Cerebellar cortical-layer-specific control of neuronal migration by pituitary adenylate cyclase-activating polypeptide

Migration of immature neurons is essential for forming the cortical layers and nuclei. Impairment of migration results in aberrant neuronal cytoarchitecture, which leads to various neurological disorders. Neurons alter the mode, tempo and rate of migration when they translocate through different cortical layers, but little is known about the mechanisms underlying this process. Here we show that endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) has short-term and cortical-layer-specific effects on granule cell migration in the early postnatal mouse cerebellum. Application of exogenous PACAP significantly slowed the migration of isolated granule cells and shortened the leading process in the microexplant cultures of the postnatal day (P)0-3 cerebella. Interestingly, in the cerebellar slices of P10 mice, application of exogenous PACAP significantly inhibited granule cell migration in the external granular layer (EGL) and molecular layer (ML), but failed to alter the movement in the Purkinje cell layer (PCL) and internal granular layer (IGL). In contrast, application of PACAP antagonist accelerated granule cell migration in the PCL, but did not change the movement in the EGL, ML and IGL. Inhibition of the cAMP signaling and the activity of phospholipase C significantly reduced the effects of exogenous PACAP on granule cell migration. The PACAP action on granule cell migration was transient, and lasted for approximately 2 h. The duration of PACAP action on granule cell migration was determined by the desensitization of its receptors and prolonged by inhibiting the protein kinase C. Endogenous PACAP was present sporadically in the bottom of the ML, intensively in the PCL, and throughout the IGL. Collectively, these results indicated that PACAP acts on granule cell migration as "a brake (stop signal) for cell movement." Furthermore, these results suggest that endogenous PACAP slows granule cell migration when the cells enter the PACAP-rich PCL, and 2 h later the desensitization of PACAP receptors allows the cells to accelerate the rate of migration and to actively move within the PACAP-rich IGL. Therefore, endogenous PACAP may provide a cue that regulates granule cell migration in a cerebellar cortical-layer-specific manner.     

Reduction by naloxone of lipopolysaccharide-induced neurotoxicity in mouse cortical neuron-glia co-cultures

An inflammatory response in the CNS mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Using mouse cortical mixed glia cultures, we have previously demonstrated that the bacterial endotoxin lipopolysaccharide induces the activation of microglia and the production of proinflammatory factors. Naloxone, an opioid receptor antagonist, inhibits the lipopolysaccharide-induced activation of microglia and the production of proinflammatory factors. Using neuron-glia co-cultures, we extended our study to determine if naloxone has a neuroprotective effect against lipopolysaccharide-induced neuronal damage and analysed the underlying mechanism of action for its potential neuroprotective effect. Pretreatment of cultures with naloxone (1 microM) followed by treatment with lipopolysaccharide significantly inhibited the lipopolysaccharide-induced production of nitric oxide and the release of tumor necrosis factor-alpha, and significantly reduced the lipopolysaccharide-induced damage to neurons. More importantly, both naloxone and its opioid-receptor ineffective enantiomer (+)-naloxone were equally effective in inhibiting the lipopolysaccharide-induced generation of proinflammatory factors and the activation of microglia, as well as in the protection of neurons. These results indicate that the neuroprotective effect of naloxone is mediated by its inhibition of microglial activity and may be unrelated to its binding to the classical opioid receptors.     

High concentrations of extracellular potassium enhance bacterial endotoxin lipopolysaccharide-induced neurotoxicity in glia-neuron mixed cultures

A sudden increase in extracellular potassium ions (K(+)) often occurs in cerebral ischemia and after brain trauma. This increase of extracellular K(+) constitutes the basis for spreading depression across the cerebral cortex, resulting in the expansion of neuronal death after ischemic and traumatic brain injuries. Besides spreading depression, it has become clear that cerebral inflammation also is a key factor contributing to secondary brain injury in acute neurological disorders. Experiments to validate the relationship between elevated levels of extracellular K(+) and inflammation have not been studied. This study aims to elucidate the roles of high concentrations of extracellular K(+) in bacterial endotoxin lipopolysaccharide-induced production of inflammatory factors. Increased concentration of KCl in the medium (20mM) significantly enhanced neurotoxicity by lipopolysaccharide in glia-neuron mixed cultures. To delineate the underlying mechanisms of increased neurotoxicity, the effects of high extracellular K(+) were examined by using mixed glial cultures. KCl at 20mM significantly enhanced nitrite, an index for nitric oxide, production by about twofold, and was pronounced from 24 to 48h, depending on the concentration of KCl. Besides nitric oxide production of tumor necrosis factor-alpha was also enhanced. The augmentative effects of high KCl on the production of inflammatory factors were probably due to the further activation of microglia, since high KCl also enhanced the production of tumor necrosis factor-alpha in microglia-enriched cultures. The increased production of nitrite by high K(+) was eliminated through use of a K(+)-blocker.Taken together, the results show that increases of extracellular K(+) concentrations in spreading depression augment lipopolysaccharide-elicited neurotoxicity, because production of inflammatory factors such as nitric oxide and tumor necrosis factor-alpha are potentiated. Since spreading depression and cerebral inflammation are important in acute neurological disorders, the present results suggest a biochemical mechanism: elevated extracellular K(+) concentrations augment glial inflammatory responses, and thus the neurotoxicity.     

垂体腺苷酸环化酶激活肽拮抗lactacystin细胞毒性的作用

背景：帕金森病的发病机制未明确,目前尚没有有效的治疗方法能从根本上阻止其病程进展。目的：分析垂体腺苷酸环化酶激活肽对lactacystin诱导的帕金森病多巴胺能PC12细胞凋亡的影响及其分子机制。方法：用神经生长因子将PC12细胞诱导分化成神经元的细胞模型,经不同浓度泛素-蛋白酶体抑制剂lactacystin处理,分别作用不同时间,取细胞存活约为50%的lactacystin作用浓度与时间,建立帕金森病细胞实验模型。实验分组：对照组、lactacystin组、垂体腺苷酸环化酶激活肽1-27干预组(干预1组)、垂体腺苷酸环化酶激活肽1-27和垂体腺苷酸环化酶激活肽6-27共同干预组(干预2组)。观察各组细胞形态变化;MTT法检测细胞活力;免疫印迹(Western blot)法检测内质网应激特异性蛋白caspase-12的表达情况。并观察垂体腺苷酸环化酶激活肽1-26及垂体腺苷酸环化酶激活肽6-27对lactacystin毒性作用的影响。结果与结论：不同浓度及作用时间的lactacystin处理PC12细胞后,细胞活力呈浓度及时间依赖性下降,其中lactacystin 20 μmol/L作用24 h使细胞活力下降约50%。在相同的lactacystin作用条件下(20 μmol/L,24 h),与对照组比较,lactacystin组细胞发生损伤性改变,细胞活力降低,caspase-12活性明显升高(P < 0.01);与lactacystin组比较,干预1组细胞损伤性改变明显好转,细胞活力增强,下调凋亡蛋白caspase-12的表达    (P < 0.01)。干预2组细胞状态则明显不如干预1组,与lactacystin组相差不大。结果提示泛素-蛋白酶体抑制剂lactacystin引起内质网应激导致细胞损伤;垂体腺苷酸环化酶激活肽1-27通过调节上述信号通路发挥保护作用。而作为垂体腺苷酸环化酶激活肽1-27的受体拮抗剂,垂体腺苷酸环化酶激活肽6-27则减弱了垂体腺苷酸环化酶激活肽1-27的这一作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

垂体腺苷酸环化酶激活肽对动脉粥样硬化形成过程中脂质过氧化的影响

目的　探讨垂体腺苷酸环化酶激活肽 (PACAP)对家兔实验性动脉粥样硬化 (AS)形成过程中脂质过氧化的影响。方法　 80只雄性新西兰家兔 ,随机分为 3组 :(1)对照组 (C组 ) :喂饲普通颗粒兔饲料 ;(2 )AS模型组 :每只兔每日喂饲含胆固醇 1 5g的颗粒兔饲料 ;(3)PACAP组 (P组 ) :喂饲与AS组同样的饲料 ,另从耳缘静脉注射PACAP。分别于实验的 0、4、8、12周末记录兔体重 ,同时从兔耳中央动脉取空腹血 ,测定丙二醛 (MDA)含量 :并取若干兔的主动脉标本作苏丹Ⅳ染色 ,测量斑块面积。结果　 (1)AS组及P组血清MDA含量均显著高于C组 ;而P组显著低于AS组。 (2 ) 4周末时P组斑块面积显著小于AS组。结论　PACAP具有抗脂质过氧化作用 ,可能与抗AS有关     

Association between pituitary adenylate cyclase-activating polypeptide and thyrotropin-releasing hormone in the rat hypothalamus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the hypothalamus including the paraventricular nucleus (PVN). In this study the anatomical relationship between PACAP- and thyrotropin-releasing hormone (TRH)-immunoreactive neuronal elements was investigated in the rat hypothalamus. Using a well-characterized mouse monoclonal antibody against PACAP and a rabbit polyclonal antiserum against TRH, we found numerous nerve fibers with PACAP-immunoreactivity (ir) closely apposed to TRH neurons in the PVN suggesting synaptic contacts. Electron microscopy confirmed the presence of synapses between PACAP-ir terminals and TRH-ir perikarya and various dendritic profiles as well as between PACAP-ir terminals and unlabeled perikarya and small- to medium-sized dendrites. Coexistence of the two peptides in perikarya of the PVN was limited to only a few neurons in the periventricular subdivision, but PACAP-ir and TRH-ir extensively coexisted in perikarya of the perifornical cell group, medial preoptic area, lateral hypothalamus and dorsomedial nucleus. The interactions between PACAP-containing neuronal processes and TRH neurons in the PVN raise the possibility that PACAP modulates the secretion of TRH destined for regulation of anterior pituitary TSH. The more general association between PACAP and TRH in other regions of the hypothalamus suggests a further role for PACAP as a cofactor in the function of TRH neurons.     

Intracerebroventricular injection of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibits feeding in chicks

Previous research has indicated an involvement of glucagon superfamily peptides in the regulation of feeding in the domestic chick brain. However the possible roles of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide-38 (PACAP) have not yet been investigated. We therefore examined the effect of intracerebroventricular (ICV) injections of VIP or PACAP on food intake in chicks. ICV injection of both VIP and PACAP significantly inhibited food intake over 4 h at doses ranging from 12 to 188 pmol. Subsequently, we compared the anorexic effect the glucagon superfamily peptides VIP, PACAP, growth hormone-releasing factor (GRF) and glucagon-like peptide-1 (GLP-1) after ICV injection at an equimolar dose (12 pmol). All four peptides significantly inhibited food intake, although the anorexic effects of VIP and PACAP were weaker than those of GRF and GLP-1. These findings support the hypothesis that glucagon superfamily peptides play an important role in the regulation of appetite in the chick brain.     

Glucose dependence of insulinotropic actions of pituitary adenylate cyclase-activating polypeptide in insulin-secreting INS-1 cells

The cAMP-elevating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin release in pancreatic B-cells. Here, we have investigated its potentiating action in rat insulinoma INS-1 cells. In intact cells, PACAP-27 (100 nM) stimulated glucose-induced insulin secretion by >60%. Using the patch-clamp technique with single-cell exocytosis monitored as increases in cell capacitance, we observed that at 10 mM and 20 mM extracellular glucose, PACAP-27 acted mainly by a >50% enhancement of depolarization-elicited Ca(2+) entry, whereas at low (3 mM) glucose, the predominant effect of the peptide was a twofold increase in Ca(2+) sensitivity of insulin exocytosis. The latter effect was mimicked by glucose itself in a dose-dependent fashion. PACAP-27 exerts a prolonged effect on insulin secretion that is dissociated from changes of cytoplasmic cAMP. Whereas an elevation of cellular cAMP content (135%) could be observed 2 min after addition of PACAP-27, after 30 min preincubation with the peptide, cAMP concentrations were not different from basal. Yet, such pretreatment with PACAP-27 stimulated subsequent insulin release by congruent with60%. This sustained action is likely to reflect an increased degree of protein-kinase-A-dependent phosphorylation, and inhibitors of the kinase largely prevented the PACAP-mediated effects.     

Expression of pituitary adenylate cyclase-activating polypeptide in the primary lymphoid organs of the duck Anas platyrhynchos

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the thymus and bursa of Fabricius of the duck Anas platyrhynchos, at different ages, using immunohistochemistry, Western blotting, RT-PCR and sequencing. In the thymus, PACAP immunoreactivity (-ir) was found in lymphoid cells. CD68/ and PGP 9.5/PACAP38 double labelling showed that PACAP was not expressed either in macrophages or in epithelial cells, suggesting that the PACAP-positive cells observed were lymphoid cells. Immunoreactive lymphocytes were observed in the interlobular septa. They increased in number with ageing. In the bursa, PACAP-ir was found in nerve fibres and in a few lymphoid cells. RT-PCR revealed PACAP mRNA expression in the thymus but not in the bursa. These results suggest that PACAP plays a role in the functions of the immune system in birds.     

Involvement of progesterone in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide gene expression in pre-ovulatory follicles of rat ovary

The present study was designed to determine whether progesterone might have a role in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide (Pacap) gene expression in rat ovary. Northern blot analysis revealed that treatment of pregnant mare's serum gonadotrophin (PMSG)-primed immature rats with the progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane, 1 h before HCG, resulted in a dose-dependent inhibition of the HCG-induced Pacap gene expression. In-situ hybridization demonstrated that the number of pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells was greatly reduced in ovaries treated with RU486. Moreover, the suppressive effect of RU486 or epostane on the LH-induced Pacap gene expression in cultured pre-ovulatory follicles was reversed by co-treatment with the synthetic progestin R5020. We further cloned the 5'-flanking region of the rat Pacap gene and identified the presence of a consensus progesterone receptor element. When luciferase fusion genes containing Pacap gene promoter were transiently transfected into granulosa cells of pre-ovulatory follicles, luciferase activity was markedly stimulated by LH. Treatment with RU486 or epostane resulted in partial suppression of LH-stimulated PACAP promoter activity. Taken together, these results indicate that progesterone, acting through progesterone receptors, plays a role in gonadotrophin induction of Pacap gene expression in granulosa cells of pre-ovulatory follicles, and thereby may be involved in the process of ovulation.     

The melanocortin system is involved in regulating autonomic nerve activity through central pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptidergic neurotransmitter that is highly expressed in the nervous system. We have previously reported that a central injection of PACAP leads to changes in the autonomic nervous system tones including sympathetic excitation and parasympathetic inhibition. An anatomical study revealed that melanocortin and PACAP are colocalized in some hypothalamic nuclei. Here, we investigated the possible role of the melanocortin system in autonomic control by PACAP using SHU9119, an antagonist of the melanocortin receptors (MC3-R/MC4-R). Pretreatment with SHU-9119 did not affect the activating neural responses of adrenal, renal, and lumbar sympathetic nerves following a PACAP injection However, SHU9119 significantly eliminated the suppressing effect of a PACAP injection on gastric vagal nerve activity and excitation effects on liver and brown adipose tissue sympathetic nerve activities. These results suggest that the brain melanocortin system might play a key role in the control of thermogenic sympathetic outflows and digestive parasympathetic outflow by PACAP, but this system does not participate in the central effects of PACAP on cardiovascular function and neural activities of renal, adrenal, and lumbar sympathetic nerves.     

Increased expression, axonal transport and release of pituitary adenylate cyclase-activating polypeptide in the cultured rat vagus nerve

The expression and axonal transport of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the cultured vagus nerve of the rat by immunocytochemistry and in situ hybridization. The number of neurons immunoreactive for PACAP increased markedly within the nodose ganglion during a 24-48 h culture period, as did the number of cells containing messenger RNA for PACAP. PACAP was found to be axonally transported and accumulated at the site of a crush injury. The peptide was also released at this site. Addition of PACAP to regenerating nerves in culture did not affect axonal outgrowth, neither did antibodies against PACAP. Separate experiments showed that neither PACAP-27 nor PACAP-38 affected proliferation of non-neuronal cells measured as the incorporation of [3H]thymidine. In contrast, forskolin, another potent stimulator of adenylate cyclase besides PACAP, dramatically decreased [3H]thymidine incorporation. The results showed that, during regeneration of peripheral nerves, PACAP expression increases and the peptide is transported into the regenerating nerve, where it is released. The functional significance of this release is unknown, but it does not seem to be directly related to the initiation of proliferation of Schwann cells or initial axonal outgrowth.     

Pituitary adenylate cyclase-activating polypeptide inhibits the cyclooxygenase pathway of rat cerebral microvessels.

The presence of nerve endings containing pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) around cerebral microvessels suggests that these peptides have regulatory roles in the cerebral microcirculation. Prostanoids synthesized by the cerebrovascular endothelium have a determining role in the regulation of the brain circulation. In the present study, the effects of PACAP and VIP on the cyclooxygenase pathway of cerebral microvessels were investigated. The isolated microvessels were incubated with 1-14C-arachidonic acid and different concentrations of the peptides. The prostanoids formed were separated by means of overpressure thin-layer chromatography, and were quantitatively determined by liquid scintillation. Higher concentrations (10-7 and 10-6 mol L-1) of PACAP significantly inhibited the activity of the cyclooxygenase pathway, whereas VIP had no significant effect on it. As regards the cyclooxygenase metabolites, the syntheses of thromboxane A2 and prostaglandin D2 were inhibited significantly. PACAP and VIP are known to increase the intracellular cAMP level in the cerebral microvessels and in the present experiments the protein kinase A inhibitor H-89 attenuated the effect of PACAP on prostanoid synthesis. It is concluded that the cyclooxygenase pathway of rat cerebral microvessels is more sensitive to PACAP than to VIP. The inhibitory effect of PACAP on prostanoid synthesis is mediated via a cAMP-dependent pathway. By inhibiting the formation of vasoactive prostanoids, PACAP can decrease the vasoreactivity of the microvessels.