NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

描述(申请人提供)：拟议的研究将研究一氧化氮(NO)介导的磷酸化机制，通过转录独立和转录依赖的途径在新生儿中导致缺氧性神经元死亡。我们认为，低氧时产生的NO导致细胞死亡抑制蛋白Bcl-2和环磷酸腺苷反应元件结合蛋白(CREB)的磷酸化增加。Bcl2和CREB蛋白的磷酸化将与脑组织缺氧的严重程度相关。我们认为，低氧时产生的NO自由基导致蛋白酪氨酸磷酸酶(PIP)和丝裂原激活的激酶磷酸酶1(MKP-1)和MKP-3失活。这些磷酸酶的失活导致BCI-2的磷酸化增加，这是一种NO介导的缺氧性神经元死亡的转录无关机制。此外，我们认为，缺氧时产生的NO增加了神经元核内钙离子的内流，导致核CaM-K的激活，导致CREB蛋白的磷酸化和细胞死亡启动子蛋白Bax的表达增加，这是一种NO介导的神经元死亡的转录依赖机制。体内脑缺氧的程度将通过31P-核磁共振波谱连续测量高能磷酸化合物并经生化证实来监测。本实验将在新生仔猪上进行：(1)定量组织缺氧与Bcl2和CREB蛋白磷酸化的关系；(2)缺氧对：(A)SH-PTP-1、MKP-1和MKP-3的活性和表达的影响；(B)MAP激酶ERK和JNK的激活和活性；(3)缺氧对SH-PTP-1、MKP-1和MKP-3中半胱氨酸残基亚硝化的影响；(4)NO介导的半胱氨酸残基体外亚硝化与抑制蛋白酪氨酸磷酸酶和MKP-1和MKP-3活性的关系；(5)NO供体对神经元核内钙内流的影响；(6)缺氧对神经元细胞核CaM-Kinase活性及随后细胞死亡促进蛋白Bax表达的影响；(7)缺氧时给予一氧化氮合酶(NOS)抑制剂对磷酸酶活性、Bcl2和CREB磷酸化、Bax表达和神经元死亡的免疫细胞化学和形态指标的影响。这些研究将提供一种新的机制，通过转录不依赖和转录依赖的途径，通过NO介导的特定蛋白的磷酸化导致缺氧性神经元死亡。阐明NO介导的细胞死亡抑制蛋白Bcl2磷酸化和细胞死亡促进蛋白Bax表达的分子机制将有助于开发新的预防新生儿缺氧性脑功能障碍的策略。

DESCRIPTION (provided by applicant): The proposed studies will investigate nitric oxide (NO)-mediated phosphorylation mechanisms that result in hypoxic neuronal death by transcription-independent and transcription-dependent pathways in the newborn. We propose that NO produced during hypoxia leads to increased phosphorylation of the cell death repressor protein Bcl-2 and cyclic AMP-response element binding (CREB) protein. Phosphorylation of Bcl-2 and CREB proteins will correlate with the severity of cerebral tissue hypoxia. We propose that NO free radicals generated during hypoxia lead to inactivation of protein tyrosine phosphatase (PIP) and mitogen-activated kinase phosphatase1 (MKP-1) and MKP-3. Inactivation of these phosphatases results in increased phosphorylation of BcI-2, a NO-mediated transcription-independent mechanism of hypoxic neuronal death. Furthermore, we propose that NO generated during hypoxia increases intranuclear Ca++-influx in neuronal nuclei leading to activation of nuclear CaM-kinase resulting in increased phosphorylation of CREB protein and increased expression of the cell death promoter protein Bax, a NO-mediated transcription-dependent mechanism of neuronal death following hypoxia. The degree of brain hypoxia in vivo will be monitored by continuous measurement of high energy phosphate compounds with 31P-nuclear magnetic resonance spectroscopy and confirmed biochemically. Experimental protocols will be carried out on newborn piglets investigating: (1) the relationship of quantitative tissue hypoxia to phosphorylation of Bcl-2 and CREB proteins, (2) the effect of hypoxia on: (a) the activity and expression of SH-PTP-1, MKP-1 and MKP-3, (b) the activation and activity of MAP kinases ERK and JNK; (3) the effect of hypoxia on nitrosylation of cysteine residues in SH-PTP-1, MKP-1 and MKP-3; (4) the relationship of NO-mediated nitrosylation of cysteine residues in vitro to inhibition of the activity of protein tyrosine phosphatase and MAP kinase phosphatases MKP-1 and MKP-3; (5) the effect of NO donors on Ca++-influx in neuronal nuclei; (6) the effect of hypoxia on the activity of neuronal nuclear CaM-kinase and subsequent expression of cell death promoter protein Bax; and (7) the effect of administration of nitric oxide synthase (NOS) inhibitors during hypoxia on activity of phosphatases, phosphorylation of Bcl-2 and CREB, expression of Bax and immunocytochemical and morphological indices of neuronal death. These studies will provide a novel mechanism of NO-mediated phosphorylation of specific proteins resulting in hypoxic neuronal death by transcription-independent and transcription-dependent pathways. The elucidation of molecular mechanisms of NO-mediated phosphorylation of the cell death repressor protein Bcl-2 and expression of the cell death promoter protein Bax in response to hypoxia will aid in the development of novel preventive strategies for hypoxia-induced brain dysfunction in the newborn.