乙酰胆碱对三种人垂体腺瘤细胞增殖及凋亡的影响

为了解乙酰胆碱（acetylcholine,ACh)在人垂体腺瘤发生、发展中的作用,本研究首先观察了人垂体腺瘤细胞内是否存在合成ACh必需的胆碱乙酰转移酶,并应用MTT实验、[^3H]TdR掺入实验、细胞周期分析和TUNEL法测定了ACh对体外培养的人垂体无功能瘤、催乳素瘤和生长激素瘤等三种瘤细胞增殖和凋亡的影响。结果发现：（1）三种垂体腺瘤细胞中均有胆碱酯酶的表达,但明显少于正常垂体;（2）ACh对三种类型的人垂体腺瘤细胞增殖代谢的影响相类似,不同浓度的ACh能明显抑制体外培养的三种人垂体腺瘤细胞的增殖,呈明显的剂量效应关系,同时ACh能减少垂体腺瘤细胞进入S、G2期的细胞比例,而使处于G1期的细胞比例增加;（3）ACh的这种作用可被阿托品阻断,但不受筒箭毒的影响;（4）ACh对体外培养的三种人垂体腺瘤细胞凋亡无明显影响。该结果提示,ACh可能以旁分泌或自分泌的方式作用于垂体前叶细胞,对垂体腺瘤细胞增殖分化有调控作用,而且是通过ACh M受体来实现的。     

青藤碱对家兔主动脉血管平滑肌细胞内游离钙浓度及蛋白激酶C的影响

观测青藤碱对培养家兔血管平滑肌细胞内游离钙浓度及正常和缺血缺氧刺激下蛋白激酶C（PKC）活性的影响。方法：Fura-2／AM作Ca^2＋指示剂,检测青藤碱对培养家兔主动脉血管平滑肌细胞静息Ca^2＋浓度及去甲肾上腺素,高K^＋,咖啡因刺激作用下的改变,并与钙拮抗剂维拉帕米进行对照研究;复制血管平滑肌细胞缺血缺氧模型,液闪仪测定PKC活性。结果：青藤碱剂量依赖性抑制高K^＋去极化引起[Ca^2＋]i升高,青藤碱10×10^-6mol.L^-1、3×10^-5mol.L^-1、10^-4mol.L^-1,对NE通过受体介导引起的[Ca^2＋]i增高也有明显抑制。但对静息状态下及咖啡因刺激的血管平滑肌细胞[Ca^2＋]i无明显影响。正常时,青藤碱处理后血管平滑肌细胞胞浆、胞膜PKC活性均升高;缺血缺氧状态下,胞浆PKC活性升高,但胞膜PKC活性降低,青藤碱处理后胞浆PKC活性下降,胞膜PKC活性上升。结论：青藤碱可能抑制血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道,降低细胞内游离钙水平。调节缺血缺氧条件下血管平滑肌细胞PKC活性。     

普罗托品对大鼠胸主动脉血管平滑肌细胞内游离钙浓度和蛋白激酶C活性的影响

本实验室先前的研究已证实,普罗托品(protopine,Pro)舒张家兔主动脉的作用,可能与其增加血管平滑肌细胞内cAMP和cGMP水平有关.为了深入探讨Pro的扩血管作用机制,实验采用等张收缩记录大鼠离体血管条张力,利用Fura-2/AM负载的大鼠胸主动脉培养细胞直接测定细胞内游离Ca2+浓度([Ca2+]i),并应用同位素γ-32p-ATP催化活性法测定蛋白激酶C(PKC)活性等方法,分别观察了Pro的相关效应.结果表明,Pro(30和100 μmol/L)明显降低去甲肾上腺素(NA)和高钾所致的动脉条收缩幅度,使二者的量效曲线呈非平行右移,最大反应压低;pD2'值分别为3.7±0.25和3.97±0.15;Pro(50和100μmol/L)对静息状态下[Ca2+]i没有任何影响,但对NA和高钾引起的[Ca2+]i升高均有明显抑制作用;Pro(30和100 μmol/L)对未经NA处理血管条的胞浆和胞膜PKC活性均无明显影响;但在NA预处理的血管条,Pro使NA所升高的胞浆内PKC的活性趋于降低,而明显升高胞膜PKC的活性,对PKC的总活性无明显影响.结果提示,在有NA存在的情况下,Pro似能促使PKC从胞浆向细胞膜转移,其扩血管效应似为其降Ca2+作用、升高cAMP和cGMP的作用及其对PKC影响等几方面的综合结果.     

乙酰胆碱对培养的人垂体腺瘤细胞增殖的影响

目的：了解乙酰胆碱（acetylcholine,Ach)对人垂体腺瘤细胞增殖的影响。方法：将Ach作用于体外培养的人垂体腺瘤细胞,测定MTT反应A值和^3H-TdR参入量及用流式细胞仪测定细胞周期。结果：10^-7-10^-5mol/LAch可剂量依赖性地使MTT反应A值和^3H-TdR参入量降低,使垂体腺瘤G1期细胞比例增加（P＜0．01）,并可被阿托品阻断。结论：Ach在体外能明显抑制培养的人垂体腺瘤细胞的增殖,这种作用是通过Ach受体来实现的。     

甲硫氨酸脑啡肽和抗δ阿片受体抗体对小鼠骨髓瘤细胞信号传递系统的作用

为了探讨阿片肽与细胞表面受体结合后所产生的生物效应及其机制 ,用不同浓度甲硫氨酸脑啡肽 ( MENK)及抗 δ阿片受体单克隆抗体处理小鼠的骨髓瘤细胞 ( NS- 1 ) ,然后测定蛋白激酶A( PKA) ,蛋白激酶 C( PKC)活性及三磷酸肌醇 ( IP3 )含量 .研究结果表明 ,NENK可升高细胞胞浆及胞膜 PKA的活性 ,且这一作用可被抗δ阿片受体抗体所拮抗 .MENK对 PKC影响呈双向反应 ,0 .1 μmol/L MENK可以升高胞浆 PKC活性 ,但却明显降低胞膜 PKC活性 ;在 MENK浓度为 1 0μmol/L时则情况刚好相反 .1 μmol/L的 MENK可明显降低胞浆及胞膜 PKC活性 ,抗体可拮抗这种下调作用 .MENK可降低细胞内 IP3 的含量 ,且这一作用可被抗δ阿片受体抗体所拮抗 .由此可以推论 :MENK在与 δ阿片受体结合后 ,可以经过多种信号传导系统来调节细胞功能 ,从而产生不同的生物效应 .     

尼氟灭酸对肝癌细胞增殖的影响

为了观察氯通道阻断剂尼氟灭酸(NFA)对人肝癌细胞(kuman hepatoma cell,HHCC)增殖的影响,我们将NFA作用于HHCC,应用细胞计数法及噻唑兰(MTT)比色分析法观察细胞增殖情况;用流式细胞仪检测细胞周期时相;并用激光扫描共聚焦显微镜检测[Ca^2 ]i的变化。结果发现,NFA使HHCC细胞数及MTT光吸收值(OD)较对照组都显著降低,去除NFA后,OD值逐渐恢复。经100μmol/L NFA处理48h的HHCC细胞G1期细胞比例比对照组明显增高,S期及G2期细胞比例明显低于对照组。细胞外应用NFA(100μmol/L)使[Ca^2 ]i快速降低,去除NFA后,[Ca^2 ]i可恢复。这些结果表明,尼氟灭酸能抑制细胞增殖,其机制可能与细胞内信号转导Ca^2 /CaM途径被抑制有关。     

依他尼酸抑制小鼠气管平滑肌收缩北大核心CSCD

本研究旨在探讨依他尼酸(ethacrynic acid, EA)抑制气管平滑肌(airway smooth muscle, ASM)收缩的作用及其机制。利用BL-420S系统测量小鼠气管环张力,用全细胞膜片钳技术记录ASM细胞通道电流,用钙成像系统测量ASM细胞内Ca^2+浓度。结果显示,EA剂量依赖性地抑制高-K^+(80 mmol/L)和乙酰胆碱(acetylcholine, ACh, 100μmol/L)引起的小鼠气管环收缩,最大舒张百分比分别为(97.02±1.56)%和(85.21±0.03)%,半数有效浓度(median effective concentration, EC50)分别为(40.28±2.20)μmol/L和(56.22±7.62)μmol/L。EA分别降低高-K^+和ACh诱导的细胞内Ca^2+浓度,分别从0.40±0.04降到0.16±0.01,0.50±0.01降到0.39±0.01。此外,EA抑制ASM细胞L-型电压依赖钙通道(L-type voltage-dependent calcium channel,LVDCC)和钙库操纵的钙离子通道(store-operated calcium channel, SOCC)电流,以及高-K^+和ACh引起的外Ca^2+内流。同时,EA可以降低小鼠呼吸系统阻力(resistance of the respiratory system, Rrs)。以上结果提示,EA通过抑制小鼠ASM细胞LVDCC和SOCC,抑制Ca^2+的内流,降低细胞内Ca^2+浓度,导致ASM舒张,提示EA是一种潜在的支气管扩张剂。     

青藤碱对培养VSMC MAPK、PKC活性和细胞[Ca^2＋]i的影响

目的：观察青藤碱对血管平滑肌细胞（VSMC）丝裂素活化蛋白激酶（MAPK）、蛋白激酶C（PKC）活性和胞内游离钙浓度（[Ca^2＋i]）的影响。方法：将VSMC正常培养液、ox-LDL诱导血管内皮细胞（VEC）损伤的条件培养基、青藤碱加ox-LDL诱导VEC损伤的条件培养基等分别作用于VSMC,采用β-放射活性法等测定MAPK及PKC活性,荧光光度法检测VSMC[Ca^2＋]i。结果：VEC损伤条件培养基作用于VSMC后,与正常培养VSMC相比,细胞MAPK、PKC活性明显增加（P〈0．01）,细胞[Ca^2＋]i增加;青藤碱作用于VEC损伤条件培养基培养的VSMC后,与模型组相比,MAPK及PKC活性明显减少（P〈0．01）、细胞[Ca^2＋]i降低。结论：青藤碱抑制VSMC增殖的作用可能与拮抗MAPK、PKC活性和细胞[Ca^2＋]i的增加有关。     

乙酰胆碱不依赖NO的释放引起耳蜗螺旋动脉平滑肌细胞的超极化

目的：乙酰胆碱（ACh）不仅是神经递质,也是一种有效的血管舒张物质参与许多血管床的调节活动。本实验观察ACh引起耳蜗螺旋动脉平滑肌细胞超极化的离子机制以及NO在超极化反应中的可能作用。方法：在豚鼠离体耳蜗螺旋动脉标本上,运用细胞内微电极技术记录外源性的ACh引起的反应。结果：在保持灌流液中含有5mmol／L K^＋以及最小纵向张力的情况下,ACh（0．1—10μmol／L）引起低静息膜电位细胞明显的超极化反应,而引起高静息膜电位细胞明显的去极化反应。ACh引起的平滑肌细胞超极化反应是浓度依赖性的（ACh的浓度是1μmol／L和10／μmol／L时,分别引起超极化的幅度是22和30mV,n=7）。ACh引起的超极化反应能被阿托品（atropine,0．1～1μmol／L,n=6）或DAMP（50～100nmol／L,n=6,一种选择性的地受体的拮抗剂）所阻断,同时也可被BAPTA—AM（10μmol／L,n=7,一种可通过细胞膜的Ca^2＋螯合剂）或eharybdotoxin＋apamin（50-100nmol／L,n=4,两种Ca^2＋激活K^＋通道的阻断剂）所阻断,但是Nω-nitro-L-arginine methyl ester（L-NAME,300μmol／L,n=8,一种NO合成酶的完全抑制剂,n≥5）或glipizide（10μmol／L,ATP敏感性的K^＋通道阻断剂,n=4）或indomethacin（10μmol／L,环氧合酶的抑制剂,n=4）不能阻断ACh引起的超极化反应。结论：ACh通过激活内皮细胞的M3受体,开放钙依赖的钾通道．进而引起耳蜗螺旋动脉平滑肌细胞产生超极化反应,并且这一超极化反应与内皮细胞NO的产生和释放无关。     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

The mechanism of ciliary stimulation by acetylcholine: roles of calcium, PKA, and PKG

Stimulation of ciliary cells through muscarinic receptors leads to a strong biphasic enhancement of ciliary beat frequency (CBF). The main goal of this work is to delineate the chain of molecular events that lead to the enhancement of CBF induced by acetylcholine (ACh). Here we show that the Ca(2+), cGMP, and cAMP signaling pathways are intimately interconnected in the process of cholinergic ciliary stimulation. ACh induces profound time-dependent increase in cGMP and cAMP concentrations mediated by the calcium-calmodulin complex. The initial strong CBF enhancement in response to ACh is mainly governed by PKG and elevated calcium. The second phase of CBF enhancement induced by ACh, a stable moderately elevated CBF, is mainly regulated by PKA in a Ca(2+)-independent manner. Inhibition of either guanylate cyclase or of PKG partially attenuates the response to ACh of [Ca(2+)](i), but completely abolishes the response of CBF. Inhibition of PKA moderately attenuates and significantly shortens the responses to ACh of both [Ca(2+)](i) and CBF. In addition, PKA facilitates the elevation in [Ca(2+)](i) and cGMP levels induced by ACh, whereas an unimpeded PKG activity is essential for CBF enhancement mediated by either Ca(2+) or PKA.     

Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.     

白介素-2促进RC-4B/C垂体瘤细胞系增殖

采用细胞培养方法,以＾３Ｈ－ＴｄＲ掺入率反映细胞增殖水平,研究了白介素－２（ＩＬ－２）对大鼠源的ＲＣ－４Ｂ／Ｃ垂体瘤细胞系增殖的影响,并初步分析了ＩＬ－２的作用机制。结果表明：⑴ＩＬ－２（１０－１０００Ｕ／ｍｌ）剂量依赖性地显著提高ＲＣ－４８／Ｃ细胞＾３Ｈ－ＴｄＲ掺入率。⑵特异性酪氨酸蛋白激酶（ＰＴＫ）抑制剂ｔｙｒｐｈｏｓｔｉｎ（１μｍｏｌ／Ｌ）可明显降低ＲＣ－４Ｂ／Ｃ细胞＾３Ｈ－ＴｄＲ掺入率,并     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Novel action of pituitary adenylate cyclase-activating polypeptide. Stimulation of extracellular acidification in rat pituitary GH4C1 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide/secretin family. Using microphysiometry, we have found that PACAP acutely (1 min) increased the extracellular acidification rate (ECAR) in GH4C1 cells approximately 40% above basal in a concentration-dependent manner. ECAR, maximally induced by PACAP, can be increased further by thyrotropin-releasing hormone (TRH), indicating that the signalling pathways for these two neuropeptides are not identical. In studies on the mechanism of PACAP-enhanced ECAR, we found that maximum stimulation of the cAMP/PKA pathway by treatment with FSK, or the PKC pathway with PMA, did not inhibit the ECAR response to PACAP. The PKC inhibitor calphostin C and the MAP kinase inhibitor PD98059 had no effect on the ECAR response to PACAP. Furthermore, PACAP induced little or no change in cytosolic Ca(2+) ([Ca(2+)](i)), while TRH induced a large increase in [Ca(2+)](i). However, the tyrosine kinase inhibitor genistein completely blocked PACAP-induced ECAR, suggesting involvement of tyrosine kinase(s). We conclude that PACAP causes an increase in ECAR in GH4C1 rat pituitary cells, which is not dependent on the PKA, PKC, MAP kinase or Ca(2+) signalling pathways, but does require tyrosine kinase activity.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.