EFFECT OF METHIONINE AND METHIONINE SULPHOXIMINE ON RAT BRAIN S-ADENOSYL METHIONINE LEVELS

Rat brain SAM levels were markedly increased after methionine administration, whereas the convulsant, L-methionine-dl-sulphoximine (MSO), produced a 35 per cent decrease in whole brain content of S-adenosyl-L-methionine (SAM). When methionine was given in combination with MSO, SAM levels were not decreased. Studies on the regional distribution of SAM revealed only a small variation between regions (from 24 nmol/g in midbrain to 49-5 nmol/g in striatum). SAM levels were reduced by about 50 per cent in the cerebellum, striatum, cortex and hippocampus 3 and 6 h after MSO. It is proposed that abberant cerebral methylation processes may be involved in the genesis of the MSO seizure.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

The effect of methionine on the uptake,distribution, and binding of the convulsant methionine sulfoximine in the rat

The effect of methionine on the uptake, distribution, and binding of the convulsant methionine sulfoximine (MSO) in 7 rat brain regions, the spinal cord, the liver, and the kidney was investigated. The administration of methionine decreased the uptake of MSO in all brain regions. The uptake of MSO by and its distribution in the nervous tissue was uniform and failed to result in any preferential accumulation of the drug. Methionine decreased the amount of MSO bound to cerebral structures and to the spinal cord. MSO bound to the spinal cord was less susceptible to release by Triton X-100 than was brain-bound MSO.     

Regulation of S-adenosylhomocysteine hydrolase in the halophilic cyanobacterium Aphanothece halophytica: a possible role in glycinebetaine biosynthesis

Aphanothece halophytica, a halophilic cyanobacterium capable of growing in saturated NaCl, accumulates high intracellular concentrations of glycinebetaine in response to increasing environmental NaCl. In this organism, intracellular levels of K⁺ rise dramatically with increasing external NaCl before an increase in glycinebetaine can be detected. Glycinebetaine synthesis requires three S-adenosylmethionine (AdoMet)-mediated transmethylations; each transmethylation reaction generates one molecule of the transmethylation inhibitor S-adenosylhomocysteine (AdoHcy). Thus, glycinebetaine synthesis should require continued removal of AdoHcy. In A. halophytica, catabolism of AdoHcy was shown to occur via the reversible reaction catalyzed by AdoHcy hydrolase (EC 3.3.1.1). Activity of AdoHcy hydrolase in the direction of synthesis of AdoHcy was inhibited by 0.4 M KCl in this organism. On the other hand, activity of AdoHcy hydrolase in the direction of AdoHcy hydrolysis was unaffected by 0.4 M KCl. Glycinebetaine increased synthesis of AdoHcy in the presence of 0.4 KCl, but had no effect on AdoHcy hydrolysis. Based upon these results, a mechanism is proposed for the regulation of glycinebetaine synthesis by K⁺ and glycinebetaine in A. halophytica. According to this mechanism, the regulatory response would be initiated by a K⁺-induced shift in the AdoMet/AdoHcy ratio.Abbreviations AdoMet S-adenosylmethionine - AdoHcy S-adenosyl homocysteine     

Decreased Transmethylation of Biogenic Amines After In Vivo Elevation of Brain S-Adenosyl-l-Homocysteine

Abstract: The ability of S -adenosyl- l -homocysteine (AdoHcy) to inhibit biologic transmethylation reactions in vitro has led us to explore the possibility of pharmacologically manipulating AdoHcy levels in vivo and examining the consequences of these alterations on the transmethylation of some biogenic amines. Swiss-Webster mice were injected intraperitoneally with different doses of adenosine (Ado) and d,l -homocysteine thiolactone (Hcy) and were killed at various times thereafter. S -Adenosyl- l -methionine (AdoMet) and AdoHcy concentrations were determined by using a modified isotope dilution-ion exchange chromatography-high pressure liquid chromatography technique sensitive to less than 10 pmol. Increasing doses of Ado + Hcy (50-1000 mg/kg of each) produced a dose-related increase in blood, liver, and brain AdoHcy levels. At a dose level of 200 mg/kg Ado + Hcy, AdoHcy levels were markedly elevated, with minimal concomitant perturbations of AdoMet. This elevation was maximal 40 min after giving Ado + Hcy, returning to control values within 6 h. Ado + Hcy treatment resulted in decreased activities of catechol- O -methyltransferase, histamine- N -methyltransferase, and AdoHcy hydrolase in vitro. The cerebral catabolism of intraventricularly administered [³H]histamine (HA) was decreased in a dose-related manner by Ado + Hcy treatment as evidenced by higher amounts of nonutilized [³H]HA in brain, concurrent decreases in [³H]methylhistamine formation, and decreases in the transmethylation conversion index. Steady state levels of HA also showed dose-related increases after Ado + Hcy treatment. It is concluded that injections of Ado + Hcy can markedly elevate AdoHcy levels in vivo , which can, in turn, decrease the rate of transmethylation reactions.     

Regional Distribution of Kininase in Rat Brain

Kininase activity, which inactivates kinins, was measured in seven regions of the rat brain (i.e., the cerebral cortex, cerebellum, striatum, midbrain, hippocampus, hypothalamus, medulla oblongata), and in the spinal cord with a bioassay method using bradykinin as the substrate. Specific kininase activities in the cerebellum and striatum were higher than those in the other five regions or the spinal cord. Angiotensin-converting enzyme activity, which was measured fluorometrically using Hip-His-Leu as substrate, showed high activity in the striatum and cerebellum. These findings suggest that the presence of high concentrations of peptidases plays a role in the degradation of kinins and/or other peptides in these areas.     

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Rate of disappearance of isethionic acid in the rat central nervous system

The rates of disappearance of tritiated isethionic acid (2-hydroxyethanesulfonic acid) in eight regions of the rat central nervous system were studied. By utilizing the technique of graphical analysis (curve peeling), it was determined that seven areas (striatum, diencephalon, pons-medulla, midbrain, hippocampus, spinal cord, and cortex) exhibited triphasic (fast, intermediate, and slow) disappearance rates while only the cerebellum displayed a biphasic (fast, slow) rate. The half-lives for the fast component in the different regions of the central nervous system varied from 0.5 hr (midbrain and cortex) to 1.5 hr (diencephalon, cerebellum, and spinal cord); the half-lives for the intermediate component of the triphasic rate varied from 3.5 hr in the midbrain and spinal cord to 5.5 hr in the hippocampus. Half-lives estimated for the slow component of the multiphasic rate of disappearance of tritiated isethionic acid ([³H]ISA) varied from 28 hr (cerebellum) to 90 hr (spinal cord).     

The Biosynthesis of Polyamines in the Brain of Audiogenic Seizure-Susceptible and -Resistant Deermice

Abstract: The biosynthesis of polyamines was investigated in the brains of the audiogenic seizure-susceptible (SS) mutant and the wild-type, seizure-resistant (SR) deermouse Peromyscus maniculatus bairdii. For this purpose a new, rapid, and economical high pressure liquid chromatography (HPLC) procedure for the quantitation of putrescine, spermidine, and spermine was developed. Benzoyl derivatives of the polyamines, prepared from a crude brain supernatant, were ether extracted and, following removal of the ether, were separated and quantitated by HPLC. The high sensitivity of the method allows quantitation of putrescine in 50 mg and of spermidine and spermine, in as little as 2-2.5 mg, of brain tissue. No differences were found in endogenous levels of the 3 polyamines in brains of SS vs SR deermice. Using [¹⁴C]putrescine as a polyamine precursor, we found the specific radioactivity of spermidine to be lower in the SS than in the SR brains following a 1 h intraventricular (i.vt.) pulse. No such differences were noted if [3,4-¹⁴C]methionine was used as the polyamine precursor. To test whether the flux of methionine through the transmethylation pathway was also different in SS and SR deermouse brain, we administered [1-¹⁴C]methionine (i.vt.) (1 h pulse). Even though the brains of SS animals contained higher methionine and lower S-adenosyl-l -methionine (AdoMet) levels than the SR brains, the specific radioactivities of methionine and AdoMet were, respectively, lower and higher in SS compared to SR brains. The latter results are in agreement with our previous findings of an accelerated utilization of AdoMet in brains of Swiss-Webster mice following administration of the chemical convulsant l -methionine-d,l-sulfoximine (MSO). Taken together, the data suggest that the SS condition, whether genetically determined (as in the SS deermouse) or chemically elicited (as after MSO), correlates positively with higher than normal rates of conversion of methionine to brain AdoMet and leads to an enhanced rate of utilization of AdoMet via the transmethylation pathway.     

The Regional Distribution of Monoamine Oxidase Activities Towards Different Substrates: Effects in Rat Brain of Chronic Administration of Manganese Chloride and of Ageing

Abstract: The effects of chronic manganese chloride administration (1 mg MnCl₂ 4H₂O/ml of drinking water) and ageing on the regional distribution of monoamine oxidase (MAO, EC 1.4.3.4) were studied in 2-month- and 24–28-month-old rats. In both the control and Mn-treated rats, the serotonin oxidation (type A) rates decreased in hypothalamus, pons and medulla, striatum, midbrain and cerebral cortex, but not in cerebellum, in ageing. On the other hand the benzylamine oxidation (type B) rates in hypothalamus, striatum and cerebral cortex increased in ageing. In all regions except the cerebellum, there was a uniform decrease in the A/B ratio. This decrease was verified by differential inhibition studies using clorgyline and l -deprenyl, specific type A and type B inhibitors respectively. The dopamine-oxidising rates decreased in all regions, except the cerebral cortex and the cerebellum, in ageing control rats. This age-related decrease was not seen in the striatum and midbrain of manganese-treated rats. In these rats the other effect was an age-related increase in the rate of oxidation of all the amines in the cerebellum, not observed in control rats. These selective effects of manganese are only seen when comparing age-related changes in both groups of animals, since comparison of manganese-treated rats with age-matched controls showed a significant difference only in the rate of serotonin oxidation in the cerebellum of 2-month-old rats. The relationship of these observations to the effects of ageing and manganese encephalopathy on specific amine systems is discussed.     

S-adenosylhomocysteine hydrolases as the primary target enzymes in androgen regulation of methylation complexes

tRNA methylation complexes consisting of S-adenosylmethionine (AdoMet) synthetase, tRNA methylases, and S-adenosylhomocysteine (AdoHcy) hydrolase have been prepared from rat Novikoff hepatoma cells. The existence of the ternary enzyme complex is supported by dissociation and reconstitution of the ternany tRNA methylation complexes. In rat prostate and testis, two isozymes each for AdoMet synthetase and AdoHcy hydrolase are detected. The K_m (methionine) values for the two AdoMet synthetases are 3.1 and 23.7 μm and the K_m (adenosine) values for the two AdoHcy hydrolases are 0.33 and 1.8 μm. Correspondingly, two groups of methylation complexes are detectable, sedimenting in a sucrose gradient as 7 S and 8 S. The 7 S complexes are composed of AdoMet synthetase and AdoHcy hydrolase with the higher K_m values, and the 8 S complexes are composed of the respective isozymes with the lower K_m values. tRNA methylation complexes belong to the 8 S group. In hormone-depleted rat prostates and testes following hypophysectomy, the specific activities of AdoMet synthetases, tRNA methylases, and AdoHcy hydrolases are decreased severely, but are restored promptly after administration of testosterone. Thus, methylation enzymes are responsive to the regulation by steroid hormone. AdoHcy hydrolases from hormone-depleted tissues are unstable, and ternary tRNA methylation complexes are easily dissociable into individual activities. The stability of AdoHcy hydrolases is markedly improved by testosterone, and the integrity of ternary tRNA methylation complexes is maintained in the presence of testosterone. These results suggest that AdoHcy hydrolases are the primary target enzymes in adrogen regulation of methylation complexes.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.     

Regional differences in the turnover of neuronal histamine in the rat brain

The turnover rate of histamine (HA) and the half-life of neuronal HA were estimated in 9 regions of the rat brain following pargyline-induced accumulation of tele-methylhistamine (t-MH). The turnover rate was the highest in the hypothalamus (108.7 ng/g/hr). The striatum also showed a high turnover rate (80.2 ng/g/hr) despite much lower levels of HA and t-MH, as compared with the levels in the hypothalamus. The turnover rate was relatively high in the thalamus, cerebral cortex, amygdala and midbrain, but it was very low in the cerebellum. t-MH accumulation in the spinal cord was nil. The HA levels were reduced to various degrees (from nil to less than 40% of the control) by (S)-alpha-fluoromethylhistidine, depending on the regions studied. The neuronal HA content of each brain region was subsequently estimated, and the half-life of neuronal HA in each region was calculated. The half-life of neuronal HA was the shortest (7.7 min) in the striatum, while it was long (about 50 min) in the hypothalamus and thalamus. Half-life values of about 20 min were obtained in other regions. These results show the high levels of histaminergic activity in some parts of the telencephalon, thalamus and midbrain as well as the hypothalamus.     

Amino acid changes in regions of the CNS in relation to function in experimental portal-systemic encephalopathy

Sustained hyperammonemia resulting from portocaval anastomosis (PCA) in the rat, is accompanied by neurological symptoms and reversible morphological changes in brain, the nature and distribution of which suggest selective vulnerability of certain brain structures. the present study was initiated to investigate the effects of increasing CNS ammonia on the distribution of amino acids in regions of the rat brain in relation to the degree of neurological impairment in PCA rats. Four weeks following PCA, rats were administered ammonium acetate (5.2 mmol/kg, i.p.) to precipitate neurological symptoms of encephalopathy which included diminished locomotor activity, loss of hindlimb extension and righting reflexes and ultimately coma. At various stages during the development of encephalopathy, rats were sacrificed and the amino acids glutamine, glutamate and aspartate measured simultaneously, using a sensitive double-isotope dansyl microassay. Homogenates of the following regions of the CNS were assayed: cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla-pons, spinal cord (gray matter) and spinal cord (white matter). Sustained hyperammonemia associated with PCA alone resulted in a non-uniform 2–4 fold increase of glutamine in all regions of the CNS. Glutamate, on the other hand, was selectively increased in striatum and cerebellum, two regions of brain shown to exhibit early morphologically-characterised astrocytic abnormalities in rats with PCA. Onset of severe neurological dysfunction was accompanied by significantly decreased glutamine and glutamate in striatum and cerebellum. Thus, sustained hyperammonemia in association with portocaval shunting results in region-selective effects with respect to glutamine-glutamate metabolism in the CNS.     

Neurochemical changes in rats chronically treated with a high concentration of manganese chloride

Several neurochemical parameters were studied in brain regions of rats chronically treated with a high concentration of manganese chloride (20 mg MnCl₂.4H₂O per ml. of drinking water) throughout development until adulthood. Large increases in Mn accumulation were found in all brain regions (hypothalamus, +530%; striatum, +479%; other regions, +152 to +250%) of Mn-treated adult rats. In these animals, Ca levels were decreased (–20 to –46%) in cerebellum, hypothalamus, and cerebral cortex but were increased (+186%) in midbrain. Mg levels were decreased (–12 to –32%) in pons and medulla, midbrain, and cerebellum. Fe levels were increased (+95%) in striatum but were decreased (–28%) in cerebral cortex. Cu levels were increased (+43 to +100%) in pons and medulla and striatum but Zn levels were decreased (–30%) in pons and medulla. Na levels were increased (+22%) in striatum but those of K and Cl remained unchanged. Type A monoamine oxidase activities were decreased (–13 to –16%) in midbrain, striatum, and cerebral cortex, but type B monoamine oxidase activities decreased (–13%) only in hypothalamus. Acetylcholinesterase activities were increased (+20 to +22%) in striatum and cerebellum. The results are consistent with out hypothesis that chronic manganese encephalopathy not only affects brain metabolism of Mn but also that of other metals.We dedicate this paper to Professor Alan N. Davison. Professor Davison has conducted pioneering research in several important areas including: brain development and myelination, aging and Alzheimer's disease, and multiple sclerosis. He encouraged us to investigate the neurochemical mechanisms of neurotoxicity of metal ions, particularly in connection with neurological diseases. His encouragement and continued support facilitated the launching of our multidisciplinary research program in the long-term effects of manganese toxicity on brain development and aging.     

100Hz电针刺受时大鼠脑和脊髓k受体结构特性的改变

西方采用放射配体结合实验研究了１００ＨＺ电针耐受发生发展过程中大鼠脑和脊髓Ｋ受体结构特性的变化。大鼠每天给予１００ＨＺ电针１次,连续７ｄ。分别在电针的第１、３、５、７天取不同脑区进行观察。     

Regional distribution of the histamine metabolite, tele-methylimidazoleacetic acid, in rat brain: effects of pargyline and probenecid

Abstract: tele -Methylimidazoleacetic acid (t-MIAA), a major brain histamine metabolite, was measured in nine rat brain regions by a gas chromatography-mass spectrometric method that also measures the precursor amine, tele -methylhistamine (t-MH). The t-MIAA concentration of cerebellum, medulla-pons, midbrain, caudate nucleus, hypothalamus, frontal cortex, hippocampus, and thalamus varied 15-fold, hypothalamus showing the highest level (2.21 nmol/g) and cerebellum the lowest (0.15 nmol/ g). The concentrations of t-MIAA and t-MH were significantly correlated in all regions except midbrain, which had relatively more t-MIAA. Probenecid did not alter whole-brain t-MIAA levels. Treatment with pargyline, an inhibitor of monoamine oxidase, lowered the t-MIAA levels in all regions.