<Emphasis Type="Italic">S</Emphasis>-adenosylmethionine and <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine levels in the aging brain of APP/PS1 Alzheimer mice

Hyperhomocysteinemia and factors of homocysteine metabolism, S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet), may play a role in Alzheimer’s disease (AD). With liquid-chromatography-tandem-mass-spectrometry AdoMet and AdoHcy were determined in brains of 8- and 15-month-old APP/PS1 Alzheimer mice, and their possible roles in AD brains investigated. The finding that AdoMet levels do not differ between the genotypes in (young) 8-month-old mice, but are different in (older) 15-month-old APP/PS1 mice compared to their wild-type littermates, suggests that alterations in AdoMet are a consequence of AD pathology rather than a cause. During aging, AdoMet levels decreased in the brains of wild-type mice, whereas AdoHcy levels diminished in both wild type and APP/PS1 mice. The finding that AdoMet levels in APP/PS1 mice are not decreased during aging (in contrast to wild-type mice), is probably related to less demand due to neurodegeneration. No effect of the omega-3 fatty acid docosahexaenoic acid (DHA) or cholesterol-enriched diets on AdoMet or AdoHcy levels were found.     

Inhibition of transmethylation disturbs neurulation in chick embryos

Periconceptional folic acid supplementation can reduce the occurrence of neural tube defects. A low folate status will result in reduced remethylation of homocysteine (Hcy) to methionine and, subsequently, in a rise of Hcy levels. Indeed, elevated Hcy concentrations have been reported in mothers of children with neural tube defects. In our previous study, we showed that treatment of chick embryos with Hcy resulted in a delay of neural tube closure in an in vitro model. In the present study, we examined whether this effect of Hcy is due to inhibition of transmethylation via elevation of S-adenosylhomocysteine (AdoHcy). Transmethylation involves methylation of DNA, RNA and proteins by donation of a methyl group from S-adenosylmethionine (AdoMet). After application of inhibitors of S-adenosylhomocysteine hydrolase and of methionine adenosyltransferase, a delay of anterior neuropore closure, comparable to that observed after Hcy treatment, was observed. The changes in AdoMet and AdoHcy concentrations confirmed the inhibition of S-adenosylhomocysteine hydrolase or methionine adenosyltransferase, respectively, and the AdoMet/AdoHcy ratio was decreased in all cases, indicating reduced transmethylation. Moreover, the inhibition of methionine adenosyltransferase was prevented by pretreatment with methionine. This study, therefore, indicates that the Hcy-induced delay of the neural tube closure is caused by the inhibition of transmethylation via elevation of AdoHcy levels and a reduction of the AdoMet/AdoHcy ratio.     

同型半胱氨酸与兔脑动脉损伤的关系及维生素B6、B12、叶酸对其预防作用的探讨

目的探讨高蛋氨酸(Met)喂饲兔引发高同型半胱氨酸(Hcy)血症与脑动脉损伤的关系,同时观察补充VitB6、VitB12、叶酸对血Hcy水平和动脉损伤的影响.方法采用纯种雄性新西兰兔26只,分为三组对照组、高蛋氨酸组、干预组,分别喂以普通兔饲料每只200g/d、普通饲料添加0.5%Met、普通饲料每天每只兔添加0.5%Met、叶酸2.5mg、VitB6 10mg、VitB12 200mg,喂养6个月,测定血浆总Hcy(tHcy),光镜检测脑动脉组织学改变.结果实验前血浆tHcy浓度三组间无明显差异,实验后断食2 h和7 h血tHcy浓度高Met组明显高于对照组(P＜0.01),而干预组血tHcy浓度明显低于高Met组(P＜0.01),但仍高于对照组.光镜组织学检测发现高Met组和干预组脑动脉可见内皮细胞坏死、脱落、溃疡形成,附壁血栓,中膜平滑肌散乱疏松.结论高Met引发高Hcy血症对脑动脉有损伤,且VitB6、VitB12、叶酸的补充可以降低高Met引发的高Hcy浓度的水平.     

Two-phase [11C]L-methionine PET in childhood brain tumors

Thirteen children (1.8-15.8 years of age) with brain tumors were studied with [11C]L-methionine positron emission tomography (METPET). Patients were injected intravenously with tracer before a baseline PET scan was obtained. To assess the sensitivity of [11C]L-methionine uptake to competitive inhibition, 10 patients received oral L-phenylalanine (100 mg/kg); 1 hour later, a second METPET was obtained. Subjective assessment of [11C]L-methionine uptake closely paralleled results of quantitative examination (r = 0.81). [11C]L-methionine uptake in tumor-containing brain was increased in 11 patients (mean ratio of [11C] radioactivity in tumor to normal brain: 1.5 +/- 0.57; range: 1.13-2.98). Increased tracer uptake occurred in ependymomas (3), medulloblastoma (1), and astrocytomas (5), but was less intense in low-grade tumors. L-phenylalanine reduced L-methionine uptake (25-69%) in 70% of studies. L-methionine uptake was not sensitive to competitive inhibition in brain radiation injury. Two-phase METPET is of potential value in difficult clinical situations evident in children with brain tumors, including the differential diagnosis of tumor recurrence and cerebral radiation injury.     

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

The accumulation of adenosine 3',5'-monophosphate in cerebral cortical slices of the quaking mouse, a neurologic mutant

Norepinephrine, adenosine, veratridine, and adenosine-biogenic amine combinations elicit an accumulation of cyclic AMP in cerebral cortical slices from C57B1/6J control (+/+), quaking (qk/qk) and heterozygous (qk/+) mice. The percent conversion of radioactive adenine nucleotides to cyclic AMP in slices previously incubated with radioactive adenine or adenosine is markedly lower in slices from quaking and heterozygous mice compared to controls with all stimulatory agents except an adenosine-norepinephrine combination. Total incorporation of radioactive adenine or adenosine into adenine nucleotides is, however, significantly higher with slices of quaking and heterozygous mice. The absolute amount of radioactive cyclic AMP and the levels of endogenous cyclic AMP are nearly identical in the 3 groups of mice following incubation with all stimulatory agents except adenosine. The absolute accumulation of both radioactive and endogenous cyclic AMP was significantly lower in quaking and heterozygous mice after incubation with adenosine. The ratio of [¹⁴C]- to [³H]cyclic AMP in slices previously incubated with [¹⁴C]adenine and [³H]adenosine is dependent upon the stimulatory agent. The ratio with each agent is consistently lower in quaking mice compared to controls. The data provide evidence for biochemical alterations in nucleotide metabolism in cortical slices ofboth quaking and heterozygous mice. These effects would not appear to be directly associated with hypomyelination and tremor of the quaking mouse, since the heterozygous mouse, with one quaking gene does not show the latter gross abnormalities.     

The behavioral effects of L-methionine and related compounds in rats and mice.

Several groups of investigators have reported that the administration of L-methionine, with or without a monoamine oxidase inhibitor, induced an acute florid psychotic reaction in 40 percent of schizophrenics tested. The mode of action of L-methionine in brain is unknown, but may be via one or more of three mechanisms: the excess methionine (i) may lead to the production by transmethylation of some psychotomimetic methylated derivative of dopamine or serotonin, or (ii) could result in an increase in the levels of a metabolite of methionine (e.g., homocysteine, cystathionine, or cysteine), or (iii) may effect the cellular uptake of other amino acids. In order to test the first two hypotheses, L-methionine, betaine (another metyl group donor), L-methionine plus L-serine, L-cysteine, L-serine, and saline (as a control) were studied on the sleep-wake cycles of random-bred Swiss mice and on the avoidance behavior or rats. L-methionine plus L-histidine, L-methionine plus nicotinamide, L-histidine, and nicotinamide were also tested in the mice. Daily injections of 250 mg/kg of these compounds were administered for at least 21 consecutive days. Schedule performance in the rat and the sleep-wake cycles of the mice were monitored during this period and compared to controls. L-Methionine induced behavioral and sleep cycle disturbances which were removed by the simultaneous administration of L-serine but not by the addition of L-histidine or nicotinamide. These data suggest that the disruption may be due to an increase in the levels of one of the metabolities of methionine, homocysteine, rather than to an increase in the number of available methyl groups.     

Specificity of axonal transport in C2, a histaminergic neuron of Aplysia californica

Several labeled neurotransmitter substances or precursors ( [3H]histamine, [3H]serotonin, and [3H]choline) were injected with pressure into the cell body of C2, an identified putative histaminergic neuron in the cerebral ganglion of Aplysia in order to study the selectivity of axonal transport. We examined transport of these substances along the posterior lip nerve which contains one of the 3 extraganglionic axons of C2. The distribution of radioactivity along this axon indicated that some of the [3H]histamine is moved by fast transport, and some by diffusion. The velocity at which [3H]histamine moved along the axon was estimated at approximately 50 mm per day at 23 degrees C. The movement was slower at lower temperatures, and was partially inhibited by colchicine. Transport was selective: 6 h after injection of [3H]histamine into the cell body of C2, most of the radioactivity that appeared in axons was in the form of histamine. In contrast, equal amounts of the labeled amine and its metabolite, gamma-glutamylhistamine, were found in the cell body. Fast transport was not observed when [3H]serotonin or [3H]choline were injected. Subcellular fractionation experiments after injection showed that [3H]histamine was enriched in particulate fractions; [3H]gamma-glutamylhistamine was recovered only in the soluble fraction. Packaging of [3H]histamine in C2 was not affected by treatment with reserpine; in contrast, subcellular fractionation experiments indicated that reserpine blocks the uptake of [3H]histamine into vesicles in the giant cerebral neuron, an identified serotonergic Aplysia cell.     

Adenosine receptors of cerebral microvessels and choroid plexus

We studied, by ligand binding methods, the two adenosine receptors, A1 and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]L-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity (Kd approximately 250 nM) and low-affinity (Kd approximately 1-2 microM) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these micro-vessels.     

Histamine turnover in rat brain

The half-life of histamine in the rat brain was measured by two independent techniques. Intravenous injection of inhibitors of histidine decar?ylase (brocresine or α-hydrazinohistidine) produced a prompt 20–25% depletion of histamine in hypothalamus and thalamus-midbrain. From the slope of the depletion curves, histamine in these regions was estimated to have a half-life of about 30 sec. A method was developed to estimate histamine half-life from the time course of [³H]histamine appearance following intraventricular injection of [³H]histidine. With this method, our data indicate a half-life for hypothalamic histamine of no more than about 1 min, much shorter than values in other parts of the body, where histamine turnover takes hours or weeks. By comparing the extent of reduction of newly synthesized [³H]histamine with that of endogenous histamine, it was determined that all histamine in the brain regions examined apparently lies within this rapidly turning over pool.     

无症状脑梗死患者胱抑素C、同型半胱氨酸和血脂的相关性分析

目的分析无症状脑梗死患者血清胱抑素C、同型半胱氨酸（Hcy）、血脂等危险因素,探讨其以上指标与无症状脑梗死的相关性。方法选取84例经头颅磁共振（MR）诊断为无症状脑梗死（SCI）患者为实验组及57例头颅MR正常患者为对照组,观察临床危险因素及相关实验室检验指标,进行统计学分析。结果无症状脑梗死血胱抑素C（Cystic C）、同型半胱氨酸（Hcy）、血脂指标中低密度脂蛋白（LDL）、脂蛋白a[Lp（a）]水平均高于对照组,两组间的差异均具有统计学意义（P〈0．05）。结论胱抑素C、同型半胱氨酸、血脂指标中LDL、Lp（a）的升高均是无症状脑梗死发病的的危险因素。     

Degeneration of CA1 neurons in hippocampus after ischemia in Mongolian gerbils: Cyclic AMP-systems

Bilateral ischemia induced by occlusion of the carotid arteries for 5 min causes a selective degeneration of CA 1 neurons of the hippocampus of Mongolian gerbils. The degeneration process is complete in 14 days as assessed by light microscopy. After one week, basal values for radioactive cyclic AMP in [3H]adenine-labeled tissue from the CA 1 region of hippocampus are greatly reduced as are the absolute magnitude of accumulations of cyclic AMP elicited by norepinephrine, 2-chloroadenosine and histamine. At 2 and 4 weeks, basal values for radioactive cyclic AMP have nearly attained control values and the response to 2-chloroadenosine is fully restored. The response of cyclic AMP-generating systems to norepinephrine is now significantly greater than in control, while the response to histamine remains reduced in magnitude. The ischemia has no effect on basal values for radioactive cyclic AMP or on responses in [3H]adenine-labeled slices from cerebral cortex. Histamine levels after ischemia are significantly increased above control in the CA 1 region. Basal and histamine-sensitive adenylate cyclase activity in the membrane preparations are slightly decreased during the first week after ischemia, followed by a recovery. There is an inverse correlation between histamine levels and adenylate cyclase activity in individual animals 4 days after ischemia.     

Effects of intraventricular injection of 6-hydroxydopamine in the developing kitten. II. On the central monoaminergic innervation

The intraventricular administration of 6-hydroxydopamine (6-OHDA) to kittens between 5 days and 4 months of age induced marked changes in the endogenous levels of DA, NE and 5-HT in various brain areas. In contrast to the well-known selective effect of 6-OHDA against catecholaminergic neurones in the rat, serotoninergic neurones were also markedly affected by 6-OHDA treatment in kittens; particularly in the hippocampus and the colliculi, 5-HT levels were markedly and permanently decreased after the intraventricular administration of 6-OHDA. A significant but less pronounced reduction in 5-HT levels was also noted in other areas such as the piriform cortex, the cerebral neocortex, the cerebellum and the septum. Only very discrete changes were detected in the caudate nucleus, the olfactory tubercle and the raphe area. The administration of chlorimipramine (10 mg/kg, i.p.) 1 h before 6-OHDA treatment completely prevented the effects of the neurotoxic agent on serotoninergic innervation. Marked regional differences were also noted concerning the effects of 6-OHDA treatment on dopaminergic neurones. Whereas DA levels in the raphe area and the hypothalamus were almost unaffected, they were permanently reduced by about 50% in the caudate nucleus and the olfactory tubercle after the intraventricular administration of 6-OHDA. In the caudate nucleus, the reduction was even much more pronounced (−90%) when 6-OHDA was administered during the first 3 postnatal weeks.

In most forebrain areas (hippocampus, piriform cortex and cerebral neocortex) and in the cerebellum, NE levels were permanently reduced to about 10% of those of control kittens as soon as the third day following the intraventricular injection of 6-OHDA. In contrast, after a transient drop, NE levels in the lateral brain stem (containing the locus coeruleus) of 6-OHDA-treated kittens returned and even surpassed (+100%) those found in age-paired controls. Analyses of the characteristics ofl-[³H]NE uptake in synaptosomes indicated that 6-OHDA treatment resulted in both a striking loss of specific uptake sites in forebrain areas and a significant increase in the V_max of the NE uptake process in synaptosomes from the lateral brain stem. However, in contrast to the rapid increase in NE levels, this change in V_max occurred much later, since it was first detected at more than one month after 6-OHDA treatment. This delay suggests that the doubling in NE levels occurring for the first month following the intraventricular administration of 6-OHDA simply resulted from an increased accumulation of the catecholamine in noradrenergic terminals in the lateral brain stem. Later on, the change in V_max might indicate a sprouting of new noradrenergic terminals in the vicinity of the locus coeruleus area. The intraventricular administration of 6-OHDA resulted in a significant increase in the maximal stimulatory effect ofl-isoproterenol on adenylate cyclase activity in homogenates of the cerebral cortex and the lateral brain stem. Therefore, not only the degeneration (in the cerebral cortex) but also the proliferation (in the lateral brain stem) of noradrenergic terminals were associated with an increase in the density of beta-adrenergic receptors. These results are discussed in relation to the possible function of the noradrenergic sprouts in the lateral brain stem.     

Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by β-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex

We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the β-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al.J. Neurosci. 14 (1994) 2953–2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 μM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely β-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.     

Effect of CDP-choline on the biosynthesis of phospholipids in brain regions during hypoxic treatment

Acute administration of CDP-choline (i.p. 100 mg/Kg b.w.), 10 min before the intraventricular injection of labeled precursors, [2-³H]glycerol and [1-¹⁴C]-palmitate, was able to correct the impairment caused by hypoxic treatment of lipid metabolism in some brain regions, ie, cerebral hemispheres, cerebellum, and brainstem. After CDP-choline treatment, an increase of the specific radioactivity of total lipids and of phospholipids was observed in mitochondria purified from the three above-mentioned brain regions of the hypoxic animals, while no effect on the other subcellular fractions was found. CDP-choline had a stimulating effect particularly on the incorporation of both precursors into mitochondrial PC, PE, and polyglycerophosphatides isolated from the three brain regions examined. The results obtained show that the action of CDP-choline in restoring lipid metabolism was more pronounced in brain mitochondria, which, among subcellular fractions, were the most affected by the hypoxic treatment.     

Attenuation by chronic imipramine treatment of [H]clonidine binding to cortical membranes and of clonidine-induced hypothermia: the influence of central chemosympathectomy

Central chemosympathectomy with intraventricular injection of 250 μg of 6-hydroxydopamine, resulting in depression of cerebral noradrenaline level by 75%, led to an increase in specific binding of [³H]clonidine to cerebral cortical membranes, but did not affect the hypothermic response to clonidine. Chronic imipramine treatment depressed [³H]clonidine binding and attenuated clonidine-induced hypothermia similarly in non-chemosympathectomized and chemosympathectomized rats.     

A comparison of the in vivo uptake of [H]GABA into rat celebral cortex and dorsal medulla following topical application

The penetration of [³H]GABA and [¹⁴C]sucrose from the dorsal surface of the rat medulla and cerebral cortex was studied in vivo. The levels of both declined exponentially from the surface of both regions and were decreased by raised potassium levels and the presence of GABA uptake inhibitors. Inhibition of GABA metabolism increased the amounts of ³H recovered particularly after long incubation periods. More [³H]GABA and [¹⁴C]sucrose was taken up into the cerebral cortex than the medulla, and more [³H]GABA than [¹⁴C]sucrose was taken up in both regions.     

The role of the cAMP-PKA system in the short-term regulation of striatal [C]-2-deoxyglucose uptake in freely moving rats

The cyclic adenosine monophosphate (cAMP)-protein kinase (PK) A system has been shown to have stimulatory effects on glucose utilization in various tissues in vitro. However, little is known about the influence of cAMP on glucose utilization in vivo. In the present study, we examined how cAMP-related compounds affected [¹⁴C]-2-deoxyglucose (DG) uptake in the striatum of freely moving rats. An intrastriatal injection of dibutyryl-cyclic adenosine monophosphate (db-cAMP), although increasing local cerebral blood flow, was found to decrease the uptake of [¹⁴C]-2-DG in the striatum. This decrease of [¹⁴C]-2-DG uptake in the striatum was completely blocked by pretreatment with Rp-adenosine-3′,5′-cyclic monophosphorothioate triethylamine (Rp-cAMPS). Moreover, intrastriatal infusion of Rp-cAMPS alone produced a striking increase of [¹⁴C]-2-DG uptake in the striatum. These results strongly suggest that transient activation of the cAMP-PKA system can depress the glucose phosphorylation process of the rat brain in vivo.     

Post- and presynaptic lesions in the CA1 region of hippocampus: Effect on [3H]forskolin and [3H]phorboldibutyrate ester binding

Summary The effect of transient cerebral ischemia and intraventricular injection of kainic acid on adenylate cyclase and protein kinase C as labeled by [³H]forskolin ([³H]FOR) and [³H]phorboldibutyrate ester ([³H]PDBU) in several rat brain microregions was investigated in a quantitative autoradiographic study. Four days after transient four vessel occlusion a 80% loss of [³H]FOR and a 35% loss of [³H]PDBU binding could be measured in the CA1 stratum radiatum of operated Wistar rats as compared to control rats. Four days after intraventricular injection of kainic acid only a marginal loss of [³H]FOR and a 30% increase of [³H]PDBU binding was seen in the CA1 stratum radiatum while in the CA3 stratum lucidum and radiatum respectively a 30% loss of [³H]FOR and no significant change in [³H]PDBU binding was observed. As transient cerebral ischemia and intraventricular kainic acid injection are depleting the hippocampal CA1 region of CA1 pyramidal cells and axons of CA3 pyramidal cells respectively in rat brain, these findings strongly suggest that both adenylate cyclase and protein kinase C are localized in CA1 pyramidal cells of rat hippocampus.Part of this study has been presented at the 16th C.I.N.P. Congress, Munich, August 15–19, 1988.