NEUROENDOCRINE FUNCTIONS OF VASOPRESSIN AND VASOPRESSIN RECEPTORS

加压素和加压素受体的神经内分泌功能

• 批准号: 7594263
• 负责人: Greti Aguilera
• 金额: $ 141.78万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/7594263
• 关键词: Acute; Address; Adrenalectomy; Anterior Pituitary Gland; Ants; Apoptosis; Apoptotic; Area; BAD gene; Bad protein; Binding; Blood Circulation; Blood Pressure; Brain; Bromodeoxyuridine; Bromouridine; CRH gene; Calmodulin; Cell Count; Cell Death; Cell Line; Cell Nucleus; Cell Proliferation; Cell membrane; Cells; Chronic; Chronic stress; Corticosterone; Corticotrope Cell; Corticotropin; Corticotropin-Releasing Hormone; Corticotropin-Releasing Hormone Receptors; Daily; Data; Extracellular Signal Regulated Kinases; Flow Cytometry; HPSE gene; Hypothalamic structure; Infusion procedures; Injection of therapeutic agent; Knockout Mice; Label; MAP Kinase Gene; Mediating; Mediator of activation protein; Minor; Mitogen-Activated Protein Kinases; Molecular; Neuronal Plasticity; Neurons; Neuropeptides; Neuroprotective Agents; Neurosecretory Systems; Noise; Numbers; PRL gene; Peptides; Phosphorylation; Physiological; Pituitary Gland; Plasma; Prolactin; Property; Purpose; Rattus; Role; SSR149415; Saline; Serum; Signal Pathway; Staining method; Stains; Stem cells; Stimulation of Cell Proliferation; Stress; System; Testing; Time; Tissues; Undifferentiated; V1 Receptors; Vasopressin Receptor; Vasopressins; Western Blotting; annexin A5; base; caspase-3; cytochrome c; day; deprivation; hypothalamic-pituitary-adrenal axis; nestin protein; neuroprotection; novel; osmotic minipump; parvocellular; prevent; pro-apoptotic protein; protein kinase C kinase; receptor; research study; response; restraint; stellate cell; stressor; transcription factor; uptake; water conservation

Summary: In addition to its recognized actions on water conservation and blood pressure, the neuropeptide vasopressin is released into the pituitary portal circulation and within distinct brain areas during stressful challenges. The neuroendocrine effects of vasopressin are mediated by specific receptor of subtype V1b in the anterior pituitary and V1a and V1b subtypes in the brain. Vasopressin produced by parvocellular neurons of the PVN potentiates the stimulatory effect of CRH on pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype (V1bR). During prolonged activation of the HPA axis there is a predominant increase in VP in parvocellular hypothalamic neurons and pituitary V1b receptors compared with CRH and CRH receptors, suggesting that VP becomes the main mediator of pituitary corticotroph responsiveness during chronic stress. To test this hypothesis, we examined the effect of pharmacologic VP receptor blockade on the ACTH and corticosterone responses of 14-day repeatedly restrained rats. In spite of the increased vasopressinergic activity, repeatedly restrained rats showed lower ACTH and corticosterone responses to 10 min white noise compared with handled controls. These responses were unchanged by the non-peptide selective V1b receptor antagonist, SSR149415, 10 g, i.v., 1h before noise application. In contrast to the response to noise stress, plasma ACTH responses to i.p. hypertonic saline injection were enhanced in the repeatedly restrained rats compared with handled controls but responses were also unaffected by SSR149415 (30mg/kg/orally) administered daily 1 h before restraint. Since SSR149415 bioactivity, assessed by corticosterone responses to exogenous VP, was low, we used minipump infusion of the peptide V1 receptor antagonist, dGlyPhaa1,D-Tyr(et), Lys, ArgVP (V1-Ant) for 14 days, which effectively blocked ACTH responses to exogenous VP. Chronic V1-Ant infusion significantly reduced plasma ACTH responses to i.p. hypertonic saline in handled controls but not in repeatedly restrained rats. These data show that VP contributes to the ACTH responses to an acute stressor but that increased vasopressinergic activity is not responsible for the enhanced ACTH responses to a novel stress. The little impact of vasopressinergic blockade on HPA axis activity during chronic stress, suggests that VP has additional roles, such as controlling the number of pituitary corticotrophs. The role of VP mediating pituitary corticotroph mitogenesis was studied in adrenalectomized rats by examining the effect of a peptide VP V1 receptor antagonist (V1-ant) on the number of cells incorporating bromouridine (BrdU). Long term adrenalectomy increased the number of both BrdU labeled cells and ACTH stained cells. Osmotic minipump infusion of V1-ant for 28 days prevented adrenalectomy-induced increases in BrdU incorporation but not the changes in the number of ACTH-stained cells. Unexpectedly, co-localization of BrdU uptake in ACTH positive cells was minor and this was unaffected by adrenalectomy or V1-antagonist infusion. No BrdU stained nuclei were observed in LH, TSH, PRL, GH, folliculo-stellate cells or nestin-labeled progenitor cells. The pituitary corticotroph exclusive transcription factor, Tpit, colocalized in >80% of ACTH containing cells but only in 5% of BrdU labeled nuclei in controls and 10% in adrenalectomized rats. In V1bR knock out mice, the number of cells incorporating BrdU following adrenalectomy was lower than in wild type, and there was no major co-localization of BrdU and ACTH. The data demonstrates that VP mediates mitogenic activity in the pituitary during long term adrenalectomy. The lack of colocalization of ACTH and BrdU, suggest that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. This data suggest that one of the functions of the marked increases in parvocellular vasopressinergic activity during adrenalectomy (and probably chronic stress) is regulating cell proliferation and remodeling of the pituitary tissue. Based on the observation that VP prevented serum deprivation-induced cell death in the neuronal cell line, H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10nM VP prevented serum deprivation-induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time and serum concentration dependent manner, and VP prevented these effects in a V1 receptor-specific manner. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen activated protein (MAP) kinase MAPK and extracellular signal-regulated kinases (ERK), Ca2+/calmodulin dependent kinase (CaMK) and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of BAD phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects prevented by 10nM VP. These data demonstrate 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. The data suggest that VP has anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain.

摘要：除了公认的节水和降压作用外，神经肽加压素在压力挑战期间还会释放到垂体门静脉循环和不同的大脑区域内。加压素的神经内分泌作用是由垂体前叶 V1b 亚型以及大脑中 V1a 和 V1b 亚型的特定受体介导的。 PVN 细小细胞神经元产生的加压素通过 V1b 亚型 (V1bR) 质膜受体增强 CRH 对垂体 ACTH 分泌的刺激作用。在 HPA 轴长时间激活期间，与 CRH 和 CRH 受体相比，小细胞下丘脑神经元和垂体 V1b 受体中的 VP 显着增加，表明 VP 成为慢性应激期间垂体促肾上腺皮质激素反应性的主要介质。为了检验这一假设，我们检查了药物 VP 受体阻断对 14 天反复抑制大鼠的 ACTH 和皮质酮反应的影响。尽管血管加压素活性增加，但与处理的对照组相比，反复抑制的大鼠对 10 分钟白噪声表现出较低的 ACTH 和皮质酮反应。在施加噪音前 1 小时，非肽选择性 V1b 受体拮抗剂 SSR149415（10 µg，静脉注射）不会改变这些反应。与对噪声应激的反应相反，血浆 ACTH 对腹腔注射的反应。与处理对照组相比，反复束缚大鼠的高渗盐水注射增强，但束缚前 1 小时每天注射 SSR149415（30mg/kg/口服）也不影响反应。由于通过皮质酮对外源性 VP 反应评估的 SSR149415 生物活性较低，因此我们使用微型泵输注肽 V1 受体拮抗剂 dGlyPhaa1、D-Tyr(et)、Lys、ArgVP (V1-Ant) 14 天，有效阻断ACTH 对外源性 VP 的反应。慢性 V1-Ant 输注可显着降低腹膜内血浆 ACTH 反应。在处理的对照中使用高渗盐水，但在反复限制的大鼠中则不然。这些数据表明，VP 有助于 ACTH 对急性应激源的反应，但增加的血管加压素活性并不导致 ACTH 对新压力的反应增强。 在慢性应激过程中，血管加压素能阻断对 HPA 轴活动的影响很小，这表明 VP 还具有其他作用，例如控制垂体促肾上腺皮质激素的数量。通过检查肽 VP V1 受体拮抗剂 (V1-ant) 对掺入溴尿苷 (BrdU) 的细胞数量的影响，研究了 VP 介导垂体促肾上腺皮质激素细胞有丝分裂的作用。长期肾上腺切除术增加了 BrdU 标记细胞和 ACTH 染色细胞的数量。 V1-ant 的渗透微型泵输注 28 天可以防止肾上腺切除术引起的 BrdU 掺入增加，但不能防止 ACTH 染色细胞数量的变化。出乎意料的是，ACTH 阳性细胞中 BrdU 摄取的共定位很小，并且不受肾上腺切除术或 V1 拮抗剂输注的影响。在 LH、TSH、PRL、GH、滤泡星状细胞或巢蛋白标记的祖细胞中未观察到 BrdU 染色的细胞核。垂体促肾上腺皮质激素专有转录因子 Tpit 共定位于超过 80% 的含有 ACTH 的细胞中，但在对照组中仅存在于 5% 的 BrdU 标记细胞核中，在肾上腺切除大鼠中共定位于 10%。在 V1bR 敲除小鼠中，肾上腺切除后掺入 BrdU 的细胞数量低于野生型，并且 BrdU 和 ACTH 不存在重大共定位。数据表明，VP 在长期肾上腺切除术中介导垂体促有丝分裂活性。 ACTH 和 BrdU 缺乏共定位，表明肾上腺切除过程中促肾上腺皮质激素的募集来自未分化细胞。该数据表明，肾上腺切除术（可能还有慢性应激）期间细小细胞加压素活性显着增加的功能之一是调节细胞增殖和垂体组织的重塑。 根据观察到 VP 可以防止表达内源性 V1 受体的神经元细胞系 H32 中血清剥夺诱导的细胞死亡，我们检验了 VP 具有抗凋亡特性的假设。流式细胞术实验表明，10nM VP 可防止血清剥夺诱导的细胞死亡和膜联蛋白 V 结合。血清剥夺以时间和血清浓度依赖性方式增加 caspase-3 活性，而 VP 以 V1 受体特异性方式阻止这些作用。介导VP抗凋亡作用的信号通路涉及丝裂原激活蛋白(MAP)激酶MAPK和细胞外信号调节激酶(ERK)、Ca2+/钙调蛋白依赖性激酶(CaMK)和蛋白激酶C(PKC)。蛋白质印迹分析显示，血清剥夺后，BAD 磷酸化呈时间依赖性下降，细胞色素 c 的胞质水平增加，而 10nM VP 可阻止这种影响。这些数据表明，内源性 V1 VP 受体的激活通过促凋亡蛋白 BAD 的磷酸化失活以及随后胞质细胞 ​​c 和 caspase-3 活化的减少来防止血清剥夺诱导的细胞凋亡。数据表明 VP 在神经元中具有抗凋亡活性，并且 VP 可能充当大脑中的神经保护剂。