乙酰胆碱信号通路在肺癌中的作用

肺癌作为当今世界最为致命的疾病之一,严重地威胁着人类的生命健康。神经递质乙酰胆碱(acetylcholine,ACh)作为肺癌的自分泌和旁分泌生长因子,可与肺癌细胞上的烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChRs)和毒蕈碱型乙酰胆碱受体(muscarinic acetylcholine receptors,mAChRs)结合,促进肺癌细胞的增殖、迁移和侵袭。近年来,全基因组数据研究表明,nAChRs与吸烟相关肺癌风险密切相关,因此其在肺癌发生发展中的作用机制的研究对肺癌药物的研发具有重要意义。本文对乙酰胆碱信号通路在肺癌进展中的作用机制进行综述,为其成为肺癌治疗新分子靶点和药物的开发提供依据。     

α7-nAChR基因敲除小鼠海马神经元突触传递和神经网络的电生理表型（英文）

研究显示,含有α7亚基的烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7-nAChR)基因敲除(α7 KO)的小鼠表现出很少的功能表型。本文旨在研究α7 KO对小鼠海马电生理特征的影响。用标准胞外场电位记录评估α7 KO对小鼠海马CA3-CA1突触传递的影响,用穿孔膜片钳记录检测小鼠海马单个神经元γ-氨基丁酸A型受体(γ-aminobutyric acid A-type receptor, GABA_A-R)的电生理学表型。结果显示,与野生型小鼠相比,α7 KO小鼠海马CA1神经元场兴奋性突触后电位(field excitatory postsynaptic potential, fEPSP)斜率显著降低,卡巴胆碱诱发的θ振荡显著减少。在给予GABA_A-R激动剂蝇覃醇(muscimol)条件下,与野生型小鼠相比,α7 KO小鼠海马CA1和CA3神经元I–V曲线均向去极化方向明显移动。以上结果提示,α7KO小鼠海马CA3-CA1突触传递显著受损,GABA_A-R成熟显著延迟,表明α7-nAChR基因缺失可显著改变海马的电生理功能,这可能为α7-nAChR在海马功能和海马相关疾病中作用的理解提供了新的认识。     

乙酰胆碱通过作用于α7烟碱型乙酰胆碱受体抑制大鼠小胶质细胞炎性反应

小胶质细胞是中枢神经系统中主要的免疫细胞。本研究旨在探讨乙酰胆碱(acetylcholine,ACh)抑制小胶质细胞炎症反应的具体机制。原代培养Sprague-Dawley(SD)大鼠小胶质细胞,脂多糖(lipopolysaccharide,LPS)诱导建立炎症反应模型,ACh处理24 h后,用Western blot检测多种炎性因子、胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)和α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)的蛋白表达,用ELISA检测多种炎性因子和IGF-1的释放情况,用慢病毒转染沉默α7nAChR后观察ACh作用的变化。结果显示,LPS可促进小胶质细胞的激活,上调诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)蛋白表达,增加白介素1β(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factorα,TNF-α)的表达和释放,减少神经营养因子IGF-1的表达和释放,ACh能逆转LPS的这些作用;LPS可下调小胶质细胞的α7nAChR蛋白表达,ACh逆转该作用;α7n AChR-shRNA慢病毒转染小胶质细胞后,ACh对抗LPS的作用消失。以上结果提示,ACh对抗LPS诱导的小胶质细胞炎症反应的保护作用是通过α7nAChR实现的,这为今后神经炎症疾病的治疗提供了新的治疗靶点。     

NIH3T3细胞中PTPα基因高表达激活Src的早期细胞表型变化

利用蛋白质酪氨酸磷酸酶α(PTPα)基因转染NIH3T3细胞 ,研究PTPα诱导前后细胞生物学行为的变化 ,为肿瘤形成早期机制的研究提供细胞模型。用携带PTPα基因的四环素调控体系转染NIH3T3细胞并诱导 2 4h后 ,用RT PCR和蛋白质印迹法证实PTPα在诱导细胞中的表达高于未诱导细胞 ,用RT PCR及蛋白质印迹法发现内源性Src的表达水平在诱导与未诱导细胞中没有变化 ,而Src激酶的活性在诱导细胞中增高 ,并且在诱导细胞中Src的酪氨酸磷酸化水平降低。再用透射式电子显微镜和流式细胞技术观察到诱导细胞的表型已发生变化 ,实验结果表明PTPα诱导 2 4h使细胞的表型开始发生转化 ,这种变化很可能与PTPα表达水平升高使SrcC末端的pTyr52 7去磷酸化而激活Src相关     

非神经元型胆碱能系统在（前）脂肪细胞的表达及nAChRα7功能状态对前脂肪细胞Visfatin基因表达的影响

原核生物、真核生物、植物体的非神经细胞和组织中,尤其是多种免疫活性细胞中,均证实乙酰胆碱酯酶（acetylcholinesterase,AChE）、胆碱乙酰转移酶（choline acetyltransferase,ChAT）和乙酰胆碱受体（acetylcholine receptor, AChR）各亚型在内的胆碱能系统组分的存在,其中烟碱样乙酰胆碱受体α7（nicotinic acetylcholine receptor α7,nAChRα7）是烟碱样胆碱能抗炎通路（nicotinic anti-inflammatory pathway）中重要的分子核心机制,同时也是机体限制宿主防御反应扩大的内源性抗炎机制之一. 本文旨在探讨（前）脂肪细胞上非神经元型胆碱能系统是否存在及初步揭示烟碱样胆碱能受体α7对前脂肪细胞功能的影响. 以体外培养的3T3-L1前脂肪细胞为研究对象,采用免疫组化和蛋白质免疫印迹技术,分别检测前脂肪细胞和成熟脂肪细胞中乙酰胆碱酯酶、胆碱乙酰转移酶和烟碱样乙酰胆碱受体α7的3种胆碱能系统主要组分的蛋白表达. 另将前脂肪细胞分为给予广谱烟碱样乙酰胆碱受体激动剂尼古丁、特异性烟碱样乙酰胆碱受体α7激动剂氯化胆碱及特异性烟碱样乙酰胆碱受体α7拮抗剂甲基牛扁亭碱干预12 h、24 h、36 h,并设立相应处理时间的空白对照组,逆转录聚合酶链反应检测前脂肪细胞visfatin mRNA表达情况. 免疫组化染色可见前脂肪细胞中AChE、ChAT及AChRα7均有阳性表达;蛋白免疫印迹检测进一步半定量证实了前脂肪细胞和成熟脂肪细胞中AChE、ChAT及AChRα7的蛋白表达;拮抗剂甲基牛扁亭碱(10⁶～10⁴mol/L)时间、剂量依赖性上调前脂肪细胞visfatin mRNA表达(1.3～1.55fold,P<0.01),与对应空白对照组相比,存在显著性统计学差异; 加入不同剂量的尼古丁和氯化胆碱,则前脂肪细胞中visfatin mRNA表达水平与对应空白对照组相比,均不同程度地下降,其中以氯化胆碱的抑制效应更为显著. 前脂肪细胞与成熟脂肪细胞中均存在有独立的胆碱能体系,其中AChRα7很可能在调节脂肪细胞因子分泌及肥胖相关的病理生理过程中发挥重要作用.     

表皮生长因子对精原干细胞的作用研究

应用不连续Percoll梯度液和选择性贴壁法分离纯化精原干细胞:c-kit细胞免疫组化鉴定细胞类型;MTT法研究EGF对精原干细胞增殖的效应;加入MAPK-ERK信号通路特异性抑制剂PD98059,探讨EGF对精原干细胞增殖作用的可能机制.证明:1)c-kit细胞免疫组化结果显示分离得到细胞为精原千细胞;2)MTT结果显示各实验组比对照组细胞数量均有显著增多(p＜0.01),且20 ng/mL剂量组的增殖作用最明显;3)与对照组相比,加入PD98095组的活细胞数有显著下降(p＜0.01).结论:EGF能够促进精原干细胞的增殖,并且可以通过MAPK-ERK信号通路起作用.     

尼古丁预防帕金森氏综合症和老年痴呆症的分子机理研究

吸烟有害健康,吸烟产生的自由基、亚硝胺和多环芳烃等是主要有害物质,而尼古丁是造成吸烟依赖的主要物质。流行病学统计显示,吸烟者患帕金森氏综合症(Parkinson's disease,PD)和老年痴呆症(Alzheimer's disease,AD)的概率远低于不吸烟者;实验和人群结果表明尼古丁可以预防PD和AD,但其机理还不十分清楚。实验发现:1)尼古丁可以有效清除活性氧自由基,能够抑制多巴胺自氧化,是一种抗氧化剂;2)尼古丁能够有效抑制6-OHDA和MPP 诱导的细胞色素C(Cytochrome C,Cyt.C)释放;3)尼古丁可以保护海马神经元抵抗β淀粉样蛋白诱导的凋亡;4)尼古丁可以防止淀粉样蛋白在转基因AD鼠脑中的沉淀;5)尼古丁可以络合金属铜和锌,防止其在脑中积聚;6)尼古丁可以通过激活烟碱型乙酰胆碱受体nAChRs(nicotin acetylcholine receptor)7和MAPK(mitogen activated protein kinase)来抑制NF-κB和C-Myc信号通路,抑制炎症和诱导型NOS表达和NO生成,预防AD。这些结果对于解释尼古丁防治神经退行性疾病AD和PD的机理具有重要意义。     

α7nAChR和nNOS在Aβ诱导的认知障碍大鼠皮质及海马表达的时间和空间变化

本研究旨在观察α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7n ACh R)与神经元型一氧化氮合酶(neuronal nitric oxide synthetase,n NOS)在Aβ诱导的认知障碍大鼠皮质和海马时间和空间的分布与变化。取Sprague-Dawley(SD)大鼠60只,随机分成6组,其中3个实验组分别经侧脑室注射凝聚态Aβ1-42(2.5μg/μL,4μL),连续观察7 d(7 d Aβ组)、14 d(14 d Aβ组)和21 d(21 d Aβ组);3个对照组则分别注射和实验组等量生理盐水并观察相同的时间。用Y迷宫刺激器检测大鼠的学习和记忆行为能力,用免疫组织化学和Western blot检测皮质和海马CA1、CA3、DG各区α7n ACh R和n NOS阳性细胞分布和蛋白表达量的变化。结果显示,与各自对照组相比,3个实验组大鼠的学习和记忆行为能力降低,前额叶皮质和海马各区α7n ACh R和n NOS的表达均显著下调,特别在前额叶皮质浅层和海马CA3区变化明显;3个实验组之间比较结果显示,Aβ诱导的认知障碍大鼠学习和记忆功能、前额叶皮质和海马α7n ACh R和n NOS表达均随着Aβ作用时间的延长呈进行性下降。以上结果提示,前额叶皮质和海马α7n ACh R和n NOS表达的共同降低可能是Aβ诱导的大鼠认知功能障碍的基础。     

可溶性Jagged-1/Fc嵌合蛋白诱导淋巴结细胞向CD4+CD25+ T细胞分化

直接用可溶性Jagged-1/Fc嵌合蛋白(Jagged-1/Fc)在体外诱导小鼠淋巴结细胞向CD4 CD25 T细胞分化.通过荧光标记单克隆抗体染色结合流式细胞术,观察不同剂量Jagged-1/Fc在不同时间对淋巴结细胞向CD4 CD25 T细胞分化的影响,观察Jagged-1/Fc诱导T细胞内细胞因子的变化;藉ELISA法检测Jagged-1/Fc诱导分化的T细胞分泌TGF-β1、IL-4和IL-10的水平.结果显示,超过500.0μg/L剂量的Jagged-1/Fc使CD4 CD25 T细胞百分比明显增高,诱导时间需要4～6天,抗Jagged-1单抗能抵消Jagged-1/Fc的诱导作用,用DAPT阻断Notch信号通路的活化也能抑制Jagged-1/Fc的诱导作用,Jagged-1/Fc诱导分化的T细胞培养上清中IL-4和IL-10的水平明显增高,TGF-β1无明显变化,胞内IL-4,IL-10,IL-2和TNF-α的水平也呈增高趋势.上述结果表明,可溶性Jagged-1/Fc嵌合蛋白在体外可诱导小鼠淋巴结细胞向CD4 CD25 调节性T细胞分化.     

电针对术后认知功能障碍大鼠认知功能及海马α7nAChR受体表达的影响

摘要 目的：探讨电针对术后认知功能大鼠认知功能及海马α7型神经烟碱胆碱能受体(nicotinic acetylcholine receptor,α7nAChR)表达含量的影响。方法：选择120只雄性SD大鼠,将其随机分为以下5组：对照组(Ctrl组)、手术组(Op组)、电针组(EA)、α7nAChR抑制剂 (EA+α-BGT)组、α7nAChR激动剂 (PHA-543,613组),每组12只,每组又分为1 d和3 d两个亚组。采用Morris水迷宫检测认知功能,ELISA检测血清肿瘤坏死因子(Tumor necrosis factor,TNF)和白细胞介素-1β(interleukin-1β,IL-1β)、高迁移率族蛋白1(High mobility group box 1,HMGB-1)含量,RT-PCR检测海马TNF-α和IL-1β、HMGB-1 m-RNA表达,蛋白印迹法检测海马α7nAChR表达,甲苯胺蓝法检测海马CA1区肥大细胞活化情况,Tunel法检测海马CA1区细胞凋亡情况。结果：与Op组相比,EA组、PHA-543,613组术后第1 d、第3 d逃避潜伏期显著缩短,穿越平台次数增加 (P<0.05);与EA组相比,EA+α-BGT组术后第1 d、第3 d逃避潜伏期显著延长,穿越平台次数明显减少(P<0.05)。与Op组相比,EA组、PHA-543,613组鼠术后第1 d、第3 d血清TNF-α和IL-1β、HMGB-1含量显著降低,海马TNF-α和IL-1β、HMGB-1 m-RNA表达亦明显下调,海马α7nAChR蛋白含量表达上调、海马CA1区肥大细胞活化数目、Tunel阳性细胞数目减少,两组比较差异有统计学意义(P<0.05)。与EA组相比,EA+α-BGT组大鼠术后第1 d、第3 d血清TNF-α和IL-1β、HMGB-1含量增加,海马TNF-α和IL-1β、HMGB-1 m-RNA表达亦明显上调,海马α7nAChR蛋白表达上调,海马CA1区肥大细胞活化数目、Tunel阳性细胞数目亦明显增加(P<0.05)。结论：电针可能通过抑制中枢肥大细胞活化,上调脑内α7nAChR蛋白表达,抑制TNF-α和IL-1β、HMGB-1表达和释放,进而改善胫骨骨折术后大鼠学习与记忆能力。     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

Voltage-independent inhibition of P/Q-type Ca2+ channels in adrenal chromaffin cells via a neuronal Ca2+ sensor-1-dependent pathway involves Src family tyrosine kinase

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.     

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.     

Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).     

Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle

Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.     

The Src tyrosine kinase pathway regulates thecal CYP17 expression and androstenedione secretion

In order to evaluate the role of Src tyrosine kinase in thecal cell steroidogenesis, a pharmacological approach was utilized by treating enriched populations of mouse ovarian theca-interstitial cells in vitro with a direct Src kinase inhibitor, PP2. Inhibition of Src with PP2 increased both basal and forskolin-stimulated androstenedione secretion, and increased cytochrome P450 17-alpha hydroxylase-lyase (CYP17) promoter activity and steady state mRNA. PP2 did not change thecal levels of StAR mRNA. Inhibition of mitogen-activated protein kinase kinase, a downstream regulator of Src activity, using PD98059 also increased forskolin-stimulated secretion of androstenedione above forskolin alone, but had no effect on basal secretion of androstenedione. Src inhibition increased mitogen-activated protein kinase phosphatase-1 protein and decreased phosphorylation of SF-1, which correlated with increased CYP17 promoter activity and mRNA levels. These results implicate Src tyrosine kinase in the regulation of CYP17 and thecal androgen secretion.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.     

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.     

Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase.

In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes.