慢性间断性低氧诱导大鼠前包钦格复合体磷酸化PKC底物表达增加
亢君君, 魏晓燕, 刘莹莹^*
空军军医大学神经生物学教研室，西安 710032
摘要
延髓腹外侧前包钦格复合体(pre-B?tzinger complex, pre-B?tC)被认为是呼吸节律产生中枢。间断性低氧可诱导呼吸长时程易化(long-term facilitation, LTF)，是呼吸可塑性的电生理特征。本研究组前期研究显示慢性间断性低氧(chronic intermittent hypoxia, CIH)诱导大鼠pre-B?tC磷酸化蛋白激酶C θ (phospho-protein kinase C θ, P-PKCθ)表达上调。本研究旨在探讨磷酸化-蛋白激酶C底物(P-PKC substrates, P-PKCsub)在大鼠pre-B?tC的超微结构分布以及CIH干预后的表达变化。应用神经激肽1受体(neurokinin-1 receptor, NK1R)免疫反应(immunoreactive, ir)产物作为pre-B?tC神经元的标志，并用抗P-PKCsub抗体双标记进行免疫荧光和免疫电镜观察，用Western blot分析延髓腹外侧区(包含pre-B?tC) P-PKCsub蛋白的表达变化。结果显示，光镜下，NK1R-ir标记主要沿pre-B?tC神经元胞膜分布，清晰勾勒胞体和突起。P-PKCsub-ir标记多呈点状分布在胞体和突起，膜下亦有分布。大部分P-PKCsub-ir神经元共表达NK1R。CIH干预诱导延髓腹外侧区P-PKCsub蛋白表达水平上调。电镜下，NK1R-ir产物主要分布在pre-B?tC神经元胞体和树突的胞膜内表面。P-PKCsub-ir金颗粒分布在pre-B?tC神经元胞体和树突，在细胞膜下有较多分布，内质网和突触后致密体亦可见。以上结果提示，CIH干预可能通过激活PKCθ，上调P-PKCsub蛋白表达，参与pre-B?tC的呼吸可塑性调控。


关键词： 前包钦格复合体; 蛋白激酶C底物; 慢性间断性低氧; 呼吸可塑性; 超微结构
分类号：R331.3; Q463

Chronic intermittent hypoxia induces expression of phospho-PKC substrates in rat pre-B?tzinger complex
KANG Jun-Jun, WEI Xiao-Yan, LIU Ying-Ying^*
Department of Neurobiology, Air Force Medical University, Xi’an 710032, China
Abstract
The pre-B?tzinger complex (pre-B?tC) residing in the ventrolateral medulla oblongata, is thought to be the kernel of respiratory rhythmogenesis. Episodic hypoxia exerts respiratory long-term facilitation, being recognized as electrophysiological characteristic of respiratory motor neuroplasticity. Our previous study demonstrated up-regulated expression of phospho-protein kinase C θ (P-PKCθ) in the pre-B?tC of rats receiving chronic intermittent hypoxic (CIH) challenge. The present study was aimed to examine subcellular distribution of P-PKC substrates (P-PKCsub) and explore PKC down-stream targeting proteins in the pre-B?tC in normoxic and CIH rats. Using neurokinin-1 receptor (NK1R) as a marker of the pre-B?tC, P-PKCsub immunoreactivity was revealed by immunofluorescence and immuno-electron microscopic double-labeling in the pre-B?tC. Western blot was applied to analyze P-PKCsub proteins in ventrolateral medulla, containing the pre-B?tC. The results showed that NK1R immunoreactivity (NK1R-ir) was expressed mainly along plasma membranes of somata and processes, outlining pre-B?tC neurons under the light microscope. P-PKCsub immunoreactive (P-PKCsub-ir) fluorophores in dot-like appearance appeared in somata and processes. Some were in close apposition to plasma membranes. A majority of P-PKCsub-ir neurons was found with NK1R-ir. CIH challenge up-regulated the expression of P-PKCsub proteins in the ventrolateral medulla. Under the electron microscope, NK1R-ir product was found to distribute along the inner membrane surfaces of somata and dendrites. P-PKCsub-ir gold particles were located in somata and dendrites, and some were distributed along the inner membrane surfaces, as well as in the endoplasmic reticulum and postsynaptic dense body. These results suggest that CIH challenge up-regulates the expression of P-PKCsub proteins, probably including some receptor proteins in the postsynaptic membrane, which may contribute to respiratory neuroplasticity via activation of PKCθ in the pre-B?tC.


Key words: Pre-Botzinger complex; PKC substrates; chronic intermittent hypoxia; respiratory plasticity; ultrastructure

收稿日期：2019-10-24　　录用日期：2020-03-19
通讯作者：刘莹莹　　E-mail: yingyliu@fmmu.edu.cn
DOI: 10.13294/j.aps.2020.0065
引用本文：
亢君君, 魏晓燕, 刘莹莹. 慢性间断性低氧诱导大鼠前包钦格复合体磷酸化PKC底物表达增加[J]. 生理学报 2020; 72 (5): 559-565.
KANG Jun-Jun, WEI Xiao-Yan, LIU Ying-Ying. Chronic intermittent hypoxia induces expression of phospho-PKC substrates in rat pre-B?tzinger complex. Acta Physiol Sin 2020; 72 (5): 559-565 (in Chinese with English abstract).



PDF全文下载地址:

点我下载PDF