NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

描述（由申请人提供）：拟议的研究将研究一氧化氮（NO）介导的磷酸化机制，通过转录非依赖性和转录依赖性途径导致新生儿缺氧神经元死亡。我们认为缺氧过程中产生的NO导致细胞死亡抑制蛋白Bcl-2和环amp反应元件结合（CREB）蛋白磷酸化增加。Bcl-2和CREB蛋白的磷酸化与脑组织缺氧的严重程度相关。我们认为缺氧时产生的NO自由基导致蛋白酪氨酸磷酸酶（PIP）和丝裂原活化激酶磷酸酶1 （MKP-1）和MKP-3失活。这些磷酸酶的失活导致BcI-2磷酸化增加，这是no介导的缺氧神经元死亡的转录不依赖机制。此外，我们提出缺氧时产生的NO增加了核内Ca++-在神经元细胞核内的内流，导致核cam -激酶的激活，导致CREB蛋白磷酸化增加，细胞死亡启动子蛋白Bax的表达增加，这是缺氧后神经元死亡的NO介导的转录依赖机制。体内脑缺氧程度将通过31p核磁共振波谱连续测量高能磷酸盐化合物并进行生化确认来监测。实验方案将在新生仔猪上进行，研究：(1)定量组织缺氧与Bcl-2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，(b) MAP激酶ERK和JNK的激活和活性的影响；(3)缺氧对SH-PTP-1、MKP-1和MKP-3中半胱氨酸残基亚硝基化的影响；(4) no介导的体外半胱氨酸残基亚硝基化与抑制蛋白酪氨酸磷酸酶和MAP激酶磷酸酶MKP-1和MKP-3活性的关系；(5)一氧化氮对神经元核内Ca++内流的影响；(6)缺氧对神经元核cam -激酶活性及细胞死亡启动子蛋白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、Bcl-2和CREB磷酸化、Bax表达及神经元死亡免疫细胞化学和形态学指标的影响。这些研究将通过转录非依赖性和转录依赖性途径提供一氧化氮介导的特定蛋白磷酸化导致缺氧神经元死亡的新机制。阐明no介导的细胞死亡抑制蛋白Bcl-2磷酸化和细胞死亡启动蛋白Bax表达在缺氧反应中的分子机制，将有助于开发新的预防新生儿缺氧诱导脑功能障碍的策略。

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.