fg-9041 and Brain-Ischemia

To investigate the effects of different antagonists on the alteration of I?B kinase (IKK) activity in rat hippocampus following global brain ischemia.. Using 4-vessel occlusion (4-VO) as brain ischemia model, IKK protein expression was examined by immunoblotting and immunoprecipitation, and IKK activity was assayed by in vitro kinase assay.. There was no alteration of IKK protein expression following ischemia or ischemia/reperfusion different time points, but IKK activity reached its peak level at ischemia 30 min. Pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, non-NMDA receptor antagonist DNQX, or NF-kappaB inhibitor PDTC decreased the IKK activity following brain ischemia 30 min. The increase in substrate myelin basic protein (MBP) phosphorylation by IKK is associated with an increase in autophosphorylation of IKK, which can also be antagonized by ketamine, DNQX, and PDTC.. NMDA receptor and non-NMDA receptor mediate the increase of IKK activity following global brain ischemia in rat hippocampus, which contributes to the alterations of expression and activity of downstream factor NF-kappaB.

Topics: Animals; Brain Ischemia; Excitatory Amino Acid Antagonists; Hippocampus; I-kappa B Kinase; Ketamine; Male; NF-kappa B; Phosphorylation; Proline; Protein Serine-Threonine Kinases; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Thiocarbamates

c-Jun N-terminal kinase-3 (JNK3), the only neural-specific isoform, may play an important role in excitotoxicity and neuronal injury. To analyze the variation of JNK3 activation, levels of phospho-JNK3 were measured at various time points of ischemia and selected time points of reperfusion, respectively. Our study illustrated that JNK3 was rapidly activated and translocated from cytosol to nucleus during ischemia. During reperfusion, two peaks of JNK3 activation occurred at 30 min and 3 days, respectively. To further define the mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC), alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptor antagonist 6,7-dinitro-quinoxaline-2,3(1H,4H)-dione (DNQX), N-methyl-D-aspartate (NMDA) receptor antagonist ketamine and L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist nifedipine were given to the rats 20 min prior to ischemia. The results showed that NAC obviously inhibited JNK3 activation during the early reperfusion, whereas DNQX preferably attenuated JNK3 activation during the latter reperfusion. Ketamine and nifedipine had no significant effects on JNK3 activation during reperfusion. Consequently, reactive oxygen species (ROS) and AMPA/KA receptor were closely associated with JNK3 activation following global ischemia.

Topics: Acetylcysteine; Animals; Brain Ischemia; Calcium Channel Blockers; Excitatory Amino Acid Antagonists; Free Radical Scavengers; Immunoblotting; Ketamine; Male; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinases; Nifedipine; Protein Transport; Protein-Tyrosine Kinases; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Time Factors

We measured the MLK3 expression, activity and backphosphorylation following cerebral ischemia. Our data showed that MLK3 protein levels were unalterable during ischemia and reperfusion. However, during ischemia MLK3 activity gradually increased and reached its peak at 30 min of ischemia. While its backphosphorylation reduced from 5 min of ischemia to 30 min of ischemia. In addition, we also detected MLK3 alteration at various time points of reperfusion after 15 min of ischemia, which showed that MLK3 activity increased twice, whereas MLK3 backphosphorylation was similarly consistent with its activity during reperfusion. To further analyze the reason of MLK3 activation, antioxidant N-acetylcysteine (NAC) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX) were given to the rats 20 min prior to ischemia. The results illustrated that NAC preferably inhibited the MLK3 activation during the ischemia and the early reperfusion, whereas DNQX effectively attenuated the MLK3 activation of the late reperfusion. We think that MLK3 activation is certainly associated with reactive oxygen species (ROS) and AMPA/KA receptor in response to ischemic insult.

Topics: Acetylcysteine; Animals; Antioxidants; Brain Ischemia; Enzyme Activation; Excitatory Amino Acid Antagonists; Hippocampus; Male; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Kainic Acid

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels, L-Type; Cytoplasm; Excitatory Amino Acid Antagonists; Hippocampus; I-kappa B Proteins; Ketamine; Male; NF-kappa B; Nifedipine; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Amino Acid; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Time Factors

The effects of three drugs including ketamine(KT), a noncompetitive antagonist of NMDA receptor (NR), nifedipine(ND), a voltage gated calcium channel (VGCC) antagonist and 6, 7dinitroquinoxaline -2, 3-dione(DNQX), a non-NMDA receptor antagonist on the contents of phospho-tyrosine proteins (p-tyr-pr) in the synaptosomal(P2), the crude membrane (P3), and the cytosolic (S3) fractions of hippocampus in forebrain ischemia of mongolian gerbils were studied.. (1) the contents of p-tyr-pr in all three fractions (P2, P3, S3) decreased 15 min after ischemia, but the contents of p-tyr-pr in S3 fraction decreased more obviously than the others did, With the increase of reperfusion time, the contents of p-tyr-pr in all of these fractions recovered gradually, but the p-tyr-pr in S3 fraction increased more rapidly among them, in the P2 fraction, the contents of p-tyr-pr increased slowly, but significantly and sustained longer during reperfusion when compared with that of P3 did. (2) The increase in p-tyr-pr contents induced by cerebral ischemia/reperfusion was partially antagonisted by KT and ND administration prior to cerebral ischemia, under these conditions, DNQX has no effect on it.. the increase of p-tyr-Pr contents induced by cerebral ischemia/reperfusion is related to NR channel and L-type VGCC, but not to non-NR channel.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels; Excitatory Amino Acid Antagonists; Gerbillinae; Hippocampus; Ketamine; Nifedipine; Phosphorylation; Phosphotyrosine; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury

The effects of ischemia/reperfusion on the levels of protein tyrosine phosphorylation in the synaptosome of gerbil hippocampus and the effects of three drugs, ketamine (KT), a noncompetitive antagonist of NMDA receptor, nifedipine (ND), a voltage gated calcium channel (VGCC) antagonist and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, on the phosphorylation were studied. The results showed that (1) 15 min of transient forebrain ischemia caused a marked decrease in the level of tyrosine phosphorylation of many protein bands, but, if followed by 15 min to 48 h of reperfusion, many protein bands including the 180 kD protein appeared to be increased; (2) the degree of tyrosine phosphorylation of the protein bands was higher than that of the sham-operated control, e.g. that of 180 kD protein was 1.8 fold of control; (3) administration of KT and ND before ischemia attenuated the increase of 180 kD protein tyrosine phosphorylation, while DNQX had no effect; and (4) immunoprecipitation and Western blot confirmed that the NR2B subunits of the NMDA receptors were among the phosphorylated 180 kD protein and ischemia /reperfusion did not affect the level of protein expression of NR2B. The above results suggest that the increase of tyrosine phosphorylation of NR2B induced by ischemia/reperfusion may further activate NR channels and aggravate neuronal injury, and that NR channels and other protein can be regulated by tyrosine phosphorylation not only through NR channels themselves but also via L-type VGCCs. Consequently, antagonists of both NR channels and L-type VGCCs may play a certain role in prevention and cure of ischemic brain injury.

Topics: Animals; Brain Ischemia; Calcium Channels, L-Type; Gerbillinae; Hippocampus; Ketamine; Nifedipine; Phosphorylation; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Synaptosomes; Tyrosine

1. Ischaemia was induced by 5 min of deprivation of glucose and an additional 5 min of deprivation of glucose and oxygen from Mg(2+)-free artificial cerebrospinal fluid in vitro. 2. During the ischaemic period, 11 +/- 1.5% of the total [3H]-dopamine ([3H]-DA) was released into the incubation medium. 3. Ischaemia-evoked release of [3H]-DA from striatal slices was attenuated by tetrodotoxin (TTX), MgSO4, dizocilpine, ketamine, 6,7-dinitroquinoxaline-2,3-dione (DNQX) or carbetapentane. 4. Release of [3H]-DA was attenuated by verapamil, omega-conotoxin GVIA and dantrolene. 5. Nomifensin inhibited the ischaemia-induced release of [3H]-DA. 6. Omission of Ca(2+) from incubation media potentiated ischaemia-evoked [3H]-DA release. The inhibitory effect of nomifensin was potentiated in Ca(2+)-free incubation media. 7. These results suggest that ischaemia induces release of [3H]-DA by dual mechanisms; one is Ca(2+)-dependent exocytosis and the other is reversal of transporter.

Topics: Animals; Brain Ischemia; Calcium; Calcium Channel Blockers; Corpus Striatum; Culture Techniques; Cyclopentanes; Dantrolene; Dizocilpine Maleate; Dopamine; Ketamine; Magnesium Sulfate; Nomifensine; omega-Conotoxin GVIA; Peptides; Quinoxalines; Rats; Tetrodotoxin; Verapamil

Since ischemic damage in the brain is linked to glutamate excitotoxicity, the effects of an acute exposure to glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) or N-methyl-D aspartate (NMDA) on the radial dendrites were compared with those occurring after a severe cochlear ischemia. Glutamate and AMPA, but not NMDA, produced a drastic swelling restricted to the radial dendrites below the inner hair cells (IHCs). At a concentration of 20 microM AMPA, a full electrophysiological recovery could be observed in some cochleas after washing the drug out. A prior perfusion of 6-7-dinitroquinoxaline-2,3-dione (DNQX, 50 microM) prevented the 25 microM AMPA-induced dendritic swelling. No protective effect of D-2-amino-5-phosphonopentanoate (D-AP5) could be observed. In the same way, ischemia (5-40 minutes) resulted in a clear swelling of the radial dendrites. While D-AP5 had no protective effects, 50 microM DNQX protected most of the radial dendrites from the ischemia-induced swelling, excepting those contacting the modiolar side of the IHCs. Finally, 50 microM DNQX + 50 microM D-AP5 resulted in a nearly complete protection of all the radial dendrites. Altogether, these results suggest that the acute swelling of radial dendrites primarily occurs via AMPA/kainate receptors. However, in radial dendrites contacting the inner hair cells on their modiolar side, NMDA receptors may be also involved.

Topics: 2-Amino-5-phosphonovalerate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids; Animals; Brain Ischemia; Cochlea; Cochlear Microphonic Potentials; Dendrites; Electrophysiology; Excitatory Amino Acid Antagonists; Female; Glutamates; Glutamic Acid; Guinea Pigs; Hair Cells, Auditory, Inner; Male; N-Methylaspartate; Neurons, Afferent; Neurotransmitter Agents; Perfusion; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

The object of this study was to investigate the role of the ventrolateral medullary pressor area in mediating the cardiovascular responses to experimentally induced global cerebral ischemia, and to test if excitatory amino acids or acetylcholine are the transmitters released in this brain region during these responses. The cerebral ischemic response was elicited in pentobarbital-anesthetized, artificially ventilated male Wistar rats by bilateral ligation of vertebral arteries followed by temporary clamping of the common carotid arteries. The pressor area was identified by microinjections of L-glutamate. Inhibition of neurons in this area by microinjections of muscimol, a gamma-aminobutyric acid receptor agonist, abolished the ischemic response, which demonstrated that this area is important in mediating these responses. Microinjections of a broad-spectrum excitatory amino acid receptor blocker (kynurenate), of specific antagonists for N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors (injected alone or in combination), and of atropine failed to block the ischemic responses. These results indicate that: 1) the ventrolateral medullary pressor area mediates pressor responses to cerebral ischemia, and 2) excitatory amino acids or acetylcholine in this area do not mediate the cardiovascular responses to cerebral ischemia.

Topics: Acetylcholine; Amino Acids; Animals; Antidiuretic Hormone Receptor Antagonists; Atropine; Blood Pressure; Brain Ischemia; Dose-Response Relationship, Drug; Glutamates; Heart Rate; Kainic Acid; Kynurenic Acid; Male; Medulla Oblongata; Muscimol; N-Methylaspartate; Quinoxalines; Quisqualic Acid; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Vasopressin; Vasopressins