PACAP促PC12细胞突起生长的分子机制

垂体腺苷酸环化酶激活肽（PACAP）具有广泛的生理功能。近年来的研究发现,PACAP具有重要的神经营养作用。PACAP可通过激活多条细胞内信号转导通路,促使PC12细胞突起生长,使其向神经元样细胞分化。本文综述了PACAP引起PC12细胞突起生长的信号转导通路,有助于深入了解PACAP神经营养作用的分子机制。     

血小板活化因子对大鼠卵巢颗粒细胞垂体腺苷酸环化酶激活肽mRNA表达的影响及调节机制

目的：探讨血小板活化因子（PAF）对大鼠卵巢颗粒细胞垂体腺苷酸环化酶激活肽（PACAP）mRNA表达的影响及其可能调节机制,旨在寻找PAF在卵巢中的作用靶点。方法：原代培养卵巢颗粒细胞,用放射免疫分析（RIA）及逆转录．聚合酶链反应（RT-PCR）方法检测颗粒细胞雌二醇分泌情况及其PACAP mRNA表达变化．结果：PAF对PACAP mRNA表达无明显影响,但与hCG共同作用可促进PACAP mRNA的表达：Forkolin可使PACAP mRNA表达升高。结论：PAF可通过对hCG的允许作用间接促进大鼠卵巢颗粒细胞PACAP mRNA表达,hCG的促进作用可能是通过cAMP—PKA途径介导的。     

垂体腺苷酸环化酶激活肽基因合成表达和产物纯化与鉴定

为利用基因工程技术获得垂体腺苷酸环化酶激活肽 (pituitaryadenylatecyclaseactivatingpolypeptide ,PACAP) ,根据大肠杆菌的密码偏好性 ,设计并人工合成编码 38个氨基酸的PACAP基因 .克隆到表达载体pET 35b(+) ,构建重组质粒pET PACAP ,转化大肠杆菌BL2 1 (DE3)pLysS+ .实现纤维素结合域 (cellulosebindingdomain ,CBD)与PACAP融合蛋白的表达 ,并在两者之间引入 (凝血 )因子Ⅹa识别位点 (Ile Glu Gly Arg↓ ) .融合蛋白CBD PACAP经纤维素亲和层析纯化后 ,因子Ⅹa酶切释放PACAP .在因子Ⅹa识别位点前引入 7个氨基酸的柔性短肽 (Gly Thr Gly Gly Gly Ser Gly)明显提高了融合蛋白对因子Ⅹa的敏感性 .HPLC进一步纯化得到纯度大于 95 %PACAP多肽 .所得的PACAP多肽的Western印迹鉴定为阳性 ;激光飞行质谱测定分子量结果与理论值相符 .生物活性分析表明 ,所制备的PACAP具有促进胰腺癌细胞株SW 1 990胞内cAMP合成的活性     

石斑鱼垂体腺苷酸环化酶激活多肽和生长激素释放激素基因的cDNA克隆及表达分析

垂体腺苷酸环化酶激活多肽 (PACAP)和生长激素释放激素 (GHRH)均属于血管活性肠肽家族成员 ,且两者前体基因在脊椎动物的鸟类、两栖类、鱼类中由同一基因编码 ,而哺乳动物是由两个不同基因编码。已有几例关于鱼类编码PACAP和GHRH基因克隆的报道 ,而关于重要海水养殖鱼类石斑鱼的PACAP和GHRH基因未见报道。克隆了PACAP GHRH前体cDNA序列 ,该前体有两种剪接方式 ,包括一个长序列和一个短序列 ,其中长序列编码PACAP和GHRH ,短序列缺失 10 5个碱基 ,仅编码PACAP而缺失编码GHRH的外显子区 ,同样情况在虹鳟和沟鲶中也有报道。通过半定量RT PCR方法对石斑鱼PACAP GHRH前体mRNA在胚胎发育和发育早期以及各部位的表达情况进行了分析。胚胎发育分析结果表明 ,从神经胚期开始 ,PACAP GHRH前体mRNA大量表达 ,提示该蛋白质在神经发育或神经营养方面具有重要作用。PACAP GHRH前体基因在中枢系统的表达量远高于外周组织。在鱼类的眼和鳃发现PACAP GHRH前体分布。     

PACAP mRNA在大鼠妊娠黄体及离体细胞的表达与调节

目的:观察垂体腺苷酸环化酶激活肽(PACAP)mRNA在大鼠妊娠黄体中的表达及调节。方法:①于妊娠不同时期收集大鼠卵巢。用RT-PCR和原位杂交方法,观察妊娠过程卵巢PACAP mRNA表达的动态变化;②未成年雌性大鼠颈部皮下注射50IU孕马血清促性腺激素,48h后注射25IU人绒毛膜促性腺激素,第6天收集培养黄体细胞。用放免法测定给予不同处理后,培养液中孕酮的含量;用RT-PCR方法检测各组PACAP mRNA表达水平。结果:从妊娠11d起,PACAP mRNA表达逐渐增强,在妊娠19d达高峰;与对照组相比,血小板活化因子(PAF)、福司考林(forskolin)、佛波酯(PMA)均使培养黄体细胞孕酮分泌量及PACAP mRNA表达显著增高(P0.05)。结论:PACAP与中、晚期妊娠的维持密切相关;PAF可促进培养黄体细胞PACAP mRNA的表达,蛋白激酶C(PKC)和蛋白激酶A(PKA)途径都有可能参与了此过程。     

垂体腺苷酸环化酶激活肽减轻大鼠缺血性脑水肿作用的实验研究

目的研究垂体腺苷酸环化酶激活肽(PACAP)在缺血性脑水肿中的作用及其可能的受体机制.方法采用大鼠四动脉结扎脑缺血模型,分别运用干湿重法和酶学法测定脑组织含水量及Na+、K+含量.结果大鼠四动脉结扎脑缺血30in,再灌流1 h,脑组织含水量明显增加,a+含量增高,而K+含量降低.缺血前经测脑室分别给予1×10-9 mol、1×1110mol及1×11-11 mol的PACAP均能抑制脑组织含水量、Na+含量的增加和K+含量的降低.特异性PACAPⅠ型受体拮抗剂PACAP6-38能完全阻断PACAP的上述作用,而单纯给予PACAP6-38对脑缺血后脑组织含水量、Na+、K+含量无显著影响.结论外源性PACAP对缺血性脑水肿具有保护作用,该作用是由I型受体介导的.     

PACAP-27抑制大鼠胃泌素引起的胃酸分泌

垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide,PACAP)是1989年发现的一种神经肽,已从下丘脑分离得到,在胃肠道神经组织中也发现有PACAP免疫活性。PACAP有两种形式,分别有38个及27个氨基酸残基,称为PACAP-38和PACAP-27,与胰高血糖素,促胰液素     

垂体腺苷环化酶激活多肽抑制β淀粉样蛋白对Neuro-2a细胞神经毒性作用的机制

通过MTT方法检测细胞活性 ,同时采用激光共聚焦显微镜技术检测细胞内游离钙离子的瞬时运动 ,研究了垂体腺苷环化酶激活多肽 (pituitaryadenylatecyclaseactivatingpolypeptide 2 7,PACAP2 7)通过调节细胞内钙对抗淀粉样蛋白Aβ2 5 35引起Neuro 2a细胞神经毒性作用的可能机制。结果表明 ,PACAP在一定浓度范围内 (<0 1μmol/L)可提高Neuro 2a细胞增殖能力并对抗Aβ引起的神经毒性 ,此作用可以被PACAP受体竞争性拮抗剂PACAP6 2 7所抑制。 2 5 μmol/LAβ使细胞内钙离子缓慢上升 ,并有一个较长时间的平台期。 0 1μmol/L的PACAP使细胞内钙离子迅速升高后下降 ,10min后回到接近基线水平 ,伴有较长时间的不应期。用PACAP预处理细胞 10min后Aβ引起细胞内钙的慢上升不再出现。推测 ,PACAP受体激活引起瞬时内向钙离子运动 ,而后伴随一个较长时间的不应期 ,可能是一个消除凋亡或阻止凋亡启动的保护机制。     

PACAP受体激活对抗Aβ25-35致神经元凋亡作用的观察

目的和方法：本研究采用体外原代培养新生大鼠海马神经元的方法探讨了垂体腺苷环化酶激活多肽（PACAP）对抗淀粉样蛋白（Aβ25-35）致凋亡的作用机理。结果：25μmol/L的Aβ蛋白处理神经元5d即有明显的凋亡特征出现。PACAP27（P27）在正常情况下对海马神经元无明显营养作用,但当Aβ导致神经元凋亡时P27有显著的保护作用,可以提高神经元的活性,减少细胞凋亡数目,降低基因组小片段DNA含量。PACAP受体拮抗剂PACAP6－27（P6－27）可以逆转P27的保护作用。结论：研究结果说明PACAP通过受体激活参与对抗Aβ的神经毒性效应。     

垂体腺苷酸环化酶激活肽的分布及其生物学功能

垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide,PACAP),是一种新发现的神经肽,具有多种生物活性。本文主要就PACAP的分子结构和基因组成、组织器官分布及其生物学功能作一综述。     

Structural analysis of pituitary adenylate cyclase-activating polypeptides bound to phospholipid membranes by magic angle spinning solid-state NMR

PACAP (pituitary adenylate cyclase-activating polypeptide) is a member of the VIP/secretin/glucagon family, which includes the ligands of class II G-protein coupled receptors. Since the recognition of PACAP by the receptor may involve the binding of PACAP to membranes, its membrane-bound structure should be important. We have carried out structural analysis of uniformly 13C,15N labeled PACAP27 and its C-terminal truncated form PACAP(1-21)NH2 (PACAP21) bound to membranes with high resolution solid-state NMR. Phosphatidylcholine bilayers and phosphatidylcholine/phosphatidylglycerol bilayers were used for PACAP27 and PACAP21, respectively. Most backbone signals were assigned for PACAP27 and PACAP21. TALOS analysis revealed that both peptides take on extended conformations on the membranes. Dilution of PACAP21 did not change the conformation of the major part. Selective polarization transfer experiment confirmed that PACAP27 is interacting with the membranes. It was concluded that the interaction of PACAP with the membrane surface causes their extended conformation. PACAP27 is reported to take an alpha-helical conformation in dodecylphosphocholine micelles and membrane-binding peptides usually take similar conformations in micelles and in membranes. Therefore, the property of PACAP27 changing its conformation in response to its environment is unique. Its conformational flexibility may be associated with its wide variety of functions.     

Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide with 38 (PACAP38) or 27 (PACAP27) amino acid residues, structurally related to vasoactive intestinal peptide (VIP). Bovine brain membrane has a PACAP specific receptor interacting with both PACAP27 and PACAP38. Affinity-labeling of the receptor with [125I]PACAP27 identified a dominant band of Mr = 60 k. The labeling density of the 60 k band decreased in the presence of unlabeled PACAP27 or PACAP38, whereas the 60 k band remained in the presence of unlabeled VIP. Binding of [125I]PACAP27 to the membrane decreased in the presence of GTP and the labeling density of the 60 k band decreased concomitantly. The results indicate that bovine brain has a specific PACAP receptor, whose apparent molecular weight is 57 k (substracting the molecular weight of [125I]PACAP27 from 60 k).     

Cardiovascular and respiratory actions of pituitary adenylate cyclase-activating polypeptides.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) and PACAP27 on the cardiovascular and respiratory systems were examined and compared to those of vasoactive intestinal polypeptide (VIP) in anesthetized beagle dogs. Intravenous PACAP27 and PACAP38 produced a decrease in mean arterial blood pressure (MBP), and an increase in both femoral arterial blood flow (ABF) and in frequency of respiration (FR) with a dose-dependent relationship between 10 and 300 pmol/kg. PACAP27 produced a dose-dependent increase in heart rate (HR) between 10 and 300 pmol/kg while PACAP38 induced tachycardia which was not dose-dependent. Administration of 300 pmol/kg PACAP38 and PACAP27 produced extreme hypertension after transient hypotension. PACAP38 produced severe bradycardia after transient tachycardia. The cardiovascular actions of PACAP38 were persistent compared to those of PACAP27. Intravenous injection of 10-300 pmol/kg VIP brought about hypotension, tachycardia and an increase in ABF and FR with a dose-dependent relationship. VIP, at 2000 pmol/kg, did not produce the biphasic response obtained by a large dose of PACAP38. The present studies demonstrate that PACAP partially possesses VIP-like cardiovascular and respiratory actions and that the C-terminal 11 amino acid residues of PACAP38 are presumably responsible for a prolongation of its actions.     

A newly developed enzyme-immunoassay for measuring the tissue contents of PACAP in fish

We have developed a novel and easy enzyme-immunoassay (EIA) for pituitary adenylate cyclase-activating polypeptide (PACAP). We used it to determine immunoreactive PACAP levels in the central nervous system (CNS) and peripheral tissues of two fishes, a teleost (the stargazer) and an elasmobranch (a stingray). An antiserum was raised in a white rabbit immunized with a conjugate of synthetic stargazer PACAP27 plus keyhole limpet hemocyanin. The EIA system used an antiserum/biotin-labeled PACAP/avidin/biotin-conjugated enzyme complex, and a double antibody method was used to precipitate the immune complexes. We call the system the avidin-biotin complex detectable EIA (ABCDEIA) for PACAP. ABCDEIA with biotin-labeled PACAP27 detected only PACAP27, recognizing neither the longer forms of PACAP nor any other peptides. PACAPs with 27, 38, and 44 residues cross-reacted in another ABCDEIA with biotin-labeled PACAP38 or PACAP44. Whole brains of both fishes contained much higher levels of PACAP, 6-30 times as high as the levels in the mammalian brain, but unexpectedly, no immunoreactive PACAP27 was found in any CNS or peripheral tissue in either fish. The gastrointestinal tracts of fish also contained lower, but significant amounts of PACAP.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.     

Effects of pituitary adenylate cyclase-activating polypeptide on the cyclooxygenase pathway of rat platelets and on platelet aggregation

Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. It can be concluded that PACAP inhibits the cyclooxygenase pathway of rat platelets via a specific PACAP receptor-activated, cAMP-dependent pathway, and these effects of PACAP are involved in the inhibition of platelet aggregation.     

Effects of PACAP(1-27) on the canine endocrine pancreas in vivo: interaction with cholinergic mechanism

The aim of the present study was to characterize the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the endocrine pancreas in anesthetized dogs. PACAP(1-27) and a PACAP receptor (PAC(1)) blocker, PACAP(6-27), were locally administered to the pancreas. PACAP(1-27) (0.005-5 microg) increased basal insulin and glucagon secretion in a dose-dependent manner. PACAP(6-27) (200 microg) blocked the glucagon response to PACAP(1-27) (0.5 microg) by about 80%, while the insulin response remained unchanged. With a higher dose of PACAP(6-27) (500 microg), both responses to PACAP(1-27) were inhibited by more than 80%. In the presence of atropine with an equivalent dose (128.2 microg) of PACAP(6-27) (500 microg) on a molar basis, the insulin response to PACAP(1-27) was diminished by about 20%, while the glucagon response was enhanced by about 80%. The PACAP(1-27)-induced increase in pancreatic venous blood flow was blocked by PACAP(6-27) but not by atropine. The study suggests that the endocrine secretagogue effect of PACAP(1-27) is primarily mediated by the PAC(1) receptor, and that PACAP(1-27) may interact with muscarinic receptor function in PACAP-induced insulin and glucagon secretion in the canine pancreas in vivo.