Division of Cardiology, Medical University of Graz, Graz, Austria ; Molecular Neurobiology and Neuroendocrinology, European Neuroscience Institute, Göttingen, Göttingen, Germany.
PLoS One. 2013 Oct 21;8(10):e78883. doi: 10.1371/journal.pone.0078883. eCollection 2013.
Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca²⁺-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca²⁺-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca²⁺] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca²⁺-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca²⁺-sensitive release-ready vesicles as determined by slow photo-release of caged Ca²⁺. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca²⁺-dependent release of α-MSH.
Rab3a 是 Rab3 亚家族的一种小 GTPase，在 Ca²⁺调节的胞吐作用的晚期发挥作用。先前在垂体黑素细胞中的功能分析表明 Rab3a 是 Ca²⁺依赖性胞吐作用的正调节剂。然而，Rab3a 同工型对调节胞吐作用的动力学和细胞内 [Ca²⁺]敏感性的确切作用，这可能影响两种主要肽激素，α-促黑素细胞刺激素（α-MSH）和 β-内啡肽在血浆中的可用性，仍然难以捉摸。我们使用 Rab3a 敲除小鼠（Rab3a KO）来探索新鲜垂体组织切片中黑素细胞的分泌表型。高分辨率电容测量显示，与野生型细胞相比，Rab3a KO 黑素细胞的 Ca²⁺触发的分泌活性受损。受阻的分泌与缺乏 cAMP 鸟嘌呤交换因子 II/Epac2 依赖性分泌成分有关。该成分归因于高 Ca²⁺敏感的释放准备好的囊泡，如通过笼状 Ca²⁺的缓慢光释放所确定的。放射免疫测定显示，α-MSH，但不是 β-内啡肽，在 Rab3a KO 小鼠的血浆中升高，表明 α-MSH 的组成型胞吐作用增加。从孵育组织切片中增加的 α-MSH 组成型分泌与 Rab3a 缺陷型垂体细胞中 α-MSH 细胞内含量减少有关。体外病毒重新表达 Rab3a 蛋白挽救了 Rab3a KO 小鼠黑素细胞的分泌表型。总之，我们认为 Rab3a 缺乏促进组成型分泌，并导致 Ca²⁺依赖性 α-MSH 释放选择性受损。
