissues after i.p. administration to rats by using a novel high-performance liquid chromatography assay. The drug reached maximal concentrations in plasma and brain within 10 to 30 min of injection (2 mg/kg) with an elimination half-life of 1.9 and 2.05 hr, respectively. Mean ratio of brain area concentration-time curve to plasma area concentration time curve was 12.5, referring to total plasma concentrations. MK-801 distributed almost equally between plasma and red cells (mean blood-to-plasma ratio averaged 1.2 +/- 0.2 when calculated 30 and 180 min from drug administration). Plasma and brain concentrations of MK-801 rose almost linearly from 0.5 to 4 mg/kg 30 min after injection and the brain-to-plasma ratio (12.9 +/- 2.8) was constant in the dose range studied. The distribution of the drug in various brain regions 30 and 180 min after 2 mg/kg i.p. showed no preferential concentration or retention in any of the areas studied. The anticonvulsant effect of MK-801 was evaluated against limbic seizures (measured by EEG) induced by intrahippocampal injection of 120 nmol of quinolinic acid, an agonist of the N-methyl-D-aspartate-type receptors, in freely moving rats. At 0.25 and 0.5 mg/kg, MK-801 significantly lowered by 71 to 77% the number of seizures and by 80% the total time spent in seizures (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aspartame on seizures in various models of experimental epilepsy.

We investigated in rats whether aspartame intake affected the susceptibility to seizures induced chemically (metrazol, quinolinic acid) or electrically (electroshock). Aspartame (0.75-1.0 g/kg), given orally as a single bolus to 16-hr fasted animals 60 min before metrazol, significantly increased the number of animals showing clonic-tonic seizures. At 1.0 g/kg the ED50 for clonic-tonic convulsions was lowered by 23%. A similar increase in seizure susceptibility was observed with 0.25-0.5 g/kg of the aspartame's metabolite phenylalanine. When aspartame was administered to fasted rats in three divided doses (0.33 g/kg) over 120 min or to fed animals after a meal, or overnight with the diet, no significant changes in the incidence of animals showing seizures was observed. One gram per kilogram aspartame and 0.5 g/kg phenylalanine did not modify the CC50 (mA) for tonic hindlimb extension induced by electroshock and the electroencephalographic seizures caused by intrahippocampal injection of 120 nmol quinolinic acid. Plasma and brain levels of phenylalanine and tyrosine significantly raised after both 1 g/kg aspartame as a single bolus (plasma: Phe 285%, Tyr 288%; brain: Phe 146%, Tyr 192%; above controls) or in three divided doses (plasma: Phe 207%, Tyr 315%; brain Phe 103%, Tyr 211%; above controls) and 0.5 g/kg phenylalanine (plasma: Phe 339%, Tyr 410%; brain: Phe 219%, Tyr 192%; above controls), but the ratio Phe/Tyr was not modified. Our data indicate that aspartame cannot be regarded as a general proconvulsant agent. The mechanisms of potentiation of seizures induced by metrazol after the administration of the sweetner in a single rapid intake will be discussed.

Quinolinic acid-induced seizures, but not nerve cell death, are associated with extracellular Ca2+ decrease assessed in the hippocampus by brain dialysis.

Seizures, neuronal damage and extracellular Ca2+ concentration were studied in rats unilaterally injected in the dorsal hippocampus with quinolinic acid, a brain metabolite with excitotoxic properties. In freely moving animals, in the first 2 h after the injection of a convulsant and neurotoxic dose (156 nmol), quinolinic acid induced a tetrodotoxin-insensitive decrease in the extracellular Ca2+ concentration (nadir 40%) in the injected area, as assessed by brain dialysis coupled to a fluorimetric method for Ca2+ detection. Blockade of quinolinic acid-induced decrements in Ca2+ by 15.6 nmol D-(-)2-amino-7-phosphonoheptanoic acid indicated that this effect was receptor-mediated. Dose-response relationships showed a close association between seizure activity (measured by EEG) and extracellular Ca2+ changes in the injected area. Changes in Ca2+ were apparent at the site of injection prior to the onset of focal seizures and they were not found in the homotypic structure where seizures were conducted. Drugs effective in blocking seizures (carbamazepine and flunarizine) prevented the fall in extracellular Ca2+, while drugs without anticonvulsant activity (ethosuximide and nifedipine) did not. Destruction of nerve cells by quinolinic acid was not prevented by treatment with carbamazepine and flunarizine. The results suggest that the fall in extracellular Ca2+ observed in the first 2 h after quinolinic acid, probably reflecting the ion influx into neurons, is involved in triggering focal seizures but is not related to the occurrence of nerve cell death.

Effect of various calcium channel blockers on three different models of limbic seizures in rats.

Voltage-dependent calcium channel-blockers were studied for their ability to modulate limbic seizures induced in rats by injection of quinolinic acid and kainic acid into the hippocampus or by hippocampal kindling. Flunarizine, at 40 mg/kg (but not 20 mg/kg), reduced the total number of seizures and total time spent in seizures induced by quinolinic acid by 75%; at 60 mg/kg, both parameters were reduced more than 90%, while at 80 mg/kg seizures induced by kainic acid were not affected. Forty and 60 mg/kg of flunarizine protected hippocampal-kindled rats from fully developed convulsions (Stage 5). Nifedipine, at 20 and 40 mg/kg, was ineffective on seizures induced by both quinolinate and kainate. However, at 20 mg/kg, 57% of the kindled animals were protected from Stage 5 and total protection was achieved at 40 mg/kg. Verapamil, at 40 mg/kg, reduced by respectively, 88% and 78%, the total number of seizures and the total time spent in seizures induced by quinolinic acid, but had no effect on seizures induced by kainate and Stage 5 seizures. The results suggest that, while seizures induced by kainic acid were refractory to all voltage-dependent calcium channel blockers, binding sites affected by flunarizine and verapamil in the brain may selectively facilitate ictal activity induced by quinolinic acid. Binding sites for dihydropyridine might contribute to the increased hippocampal excitability in kindled animals. The role of calcium entry through voltage-dependent calcium channels in the occurrence of seizures in these models of limbic epilepsy is discussed.