NEUROENDOCRINE FUNCTIONS OF VASOPRESSIN AND VASOPRESSIN RECEPTORS

加压素和加压素受体的神经内分泌功能

基本信息

批准号：
7968730
负责人：
Greti Aguilera
金额：
$ 15.43万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7968730
关键词：
1-Phosphatidylinositol 3-Kinase Address Affect Apoptosis Apoptotic Brain Cell Death Cell Line Cell Survival Cells Chronic Data Exposure to Extracellular Signal Regulated Kinases Glutamates Hippocampus (Brain)Hour Laboratory Study MAPK Signaling Pathway Pathway Mediating Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Mitogens Molecular Neuronal Plasticity Neurons Neuroprotective Agents Neurosecretory Systems Nutrient Pathway interactions Peptides Phosphatidylinositide 3-Kinase Inhibitor Phosphorylation Physiological Play Protein Kinase C Proteins Proto-Oncogene Proteins c-akt Role Serum Signal Pathway Staining and Labeling System Testing U-0126 V1 Receptors Vasopressin Receptor Vasopressins Western Blotting caspase-3 deprivation hypothalamic-pituitary-adrenal axis parvocellular prevent pro-apoptotic protein protective effect receptor

项目摘要

Summary: Recent studies from this laboratory using the neuronal cell line H32, which expresses endogenous V1 VP receptors, showed that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. These actions of VP are largely mediated by mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), but also involves other signaling pathways. Studies during the past year have examined the role of protein kinases C (PKC) and B (PKB) mediating VP induced antiapoptosis in H32 cells. Serum deprivation increased PKCδ but not PKC or PKC activity, while VP increased PKC and PKC without affecting PKCδ activity. Inhibition of PKCδ prevented caspase-3 activation, indicating that PKCδ mediates the proapoptotic actions of serum deprivation. Simultaneous inhibition of PKC & and MAP kinase abolished VP-induced Bad phosphorylation, but it only partially prevented caspase-3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of PI3K/protein kinase B (Akt). The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKC& together with ERK/MAP kinase activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway. The studies in H32 cells demonstrate that VP has antiapoptotic actions in a neuronal cell line and suggest that VP has neuroprotective actions. This hypothesis was tested in primary cultures of hippocampal neurons by examining the ability of VP to prevent nutrient deprivation- or glutamate induced apoptosis, and the signaling pathways involved in this effect. Deprivation of trophic factors (by changing the defined media supplement by 0.1%BSA) for 24 hours or exposure to glutamate (3 to 10 M) caused significant decrease in neuronal cell viability and an increase in caspase-3 activity, consistent with apoptotic cell death. The effect of trophic factors deprivation or glutamate was significantly reduced by addition of 10nM VP, suggesting that VP exerts anti-apoptotic effects in neurons. This was confirmed by the ability of VP to prevent trophic factor deprivation-induced Tdt-mediated dUTP nick-end labeling (TUNEL) staining neurons. The protective effect of VP was completely blocked by V1 receptor antagonist, (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-Vasopressin, indicating that it is mediated via V1 VP receptors. The anti-apoptotic effect of VP in neurons involves mitogen activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) and the PI3 kinase/Akt signaling pathways. Co-incubation of nutrient deprived neurons with VP and the selective MAPK inhibitor, U0126, or the PI3 kinase inhibitor, LY294002, reduced the inhibitory action of VP on caspase-3 activity and TUNEL staining neurons. Western blot analysis revealed biphasic increases in phosphorylation/activation of ERK during incubation with VP, increasing at from 2.5 to 5 min returning to basal by 10 min and increasing again from 30 min to 1 h. This data shows that VP has anti-apoptotic actions in neurons, an effect which is partially mediated by the MAPK signaling pathway. The study supports the view that VP plays a role as a neuroprotective agent in the brain.

Summary: Recent studies from this laboratory using the neuronal cell line H32, which expresses endogenous V1 VP receptors, showed that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. VP的这些作用在很大程度上是由有丝分裂原活化蛋白（MAP）激酶和细胞外信号调节激酶（ERK）介导的，但也涉及其他信号通路。过去一年的研究研究了蛋白激酶C（PKC）和B（PKB）介导VP诱导的H32细胞中抗凋亡的作用。血清剥夺增加了PKCδ，但不增加PKC或PKC活性，而VP增加了PKC和PKC而不会影响PKCδ活性。 PKCδ的抑制阻止了caspase-3激活，表明PKCδ介导了血清剥夺的促凋亡作用。同时抑制PKC＆和MAP激酶消除了VP诱导的不良磷酸化，但仅部分阻止了caspase-3抑制作用。完全废除VP对血清剥夺诱导的caspase 3活性的保护作用，需要对PI3K/蛋白激酶B（AKT）的额外阻断。数据表明，VP通过多种途径施加抗凋亡。尽管PKC＆与ERK/MAP激酶一起激活介导了不良的磷酸化（灭活），但VP的完整保护作用需要额外激活PKB（PI3K/AKT）途径。 H32细胞中的研究表明，VP在神经元细胞系中具有抗凋亡作用，并表明VP具有神经保护作用。通过检查VP预防养分剥夺或谷氨酸诱导的凋亡的能力以及参与此作用的信号传导途径，在海马神经元的原发性培养物中进行了检验。剥夺营养因子（通过将定义的培养基补充剂更改为0.1％BSA）24小时或暴露于谷氨酸（3至10毫米）导致神经元细胞活力的显着降低，caspase-3活性增加，与凋亡细胞死亡一致。通过添加10nm VP可显着降低营养因子剥夺或谷氨酸的影响，这表明VP在神经元中发挥抗凋亡作用。 VP能够预防营养因子剥夺引起的TDT介导的DUTP Nick-End标记（TUNEL）染色神经元的能力证实了这一点。 VP的保护作用被V1受体拮抗剂（苯克1，d-tyr（et）2，lys6，arg8，des-gly9）-Dasopressin完全阻断，表明它是通过V1 VP受体介导的。 VP在神经元中的抗凋亡作用涉及有丝分裂原活化蛋白（MAP）激酶和细胞外信号调节激酶（ERK）和PI3激酶/AKT信号通路。养分剥夺神经元与VP和选择性MAPK抑制剂U0126或PI3激酶抑制剂LY294002的共结合减少了VP对Caspase-3活性和TUNEL染色神经元的抑制作用。 Western印迹分析显示，与VP孵育过程中ERK的磷酸化/激活的双相增加，在2.5分钟至5分钟恢复到基础上的磷酸化/激活增加，从30分钟恢复到基础，并从30分钟增加到1小时。该数据表明，VP在神经元中具有抗凋亡作用，该作用由MAPK信号通路部分介导。该研究支持以下观点，即VP在大脑中起着神经保护剂的作用。