Effect of 2-guanidinoethanol on levels of monoamines and their metabolites in the rat brain

The contents of monoamines and their metabolites in rat brains 3 hours after the intracerebroventricular injection of 6 mol of 2-guanidino-ethanol (GEt) were measured by HPLC. GEt which is a configurational analogue of 4-aminobutanoic acid (GABA) induced severe running fits and tonic-clonic convulsions as well as epileptic discharges. In GEt-administered rats, dopamine (DA) decreased in the cortex, hippocampus and hypothalamus. 3,4-Dihydroxyphenylacetic acid (DOPAC) increased to about the same level in all brain regions, therefore the distribution of DOPAC appeared to be homogeneous in the brain. The homovanillic acid levels also increased in the striatum and hippocampus. No significant change in the norepinephrine contents was observed in any region. The turnover ratio of DA increased significantly except in the striatum. Serotonin levels increased in the hypothalamus and midbrain by GEt administration, though 5-hydroxyindoleacetic acid levels showed no change in any of the brain regions. These data suggest that the activity of dopaminergic and serotonergic neurons are increased by GEt.     

Effects of Aging and Amyloid-β Peptides on Choline Acetyltransferase Activity in Rat Brain

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), involved in the learning and memory processes is responsible for the synthesis of acetylcholine. There are many discrepancies in literature concerning ChAT activity during brain aging and the role of amyloid beta peptides in modulation of this enzyme. The aim of the study was to investigate the mechanism of ChAT regulation and age-related alteration of ChAT activity in different parts of the brain. Moreover the effect of A peptides on ChAT activity in adult and aged brain was investigated. The enzyme activity was determined in the brain cortex, hippocampus and striatum in adult (4-months-old), adult-aged (14-months-old) and aged (24-months-old) animals. The highest ChAT activity was observed in the striatum. We found that inhibitors of protein kinase C, A, G and phosphatase A2 have no effect on ChAT activity and that this enzyme is not dependent on calcium ions. About 70% of the total ChAT activity is present in the cytosol. Arachidonic acid significantly inhibited cytosolic form of this enzyme. In the brain cortex and striatum from aged brain ChAT activity is inhibited by 50% and 37%, respectively. The aggregated form of A 25-35 decreased significantly ChAT activity only in the aged striatum and exerted inhibitory effect on this enzyme in adult, however, statistically insignificant. ChAT activity in the striatum was diminished after exposure to 1 mM H₂O₂. The results from our study indicate that aging processes play a major role in inhibition of ChAT activity and that this enzyme in striatum is selectively sensitive for amyloid beta peptides.     

Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-γ-glutamyl hydrazone in the developing rat

We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate--glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B₆ depletion, but alterations of other B₆-dependent biochemical pathways cannot be discarded.     

Developmental changes in choline acetyltransferase and glutamate decarboxylase activity in various regions of the brain of the male,female, and neonatally androgenized female rat

In an attempt to discern effects of sex hormones on the development of neurotransmitter systems in the rat brain, choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) have been measured at postnatal days 8, 12, 25, and 60 in five regions (amygdala, anterior hypothalamus, hippocampus, olfactory bulbs, and cerebral cortex) of the brains of normal male, normal female, and neonatally androgen-treated female rats. Essentially no associations between sex or of neonatal androgenization on either enzyme were found. The data, however, provide new information on the relative rates of development of ChAT and GAD in five regions of the rat brain which supplement the limited information already available in the literature. ChAT activity was highest in amygdala and hypothalamus, but developed most rapidly in hippocampus and cerebral cortex. The relative activities and patterns of development of GAD activity were similar to those of ChAT.     

Effects of the γ-aminobutyrate transaminase inhibitors gabaculine and γ-vinyl GABA on γ-aminobutyric acid release from slices of rat cerebral cortex

The release of [³H]-aminobutyric acid (GABA) from pre-loaded slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitors gabaculine and -vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 M) substantially reduced the Ca²⁺-dependence of the release of [³H]GABA evoked by a 4 min 30 mM K⁺ pulse, whereas this was not appreciably reduced by the presence of -vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of [³H]GABA release were not identical in the presence and absence of either inhibitor.     

Developmental change of an enzyme activity oxidizing γ-aminobutyraldehyde to γ-aminobutyric acid in the chick embryonic brain

An enzyme activity oxidizing -aminobutyraldehyde (ABAL) to GABA reflecting an alternative pathway for GABA synthesis was assayed in the developing chick embryonic brain and was compared with glutamate decarboxylase (GAD) activity. An enzyme activity oxidizing ABAL to GABA showed almost constant level during development in the chick embryonic brain, and was present at low levels compared with GAD activity. The results indicate that GABA synthesis via an alternative pathway is always much less than synthesis via the GAD-dependent pathway in the developing chick embryonic brain.     

Effect of cytochrome P450 1A induction on oxidative damage in rat brain

Polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs) can enhance the generation of reactive oxygen species (ROS) by inducing cytochrome P450 1A (CYP 1A) in vivo and in vitro. While the brain is vulnerable to oxidative injury, whether or not CYP 1A induction in the brain can produce measurable levels of oxidative damage has not been reported. The objective of this study was to investigate the effect of this induction on oxidative damage to the CNS. Time course changes in rat brain CYP 1A activity were determined by measurement of ethoxyresorufin Odeethylase (EROD) activity in whole brain homogenates. Three days after exposure of rats to five daily injections of 3methylcholanthrene (3MC) an approximately sevenfold increase in EROD activity was observed. Hepatic levels were increased 60–100 fold. This increase in CYP 1A activity was not accompanied by increased protein or lipid oxidation as measured by tryptophan fluorescence and TBAR formation, or decreased glutamine synthetase (GS) activity. These findings indicate that if increased CYP 1A activity in the brain following 3MC treatment leads to increased ROS generation, the increase is insufficient to overwhelm the endogenous antioxidant defense system, produce detectable oxidative damage, and alter glutamate homeostasis.     

Autoradiographic localization of 3H-GABA uptake in the thyroid gland of the rat

Summary The uptake of -aminobutyric acid (GABA) in the thyroid gland of the rat was studied autoradiographically following in vitro incubation.High-affinity GABA uptake was localized in follicle cells, whereas C cells (parafollicular cells) in general did not accumulate GABA by high-affinity transport. The follicle cells were also the main sites of low-affinity GABA uptake. Additionally, some nerve fibres were found to accumulate GABA.The predominant localization of GABA uptake in follicle cells is discussed in view of a presumed role of GABA in thyroid function.     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

Modification of chloride flux across brain membranes by inhibitory amino acids in developing and adult mice

The influx of³⁶Cl^– was studied in membrane vesicles prepared from different brain regions from 3-day-old and adult mice. In both age groups the influx was enhanced about threefold by -aminobutyric acid (GABA), which effect was blocked by bicuculline and picrotoxin but not by baclofen, characteristic of a GABA_A receptor-mediated event. In samples from the adult brain stem the GABA stimulation was smaller than in samples from the other brain regions. Most of the compounds studied apparently act at the same receptor site with the following order of efficacy: muscimol > GABA > -alanine > hypotaurine > taurine. A number of anticonvulsant taurine derivatives were not effective and glycine only in the brain stem. The weak modulatory effects of taurine could be of significance in vivo since depolarizing stimuli release massive amounts of taurine in developing brain tissue.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Postresuscitation Changes in Brain Free Radical-Mediated Processes and Nitric Oxide Synthase Activity in Rats: Effects of Individual Behavior in "Emotional Resonance” Test

Effects of 7-min cardiac arrest and individual behavior on free radical-mediated processes and nitric oxide synthase (NOS) activity was evaluated in brains of male Wistar rats one hour and one week after resuscitation. "Emotional resonance test was used for the behavioral selection of rats. The test includes factors of significance for rats: the choice between large and lighted or small and dark space as well as signals of pain of another rat. Free radical generation (using chemiluminescence method), superoxide scavenging/generating activity, substances reacting with 2-thiobarbituric acid and NOS activity (by measuring mononitrosyl iron complex of NO with diethyl dithiocarbamate and endogenous brain Fe²⁺ by electron spin resonance spectroscopy) were determined in cerebral cortex, cerebellum and hippocampus. Cardiac arrest induced oxidative stress accompanied by the loss of NOS activity, as well as compensatory changes of free radical-mediated processes in cerebral cortex. Oxidative stress was also evident in cerebellum and, to a lesser extent, in hippocampus. Most of neurochemical differences between behavioral groups were induced by cardiac arrest. These differences were global, related to a specific brain region or became apparent in cerebral lateralization of biochemical indices.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

GABA-gated chloride ion influx in brains of epileptic El mice

GABA-gated chloride ion influx was measured in brain microsac preparations of epileptic El mice. There was significantly greater sensitivity to GABA in stimulated El mice (which had 14–18 convulsions induced at weekly intervals) than in unstimulated El mice (which had not experienced convulsions) or ddY mice. GABA-gated chloride ion influx was significantly decreased 20 min after a single convulsion, and returned to the preconvulsion level 60 min after a convulsion. These findings suggest that the functional state of GABA-gated chloride channel in El mice is changed secondarily by single or repeated convulsions.     

Depolarization- and α-Latrotoxin-Induced [3H]GABA Release from the Rat Brain Synaptosomes: Effect of Phenylarsine Oxide

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of cellular processes, including neurotransmitter release. Here we present evidence for the strong influence of an inhibitor of phosphatidylinositol 4-kinase, phenylarsine oxide, on depolarization- and -latrotoxin-evoked exocytotic release of [³H]GABA from the rat brain synaptosomes. Our data also show that subnanomolar concentrations of the toxin stimulate the process of exocytosis per se, while nanomolar toxin concentrations in addition cause neurotransmitter outflow from the cytosolic pool.     

Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific¹²⁵I-omega-conotoxin (-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited¹²⁵I--CgTX binding, but not that of [³H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of¹²⁵I--CgTX, because the venom also inhibited the binding of [³H](+)PN200-110 to a similar degree. The amount of specific binding of¹²⁵I--CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with¹²⁵I--CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of -CgTX-sensitive Ca channels in crude membranes from rat whole brain and that -CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.     

ω-Aga IVA selectively inhibits the calcium-dependent fraction of the evoked release of [3H]GABA from synaptosomes

The effect of -Aga IVA, a P-type Ca²⁺ channel blocker, on the release of the inhibitory neurotransmitter GABA and on the elevation of Ca_i induced by depolarization was investigated in [³H]GABA and fura-2 preloaded mouse brain synaptosomes, respectively. Two strategies (i.e. 20 mM external K⁺ and veratridine) that depolarize by different mechanisms the preparation were used. High K⁺ elevates Ca_i and induces [³H]GABA release in the absence of external Na⁺ and in the presence of TTX, conditions that abolish veratridine induced responses. The effect of -Aga IVA on the Ca²⁺ and Na⁺ dependent fractions of the depolarization evoked release of [³H]GABA were separately investigated in synaptosomes depolarized with high K⁺ in the absence of extermal Na⁺ and with veratridine in the absence of external Ca²⁺, respectively. The Ca²⁺ dependent fraction of the evoked release of [³H]GABA and the elevation of Ca²⁺ induced by high K⁺ are markedly inhibited (about 50%) in synaptosomes exposed to -Aga IVA (300 nM) for 3 min before depolarization, whereas the Na⁺ dependent, Ca²⁺ independent carrier mediated release of [³H]GABA induced by veratridine, which is sensitive to verapamil and amiloride, is not modified by -Aga IVA. Our results indicate that an -Aga IVA sensitive type of Ca²⁺ channel is highly involved in GABA exocytosis.     

Effects of taurine on GABA release from synaptosomes of rat olfactory bulb

Summary Superfusion of synaptosomes prepared from rat olfactory bulb revealed constant basal release of endogenous taurine (Tau), aspartate (Asp), glutamate (Glu) and-aminobutyrate (GABA): their release rates were 110.4 ± 13.0, 30.3 ± 6.7, 93.7 ± 13.1, and 53.3 ± 8.8 pmol/min/mg protein, respectively. The depolarizing-stimulation with 30mM KCl evoked 1.17-, 2.18-, 2.55- and 1.53-fold increases, respectively. Tau release was calcium-independent. However, the perfusion of synaptosomes with Tau (10µM) inhibited the evoked increase in GABA release by 63% without changing basal release, although it did not affect release of Asp and Glu. Phaclofen (10µM, a GABA_B receptor antagonist), but not bicuculline (10µM, a GABA_A receptor antagonist), counteracted the Tau-induced reduction in GABA release. These data suggest that Tau may be abundantly released from nerve endings of rat olfactory bulb and that it may regulate GABA release through the activation of presynaptic GABA_B autoreceptors.     

Measurement of Acute Phase Proteins in the Rat Brain: Contribution of Vascular Contents

A localized acute phase response occurs in the brain in Alzheimer's disease. Acute phase proteins have previously been measured in brain homogenates to quantify this response. The extent to which measurements of these proteins reflect brain parenchymal contents, as opposed to vascular contents, is unknown. In this study, the acute phase proteins ceruloplasmin (CP), complement factor 3 (C3), haptoglobin (HP), and albumin were measured in regional brain homogenates from phosphate buffered saline-perfused and sham-perfused rats (n = 7–9/group). Interleukin 1- (IL1-) and copper were also measured. Mean CP, C3, HP, and albumin concentrations in perfused specimens decreased by 94%, 88%, 90%, and 81% vs. sham-perfused specimens (all p < 0.001), while ILl- and copper were unchanged. These results suggest that acute phase protein measurements in brain homogenates reflect primarily vascular contents. However, IL1- and copper concentrations in brain homogenates are minimally influenced by vascular contents.