Effect of 4-aminopyridine on transmitter release in synaptosomes

The effect of 4-aminopyridine (4-AP) on the release of labeled transmitters in mouse brain synatosomes was studied in a superfusion system. 4-AP at μM concentrations notably stimulated the spontaneous release of labeled GABA and glutamate, and of acetylcholine (ACh) derived from tritiated choline. No effects on the release of labeled α-aminoisobutyric acid were observed. The stimulation of GABA and ACh release was dependent on the presence of Ca²⁺ in the superfusion media, whereas the effect on glutamate release was more variable and no clear Ca²⁺-dependence was observed. In contrast to these results, 4-AP did not have any effect on the release of the above transmitters by K⁺-depolarization in the presence of Ca²⁺. These results are discussed in terms of the possible participation of Ca²⁺ in the action of 4-AP on spontaneous transmitter release in isolated nerve endings.     

Effect of hyperbaric oxygen on GABA transport in rat brain synaptosomes

Summary Initial velocities of uptake of GABA have been measured in rat brain synaptosomes from animals which had been exposed to oxygen at high pressure (OHP) and compared to similar measurements in normobaric controls. For hypothalamus, no changes in GABA uptake occurred subsequent to exposure to OHP. For cortical synaptosomes, however, exposure to OHP resulted in a decreased velocity of GABA uptake at all combinations of [Na] and [GABA] used. The OHP data were found to fit the same transport model as found previously for control data. Thus, OHP exposure did not alter the basic mechanism by which sodium and GABA interact with the carrier in the process of transport. However, the constants which quantitate the model were changed by OHP exposure. As a consequence, the several kinetic parameters which are calculated from the model change in the OHP animals. These kinetic parameters are compared to similar calculations for both normobaric control animals and normobaric aged animals. Although the effects of OHP do not precisely parallel the effects of aging, the alterations in kinetic parameters are in several ways similar in the aged and OHP animals.     

Effect of phenytoin on [H]norepinephrine release from synaptosomes

Phenytoin reduces depolarization-linked [³H]norepinephrine release from rat brain synaptosomes. When choline chloride was substituted for NaCl the phenytoin effect was attenuated but still significant. This is consistent with the theory that phenytoin reduces both Na and Ca influx during depolarization.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Effect of acetyl-L-carnitine on the dopaminergic system in aging brain.

We studied the effect of acetyl-L-carnitine (ALCAR) on dopamine release and the effect of long-term acetyl-L-carnitine treatment on age-related changes in striatal dopamine receptors and brain amino acid levels. In striatal tissue that had been incubated with [3H]dopamine, acetyl-L-carnitine increased the release of [3H]dopamine evoked by electrical stimulation. In striatal tissue from aged mice administered acetyl-L-carnitine for 3 months, the release of [3H]dopamine evoked by electrical stimulation was higher than that of its aged control; the release after a second stimulation was similar in the two groups. There was a significant decline in the number of D1 striatal dopamine receptors with age. The Bmax was 51% lower in 1.5-year-old mice than in 4-month-old animals. Administration of acetyl-L-carnitine for 3 months diminished the reduction in the binding of [3H]SCH-23390. [3H]Spiperone binding to D2 receptors was not decreased with age and was not affected by acetyl-L-carnitine treatment. Age-related decreases in levels of several amino acids were observed in several brain regions. Acetyl-L-carnitine lessened the reduction in the level of taurine only in the striatum. The findings confirm the multiple effects of acetyl-L-carnitine in brain, and suggest that its administration can have a positive effect on age-related changes in the dopaminergic system.     

A comparison of sodium dependent GABA transport in cortical synaptosomes from Long-Evans and Sprague-Dawley rats

Summary The sodium dependent, high affinity transport of GABA has been studied in cortical synaptosomes from Sprague-Dawley (SD) rats and the results compared to previous results from Long-Evans (LE) rats. Initial velocity of uptake was measured as a function of both GABA and sodium concentration. These data were then fitted to the rate equation for the model which was found to give minimal best fit to similar data from Long-Evans animals. An excellent fit was obtained; the average per cent error between experimental data and model predictions is only 2.55%. No simplification of the model could be made without lessening the goodness of fit. Thus there are no fundamental differences between the two groups in the mechanism by which carrier, sodium, and GABA interact in the process of transport.Although the same model was found to fit the velocity data from both groups of animals, there are differences in the constants which quantitate the model. As a consequence, uptake at a given sodium and GABA concentration may be different for the two groups. The rate equation for the model permits certain functions to be defined in terms of dissociation and translocation constants, GABA, sodium, and total carrier concentrations. The best fit constants were used to calculate these functions, which were then utilized to demonstrate further the quantitative similarities and differences in the transport mechanism in the two groups of animals. In general, those parameters reflective of carrier affinity for sodium or GABA indicated greater affinities in the LE group, while those parameters reflective of carrier concentration or rate constants indicated greater carrier concentration, rate constants, or both, for SD animals.     

Release-regulating GABAA receptors are present on noradrenergic nerve terminals in selective areas of the rat brain

The effects of gamma-aminobutyric acid (GABA) on the spontaneous release of [3H]-norepinephrine ([3H]-NE) were investigated by means of superfused synaptosomes prepared from different areas of the rat brain and prelabeled with [3H]-NE. GABA increased in a concentration-dependent way (1-300 microM) the release of [3H]-NE in hippocampal synaptosomes. The effect of GABA was mimicked in part by muscimol. Similar effects were observed in cerebral cortex synaptosomes where GABA and muscimol were however less potent than in hippocampus. No effect could be observed in hypothalamic synaptosomes. Bicuculline antagonized the effect of muscimol and that of low concentrations of GABA (below 10 microM). Above 10 microM, the [3H]-NE releasing effects of GABA became progressively less sensitive to bicuculline. (-)-Baclofen did not affect the spontaneous release of [3H]-NE. It is concluded that release-regulating receptors of the GABAA subtype are present on NE nerve terminals in selective areas of the rat brain.     

A 23187-stimulated calcium uptake and GABA release by cerebrocortical synaptosomes: effects of high pressure

Summary Guinea pig cerebrocortical synaptosome preparations were used to study the effect of compression to 62 ATA on⁴⁵Ca²⁺ uptake and [³H]GABA release using a calcium ionophore A23187, which bypasses the voltage-sensitive calcium channel. Pressure was found to exert a suppressive effect on the A 23187-induced release of [³H]GABA, while having no significant effect on A 23187-stimulated⁴⁵Ca²⁺ uptake. On the other hand, both depolarization-induced⁴⁵Ca²⁺ uptake and [³H]GABA release were inhibited by pressure exposure. These results suggest that pressure may suppress GABA release by affecting pre-synaptic events subsequent to calcium influx.     

Chronic fluoxetine treatment upregulates 5-HT uptake sites and 5-HT2 receptors in rat brain: An autoradiographic study

This study was undertaken to investigate the effect of chronic treatment with fluoxetine, a selective serotonin uptake inhibitor used widely in the treatment of depression, on the distribution and density of 5-HT uptake sites, 5-HT₂ receptors, and vesicular amine uptake sites in rat brain. Fluoxetine (10 mg/kg i. p.) was administered daily for 21 days. The density of 5-HT uptake sites labelled by [³H]paroxetine, 5-HT₂ receptors labelled by [³H]ketanserin in presence of tetrabenazine and vesicular amine uptake sites labelled by [³H]ketanserin in the presence of mianserin were measured by quantitative autoradiography in 22 areas of rat brain, using coronal tissue sections. Chronic administration of fluoxetine produced significant increases in the density of 5-HT uptake sites in layers of frontoparietal cortex (by 32–43%), of striate cortex (by 55%), in CA1 field of hippocampus (by 111%) and in superior colliculus (by 20%). Fluoxetine treatment also resulted in upregulation of 5-HT₂ receptors in layers of frontparietal cortex (31–38%) and in CA2-3 fields of hippocampus (by 39%). The density of tetrabenazine-sensitive vesicular amine uptake sites in the caudate-putamen was also significantly increased (by 66%). The observed alterations in 5-HT uptake site and 5-HT₂ receptor densities are likely a part of adaptive neuronal changes that occur after chronic administration of fluoxetine and may be related to the antidepressant effect of the drug. © 1993 Wiley-Liss, Inc.     

Autoradiographic localization of calcium channel antagonist receptors in rat brain with [H]nitrendipine

In vitro autoradiographic techniques have been used to localize [³H]nitrendipine binding sites in the rat brain. The superficial cerebral cortex, the ventral, lateral and posterior nuclei of the thalamus, the molecular layer of the dentate gyrus, the substantia nigra and the external plexiform layer of the olfactory bulb, all contain high densities of silver grains. The level of binding sites are greatly reduced in areas low in synaptic connections. The corpus callosum, the alveus hippocampi and the dorsal commissure of the fornix all lack specific grains, as does the lateral olfactory tract of theolfactory bulb. Specific silver grains are not found in the habenula or the hypothalamus. Grains are not associated with blood vessels profiles. The discrete localizations of [³H]nitrendipine bindings sites suggest a specific synaptic role.     

Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain

We investigated the effect of 10 months ovariectomy and a correction therapy, 2 weeks before the rats were killed, of oestradiol, progesterone or their combination on NMDA and AMPA receptor binding in the hippocampus, dentate gyrus, striatum, nucleus accumbens and frontal cortex of the rat brain as well as on amino acid levels in frontal cortex. NMDA and AMPA binding densities were assayed by autoradiography using, respectively, L-[3H]glutamate and [3H]AMPA; amino acid concentrations were measured by high performance liquid chromatograhy (HPLC) coupled with UV detection. Ovariectomy was without effect on NMDA and AMPA binding density in all brain regions assayed except in the hippocampal CA1 region and dentate gyrus where it decreased NMDA binding density compared to intact rats values. Oestradiol restored and increased NMDA binding density in the CA1 subfield and the dentate gyrus of ovariectomized rats but, by contrast, it decreased binding density in the striatum and in the frontal cortex while having no effect in the CA2/3 subfield of the hippocampus and in the nucleus accumbens. Oestradiol was without effect on AMPA binding density in the hippocampus and the dentate gyrus but it reduced AMPA binding density in the striatum, the frontal cortex and the nucleus accumbens. Progesterone, and oestradiol combined with progesterone, decreased NMDA but not AMPA binding density in the frontal cortex of ovariectomized rats, and they were without effect on these receptors in the other brain regions assayed. Amino acid concentrations in the frontal cortex were unchanged after ovariectomy or steroid treatments. The effect of oestradiol in the hippocampus confirmed in the present study and our novel findings in the frontal cortex, striatum and nucleus accumbens may have functional significance for schizophrenia and neurodegenerative diseases.     

Heterogeneity of opioid receptor binding in brain slices

A methodological approach was established for the study of ligand binding to multiple opioid receptors in slices from rat brain striatum. Specific binding of radiolabeled opiates was resolved from total binding with enantiomers or excess unlabeled ligand. Equilibrium binding of triated etorphine, dihydromorphine, and ethylketocyclazocine, and competitive displacement of [3H]etorphine and [3H]dihydromorphine by the unlabeled opiates were used to assess both high and low affinity receptor sites. The high-affinity binding components of the radiolabeled opiates were characterized by linear Scatchard plots, Kd values of 2.8-3.7 nM, and binding site densities of 180-297 fmol/mg protein. The displacement of [3H]etorphine by morphine and ethylketocyclazocine displayed Hill coefficients of 0.62 and 0.47, respectively, and revealed receptor sites with much lower affinities than those described by the direct binding of these opiates. On the other hand, both morphine and ethylketocyclazocine displaced [3H]dihydromorphine with similar high potencies (apparent Kd's, 3-4 nM). The results support the feasibility of using brain slices as a cellular preparation to study opioid receptor mechanisms.     

胞浆型磷脂酶A2在实验性自身免疫性脑脊髓炎小鼠脊髓中的表达

目的研究胞浆型磷脂酶A2（cPLA2）在实验性自身免疫性脑脊髓炎（EAE）小鼠发病不同时期的表达。方法8～10周龄的雌性C57BL／6小鼠30只随机分为2组，EAE组（n=20）以髓鞘少突胶质细胞糖蛋白（MOG）35-55多肽加福氏完全佐剂皮下注射诱发EAE，对照组（n=10）则用PBS液取代MOG35-55多肽。用免疫组织化学方法观察EAE小鼠发病后第0天（初期）、第7天（高峰期）及第30天（恢复期）脊髓中cPLA2的表达情况。结果cPLA2在EAE发病初期及高峰期脊髓内表达明显增多，初期在绝大多数血管内皮细胞中表达，高峰期时血管周围炎性细胞中表达相对增多，恢复期时表达下调；对照组小鼠脊髓中则没有cPLA2表达。结论cPLA2在EAE发病不同时期表达存在差异，可能与EAE发生及发展有密切联系。     

铝对大鼠脑细胞三磷酸肌醇合成的影响

目的:通过铝对大鼠脑细胞内三磷酸肌醇(IP3)合成的影响,探讨铝的神经毒性机制。方法:取摄用含Al2(SP4)32.5%饮水6个月大鼠及未摄铝同龄对照组大鼠大脑皮质,以AmershamIP3测试系统测定各组脑组织IP3含量并观察M受体激动剂carbachol及KCl(40mM)对IP3合成的影响。结果:摄铝大鼠脑组织基础IP3含量明显低于对照组(P<0.01),铝抑制carbachol及K+诱发的IP3的合成(P<0.01)。结论:高浓度铝降低基础IP3的含量,并抑制carbachol及K诱发的IP3的生物合成。抑制G蛋白磷脂酶C偶连及IP2敏感的磷脂酶C为铝对脑细胞毒性的机制之一。     

Secreted phospholipase A2 potentiates glutamate-induced calcium increase and cell death in primary neuronal cultures

Secreted phospholipases A2 (sPLA2s) modulate neuronal survival and neurotransmitter release. Here we show that sPLA2 (group III) synergistically increases glutamate-induced cell death and intracellular calcium ([Ca2+]i) in cultured primary cortical and hippocampal neurons. Whereas 1 microM glutamate elicited transient [Ca2+]i increases in all neurons that recovered 66% to baseline, 25 ng/ml sPLA2 pretreatment resulted in sustained [Ca2+]i increases, with only 5% recovery. At 250 nM glutamate, 25% of neurons failed to respond, and the average recovery time was 101 +/- 12 sec; sPLA2 increased recovery time to 158 +/- 6 sec, and only 2% of cells failed to respond. Both the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the calcium-channel blocker cobalt inhibited this effect. Experiments with the glutamate uptake inhibitor L-trans-pyrollidine-2,4-dicarboxylic acid (2.5 microM) indicated that glutamate uptake sites are not a likely modulation point by sPLA2, whereas arachidonic acid (AA) potentiated calcium responses to glutamate. Thus the enhancement of glutamate-induced [Ca2+]i increases by sPLA2 may be due to modulation at NMDA receptors and/or calcium channels by AA. These results indicate that sPLA2 affects neuronal responses to both nontoxic (0.1-10 microM) and toxic (=25 microM) concentrations of glutamate, implicating this enzyme in neuronal functions in pathology.     

血浆脂蛋白相关磷脂酶A2水平对进展性脑梗死的预测价值

目的研究血浆脂蛋白相关磷脂酶A2(Lp-PLA2)水平对进展性脑梗死的预测价值。方法采用双抗体夹心酶联免疫吸附法检测168例急性脑梗死患者和72名正常对照者的血浆Lp-PLA2水平。ACI患者每天进行美国国立卫生研究院卒中量表(NIHSS)评分,发病第3 d NIHSS评分比发病第1 d增加≥2分者为进展性脑梗死(进展性脑梗死组),<2分者为稳定性脑梗死(稳定性脑梗死组)。结果 168例ACI患者中进展性脑梗死40例,稳定性脑梗死128例。ACI组血浆Lp-PLA2水平明显高于正常对照组(P<0.01),进展性脑梗死组明显高于正常对照组及稳定性脑梗死组(均P<0.01);稳定性脑梗死组与正常对照组的差异无统计学意义。结论进展性脑梗死患者发病后血浆Lp-PLA2水平明显升高,其可能为早期预测进展性脑梗死发生的生物学指标。     

H型高血压和脂蛋白相关磷脂酶A2对脑白质疏松症严重程度及预后的影响

目的 探讨H型高血压和脂蛋白相关磷脂酶A2(Lp-PLA2)对脑白质疏松症(LA)严重程度及预后的影响.方法 前瞻性入选沧州市中心医院2016年3月至2018年3月行头颅CT检查诊断有LA的住院患者,入院后24h内空腹化验血清Lp-PLA2;根据有无高血压和高同型半胱氨酸血症(Hhcy),把LA患者分为:A.同时伴高血...     

Oxygen dependence of dopamine-β-hydroxylase activity and lactate metabolism in synaptosomes from rat brain

Dopamine-β-hydroxylase activity can be assayed in vitro in suspensions of rat hypothalamic synaptosomes by following tritium release from [2-³H]dopamine in the presence of monoamine oxidase inhibitors. The activity is inhibited by diethyldithiocarbamate, fusaric acid, amphetamine, reserpine, and desipramine. It is not sensitive to addition of tyrosine at the concentration present in cerebrospinal fluid, to added fumarate or to a rat heart extract known to contain potent inhibitors of the isolated enzyme. The reaction in air follows simple saturation kinetics with respect to variation in dopamine concentration with aK_m of 0.075 μM. There is reversible inhibition of activity when it is assayed at low oxygen tension. The apparent oxygen affinity is such that the enzyme does not appear saturated with respect to oxygen even at arterial oxygen tensions. Other reactions assayed exhibit less sensitivity to hypoxia: production of lactic acid from glucose becomes stimulated at 10–12 mm Hg; and oxidation of lactic acid is not inhibited at oxygen tensions above 3–4 mm Hg. Comparison of these data with the distribution of oxygen tensions present in brain suggests a basis for classifying oxygen dependent neurochemical reactions.     

Dopamine D2 receptors mapped in rat brain with [3H](+)PHNO

Previous work with membrane preparations had demonstrated that the agonist (+)-4-propyl-9-hydroxynaphthoxazine (PHNO) labels the high-affinity state of dopamine D₂ receptors with 67-fold selectivity over D₁ sites. In this study, quantitative autoradiography was used to examine the binding of [³H](+)PHNO to rat brain sections. Highest binding densities were found in caudate-putamen, accumbens, and olfactory tubercles, as expected, and also in specific layers of the olfactory bulb. In addition, a second group of brain regions, including lateral septum, entorhinal cortex, molecular layer of hippocampus, and several brainstem structures showed low but consistent levels of binding. In all brain regions [³H](+)PHNO binding (2 nM) was completely displaced by 10 μM sulpiride (>99%). Addition of 150 μM guanilylimidodiphosphate, which normally converts D2 receptors from high to low affinity states, abolished [³H](+)PHNO binding in all brain regions (>99%), except for the islands of Callejas. This is likely to reflect binding to D₃ sites in this area. Omission of preincubation in binding assays decreased [³H](+)PHNO binding in a regionally dependent manner, with strongest effects (22%) seen in high-density areas. These preincubation results confirm that (+)PHNO may have limitations for in vivo imaging studies. On the other hand, [³H](+)PHNOs negligible levels of non-specific binding compared to other agonists and overall selectivity would make it an excellent tool for in vitro autoradiographic monitoring of the high affinity state of D₂ receptors. © 1994 Wiley-Liss. Inc.     

Purification and characterisation of a snake venom phospholipase A2: A potent inhibitor of platelet aggregation

An inhibitor of human platelet aggregation was identified from the venom of an Australian Copperhead snake, Austrelaps superba, as a novel phospholipase A₂. The inhibitor was purified to homogeneity by chromatography on Q-Sepharose, S-Sepharose and C8 reverse phase HPLC. The purified phospholipase A₂ has a molecular weight of 15 kDa as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence analysis of the platelet inhibitor revealed 70–80% sequence identity to other previously described secretory phospholipase A₂. Phospholipase activity of the purified protein was confirmed by the ability of the enzyme to hydrolyse lecithin. Pretreatment of the purified protein with the specific phospholipase A₂ inhibitor p-bromophenacyl bromide, resulted in abrogation of both its enzyme and platelet inhibitory activity. The phospholipase A₂ inhibited platelet aggregation and serotonin release, induced by a variety of platelet agonists, in a time and dose dependent manner.