精氨酸加压素C端片段（4—8）对大鼠海马神经元CCT的影响

为了研究精氨酸加压素C端片断 4～ 8(AVP4～ 8)对大鼠海马神经元中胞苷三磷酸 :磷酸胆碱胞苷转移酶 (CCT)的mRNA水平和酶活性的影响及其作用机制。将大鼠海马神经元用不同药物处理一定时间后 ,用RT PCR结合Southern杂交的方法测定CCT的mRNA水平 ,用测定胞苷二磷酸胆碱中 [14 C]的参入率来确定CCT活性。结果表明 :AVP4～ 8能上调海马神经元内CCT的mRNA水平 ,AVP4～ 8的拮抗剂ZDC(C)PR对此有抑制作用 ,当用放线菌素D中止基因转录 ,发现用AVP4～ 8共处理的细胞 ,比单独用放线菌素D处理的细胞 ,其CCTmRNA的半衰期较长 ;AVP4～ 8处理后 ,神经元内CCT的酶活性也有一定程度的上升。由此得出结论 :AVP4～ 8通过提高神经元内CCTmRNA的稳定性 ,提高了CCTmRNA的水平 ,进而影响了胞内CCT的酶活性。     

精氨酸加压素对大鼠背根神经节神经元GABA激活电流的增强作用（英文）

越来越多的研究表明,精氨酸加压素(arginine vasopressin,AVP)在痛觉调制中具有镇痛作用。已报道的研究专注于AVP镇痛的中枢作用机制,而本研究旨在研究AVP镇痛的的外周作用机制。应用全细胞膜片钳技术,在急性分离的大鼠背根神经节(DRG)神经元上,观察AVP对GABA激活电流(IGABA)的增强作用以及AVP对GABAA受体功能的影响。结果显示,AVP(1×10-10~1×10-5 mol/L)预处理后,IGABA增大,GABA剂量效应曲线上移,IGABA的最大值较之对照增加约49.1%;而EC50值几乎不变,表示此种加强为非竞争性的,而且AVP对GABA电流的作用可能是电压非依赖性的。AVP对IGABA的加强作用几乎完全被V1a受体的拮抗剂SR49059(3×10-6 mol/L)阻断。二次钳压技术胞内透析非水解GDP类似物GDP-β-S(5×10-4 mol/L)或PKC抑制剂GF109203X(2×10-6 mol/L)也可以阻断AVP对IGABA的加强作用。以上结果提示,AVP经由G蛋白耦联受体以及PKC信号通路上调DRG神经元GABAA受体的功能,可能是其诱导镇痛作用的基础。     

发现一种高效、高特异性的促进学习、记忆的神经肽

脑中九肽精氨酸加压素(AVP)对提高学习和记忆的作用已被证实。AVP可保留大鼠的条件性回避反应,使主动回避反应消退的时间减慢,促进被动回避反应和脑的发育。临床上,AVP对老年性记忆力减退及遗忘症的治疗都取得了满意的效果。近来实验发现,将AVP与鼠脑突触膜孵育,经膜内蛋白水解酶水解后,由反相高压液相色谱仪分离,得到AVP代谢产物:p谷-天冬酰-半胱(半胱)-脯-精-甘-NH_2([p谷~4,半胱~?]AVP-(4一9))以及它的脱甘氨酰胺衍生物,对大鼠具有比AVP更高的选择性和更强的促进记忆巩固作用。当大鼠建立了被动回避反应的实验模型后,向两侧侧脑室注射[p谷~4,半胱~6]AVP-(4-9),发现它可促进大鼠的被动回避行为,有效剂量范围极小(0.01～1pg);在羧基端脱甘氨酰胺的衍生物[p谷~4,半胱~6]AVP-(4-8)的有效剂量范围在     

胆囊收缩素8对大鼠大脑皮质细胞钙调素和蛋白激酶C活性的影响

 为阐明中枢神经系统中胆囊收缩素 8(CCK8)受体的信号传递机制 ,以分离的大鼠大脑皮质神经细胞为材料 ,观察了CCK8对细胞内钙调素 (CaM)、3′ ,5′ 环腺苷酸 (cAMP)、蛋白激酶C(PKC)活性的影响 研究结果表明 ,CCK8可刺激大脑皮质细胞CaM、PKC活性的增加 ,并有剂量依赖关系 但CCK8在 10 -12 ～ 10 -6mol L范围内 ,细胞内cAMP含量无显著变化 利用受体亚型L 364,718和L 365,2 60的研究表明 ,两种拮抗剂均可抑制CCK8引起的CaM和PKC活性变化 ,但两者IC50 不同 对于CaM ,CCKB 受体拮抗剂L 365,2 60的IC50 比CCKA 受体拮抗剂L 364,718低 4 0倍 ;而对于PKC ,L 365,2 60的IC50 比L 364,718低 60倍 因此认为 ,CCK8主要是通过CCKB 受体介导了CaM和PKC活性的变化     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca2+浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 ]i 升高 ,即 PKC和 Ca2 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用.我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖.本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用,并探计其机制.采用组织块法培养成年大鼠CFs,用[3H]-TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布.根据特异性底物髓磷脂基质蛋白(myelin basic protein, MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)的活性.用Western blot检测ERK1/2的磷酸化和p27Kip1、细胞周期蛋白D1、 A、 E的表达.结果显示,AVP(0.1μmol/L)可促进成年大鼠CFs的DNA合成,该作用可被V1受体拮抗剂d(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)阻断,而不受V2受体拮抗剂desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/L)的影响.AVP可激活ERK1/2,用蛋白激酶C(protein kinase C, PKC)激动剂佛波酯(phorbol 12-myristate 13-acetate, PMA, 30nmol/L, 5min)急性刺激可模拟该作用,而PMA持续慢性作用(2.5μmol/L,24h)耗竭PKC后则抑制AVP对ERK1/2的激活.AVP可抑制p27Kip1的蛋白表达,升高细胞周期蛋白D1、 A和E的表达,同时促进细胞周期由G0/G1期进入S期.ERK1/2抑制剂PD98059 (30μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用,并抑制细胞周期进程.以上结果表明,AVP可促进成年大鼠CFs增殖,该作用由V1受体和PKC-ERK1/2通路介导.AVP可通过ERK1/2调控p27Kip1、细胞周期蛋白D1、A和E的表达,从而促进成年大鼠CFs的细胞周期进程.     

大鼠运动病敏感性性别差异与相关脑区AVP及其V1b受体表达的关系

目的：检测不同性别大鼠旋转刺激后脑内相关区域精氨酸加压素（AVP）含量及V1b受体表达的变化,探讨AVP及受体参与运动病的可能机制。方法：给予SD大鼠30 min绕水平轴的旋转刺激,然后采用放免法检测相关脑区AVP含量,并通过荧光免疫组化方法测定相应脑区V1b受体的表达情况。结果：①在雌性大鼠,旋转刺激组各脑区AVP含量无显著性改变;对于雄性大鼠,对照组各检测脑区AVP含量高于雌性,旋转刺激组小脑、延髓内AVP含量的变化无显著性意义,但前脑、间脑、脑桥内AVP含量较对照组明显降低（P〈0.05）。②雌性大鼠视上核AVP的V1b受体表达阳性神经元数量旋转刺激组显著低于对照组（P〈0.05）,而前庭核、最后区V1b受体表达阳性神经元数量明显多于对照组（P〈0.05）;在雄性大鼠,旋转刺激组视上核与前庭核V1b受体表达阳性神经元数量无显著性改变,而最后区V1b受体表达阳性神经元数量有所增加（P〈0.05）,但增加幅度没有雌性大鼠明显。结论：前脑、间脑、脑桥内AVP含量与前庭核和最后区V1b受体表达及对旋转刺激反应的差异可能与运动病敏感性性别差异有关,并且前庭核、最后区可能是AVP-V1受体拮抗剂抗运动病作用的靶点。     

蛋白激酶C活性变化对Adipophilin介导THP-1巨噬细胞脂质蓄积的影响

以THP-1巨噬细胞为研究对象,观察蛋白激酶C(PKC)激动剂佛波酯(PMA)和抑制剂钙磷酸结合蛋白C(CalphostinC)对胞膜PKC活性、胞膜PKCα及胞浆内过氧化物酶体增殖物激活受体(PPARγ)和adipophilin表达以及细胞内脂质蓄积的影响,初步探讨PKC调控adipophilin表达及脂质蓄积的作用机制.采用PepTagRAssay、RT-PCR、蛋白质印迹、油红O染色和高效液相色谱法,观察到100nmol/LPMA在激活胞膜PKC((0.2514±0.0154)U/ml)的同时可以与氧化低密度脂蛋白(oxLDL)协同增强PKCα、PPARγ和adipophilin表达并使细胞内脂滴的蓄积极大地增强.细胞内胆固醇酯/总胆固醇比值增至(69.8±9.5)%;300nmol/L CalphostinC对荷脂THP-1巨噬细胞的处理则抑制酶活性至((0.0927±0.0056)U/ml,细胞内脂滴减少,胆固醇酯/总胆固醇比值降至(40.1±9.1)%;CalphostinC呈剂量依赖性的方式下调酶活性、PKCα、PPARγ和adipophilin表达,400nmol/LCalphostinC基本上可以逆转50mg/LoxLDL诱导的酶活化和PKCα、PPARγ和adipophilin表达的上调.结果提示,蛋白激酶C活性的改变可以影响adipophilin介导的脂质蓄积,其中PPARγ可能在这一调控机制中发挥了重要作用.     

弓状核内胱硫醚β-合成酶通过PKC的上调和GluN2B磷酸化参与慢性胰腺炎大鼠腹部痛觉过敏（英文）

有报道显示初级感觉神经元中的硫化氢(H2S)参与内脏痛敏的形成,但其在中枢神经系统中的作用却鲜为人知。该研究旨在探讨下丘脑弓状核(arcuate nucleus,ARC)内H2S和其内源性合成酶是否参与慢性胰腺炎(chronic pancreatitis,CP)腹部痛觉过敏及其潜在机制。成年雄性Sprague-Dawley大鼠胰管内注射三硝基苯磺酸(trinitrobenzene sulfonic acid,TNBS)诱导产生CP模型,运用von Frey filament(VFF)评测大鼠腹部对机械刺激的反应频率,运用Western blot检测ARC内蛋白表达水平。TNBS注射4周后ARC内胱硫醚-β-合成酶(cystathionineβ-synthetase,CBS)表达显著上调,而胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)的表达没有明显改变;CP显著提高了NMDA受体Glu N2B亚单位的磷酸化水平,而总Glu N2B不变;CP也显著上调了蛋白激酶Cγ(PKCγ)在ARC的表达。ARC内微量注射CBS的抑制剂O-(羧甲基)羟胺半盐酸盐(AOAA)能显著降低CP大鼠的腹部疼痛,也翻转了CP大鼠ARC中p-Glu N2B和PKCγ的上调;ARC内微量注射Glu N2B抑制剂或PKC特异性抑制剂白屈菜赤碱能显著减轻CP大鼠的腹部痛觉过敏,PKC特异性抑制剂能减少p-Glu N2B表达。总之,以上数据表明,CP引起了ARC内CBS的上调,并可能通过PKCγ介导了Glu N2B的活化,参与CP痛觉过敏。本研究在一定程度上揭示了CP痛觉过敏产生的中枢机制,并有助于发现CP疼痛治疗的新靶标。     

Phorbol ester-induced down-regulation of protein kinase C abolishes vasopressin-mediated responses in rat anterior pituitary cells

The role of protein kinase C (PKC) in the multihormonally regulated ACTH secretory responses of rat anterior pituitary cells was examined in control cells or after pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC. Using affinity-purified polyclonal antiserum raised against purified rat brain PKC, immunoprecipitable PKC was demonstrated in [35S]methionine-labeled cells appearing as a doublet of 78/80 kilodaltons. Long-term treatment (24 h) of cells with 0.6 microM TPA caused the specific loss of immunologically reactive PKC. Consistently, TPA pretreatment decreased the amount of phosphatidylserine-dependent protein kinase activity measured in vitro by 90%. In control cells, vasopressin (AVP) stimulated ACTH secretion and potentiated ACTH secretion stimulated by CRF. After a 24-h treatment with 0.6 microM TPA, secretory responses to AVP and the potentiating effect of AVP on CRF action were completely abolished. In contrast, CRF action on ACTH secretion, thought to be mediated by cAMP, was unaffected. Similarly, forskolin- and 8 bromo-cAMP-induced ACTH secretion remained unchanged after TPA pretreatment. These results indicate a crucial role for PKC in mediating the effects of AVP on ACTH secretion and on the potentiating action of AVP on CRF-induced secretion from corticotropic cells of the anterior pituitary.     

AVP_（4－8）结合点在大鼠海马内的分布和AVP_（4－8）对其受体发育的影响：放射自显影研究

本文利用放射自显影方法结合神经毒对海马神经元的选择性损毁观察ＡＶＰ（４－８）结合点在大鼠海马内的分布和定位;利用外源性ＡＶＰ（４－８）对新生大鼠的处理,观察海马ＡＶＰ（４－８）结合点的发育调节。在成年大鼠海马内,ＡＶＰ（４－８）结合点集中分布在整个海马的锥体细胞层和齿回的颗粒细胞层。秋水仙碱处理后,齿回颗粒细胞层消失,齿回区的ＡＶＰ（４－８）结合点也消失。红藻氨酸（Ｋａｉｎｉｃａｃｉｄ）处理后海马ＣＡ３－ＣＡ４的锥体细胞层消失,该区的ＡＶＰ（４－８）结合点也消失。新生大鼠海马锥体细胞层的ＡＶＰ（４－８）结合点在出生后第６天开始出现,齿回颗粒细胞层的ＡＶＰ（４－８）结合点在出生后第７天开始出现。然而,新生大鼠每天经外源性ＡＶＰ（４－８）处理,海马锥体细胞层和齿回颗粒细胞层的结合点均在出生后第５天已变得十分稠密。本文就大鼠海马ＡＶＰ（４－８）结合点的特异性分布和ＡＶＰ（４－８）处理促进海马ＡＶＰ（４－８）结合点的发育与成年后大鼠学习能力的提高的相互关系作了讨论。     

Involvement of ERK and AKT signaling in the growth effect of arginine vasopressin on adult rat cardiac fibroblast and the modulation by simvastatin

Arginine vasopressin (AVP) has been shown to directly induce neonatal rat cardiac fibroblasts (CFs) proliferation, a major component involved in cardiac hypertrophy. Herein, we explored whether AVP is also a growth factor for adult rat CFs and, if so, whether the growth effect could be inhibited by simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. AVP significantly increased DNA synthesis in adult rat CFs by 73.5 +/- 5.1% (P < or = 0.05), an effect inhibited by V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (ERK1/2) as assessed by MBP phosphotransferase activity (5.1 +/- 0.6 fold over basal level, P < or = 0.05) and Western blot analysis, and effects were mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by inhibiting cellular PKC through chronic PMA incubation. In addition, AVP induced PKC activation (27.2 +/- 3.8% from a basal value of 9.3 +/- 0.7%, P < or = 0.05). AVP-induced increase in DNA synthesis could be attenuated by the specific inhibitors of ERK1/2 (PD98059), PI3K (LY294002), and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, HIMO). Simvastatin inhibited the effects of AVP on DNA synthesis, ERK1/2, and PKC activation in a dose-dependent manner. Phosphatidylinositol-3-kinase (PI3K)-dependent AKT activation induced by AVP was also inhibited by simvastatin. The effects of simvastatin on ERK1/2, PKC, and AKT activation and DNA synthesis could be reversed by mevalonate. These results support a growth-inducing effect of AVP on adult rat CFs through ERK and AKT signalings and the growth effect could be attenuated by simvastatin via inhibiting these two pathways.     

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24h resulted in a reduction in ANP receptor binding activity by about 50% (B(max); control cells, 22.9+/-2.5 fmol/mg protein, AVP-treated cells, 11.4+/-1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Gialpha-2 and Gialpha-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Gialpha proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsalpha protein and Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gbeta were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V(1) and ANP-C receptor-mediated signaling pathways.     

Protein Kinase C-Mediated Phosphorylation of Kvβ2 in Adult Rat Brain

The phosphorylation of Kvβ2 was investigated by different protein kinases. Protein kinase A catalytic subunit (PKA-CS) yielded the greatest phosphorylation of recombinant Kvβ2 (rKvβ2), with limited phosphorylation by protein kinase C catalytic subunit (PKC-CS) and no detectable phosphorylation by casein kinase II (CKII). Protein kinase(s) from adult rat brain lysate phosphorylated both rKvβ2 and endogenous Kvβ. The PKA inhibitor, PKI 6-22, fully inhibited PKA-mediated phophorylation of rKvβ2 yet showed minimal inhibition of kinase activity present in rat brain. The inhibitor Gö 6983, that blocks PKCα, PKCβ, PKCγ, PKCδ and PKCζ activities, inhibited rKvβ2 phosphorylation by rat brain kinases, with no inhibition by Gö 6976 which blocks PKCα and PKCβΙ activities. Dose-response analysis of Gö 6983 inhibitory activity indicates that at least two PKC isozymes account for the kinase activity present in rat brain. Τhus, while PKA was the most active protein kinase to phosphorylate rKvβ2 in vitro, Kvβ2 phosphorylation in the rat brain is mainly mediated by PKC isozymes.     

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.     

加压素(4 -8)对大鼠脑内Ca2+/CaM依赖的蛋白激酶Ⅱ自身磷酸化的影响

大鼠皮下注射加压素（ＡＶＰ）（４－８）１ｈ后,大脑皮层中Ｃａ＾２＋／ＣａＭ依赖的蛋白激酶Ⅱ自身磷酸化程度与对照组比较增高１９２％,Ｐ〈０．００１;海马中增高４０％,Ｐ〈０．０５。ＣａＭＫⅡ的自身磷酸化程度依赖于Ｃａ＾２＋及ＣａＭ浓度。在用抗 ＣａＭＫⅡα单克隆抗体对给药１ｈ组样品和对照组样品进行免疫印迹检测时,发现皮下注射ＡＶＰ（４－８）１ｈ后,大脑皮层中ＣａＭＫⅡα亚基的蛋白量没有明显差异。ＡＶ     

Interaction of putative vasopressin receptors in rat brain and bovine pituitary gland with a vasopressin anti-idiotype antibody as revealed by immunoblotting

Vasopressin (AVP)-binding proteins were obtained from rat brain and the anterior and posterior lobes of bovine pituitary glands by (a) preparation of crude membranes, (b) solubilization of membrane proteins, (c) passage through an affinity column containing immobilized AVP, and (d) elution from the column with excess AVP. Gel electrophoresis revealed protein bands of 55 and 62 kilodaltons in rat brain, bovine posterior lobe and, to a far lesser extent, in the anterior lobe, which were similar to those previously identified in rat brain to be associated with AVP binding. Immunoblotting demonstrated that the 55 kilodalton bands of rat brain and bovine pituitary gland were selectively immunoreactive with an AVP anti-idiotype antibody. In addition, immunoreactivity occurred with a 62 kilodalton component of rat brain.     

Characterization of 3H-AVP binding sites in particulate preparations of rat brain

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)