Nicotinic receptors differentially regulate N-methyl-D-aspartate damage in acute hippocampal slices

Although in neuronal cultures nicotine was reported to prevent early and delayed excitotoxic death, no studies with nicotinic drugs have been done with acute hippocampal slices. We investigated the effect of nicotine and methyllycaconitine (MLA) on the toxicity of N-methyl-d-aspartate (NMDA) in the CA1 area of hippocampal slices. The excitotoxic effect of NMDA was assessed as decreased recovery of the capability to produce synaptically evoked population spikes (PSs). Application of nicotine or MLA before NMDA application increased the recovery of PSs. This electrophysiological recovery was used as a measure of the early neuroprotective events. The neuroprotection conferred by both nicotine and MLA was inhibited by dihydro-beta-erythroidine, showing mediation of neuroprotection by alpha 4 beta 2 neuronal nicotinic receptors (nAChRs). Because nicotine activates alpha 4 beta 2 and other nAChR subtypes, whereas 10 nM MLA inhibits the alpha 7 subtype, we propose the involvement of a neuronal circuitry-dependent mechanism for nicotinic neuroprotection. The effect of nicotine downstream from the receptors was investigated using inhibitors of cell signaling. The results suggest that the effect of nicotine is mediated by tyrosine receptor kinases, 1,2-phosphatidylinositol-3 kinase, and the mitogen-activated extracellular signal-regulated kinases. Although nicotine neuroprotection is Ca2+-dependent, neither L-type Ca2+ channels nor calmodulin-dependent protein kinase is involved in the effect of nicotine. In summary, these results suggest that in acute slices nicotinic protection is initiated either by direct activation of alpha 4 beta 2 or indirectly by inhibition of alpha 7 followed by signal transduction involving tyrosine kinases, phospholipid-dependent kinases, and mitogen-activated kinases.     

Nicotinic receptor subtypes in rat hippocampal slices are differentially sensitive to desensitization and early in vivo functional up-regulation by nicotine and to block by bupropion

To identify the brain nicotinic acetylcholine receptor (nAChR) subtypes that may be involved in nicotine addiction, we investigated the actions of bupropion, a drug used in cigarette smoking cessation programs, and nicotine on three pharmacologically identified nAChRs in rat hippocampal slices, namely, type IA, type II, and type III nAChRs, likely representing alpha7, alpha4beta2, and alpha3beta4 subunits, respectively. Using whole-cell patch-clamp recordings from interneurons of acute hippocampal slices prepared from male rat pups, we studied the effect of nicotine on in vivo up-regulation and in vitro desensitization of nAChRs. Two subcutaneous injections of nicotine (0.586 mg/kg free base, in less than a day) to rats at postnatal days 14 to 15 significantly enhanced the magnitude of functional responses arising from type III and type II, but not type IA nAChRs. This treatment did not increase the functional affinity for acetylcholine at type II nAChRs. A single injection of nicotine also produced a significant increase in type III nAChR response. In addition, type III and type II, but not type IA nAChRs, are desensitized by in vitro exposure to nicotine at concentrations found in the venous blood of cigarette smokers. Bupropion at 1 muM produced 56, 15, and 0% inhibition of type III, type II, and type IA nAChR responses, respectively, in the slices. Our results suggest that in vivo-nicotine-induced nAChR up-regulation observed in neurons of intact brain tissue is a physiologically relevant phenomenon and that early up-regulation of type III and type II nAChRs could be an important biological signal in nicotine addiction.     

丙泊酚对缺氧无糖损伤大鼠海马脑片的保护作用

目的探讨丙泊酚对缺氧无糖(OGD)损伤脑片的保护作用。方法采用缺氧无糖的人工脑脊液灌流离体大鼠海马脑片模拟缺血损伤,根据给药方式不同分为:OGD前给药组和OGD给药组,均给予1、10和20μmol·L-1的丙泊酚和脂肪乳(溶剂对照)。记录海马脑片顺向群峰电位(OPS)和缺氧损伤电位(HIP)的变化。结果OGD给药组应用10μmol·L-1的丙泊酚,与应用脂肪乳相比OPS消失时间推迟(P<0.05),OPS恢复率升高(P<0.05),HIP出现时间推迟(P<0.01),HIP出现率降低(P<0.05)。结论在缺氧过程中应用10μmol·L-1的丙泊酚对缺氧无糖损伤海马脑片具有保护作用。     

Endogenous D-serine is involved in induction of neuronal death by N-methyl-D-aspartate and simulated ischemia in rat cerebrocortical slices

Emerging evidence indicates that D-serine rather than glycine serves as an endogenous agonist at glycine site of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, in several nervous tissues, including the developing cerebellum and the retina. Here, we examined whether endogenous D-serine plays a significant role in neuronal damage resulting from excitotoxic insults in the cerebral cortex, using rat brain slices maintained in a defined salt solution. Neuronal cell death induced by application of NMDA or by oxygen-glucose deprivation (simulated ischemia) was markedly suppressed by a competitive glycine site antagonist 2,7-dichlorokynurenic acid. Addition of glycine or D-serine did not augment neuronal damage by NMDA or simulated ischemia, indicating that sufficient amount of glycine site agonist(s) is supplied endogenously within the slices. Application of D-amino acid oxidase, an enzyme that degrades D-serine, markedly inhibited neuronal damage by NMDA and simulated ischemia, which was reversed by addition of excess D-serine or glycine. Sensitivity to the glycine site antagonist of NMDA- or ischemia-induced damage was not affected by the presence of a non-NMDA receptor antagonist, suggesting that kainate receptor-stimulated D-serine release as demonstrated in primary cultured astrocytes does not contribute significantly to the extent of neuronal injury in these settings. The present results suggest that endogenous supply of D-serine as a glycine site agonist is important for neuronal injury involving NMDA receptor overactivation in the cerebral cortex.     

N-甲基-D-天冬氨酸受体在缺氧缺血性脑损伤新生大鼠脑海马中的远期表达

目的:观察新生大鼠缺氧缺血性脑损伤后,海马发育过程中N-甲基-D-天冬氨酸受体的远期表达变化。方法:实验于2006-03/06在解放军第三军医大学新桥医院中心实验室完成。选用新生7日龄SD大鼠72只,按随机数字表法分为缺氧缺血性脑损伤组和假手术组,每组36只。各组又分为生后15d(n=6)、22d(n=6)、29d(n=6)、36d(n=12)及43d(n=6)5个时相点。①参照Rice法通过结扎7日龄大鼠左侧颈总动脉,吸入氧气体积分数为0.08的氮氧混合气,制成缺氧缺血性脑损伤模型,出现自发或夹尾左旋则证明模型制作成功。假手术组仅切开颈部皮肤,暴露左侧颈总动脉。②应用免疫组织化学法检测缺氧缺血后不同时点两组大鼠脑海马CA1区N-甲基-D-天冬氨酸受体NR1亚单位的表达(n=6),测量平均灰度值,灰度值越低,蛋白表达越强;生后36d时相点大鼠(n=6)利用Morris水迷宫测定学习记忆能力;最后应用电镜观察生后36d大鼠海马突触结构。结果:72只大鼠,全部进入结果分析,无脱失。①生后22-43d缺氧缺血性脑损伤组大鼠(缺氧缺血后15-36d)脑海马CA1区NR1平均灰度值分别为167.69±6.48,174.57±4.81,179.30±5.92,176.50±5.93,均显著高于同日龄假手术组148.96±4.91,151.17±6.37,152.06±9.86,156.32±6.86(P均<0.01)。②Morris水迷宫实验中,缺氧缺血性脑损伤组大鼠逃避潜伏期显著长于假手术组(48.87±9.47)s,(11.97±2.20)s,(P<0.01);原平台象限游泳距离/总游泳距离的百分比显著低于假手术组(14.45±3.85)%,(62.20±8.74)%(P<0.01)。③生后36d(缺氧缺血后29d),透射电镜下缺氧缺血性脑损伤组大鼠患侧海马突触后膜致密物较假手术组明显减少。结论:新生大鼠缺氧缺血性脑损伤后期存在海马N-甲基-D-天冬氨酸受体的表达下调,可能对大鼠远期空间学习记忆产生一定影响。     

离体海马脑片CA1区锥体细胞缺氧期间电生理指标变化与N-甲基-D-天冬氨酸受体阻断剂MK801干预后的变化

背景：海马CA1区锥体细胞是对缺血缺氧性损伤最为敏感的神经元，缺血缺氧后海马CA1区锥体细胞膜电位表现为缺氧早期细胞膜的超极化，随着缺氧时间的延长，细胞膜发生缓慢去极化及快速去极化，引起神经元不可逆性损伤。目的：应用细胞内记录技术，观察N-甲基-D-天冬氨酸受体阻断剂MK801对离体海马脑片CA1区锥体细胞缺氧期间电生理指标的变化。设计：观察对比实验。单位：解放军第九七医院，徐州医学院江苏省麻醉学重点实验室，Healthy-Science Center,State University of New York．材料：实验于2002—09／2003—02在美国纽约州立大学医学中心完成。选择成年雄性SD大鼠5只，预吸纯氧3min后以体积分数为0．02的异氟醚麻醉，快速断头取脑，制备离体海马脑片。方法：大鼠海马脑片随机分为单纯缺氧组和MK801组，每组10个。单纯缺氧组海马脑片给予10min缺氧；而MK801组的海马脑片在缺氧前10min及10min的缺氧期间分别应用100μmol／L的MK801。所有脑片均给予60min的复氧。应用细胞内记录技术记录海马CA1区神经元缓慢去极化及快速去极化的时间、快速去极化的幅度。在复氧末，应用细胞内注入电流及经Schaffer通路刺激，观察神经元对刺激的反应。主要观察指标：①两组海马CA1区锥体神经元缓慢去极化速率。②两组海马CA1区神经元的快速去极化时间。③两组海马CA1区神经元的快速去极化幅度。④MK801对海马CA1区神经元功能恢复的影响。结果：①海马CA1区锥体神经元缓慢去极化速率：单纯缺氧组显著高于MK801组[（0．20&;#177;0．05）mV／s，（0．08&;#177;0．03）mV／s，（P〈0．05）]。②海马CA1区神经元的快速去极化时间：MK801组显著高于单纯缺氧组[（537&;#177;139）s，（261&;#177;26）s，（P〈0．05）]。③海马CA1区神经元的快速去极化的幅度：MK801组显著小于单纯缺氧组[（4&;#177;13）mV，（53&;#177;7）mV，（P〈0．05）。④在复氧末期，MK801组的10个神经元中，9个神经元恢复对刺激的反应。结论：N-甲基-D-天冬氨酸受体阻断剂MK801可以显著降低缺氧引起的神经元缓慢去极化速率，延缓神经元快速去极化的发生及降低快速去极化的幅度，说明N-甲基-D-天冬氨酸受体阻断剂可显著减轻神经元的缺氧性损伤，促进复氧后神经元功能的恢复。     

抑制p38 MAPK通路对大鼠癫痫发作引起海马神经元损伤的保护作用

目的探讨抑制p38 MAPK通路对减轻红藻氨酸诱导的颞叶癫痫发作引起大鼠海马神经元所造成损害的作用和机制.方法经侧脑室给予不同剂量(0,0.01,0,1,1 μg)SB203580(p38 MAPK特异性抑制剂)预处理,30 min后再予红藻氨酸制作大鼠颞叶癫痫持续状态模型.7 d后采用Nissl染色方法观察海马各区的锥体细胞和颗粒细胞的情况.结果红藻氨酸注射7 d后CA3区可见大量锥体细胞缺失,尚存细胞变性,伴局限性颗粒细胞增多,呈放射状分布.CA1区受累较轻,但也存在一定程度的细胞脱失.预先给予SB203580(0.1μg以上剂量)处理的动物以上变化明显减轻.假手术大鼠海马各区有大量正常致密锥体细胞,未见细胞脱失.结论大鼠侧脑室内注射红藻氨酸引起全身痉挛性癫痫持续状态对海马神经元造成损害,而抑制p38 MAPK通路,对海马神经元起一定保护作用.     

离体海马脑片CA1区锥体细胞缺氧期间电生理指标变化与N-甲基-D-天冬氨酸受体阻断剂MK801干预后的变化

背景海马CA1区锥体细胞是对缺血缺氧性损伤最为敏感的神经元,缺血缺氧后海马CA1区锥体细胞膜电位表现为缺氧早期细胞膜的超极化,随着缺氧时间的延长,细胞膜发生缓慢去极化及快速去极化,引起神经元不可逆性损伤.目的应用细胞内记录技术,观察N-甲基-D-天冬氨酸受体阻断剂MK801对离体海马脑片CA1区锥体细胞缺氧期间电生理指标的变化.设计观察对比实验.单位解放军第九七医院,徐州医学院江苏省麻醉学重点实验室,Healthy-Science Center,State University of New York.材料实验于2002-09/2003-02在美国纽约州立大学医学中心完成.选择成年雄性SD大鼠5只,预吸纯氧3 min后以体积分数为0.02的异氟醚麻醉,快速断头取脑,制备离体海马脑片.方法大鼠海马脑片随机分为单纯缺氧组和MK801组,每组10个.单纯缺氧组海马脑片给予10 min缺氧;而MK801组的海马脑片在缺氧前10 min及10 min的缺氧期间分别应用100 μmol/L的MK801.所有脑片均给予60 min的复氧.应用细胞内记录技术记录海马CA1区神经元缓慢去极化及快速去极化的时间、快速去极化的幅度.在复氧末,应用细胞内注入电流及经Schaffer通路刺激,观察神经元对刺激的反应.主要观察指标①两组海马CA1区锥体神经元缓慢去极化速率.②两组海马CA1区神经元的快速去极化时间.③两组海马CA1区神经元的快速去极化幅度.④MK801对海马CA1区神经元功能恢复的影响.结果①海马CA1区锥体神经元缓慢去极化速率单纯缺氧组显著高于MK801组[(0.20±0.05)mV/s,(0.08±0.03)mV/s,(P＜0.05)].②海马CA1区神经元的快速去极化时间MK801组显著高于单纯缺氧组[(537±139)s,(261±26)s,(P＜0.05)].③海马CA1区神经元的快速去极化的幅度MK801组显著小于单纯缺氧组[(4±13)mV,(53±7)mV,(P＜0.05).④在复氧末期,MK801组的10个神经元中,9个神经元恢复对刺激的反应.结论N-甲基-D-天冬氨酸受体阻断剂MK801可以显著降低缺氧引起的神经元缓慢去极化速率,延缓神经元快速去极化的发生及降低快速去极化的幅度,说明N-甲基-D-天冬氨酸受体阻断剂可显著减轻神经元的缺氧性损伤,促进复氧后神经元功能的恢复.     

骨髓间充质干细胞及补脑Ⅰ号处理后血清对小鼠海马神经元缺氧缺糖模型ICAM-1、NF200表达的影响

目的观察骨髓间充质干细胞（BMSCs）、补脑Ⅰ号血清对小鼠海马神经元缺氧缺糖（OGD）模型神经中丝200（NF200）、细胞间黏附因子-1（ICAM-1）的影响。 方法分离培养BMSCs、海马神经元,对海马神经元进行OGD造模。制备补脑Ⅰ号血清与正常血清。经MTT法筛选补脑Ⅰ号血清对海马神经元OGD模型干预的最佳浓度为10%,时间为72 h。实验分为正常组、模型组、BMSCs组、正常血清组、中药血清组、BMSCs＋正常血清组、BMSCs＋中药血清组。qRT-PCR检测细胞NF200、ICAM-1 mRNA表达;Western Blot检测细胞NF200、ICAM-1蛋白表达。 结果与正常组比较,模型组、BMSCs组、正常血清组、BMSCs＋正常血清组、中药血清组、BMSCs＋中药血清组ICAM-1、NF200 mRNA及蛋白表达升高（P＜0.05）;与模型组比较,BMSCs组、BMSCs＋正常血清组、中药血清组、BMSCs＋中药血清组NF200 mRNA及蛋白表达升高,ICAM-1 mRNA及蛋白表达降低,差异有统计学意义（P＜0.05）;BMSCs＋中药血清组较BMSCs组、正常血清组、中药血清组及BMSCs＋正常血清组的NF200 mRNA及蛋白表达升高,ICAM-1 mRNA及蛋白表达减少,差异有统计学意义（P＜0.05）。 结论BMSCs和补脑Ⅰ号血清可能通过抑制炎症、促进神经再生及双重作用修复OGD损伤的海马神经元,两者联合应用效果优于单独使用。     

诺维本致大鼠组织损伤后处理的实验研究

目的 :运用大鼠模型探讨诺维本致组织损伤后的最佳处理方法。方法 :将 4 8只SD大鼠建立诺维本组织损伤模型后随机分为 4组 ,即对照组、局封加冷敷组、局封加热敷组、透明质酸酶加红外线照射组。于 2 4h、1周、2周分别进行局部组织病理学检查。结果 :透明质酸酶加红外线照射组对诺维本致大鼠组织病理改变有明显减轻作用 ,局封加冷敷组未见明显疗效。结论 :透明质酸酶加红外线照射用于治疗诺维本外渗所引起的组织损伤效果最显著     

Pulse of nitric oxide release in response to activation of N-methyl-D-aspartate receptors in the rat striatum: rapid desensitization, inhibition by receptor antagonists, and potentiation by glycine

Increased activity of glutamate N-methyl-d-aspartate (NMDA) receptors is the dominant mechanism by which nitric oxide (NO.) is generated. By using a selective direct-current amperometry method, we characterized real time NO* release in vivo in response to chemical stimulation of NMDA receptors in the rat striatum. The application of NMDA caused the appearance of a sharp and transient oxidation signal. Concentration-response studies (10-500 microM) indicated an EC(50) of 48 microM. The NMDA-induced amperometric signal was suppressed by focal application of the nitric-oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME, 100 microM) or D-(-)-2-amino-5-phosphonopentanoic acid (AP-5, 100 microM) or by systemic injection of dizocilpine (1 mg/kg i.p.), drugs that, when given alone, had no effect on baseline oxidation current. Repeated injections of NMDA at short intervals (approximately 80 s) resulted in a progressive reduction of the amperometric signal with a decay half-life of 1.36 min. Sixty min after the last NMDA application the amperometric response was restored to initial levels. Finally, the coapplication of glycine (50 or 100 microM), which, when given alone had no effect, potentiated the NMDA-induced response. Thus, NMDA receptor-mediated activation of striatal NO* system shuts off quickly and undergoes rapid desensitization, suggesting a feedback inhibition of NMDA receptor function. To the extent of NO* release can represent a correlate of NMDA receptor activity, its amperometric detection could be useful to assess in vivo the state of excitatory transmission under physiological, pharmacological, or pathological conditions.     

Stereoselective inhibition of amantadine accumulation by quinine and quinidine in rat renal proximal tubules and cortical slices

The renal organic cation transport system was examined. The accumulation of a nonchiral cation, amantadine, by rat renal proximal tubules and cortical slices was investigated, together with the effects of two diastereoisomers, quinine and quinidine. The proximal tubules actively concentrated amantadine with a tissue/medium ratio of 96.3 +/- 1.7 (mean +/- S.E.M., n = 18). Apparent Km was 85 +/- 2 microM and Vmax was 8.0 +/- 0.2 nmol/mg of tubular protein per min. Amantadine accumulation was inhibited competitively by quinine and quinidine with Ki values of 32 +/- 3 and 84 +/- 11 microM, respectively (n = 4). Amantadine was also concentrated by renal cortical slices with tissue/medium ratio of 3.3 +/- 0.3 (n = 4). Apparent Km and Vmax were 94.0 +/- 5.2 microM and 1.27 +/- 0.08 nmol/mg of tubular protein per min, respectively (n = 10). Quinine and quinidine again inhibited amantadine accumulation competitively by the slices, with Ki values of 368 +/- 28 and 780 +/- 84 microM, respectively (n = 4). A similar affinity (Km) for amantadine was observed in both preparations. However, the lower Vmax value in the slice system may be due to additional amantadine transport sites with lower capacity, lesser luminal accumulation and/or limited substrate(s) penetration in the cortical slices. In either preparation, quinine and quinidine functioned as competitive inhibitors and stereoselectivity was observed for the (-)-isomer, quinine, over the (+)-isomer, quinidine. Additional transport sites, reduced luminal substrate accumulation and/or diffusional restraints in the slices are also feasible mechanisms in explaining the differences in Ki values between the two preparations, and their relative contributions await further investigation.     

Enhanced vascular neuronal nitric-oxide synthase-derived nitric-oxide production underlies the pressor response caused by peripheral N-methyl-D-aspartate receptor activation in conscious rats

Although the N-methyl-D-aspartate (NMDA) receptor (NMDAR) obligatory unit NMDAR1 is expressed in the vasculature and myocardium, the impact of peripheral NMDAR activation on blood pressure (BP) has received little attention. We demonstrate, for the first time, dose-related pressor responses elicited by systemic NMDA (125, 250, 500, and 1000 μg/kg) in conscious rats. The pressor response was peripheral NMDAR-mediated because: 1) it persisted after ganglion blockade (hexamethonium; 5 mg/kg i.v.); 2) it was attenuated by the selective NMDAR blocker DL-2-amino-5-phosphonopentanoic acid (5 mg/kg, i.v.) or the glycine/NMDAR antagonist R-(+)-3-amino-1-hydroxypyrrolid-2-one [R-(+)-HA-966; 10 mg/kg i.v.]; and 3) NMDA (1.25-10 mM) increased contractile force of rat aorta in vitro. It is noteworthy that ex vivo studies revealed enhanced nitric oxide (NO) and reactive oxygen species (ROS) generation in vascular tissues collected at the peak of the NMDAR-mediated pressor response. Pharmacological, ex vivo, and in vitro findings demonstrated attenuation of the NMDAR-mediated increases in BP and vascular NO and ROS by the nonselective NO synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride (10 mg/kg i.v.) or the neuronal NOS (nNOS) inhibitor N(ω)-propyl-L-arginine hydrochloride (150 μg/kg i.p.) but not by the endothelial NOS inhibitor N(5)-(1-iminoethyl)-L-ornithine (4 or 10 mg/kg i.v.). Furthermore, R-(+)-HA-966 attenuated NMDA-evoked generation of vascular NO and ROS. The findings suggest a pivotal role for enhanced vascular nNOS-derived NO in ROS generation and in the subsequent pressor response elicited by peripheral NMDAR in conscious rats.     

Heterogeneity of the N-methyl-D-aspartate receptor ionophore complex in rat brain, as revealed by ligand binding techniques.

The polyamine spermidine markedly potentiated the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to open ion channels associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in the presence of a maximally effective concentration of L-glutamic acid (Glu) and glycine (Gly), in Triton-treated preparations of synaptic membranes from the cerebral cortex as well as hippocampus of rat brain, without significantly affecting that in cerebellar synaptic membranes. Among several ligands for the binding sites of [3H]MK-801, the binding was most potently displaced by (+)-MK-801, followed by N-[1-(2-thienyl)cyclohexyl]piperidine, (-)-MK-801, phencyclidine, cyclazocine, ketamine, ketocyclazocine, N-allylnormetazocine and pentazocine, in a rank order of decreasing potency, in hippocampal synaptic membranes. However, the abilities of these ligands to displace the binding were much lower in cerebellar membranes than in hippocampal membranes. Competitive NMDA antagonists induced a much more potent inhibition of [3H] MK-801 binding in the hippocampus than in the cerebellum, whereas competitive Gly antagonists elicited a similarly potent inhibition of the binding in both structures. Moreover, NMDA antagonists caused a greater than 10 times more potent displacement of [3H]Glu binding to the NMDA recognition site in hippocampal membranes than in cerebellar membranes, whereas NMDA agonists similarly displaced [3H]Glu binding in both central regions. Gly agonists elicited an equivalent displacement of strychnine-insensitive [3H]Gly binding in both membrane preparations, whereas Gly antagonists more potently displaced [3H]Gly binding in the hippocampus than in the cerebellum. These results suggest possible heterogeneity of the respective domains within the NMDA receptor ionophore complex, in terms of differential sensitivity to isosteric and allosteric ligands.     

Lung damage caused by phospholipase C and the changes in phospholipids in the rat lung.

The lipid and protein fractions of the endobronchial lavage fluid from the normal rats which contained the lung surfactant were analysed. Lecithin, the main main component of the lung surfactant, was exclusively combined with a dextran precipitable protein. This protein then behaved as beta-globulin on cellulose acetate electrophoresis or low density lipoprotein on agarose gel filtration. After administration of phospholipase C (from Clostridium Welchii), the protein content of the lavage fluid increased markedly. The amount of dextran precipitable protein also increased markedly and its properties remained the same on gel filtration after treatment with phospholipase C. The phospholipids in the lavage fluid were not affected, although the total phospholipids in the lung tissue, especially lecithin, did decrease during the 10 days after treatment. Histopathologically, an eosinophilic dense exudative fluid appeared in both the interstitium and the broncho-alveolar spaces. A large number of the alveolar lining cells disappeared and a few of them were desquamated into the alveolar spaces. Thus, the immediate destruction of the alveolar lining cells after the administration of phospholipase C resulted in interstitial pneumonia in 10 days. The significance of phospholipase in pulmonary inflammation is discussed.     

Protein kinase C-dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons

Intracellular calcium concentration ([Ca2+]i) plays a major role in neuronal excitability, especially that triggered by the N-methyl-d-aspartate (NMDA)-sensitive glutamatergic receptor. We have previously shown that sigma1 receptor agonists potentiate NMDA receptor-mediated neuronal activity in the hippocampus and recruit Ca2+-dependent second messenger cascades (e.g., protein kinase C; PKC) in brainstem motor structures. The present study therefore assessed whether the potentiating action of sigma1 agonists on the NMDA response observed in the hippocampus involves the regulation of [Ca2+]i and PKC. For this purpose, [Ca2+]i changes after NMDA receptor activation were monitored in primary cultures of embryonic rat hippocampal pyramidal neurons using microspectrofluorometry of the Ca2+-sensitive indicator Fura-2/acetoxymethyl ester in the presence of sigma1 agonists and PKC inhibitors. We show that successive activations of the sigma1 receptor by 1-min pulses of (+)-benzomorphans or (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784) concomitantly with glutamate time dependently potentiated before inconstantly inhibiting the NMDA receptor-mediated increase of [Ca2+]i, whereas 1,3-di-o-tolyl-guanidine, a mixed sigma1/sigma2 agonist, did not significantly modify the glutamate response. Both potentiation and inhibition were prevented by the selective sigma1 antagonist N,N-dipropyl-2-[4-methoxy-3-(211phenylethoxy) phenyl]-ethylamine monohydrochloride (NE-100). Furthermore, only (+)-benzomorphans could induce [Ca2+]i influx by themselves after a brief pulse of glutamate. A pretreatment with the conventional PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole (G?-6976) prevented the potentiating effect of (+)-benzomorphans on the glutamate response. Our results provide further support for a general mechanism for the intracellular sigma1 receptor to regulate Ca2+-dependent signal transduction and protein phosphorylation.     

白细胞介素1 β、白细胞介素1受体拮抗剂对癫痫大鼠行为、海马超微结构及NMDAR活性的影响

目的探讨白细胞介素1β(IL-1β)及白细胞介素1受体拮抗剂(IL-1ra)对癫痫的影响,揭示癫痫发作的免疫学机制.方法实验于2003-10/2004-06在哈尔滨医科大学第二临床医学院实验中心完成,侧脑室注射生理盐水,L-1β及IL-1ra 30 min后戊四氮致痫观察大鼠行为,分别于致痫后2,,24 h处死大鼠,通过放射配基结合实验测定NMDA受体(NMDAR)活性,取海马CA3区电镜下观察超微结构的变化.结果外源性IL-1β能够缩短癫痫发作的潜伏期,与生理盐水对照组相比差异有显著性意义(t=3.85,＜0.05),对癫痫发作强度的影响与对照组相比差异无显著性意义,观察到明显的神经元变性坏死,大鼠脑内NMDAR活性在致痫后个时间点均显著增强;IL-Ira对发作潜伏期的影响及发作强度的影响与对照组相比差异无显著性意义,但可明显减轻戊四氮致痫所导致的神经元损害,致痫大鼠脑内NMDAR活性各时间与对照组相比明显减低.结论IL-1 β通过提高NMDAR活性促进癫痫发生及发展,加重癫痫所导致的神经元损伤的程度,L-1ra可以通过抑制IL-1β的活性,减轻其上述作用.     

Rapid inhibition of thyroxine-induced bone resorption in the rat by an orally active vitronectin receptor antagonist

An excess of thyroid hormone results in increased bone turnover and loss of bone mass in humans. Exogenous administration of thyroid hormone to rats has served as a model of human hyperthyroidism in which antiresorptive therapies have been tested. We have further refined this model of thyroxine (T4)-induced turnover in the rat. Daily administration of T4 to aged rats for as short as 1 week resulted in elevated bone resorption determined by significantly higher urinary deoxypyridinoline (Dpd) compared with vehicle controls or animals receiving T4 plus estradiol. Three weeks of daily administration of T4 led to significantly lower bone mineral density compared with untreated controls or animals receiving T4 plus estradiol. In a follow-up study, a depot formulation of T4 caused an increase in Dpd identical to that achieved with a bolus dose. SB-273005 [(4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl] ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine-4- acetic acid] a potent antagonist of the integrins alpha(v)beta(3) and alpha(v)beta(5), has been shown previously to inhibit bone resorption in cultures of human osteoclasts and to protect bone in ovariectomized rats. The effect of SB-273005 by oral administration was evaluated in this thyroxine-induced turnover model. Dose-dependent inhibition of resorption was seen with SB-273005 after 7 days of dosing using Dpd as a measure of bone resorption. In summary, it has been demonstrated that the antiresorptive activity of a vitronectin receptor antagonist can be measured after only 7 days of treatment in this refined rat model of thyroxine-induced bone turnover. These data suggest that SB-273005 may be useful for the treatment of metabolic bone diseases, including those resulting from hyperthyroidism.     

Long-lasting facilitation of 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release from rat hippocampal slices by nicotinic receptor activation

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.     

Stimulation of adenosine 3',5'-monophosphate formation in rat cerebral cortical slices by methoxamine: interaction with an alpha adrenergic receptor.

Methoxamine elicits a rapid accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in rat cerebral cortical slices with maximal effects at 100 muM. The accumulations of cyclic AMP elicited by this amine are completely blocked by the alpha adrenergic antagonists, phenoxybenzamine and dihydroergokryptine, partially blocked by the alpha antagonist, phentolamine, and unaffected by the beta blocking agent, propranolol, or by the local anesthetic, tetracaine. The magnitude of the accumulations of cyclic AMP elicited by methoxamine in cerebral cortical slices of four rat strains (F-344, ACI, BUF, and Sprague-Dawley) exhibit a strong negative correlation with spontaneous motor activity and a positive correlation with the magnitude of norepinephrine-elicited accumulations of cyclic AMP. The stimulatory interaction of methoxamine with alpha adrenergically regulated cyclic AMP-generating systems differs from the interaction of norepinephrine with alpha receptors as evidenced by the following observations: 1) the stimulatory effects of methoxamine and norepinephrine are nearly additive; 2) the stimulatory effects of methoxamine and adenosine are nearly additive, whereas the effects of norepinephrine and adenosine are much more than additive. Methoxamine, however, does not increase further the magnitude of accumulation of cyclic AMP elicited by a combination of norepinephrine and adenosine. The results are consonant with the interaction of methoxamine with alpha adrenergic receptors which are normally activated by norepinephrine only to a marginal extent. However, in the presence of adenosine, these receptors are now sensitive to activation by norepinephrine.