ERKⅠ/Ⅱ在脊髓背角LTP的诱导和维持中的作用

目的：探讨胞外信号调节激酶（ERKⅠ/Ⅱ）在大鼠脊髓背角C-纤维诱发电位长时程增强（LTP）的诱导和维持中的作用。 方法： 细胞外记录技术在脊髓腰膨大部记录背角浅层神经元C-纤维诱发电位。 结果： (1) MEK的选择性抑制剂PD98059（100 μmol/L）或SL327 (200 μmol/L) 对脊髓背角C-纤维诱发电位的基础电位没有影响，但可阻断脊髓背角LTP的诱导。(2) PD98059或SL327呈时间依赖性逆转脊髓背角LTP。在LTP 诱导后15 min，脊髓局部给予PD98059（100 μmol/L）或SL327(200 μmol/L)可完全逆转LTP。在LTP 诱导后30 min，单独给予PD98059或SL327，LTP的抑制率分别为62.5％与75.0％。但同样浓度的PD98059或SL327在LTP 诱导后1 h，均不能逆转业已建立的LTP。 结论： 脊髓背角ERKⅠ/Ⅱ的激活参与C-纤维诱发电位LTP的诱导和早期维持。     

脊髓背角长时程增强中CaMKⅡ磷酸化水平的变化

目的探讨长时程增强诱导和维持过程中脊髓背角钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)磷酸化水平的变化。方法(1)细胞外记录脊髓腰膨大部背角浅层神经元C-纤维诱发电位;(2)免疫组化技术观察脊髓背角CaMKⅡ磷酸化水平的变化。结果(1)LTP30min、LTP3h脊髓背角CaMKⅡThr286的磷酸化水平明显高于对照组;(2)强直刺激前30min脊髓局部给予KN-93(CaMKⅡ选择性抑制剂,100μmol/L),LTP的诱导被完全阻断,CaMKⅡ磷酸化水平与对照组无明显差别;(3)强直刺激后30min给予KN-93,明显抑制LTP,CaMKⅡ的磷酸化水平也显著降低;(4)LTP3h后给予KN-93,LTP幅度和CaMKⅡ磷酸化水平与用药前相比,差异没有统计学意义。结论CaMKⅡ磷酸化可能在脊髓背角C-纤维诱发电位长时程增强诱导和早期维持中发挥重要作用。     

PKA参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持

目的: 探讨环一磷酸腺苷依赖的蛋白激酶 (PKA) 在脊髓背角C-纤维诱发电位长时程增强(LTP)的诱导和维持中的作用。方法:细胞外记录技术在脊髓腰膨大部记录背角浅层神经元C-纤维诱发电位。 结果:(1) 8-Br-cAMP诱发脊髓背角C-纤维诱发电位LTP,且8-Br-cAMP-诱导的 LTP 遮蔽强直刺激诱导的LTP。(2) PKA的选择性抑制剂Rp-CPT-cAMPS阻断C-纤维诱发电位LTP的诱导和时间依赖性翻转C-纤维诱发电位LTP。(3)蛋白质合成抑制剂茴香霉素彻底阻断8-Br-cAMP诱发的 LTP。(4)MAPK选择性抑制剂PD98059阻断8-Br-cAMP诱发的LTP。 结论:脊髓背角神经元存在PKA信号途径,并参与脊髓背角C-纤维诱发LTP的诱导和早期维持。     

小胶质细胞SFKs在ATP诱导的脊髓背角LTP中的作用

目的: 研究Src家族激酶(SFKs) 在5'-三磷酸腺苷(ATP)诱导的脊髓背角长时程增强(LTP)中的作用。方法: 雄性SD大鼠(250-280 g)。在体电生理记录脊髓腰膨大部背角浅层神经元C-纤维诱发电位,Western blotting和免疫组织化学观察脊髓背角SFKs的磷酸化水平和表达部位。结果: ATP应用后30 min和60 min,磷酸化的Src-家族激酶(p-SFKs)的水平明显升高;p-SFKs只表达于小胶质细胞中,星型胶质细胞和神经元中没有表达。脊髓表面应用SFKs抑制剂阻断ATP诱导的LTP。结论: 小胶质细胞SFKs可能在ATP诱导的脊髓背角C-纤维诱发电位LTP中发挥重要作用。     

酪氨酸家族激酶在大鼠脊髓背角LTP中的作用及其机制

目的: 探讨酪氨酸家族激酶(SFKs)在大鼠脊髓背角C纤维诱发电位的长时程增强(LTP)中的作用及其机制。 方法: 用在体电生理方法检测SFKs抑制剂对高频刺激坐骨神经诱导的脊髓背角LTP的影响;用Western blotting方法检测高频刺激诱导脊髓LTP的不同时点大鼠脊髓背角磷酸化SFKs的表达情况;用免疫荧光双染方法检测高频刺激诱导脊髓LTP后大鼠脊髓背角磷酸化SFKs表达的细胞学定位。 结果: SFKs的抑制剂(PP2或SU6656)可以完全阻断LTP的诱导,甚至将LTP逆转为长时程抑制(LTD);高频电刺激大鼠坐骨神经诱导脊髓LTP后15 min开始,刺激同侧的脊髓背角中磷酸化SFKs表达明显增加,并且这些高表达的磷酸化SFKs仅仅存在于脊髓小胶质细胞中。 结论: 在脊髓背角,小胶质细胞中的SFKs 是诱导LTP的必要条件,抑制SFKs及其下游分子可能有助于治疗病理性疼痛。     

PKC/Raf-1/NF-κB信号通路在低氧诱导大鼠单核细胞表达TNF-α中的作用

目的：研究PKC/Raf-1/NF-κB信号通路在低氧诱导大鼠外周血单核细胞（PBMCs）肿瘤坏死因子α（TNF-α）表达中的作用，探讨低氧与全身炎症反应综合征(SIRS)的关系，为进一步研究老年多器官功能障碍综合征(MODSE)的肺启动机制奠定基础。方法:采用明胶法分离大鼠外周血单核细胞，分为chelerythrine+低氧组、forskolin+低氧组和单纯低氧组。Chelerythrine+低氧组和forskolin+低氧组细胞在低氧前分别予10 μmol/L chelerythrine和50 μmol/L forskolin预处理。然后各组均于低氧条件下（3 % O2, 5 % CO2, 92 % N2）培养0、1、3、6、9、12、24 h后，收集细胞及培养液上清，分别采用PKC、Raf活性检测试剂盒、电泳迁移分析法（EMSA）、逆转录PCR（RT-PCR）和酶联免疫吸附试验（ELISA）检测PKC、Raf-1、NF-κB活性及TNF-α表达量。结果:低氧1-9 h，PKC、Raf-1活性、NF-κB结合活性及TNF-α表达量显著高于正常对照组（P<0.01）。低氧后1-24 h，PKC、Raf-1活性、NF-κB结合活性与TNF-α mRNA和蛋白表达水平间呈显著正相关（P<0.01，P<0.05）。10 μmol/L chelerythrine可显著抑制低氧诱导的PKC、Raf-1、NF-κB活性升高和TNF-α表达。50 μmol/L forskolin可显著抑制低氧诱导的Raf-1、NF-κB活性升高及TNF-α表达。结论:低氧可显著增强大鼠外周血单核细胞的PKC、Raf-1活性及NF-κB结合活性,并可诱导其产生大量的促炎症因子TNF-α，这些变化可能与急性呼吸窘迫综合征（ARDS）时血浆中大量促炎症因子持续存在密切相关。     

白细胞介素-1β在病理性疼痛大鼠脊髓LTP中的作用及机制

目的: 探讨白细胞介素-1β (interleukin-1β, IL-1β)在神经病理性疼痛大鼠脊髓背角C纤维诱发电位长时程增强(long-term potentiation, LTP)中的作用及其机制。方法: 用坐骨神经部分损伤(spared nerve injury, SNI)和腰5前根切断(lumbar 5 ventral root transection, L5 VRT)方法复制大鼠病理性疼痛模型,观察外源性IL-1β对正常大鼠及病理性疼痛模型大鼠脊髓背角C纤维诱发电位的影响,并且检测p38 MAPK (p38 mitogen-activated protein kinase)和NF-κB (nuclear factor-kappa B)信号通路在其中的作用。结果: 500 μg/L IL-1β对正常大鼠C纤维介导的基本突触传递和高频刺激诱导的LTP都没有影响,而5 μg/L的IL-1β可以在神经病理性疼痛模型的大鼠上诱导出LTP。预先用p38 MAPK或NF-κB的抑制剂(SB203580或PDTC)可以完全阻断IL-1β诱导的LTP。结论: 外源性IL-1β可诱导神经病理性疼痛大鼠脊髓背角C纤维诱发电位的LTP。p38 MAPK和NF-κB信号通路可能参与这一过程。     

蛋白激酶C活性下调对钙库操纵的钙通道活性和气道平滑肌细胞增殖的作用

目的:研究蛋白激酶C(PKC)活性下调对大鼠气道平滑肌细胞(ASMCs)的Ca2+库操纵的Ca2+通道(SOC)和ASMCs增殖的影响。方法:分离培养大鼠支气管平滑肌细胞;利用激光共聚焦显微镜测定ASMCs的fluo-3/AM的荧光信号;利用长时间暴露于PKC活化剂诱导PKC活性下调,观察PKC活性下调对ASMCs的SOC通道和增殖的影响;Alamar blue还原率测定法测定ASMCs的增殖。结果:PKC激活剂PMA(10μmol/L)和PDBu(1μmol/L)作用24h下调PKC活性后可以抑制ASMCs的增殖,PKC抑制剂chelerythrine同样抑制ASMCs的增殖;PKC活性下调和chelerythrine均可以抑制ASMCs的SOC通道活性;小剂量PKC激活剂PMA(100nmol/L)可以促进ASMCs的增殖,这种作用被SOC通道抑制剂SKF-96365抑制。结论:PKC活性下调或抑制导致SOC通道的活性下降,表明PKC可能参与了SOC通道功能调控;SOC通道开放可能参与了PKC促进ASMCs增殖的作用。     

PKC抑制剂灯盏花素乙对甲醛炎性痛大鼠脊髓NOS表达及NO含量的影响

目的：观察蛋白激酶C（PKC）选择性抑制剂灯盏花素乙（CH）对甲醛炎性痛时大鼠自发痛反应、脊髓一氧化氮合酶（NOS）表达和一氧化氮（NO）含量的影响，探讨炎性痛时脊髓内NO产生是否受PKC调控。 方法： 采用右后掌足底注射甲醛复制炎性痛模型；计数缩足反射次数反映自发痛程度；应用NADPH-d组织化学法测定脊髓NOS表达；硝酸/亚硝酸还原法测定脊髓腰膨大部位NO2-/NO3-含量。 结果： 甲醛炎性痛大鼠L5脊髓后角浅层和中央管周围灰质NADPH-d阳性细胞的数目、阳性细胞胞体及纤维的染色深度均明显大于正常对照组，脊髓腰膨大部位 NO2-/NO3-含量明显增高。预先鞘内给予CH，可明显抑制甲醛炎性痛诱导的大鼠第二相自发痛反应以及脊髓NOS表达和NO2-/NO3-的含量。 结论： 炎性痛时，脊髓伤害性感受神经元内PKC激活可以促进NOS表达和NO的产生。     

瑞芬太尼诱导大鼠脊髓背角痛觉过敏及利多卡因的抑制作用

目的探讨大鼠脊髓背角细胞蛋白激酶Cγ(PKCγ)膜转位/激活在瑞芬太尼诱导痛觉过敏中的作用及利多卡因的抑制作用。方法将大鼠随机分为4组:(1)丙泊酚组(P组),(2)瑞芬太尼组(R组),(3)瑞芬太尼-利多卡因组(RL组)及(4)利多卡因组(L组)。比较4组麻醉后累积疼痛评分和机械性刺激缩足阈值。用免疫印迹(每组n=8)和免疫荧光法测量脊髓背角PKCγ膜转位/激活。结果 (1)累积疼痛评分P、RL和L组相似;R组18(15-20.75)高于P组11.5(8-13.5)、RL组11(9-14)和L组8.5(11-16.25)(P<0.05)。(2)手术侧机械性刺激缩足阈值R组39.2(39.2-68.6)mN低于P组117.6(58.8-137.2)mN、RL组588(588-588)mN和L组588(588-588)mN组(P<0.05)。(3)蛋白免疫印迹显示脊髓背角PKCγ膜转位/激活R组(假手术组153%±35%,手术对侧为160%±41%,手术侧为157%±36%)高于其他3组,R组PKCγ免疫阳性显色在细胞边缘增强。术后短时间内,未发现组织损伤对脊髓背角PKCγ膜转位/激活的影响。结论脊髓背角PKC...     

Roles of CaMKII, PKA, and PKC in the induction and maintenance of LTP of C-fiber-evoked field potentials in rat spinal dorsal horn

Long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn may be relevant to hyperalgesia, an increased response to noxious stimulation. The mechanism underlying this form of synaptic plasticity is, however, still unclear. Considerable evidence has shown that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC) are important for LTP in hippocampus. In this study, the roles of these three protein kinases in the induction and maintenance of LTP of C-fiber-evoked field potentials were evaluated by application of specific inhibitors of CaMKII (KN-93 and AIP), PKA (Rp-CPT-cAMPS), and PKC (chelerythrine and G? 6983) at the recording segments before and after LTP induction in urethane-anesthetized Sprague-Dawley rats. We found both KN-93 and AIP, when applied at 30 min prior to tetanic stimulation, completely blocked LTP induction. At 30 min after LTP induction, KN-93 and AIP reversed LTP completely, and at 60 min after LTP induction, they depressed spinal LTP in most rats tested. Three hours after LTP induction, however, KN-93 or AIP did not affect the spinal LTP. Rp-CPT-cAMPS, chelerythrine, and G? 6983 blocked the spinal LTP when applied at 30 min before tetanic stimulation and reversed LTP completely at 15 min after LTP induction. In contrast, at 30 min after LTP induction, the drugs never affected the spinal LTP. These results suggest that activation of CaMKII, PKA, and PKC may be crucial for the induction and the early-phase but not for the late-phase maintenance of the spinal LTP.     

Protein synthesis inhibition blocks the late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn

Previous studies have demonstrated that in the hippocampus the maintenance of long-term potentiation (LTP) requires de novo protein synthesis. To investigate the role of protein synthesis in the maintenance of LTP of C-fiber evoked field potentials in spinal dorsal horn, which may be relevant to hyperalgesia, protein synthesis inhibitor (either cycloheximide or anisomycin) was applied locally to the recording segments of spinal cord in anesthetized rats, 30 min prior to tetanic stimulation to the sciatic nerve. We found that both cycloheximide and anisomycin selectively inhibited late-phase maintenance of the spinal LTP but affected neither LTP induction nor baseline responses of C-fiber evoked field potentials. In the presence of cycloheximide, LTP of C-fiber evoked field potentials was 281.5 +/- 16.5% (n = 6) of baseline 1 h after tetanic stimulation and the potentiation significantly decreased to 235.5 +/- 18.5% at 145 min after tetanic stimulation (P < 0.05). Afterward, LTP of C-fiber evoked field potentials decreased continuously and at 270 min after tetanic stimulation reached 130.8 +/- 18.0%, which was no longer different from baseline (P > 0.05). Spinal application of anisomycin at 30 min before tetanic stimulation yielded similar results (n = 6). These results suggest that protein synthesis may be crucial for the late-phase maintenance of LTP of C-fiber evoked field potentials in spinal dorsal horn.     

Evidence for a role of protein kinase C in the sustained activation of rat dorsal horn neurons evoked by cutaneous mustard oil application

The intracellular mechanisms involved in the sensitisation of spinal dorsal horn neurons brought about by sustained or repeated nociceptive inputs are unknown. The present experiments addressed any role of protein kinase (PKC) in sustained nociceptive responses of rat dorsal horn neurons by: (i) ionophoretic administration of PKC inhibitors whilst recording activity evoked by repeated cutaneous application of mustard oil; and (ii) assessing subcellular translocation of PKC evoked in spinal cord by cutaneous application of mustard oil. Both marked attenuation of mustard oil-induced neuronal activity by PKC inhibitors and selective translocation of PKC in spinal cord tissue ipsilateral to mustard oil application strongly supported a critical role of PKC in sustained nociceptive responses to mustard oil.     

脑动脉内皮细胞缺氧时内皮型一氧化氮合酶基因表达的变化及蛋白激酶C的作用

目的：研究缺氧时脑动脉内皮细胞(CAECs)内皮型一氧化氮合酶(eNOS)基因表达的变化，并探讨可能的分子机制。方法： 分别采用RT-PCR和蛋白质免疫印迹技术检测原代培养的猪脑动脉内皮细胞缺氧2、6、12、24、48 h后eNOS mRNA和蛋白质表达的变化，并观察蛋白激酶C(PKC)抑制剂对缺氧24 h引起的eNOS mRNA和蛋白质变化的影响。加入转录抑制剂放线菌素D后观察缺氧24 h对eNOS mRNA稳定性的影响。结果：缺氧2 h后脑动脉内皮细胞eNOS mRNA和蛋白质表达均增加，12 h达到高峰，约分别为常氧组的2.5倍和2.0倍，缺氧48 h仍高于常氧组。缺氧对eNOS mRNA稳定性无明显影响。选择性PKC抑制剂BIM I(1 μmol/L)、G6983(1 μmol/L)均能降低缺氧24 h所引起的eNOS基因表达的上调。结论： 脑动脉内皮细胞缺氧时可通过PKC信号途径上调eNOS基因的表达，并可能由此介导缺氧时脑血管的扩张反应，发挥其神经保护作用。     

Activation of spinal d1/d5 receptors induces late-phase LTP of C-fiber-evoked field potentials in rat spinal dorsal horn

Long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn may be relevant to pathological pain. Our previous work has shown that the late phase of the spinal LTP is protein synthesis-dependent. Considerable evidence has accumulated that dopamine D1/D5 receptors are important for late-phase LTP in hippocampus. In this study, the role of D1/D5 receptors in LTP of C-fiber-evoked field potentials in spinal dorsal horn was evaluated in urethan-anesthetized Sprague-Dawley rats. We found the following. 1) Spinal application of SKF 38393, a D1/D5 receptor agonist, induced a slowly developed LTP of C-fiber-evoked field potentials, lasting for >10 h, and the effect was blocked by the D1/D5 antagonist SCH 23390, whereas a D2 receptor agonist (quinpirole) induced depression of C-fiber responses, lasting for 2 h. 2) The potentiation produced by D1/D5 receptor agonist occluded the late phase but not the early phase of the spinal LTP produced by tetanic stimulation. 3) SCH 23390 selectively depressed the late-phase LTP, when applied 40 min before tetanic stimulation. 4) The D1/D5 agonist-induced potentiation is blocked by the protein synthesis inhibitor anisomycin. 5) Activation of protein kinase A by spinal application of 8-Br-cAMP also induced spinal LTP, and the action occluded the potentiation induced by the D1/D5 receptor agonist. These results suggest that the spinal D1/D5 receptors participate in the protein synthesis-dependent late-phase LTP of C-fiber-evoked field potentials in spinal dorsal horn through the cAMP signaling pathway.     

Mu opiates inhibit long-term potentiation induction in the spinal cord slice

Long-term potentiation (LTP) involves a prolonged increase in neuronal excitability following repeated afferent input. This phenomenon has been extensively studied in the hippocampus as a model of learning and memory. Similar long-term increases in neuronal responses have been reported in the dorsal horn of the spinal cord following intense primary afferent stimulation. In these studies, we utilized the spinal cord slice preparation to examine effects of the potently antinociceptive mu opioids in modulating primary afferent/dorsal horn neurotransmission as well as LTP of such transmission. Transverse slices were made from the lumbar spinal cord of 10- to 17-day-old rats, placed in a recording chamber, and perfused with artificial cerebrospinal fluid also containing bicuculline (10 microM) and strychnine (1 microM). Primary afferent activation was achieved in the spinal slice by electrical stimulation of the dorsal root (DR) or the tract of Lissauer (LT) which is known to contain a high percentage of small diameter fibers likely to transmit nociception. Consistent with this anatomy, response latencies of LT-evoked field potentials in the dorsal horn were considerably slower than the response latencies of DR-evoked potentials. Only LT-evoked field potentials were found to be reliably inhibited by the mu opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)] enkephalin-ol (DAMGO, 1 microM), although evoked potentials from both DR and LT were blocked by the AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione. Moreover repeated stimulation of LT produced LTP of LT- but not DR-evoked potentials. In contrast, repeated stimulation of DR showed no reliable LTP. LTP of LT-evoked potentials depended on N-methyl-D-aspartate (NMDA) receptor activity, in that it was attenuated by the NMDA antagonist APV. Moreover, such LTP was inhibited by DAMGO interfering with LTP induction mechanisms. Finally, in whole cell voltage-clamp studies of Lamina I neurons, DAMGO inhibited excitatory postsynaptic current (EPSC) response amplitudes from LT stimulation-evoked excitatory amino acid release but not from glutamate puffed onto the cell and increased paired-pulse facilitation of EPSCs evoked by LT stimulation. These studies suggest that mu opioids exert their inhibitory effects presynaptically, likely through the inhibition of glutamate release from primary afferent terminals, and thereby inhibit the induction of LTP in the spinal dorsal horn.