斜带石斑鱼PACAP的原核表达及活性分析

自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽（Pituitary adenylate cyclase activating polypeptide,PACAP）以来（Miyata et al．,1989）,已证明它能促进垂体激素释放,同时还具有神经递质、神经调质和神经营养等作用,使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽（VIP）-胰高血糖素-生长激素释放因子-分泌素家族（Campbell and Scanes,1992）成员,已鉴别出包含27和38个氨基酸两种类型。对原索动物（McRory et al．,1997）、两栖类（蛙）（Alexandre et al．,2000）、爬行类（蜥蜴）（Pohland Wank,1998）、鸟类（鸡）（McRory et al．,1997）,啮齿类（鼠）（Ghatei et al．,1993）等脊椎动物PACAP的研究多集中在结构与进化方面,对功能了解甚少。     

PACAP对神经系统疾病保护作用的研究进展

垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase-activating polypeptide,PACAP)存在于中枢神经系统内,作为神经递质、神经调质、神经营养因子及生长因子通过多种途径发挥神经保护作用.对脑缺血-再灌注损伤、创伤性脑损伤、精神分裂症、焦虑症、帕金森病、阿尔茨海默病等重大疾病具有重要的神经保护作用.本文就PACAP对神经系统疾病的保护作用及其机制的研究进行综述.     

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 (PituitaryAdenylateCyclaseActivitingPoly peptide ,PACAP) ,是一种新发现的神经肽 ,具有多种生物活性。主要综述PACAP的分子结构和基因组成、组织器官分布及其生物学功能。     

The biological role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase‐activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth‐promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Hyperpolarization-activated Cl current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1–10 μM) activated an inward rectifier current at membrane potentials more negative than −60 mV without causing any significant change in currents at potentials more positive than −60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at −22.5 mV, close to the theoretical value of Cl⁻ equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl⁻]_o. This current was blocked by Cl⁻ channel blocker SITS and Ba²⁺. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl⁻ current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

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。     

Local regulation of anion secretion by pituitary adenylate cyclase-activating polypeptide in human colonic T84 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel hypothalamic peptide, which has been shown to exert various functions in a number of tissues, including exocrine and endocrine tissues. The present study investigated the role of local PACAP in the control of anion secretion by the human colonic T84 cell. Both bioactive forms of PACAP-27 and PACAP-38 gave rise to a dose-dependent increase in the short-circuit current (I(SC)). However, there was a reversal in the order of potency observed at different concentration ranges for the two bioactive forms. PACAP-27 was greater than PACAP-38 when the peptide concentrations were below 10 n m; PACAP-38 was greater than PACAP-27 in the range of 10-80 n m. The effects of both PACAP forms were restricted to the apical aspect of the T84 cell. The I(SC)responses to both PACAP-27 and PACAP-38 were suppressed respectively by the non-selective Cl(-)channel blocker, diphenylamine-dicarboxylic acid (DPC), by the Ca(2+)dependent Cl(-)channel blocker, diisothiocyanatostilbene-disulfonic acid (DIDS) and by the Ca(2+)chelator, BAPTA-AM, indicating the involvement of Ca(2+). The expression of PACAP was demonstrated and localized specifically to the perinuclear cytoplasm of the T84 cell using immunocytochemistry, indicating its epithelial origin. Thus, the present data suggest that, in addition to the well-known cAMP-dependent pathway, PACAP may play a role in regulating colonic Cl(-)secretion via a Ca(2+)-dependent pathway, perhaps through two distinct PACAP receptor subtypes. Moreover, the regulation of anion secretion by T84 cells may be mediated by locally formed PACAP in an autocrine or paracrine fashion.     

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。     

Pituitary adenylate cyclase‐activating polypeptide in the testis of the quail Coturnix coturnix: Expression,localization, and phylogenetic analysis

To evaluate the involvement of pituitary adenylate cyclase‐activating polypeptide (PACAP)/receptors system in the control of testis activity, we have investigated the expression and localization of PACAP and the distribution of its receptors in the testis of mature samples of quail Coturnix coturnix, and we have performed a phylogenetic analysis of PACAP in birds. Using histological, molecular, and bioinformatics tools, we demonstrated that (a) PACAP messenger RNA shows a high sequence identity with that reported in other birds studied so far and in other vertebrates. Furthermore, we showed that purifying selection acts on PACAP; (b) the PACAP peptide is present only in Leydig cells, whereas its receptors are localized within both Leydig and germ cells; (c) the synthesis of PACAP does not take place in seminiferous tubules. The role of PACAP in the control of spermatogenesis and steroidogenesis in birds is discussed. Finally, we talk about the phylogenetic and evolutionary relationships between PACAP in birds and in other vertebrates.     

LOCALIZATION AND DISTRIBUTION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN THE RAT EPIDIDYMIS

Previous investigation has provided evidence for the control of electrogenic chloride secretion by pituitary adenylate cyclase-activating polypeptide (PACAP) across the rat epididymal epithelium using electrophysiological measurement of transepithelial transport in cultured epididymal system. Hence, it suggests that epididymal and sperm functions are subject to control by a local PACAP system in the rat epididymis. In the present study, localization and distribution of PACAP in the rat epididymal duct was studied by an indirect immunofluorescence technique in conjunction with confocal laser scanning microscopy. Immunoreactivity for PACAP was found in all regions of the epididymal duct. However, the intensity of immunoreactivity for PACAP was stronger in the caput and corpus regions when compared to that of the cauda epididymidis. Much weaker immunostaining for PACAP, as compared to those found in other regions, was observed in the cauda epididymidal tubules which are in close proximity to the vas deferens. No immunoreactivity for PACAP was found in epididymal spermatozoa. Together with the previous finding, the present results suggest that PACAP may exhibit a regional difference in its expression along the epididymal duct and it may act in a paracrine or autocrine fashion in the regulation of epididymal chloride secretion and hence fluid secretion, thus regulating epididymal and sperm functions along the epididymal duct.     

Expression patterns of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and is known to act as a tropic factor in various cells. Recent report revealed the expression of PACAP and the PACAP type I (PAC(1)) receptor in human and rat placentas at term. Placenta is a critical organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. However, there is little information regarding the expression pattern and cellular localization of PACAP and PAC(1) during pregnancy. The aim of this study was to define the expression and distribution of PACAP and PAC(1) receptor mRNAs in the rat placenta during pregnancy. PACAP and PAC(1) receptor mRNAs were expressed in decidual cells, chorionic vessels, and stromal cells of the chorionic villi. Interestingly, the expression of these genes varied with the day of gestation. For example, PACAP and PAC(1) receptor mRNAs expressed in decidual cells on day 13.5 and 15.5, their expression was strong in chorionic vessels and stromal cells of the chorionic villi within the labyrinth zone on day 17.5, 19.5, and 21.5. In fact, as gestation advanced, the expression of PACAP and PAC(1) receptor mRNAs in the decidua cells disappeared, as their high expression became evident in the chorionic vessels and stromal cells of the chorionic villi. Our finding that PACAP and the PAC(1) receptor are co-localized and their genes seemingly co-regulated within specific placental areas, strongly suggest that this peptide may play an important role, as an autoregulator or pararegulator via its PAC(1) receptor, in physiological functioning of the placenta for gestational maintenance.     

共表达生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)对动物生长的影响

生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)在序列及功能方面均相似,且同为PACAP/胰高血糖素超家族成员．研究了这二者对生长激素释放的刺激作用,以及对动物生长的影响．构建了3个表达载体,pIRES1- GHRH-PACAP(P-G-P),pIRES1-GHRH(P-G) 及 pIRES1-PACAP(P-P),并转染到CHO细胞中,进行RT-PCR,Dot-ELISA以及Westen-blot检测．此外,给大鼠注射细胞上清表达产物,检测其生物学活性．注射8 h后,注射表达P-G-P上清的大鼠血清中IGF-Ⅰ浓度显著高于其他组(P < 0.05)．用PLGA微球包裹各种质粒,并注射到家兔后肢胫前肌．观察家兔生长情况,并于注射后0,15,30,45天时分别采集家兔血液,检测血液中IGF-Ⅰ浓度．结果显示,三质粒注射组动物体重变化及血液中IGF-Ⅰ浓度均高于对照组．注射后30天时,P-G-P组增重较对照组提高81% (P < 0.01),P-G组比对照组提高15%(P > 0.05),P-P组比对照组高7%(P > 0.05)．另一方面,P-G-P组动物血液中IGF-Ⅰ含量比分别比P-G、P-P及对照组提高16.68% (P > 0.05),17.14%(P > 0.05),50.46%(P < 0.05)．以上结果揭示：给动物注射PLGA微球包裹的共表达GHRH与PACAP质粒,可以增强动物体内生长激素(GH)的分泌,并促进动物生长．通过上述研究发现,肌肉注射PACAP表达质粒可以促进家兔的生长,PACAP和GHRH 共表达可以起到协同作用．这可能为动物的促生长研究提供新的方法．     

Phosphatidylinositol 3'-OH kinase and protein kinase A pathways mediate the anti-apoptotic effect of pituitary adenylyl cyclase-activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol

Cerebellar granule neurons cultured in the presence of 5 mm KCl undergo spontaneous apoptosis, which is reduced by exposure to pituitary adenylyl cyclase-activating polypeptide (PACAP). Previous work has suggested roles for the cyclic AMP/PKA and MAP kinase signaling pathways in the anti-apoptotic effect of PACAP. In the present study, the use of specific inhibitors confirmed the role of the cyclic AMP/PKA pathway, and also demonstrated a role for the phosphatidylinositol 3'-OH kinase (PI 3-kinase) neuroprotective pathway in the action of PACAP. Ethanol exposure accelerates the anti-apoptotic effect of PACAP by a mechanism that involves the PKA and PI-3 kinase pathways. The results demonstrate that ethanol can increase neuroprotection induced by PACAP. As previous work has shown that ethanol can increase apoptosis of cerebellar granule neurons by inhibiting the protective effect of agents such as NMDA or IGF-1, the overall effect of ethanol on cerebellar neuron apoptosis during development may reflect the balance between inhibition and enhancement of the actions of various endogenous neuroprotective agents.     

Comparative gene expression profiles in pancreatic islets associated with agouti yellow mutation and PACAP overexpression in mice

In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic β-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F₁ offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with >16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.     

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.     

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice

Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P<0.01), decrease in body fat (P<0.01), down-regulation of basal plasma glucose (P<0.01), upregulation of basal plasma insulin (P<0.01) and improved glucose tolerance (P<0.01) and insulin sensitivity (P<0.01). An elevation in plasma LDL (P<0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P<0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.     

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。     

Modulation of IL-1β and VEGF expression in rat diabetic retinopathy after PACAP administration

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Hyperglycemic/hypoxic microenvironment concurs to aberrant angiogenesis characterizing the pathology and activates many downstream target genes including inflammatory cytokines and vasoactive peptides, such as interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF).It has been largely demonstrated that pituitary adenylate cyclase-activating peptide (PACAP) plays a protective effect in DR. In the present study, we investigated the role of PACAP to protect retinal tissue through IL-1β and VEGF expression. Diabetes was induced in rats by streptozotocin (STZ) injection, and one week later a single intravitreal injection of 100 μM PACAP was administrated. Analyses of IL-1β and VEGF levels were performed three weeks after diabetes induction.The results demonstrated that a single intraocular administration of PACAP significantly reduced the expression of IL-1β in diabetic animals. Moreover, it affects VEGF and its receptors (VEGFRs) levels and interferes with their retinal layers distribution as showed by confocal microscopy analysis. In particular, PACAP treatment downregulates VEGF and VEGFRs that are increasingly expressed in STZ-treated animals as compared to controls. These results indicate that PACAP plays an important role to attenuate the early phase of DR.     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究——PACAP与动脉粥样硬化关系的探讨

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象,研究了垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造成的ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质过氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细胞保护作用,因此提示ＰＡＣＡＰ可能具有一定的抗ＡＳ作用。