Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

大鼠坐骨神经切断后外源性bcl-2对脊髓运动神经元的保护作用

采用大鼠坐骨神经切断损伤模型,行神经外膜端端对线缝合,术中依不同组别,动物于神经缝合处远端0．5cm处分别注射人的正义和反义bcl-2重组腺病毒(Ad／s-bcl-2、Ad／as-bcl-2),报道基因重组腺病毒(Ad／lacZ)和生理盐水。术后48h,7d,15d和30d常规灌注固定大鼠,取L4-L6脊髓节段,应用X-gal染色、bel-2原位杂交和免疫组化染色、TUNEL染色以及乙酰胆碱酯酶(AChE)组织化学染色方法,观察到外源基因能在脊髓中表达,同时外源性Ad／s-bcl-2能显著减少L4到L6节段脊髓前角运动神经元凋亡的数目,减少脊髓前角运动神经元中因坐骨神经切断导致的AChE活性的降低幅度,并加快其恢复。而Ad／as-bcl-2可显著增加坐骨神经切断诱导的脊髓前角运动神经元凋亡数目以及AChE活性降低幅度,并延缓其恢复。这些观察结果表明,外源性bcl-2能保护周围神经切断后引起的脊髓运动神经元损伤。     

Hippocampal Long-Term Potentiation Attenuated by Lesions in the Marginal Division of Neostriatum

The marginal division (MrD) is a spindled-neurons consisted zone at the caudal border of the neostriatum in the mammalian brain and has been verified as contributing to associative learning and declarative memory in the rat and human with behavior and functional magnetic resonance imaging methods. It was proved to have functional connections with the limbic system. Whether the MrD has influence on the hippocampal long-term potentiation (LTP) was investigated in this study. LTP was induced from the dentate gyrus (DG) in the hippocampus by high-frequency stimulation (HFS) to the perforant path (PP). The amplitude of the population spike (PS) and the slope of the excitatory postsynaptic potential (EPSP) increased significantly to form LTP in the DG of the hippocampus after HFS of PP in normal and saline-injected control groups of rats. Lesions introduced in the MrD reduced significantly both the amplitude of PS and the slope of the EPSP following HFS of the PP. The results indicated that lesions in the MrD could attenuate LTP formation in the hippocampus. Our data suggest that the MrD might very possibly have excitatory functional influence on the hippocampus and therefore might influence the function of the hippocampus.     

利多卡因和硫喷妥钠对培养的大鼠海马脑片神经元损伤的影响

目的 :观察利多卡因和硫喷妥钠对生后 2 2d大鼠培养海马脑片的实验型缺血后神经元损伤的影响。方法 :将培养的SD大鼠海马脑片实验型缺血 (缺氧缺糖 ) 1 0min ,给药组在缺血前 1 0min给予 1 0nmol/L、1 0 0nmol/L的利多卡因或 2 50nmol/L、60 0nmol/L的硫喷妥钠 ,缺血后换用正常培养基继续培养 7d ,并用荧光染料PropidiumIo dide(PI)连续观察海马CA1区和齿状回神经元的损伤。结果 :缺血后第 1d缺血组即出现神经元损伤高峰 ,CA1区和齿状回的PI指数显著增加 (P <0 .0 1 ) ;直至缺血后第 7d其损伤指数仍显著高于缺血前水平 (P <0 .0 1 )。两浓度的利多卡因和硫喷妥钠均可降低缺血后CA1区和齿状回神经元损伤的程度 (P <0 .0 1 ) ,并可将CA1区和齿状回的神经元损伤高峰推迟至缺血后第 3d。结论 :利多卡因和硫喷妥可减轻缺血后海马CA1区和齿状回的神经元损伤 ,推迟神经元的损伤高峰。     

代谢性谷氨酸受体配体(s)-4C3HPG对大鼠脑缺血耐受性诱导的影响

目的：观察侧脑室注射代谢型谷氨酸受体1／5亚型（mGluR1/5)配体（s)-4C3HPG对海马脑缺血耐受（BIT）诱导的影响,以探讨mGLUR1/5在BIT诱导中的作用。方法：采用大鼠四血管闭塞全脑缺血模型（4－vessel occlusion,4VO),应用硫堇染色和GFAP免疫组化法。36只大鼠椎动脉凝闭后分为sham组、单纯缺血组、BIT组和（s)-4C3HPG组,其中（s)-4C3HPG组又按所给药物剂量不同,分为0．2、0．04和0．008mg三个亚组。所有动物均在手术后或末次缺血后7d处死取材观察。结果：（1）单纯8min缺血可使海马CA1区组织学分级升高、锥体神经元密度降低和胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP)阳性表达增加（P＜0．05vs sham）.(2)BIT组未见单纯缺血组的上述变化,表明CIP可防止后续8min缺血造成的神经元损伤。（3）CIP的这种保护作用可被（s)-4C3HPG阻断,表现为海马CA1区组织学分级升高和锥体神经元密度降低（P＜0．05 vs sham)。这种变化与（s)-4C3HPG的剂量呈现明显的相关性,即剂量越大,上述改变越明显。结论：（s)-4C3HPG可阻断CIP诱导BIT的作用,提示mGluR1/5参与BIT的诱导。     

Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca(2+) removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.     

Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells

Apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. The present study uses an in vitro tissue culture model to investigate whether free fatty acids (FFAs), at concentrations comparable to those found following traumatic brain injury, trigger cell death. Nerve growth factor (NGF)-differentiated PC12 cells exposed to oleic and arachidonic acids (2 : 1 ratio FFA/BSA) showed normal cell survival. However, when cells were exposed to stearic and palmitic acids, there was a dramatic loss of cell viability after 24 h of treatment. The cell death induced by stearic acid and palmitic acid was apoptotic as assessed by morphological analysis, and activation of caspase-8 and caspase-3-like activities. Western blotting showed that differentiated PC12 cells exposed to stearic and palmitic acids exhibited the signature apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP). Interestingly, blockade of caspase activities with the pan-caspase inhibitor z-VAD-fmk failed to prevent the cell death observed induced by palmitic or stearic acid. RT-PCR and RNA blot experiments showed an up-regulation of the Fas receptor and ligand mRNA. These findings are consistent with our hypothesis that FFAs may play a role in the cell death associated with trauma in the CNS and PNS.     

Chronic lithium administration potentiates brain arachidonic acid signaling at rest and during cholinergic activation in awake rats

Studies were performed to determine if the reported 'proconvulsant' action of lithium in rats given cholinergic drugs is related to receptor-initiated phospholipase A2 signaling via arachidonic acid. Regional brain incorporation coefficients k* of intravenously injected [1-14C]arachidonic acid, which represent this signaling, were measured by quantitative autoradiography in unanesthetized rats at baseline and following administration of subconvulsant doses of the cholinergic muscarinic agonist, arecoline. In rats fed LiCl for 6 weeks to produce a therapeutically relevant brain lithium concentration, the mean baseline values of k* in brain auditory and visual areas were significantly greater than in rats fed control diet. Arecoline at doses of 2 and 5 mg/kg intraperitoneally increased k* in widespread brain areas in rats fed the control diet as well as the LiCl diet. However, the arecoline-induced increments often were significantly greater in the LiCl-fed than in the control diet-fed rats. Lithium's elevation of baseline k* in auditory and visual regions may correspond to its ability in humans to increase auditory and visual evoked responses. Additionally, its augmentation of the k* responses to arecoline may underlie its reported 'proconvulsant' action with cholinergic drugs, as arachidonic acid and its eicosanoid metabolites can increase neuronal excitability and seizure propagation.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.