guanosine-triphosphate and Seizures

Tissue transglutaminase (tTG) post-translationally modifies proteins in a calcium-dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild-type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid-treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid-induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.

Topics: Animals; Blotting, Western; Calcium; Cell Count; Excitatory Amino Acid Agonists; Gene Expression; Genotype; Guanosine Triphosphate; Hippocampus; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Kainic Acid; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Phosphopyruvate Hydratase; Phosphorus Isotopes; Photobleaching; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Seizures; Staining and Labeling; Time Factors; Transglutaminases

gamma-Hydroxybutyric acid (GHB) is a naturally occurring compound that has the ability to induce generalized absence seizures when given to animals. GHB has been hypothesized to induce this effect via the postsynaptic gamma-aminobutyric acidB (GABAB) receptor. We sought to test this hypothesis by examining the affinity of GABAB agonists and antagonists for the [3H]GHB binding site, the affinity of GHB and a GHB antagonist for the [3H]GABAB binding site, and the effect of guanine nucleotides and pertussis toxin on both, using autoradiographic binding assays. GHB and its antagonist, NCS 382, did not compete for [3H]GABAB binding, nor did (-)-baclofen or the [3H]GABAB antagonists, CGP 35348 or SCH 50911, compete for [3H]GHB binding; however, the GABAB agonist 3-amino-propylphosphinic acid (3-APPA), and the GABAB antagonists phaclofen and 2-hydroxysaclofen (2-OH saclofen) did show a weak affinity for [3H]GHB binding in frontal cortex. GTP and the nonhydrolyzable GTP analogues, GTP gamma S and Gpp(NH)p, depressed [3H]GABAB binding throughout the brain, but increased [3H]GHB binding in frontal cortex and thalamus, those regions involved in GHB-induced absence seizures. Pertussis toxin significantly depressed [3H]GABAB binding throughout the brain, but attenuated [3H]GHB binding only in frontal cortex, and to a lesser degree than [3H]GABAB binding. The guanine nucleotide-induced changes in [3H]GHB and [3H]GABAB binding were due to a change in KD for both. Moreover, GTP gamma S reversed the ability of 3-APPA, phaclofen, and 2-OH saclofen to compete for [3H]GHB binding. These data do not support the hypothesis that GHB acts through the postsynaptic GABAB receptor to produce absence seizures. Rather, they raise the possibility either that the [3H]GHB binding site may be an isoform of the presynaptic GABAB receptor or that an independent GHB site is operative in the GHB model of absence seizures.

Topics: Animals; Autoradiography; Baclofen; Benzocycloheptenes; Binding Sites; Brain; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Guanosine Triphosphate; In Vitro Techniques; Male; Organophosphorus Compounds; Rats; Rats, Sprague-Dawley; Receptors, GABA-B; Seizures; Sodium Oxybate; Stereoisomerism

Neuronal activity results in long term cellular changes that underlie normal brain development and synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity. Here we describe an inducible novel member of the Ras family of small GTP-binding proteins we have termed Rheb. rheb mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity in the long term potentiation paradigm. The predicted amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2. The putative GTP binding regions are highly conserved. A bacterial fusion protein of Rheb binds GTP and exhibits intrinsic GTPase activity. Like Ha-Ras, the carboxylterminal sequence encodes a CAAX box that is predicted to signal post-translational farnesylation and to target Rheb to specific membranes. rheb mRNA is expressed at comparatively high levels in normal adult cortex as well as a number of peripheral tissues, including lung and intestine. In the developing brain, rheb mRNA is expressed at relatively high levels in embryonic day 19 cortical plate, and expression remains at stable levels throughout the remainder of prenatal and postnatal development. Its close homology with ras and its rapid inducibility by receptor-dependent synaptic activity suggest that rheb may play an important role in long term activity-dependent neuronal responses.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Brain; DNA, Complementary; Escherichia coli; Gene Expression Regulation; Gene Library; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hippocampus; In Situ Hybridization; Long-Term Potentiation; Mice; Molecular Sequence Data; Monomeric GTP-Binding Proteins; N-Methylaspartate; Neuropeptides; Ras Homolog Enriched in Brain Protein; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Seizures; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Synapses; Tissue Distribution

In the central nervous system, adenosine has been shown to be a major regulator of neuronal activity in convulsive disorders, mainly via the A1 receptor subtype. In a previous work, we have shown that seizures lead to an age-dependent upregulation of cerebral adenosine A1 sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was so far lacking. In the present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central adenosine A1 receptors in developing rats and in adults. Animals were sacrificed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Adenosine A1 receptors in brain sections were labeled by [3H]N6-cyclohexyladenosine (CHA), a potent receptor agonist. Generalized seizures induced a widespread increase in CHA-specific binding, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum and hippocampus. Moreover, the addition of guanylyl-5'-imidodiphosphate, a GTP analogue, to the incubation medium reduced CHA binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional adenosine A1 receptors are also functionally linked to G proteins. The age-related postictal increase in adenosine receptors might contribute to facilitate adenosine anticonvulsant effect, especially in newborns.

Topics: Adenosine; Animals; Autoradiography; Bicuculline; Brain; Brain Chemistry; Female; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Seizures