Department of Integrative Physiology, and Cardiovascular Research Institute, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
J Neuroendocrinol. 2011 Oct;23(10):894-905. doi: 10.1111/j.1365-2826.2011.02209.x.
We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain-derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. Western blot analysis of brain punches containing the SON revealed that tyrosine phosphorylation of TrkB (pTrkBY(515)), serine phosphorylation of NR1 (pNR1S(866) or pNR1) and tyrosine phosphorylation of NR2B subunits (pNR2BY(1472) or pNR2B) were significantly increased in WD animals compared to controls. Access to water for 2 h reduced pTrkBY(515) content to control levels without affecting pNR1 or pNR2B. Four hours of rehydration was needed to reduce pNR1 and pNR2B to control levels. To test whether increased phosphorylation of TrkB in the present study is mediated by BDNF, a group of animals were instrumented with right SON cannula coupled to mini-osmotic pumps filled with vehicle or TrkB-Fc fusion protein, which prevents BDNF binding to TrkB. In the left SON contralateral to the cannula, TrkB phosphorylation was significantly enhanced following WD. Separate analysis of the right SON, which received TrkB-Fc, showed that the TrkB receptor phosphorylation following WD was significantly attenuated. Although increased pNR1S(866) following WD was not affected by local infusion of TrkB-Fc, pNR2BY(1472) was significantly reduced. Co-immunoprecipitation revealed an increased physical interaction between Fyn kinase and NR2B and TrkB in the SON following WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.
我们研究了水剥夺（WD）对大鼠视上核（SON）酪氨酸激酶 B（TrkB）和 NMDA 受体亚单位磷酸化的影响。使用激光捕获显微切割和定量逆转录聚合酶链反应来证明血管加压素 SON 神经元中的脑源性神经营养因子（BDNF）和 TrkB 基因表达。免疫组织化学证实 BDNF 在血管加压素神经元中的染色，而 WD 后磷酸化 TrkB 的染色增加。包含 SON 的脑片的 Western blot 分析显示，WD 动物中 TrkB 的酪氨酸磷酸化（pTrkBY(515)）、NR1 的丝氨酸磷酸化（pNR1S(866)或 pNR1）和 NR2B 亚单位的酪氨酸磷酸化（pNR2BY(1472)或 pNR2B）明显增加。与对照组相比。给予 2 小时的水可使 pTrkBY(515)含量降低至对照水平，而不影响 pNR1 或 pNR2B。需要 4 小时的再水合作用才能使 pNR1 和 pNR2B 降低至对照水平。为了测试本研究中 TrkB 的磷酸化增加是否由 BDNF 介导，一组动物被植入右侧 SON 套管，套管连接到装有载体或 TrkB-Fc 融合蛋白的微型渗透泵，TrkB-Fc 融合蛋白可防止 BDNF 与 TrkB 结合。在与套管相对的左侧 SON 中，WD 后 TrkB 磷酸化明显增强。对接受 TrkB-Fc 的右侧 SON 的单独分析表明，WD 后 TrkB 受体磷酸化明显减弱。尽管 WD 后 pNR1S(866)的增加不受局部输注 TrkB-Fc 的影响，但 pNR2BY(1472)明显减少。共免疫沉淀显示 WD 后 SON 中 Fyn 激酶与 NR2B 和 TrkB 的物理相互作用增加。因此，WD 后 SON 中 TrkB 的激活可能通过 NR2B 亚单位的磷酸化影响细胞兴奋性。
