NO-MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

描述（由申请人提供）：拟定的研究将研究一氧化氮（NO）介导的磷酸化机制，该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性神经元死亡。我们建议，在缺氧过程中产生的NO导致细胞死亡阻遏蛋白Bcl-2和环AMP反应元件结合（CREB）蛋白的磷酸化增加。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，在缺氧过程中产生的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的失活。这些磷酸酶的失活导致Bcl-2磷酸化增加，这是一种NO介导的缺氧神经元死亡的转录非依赖性机制。此外，我们建议，在缺氧过程中产生的NO增加核内Ca++流入神经细胞核，导致核钙调素激酶的激活，导致CREB蛋白的磷酸化增加和细胞死亡促进蛋白Bax的表达增加，一个NO介导的转录依赖性机制的神经元缺氧后死亡。将通过使用31 P-核磁共振光谱连续测量高能磷酸盐化合物来监测体内脑缺氧的程度，并进行生化确认。本研究以新生仔猪为实验对象，研究了缺氧对Bcl-2和CREB蛋白磷酸化的影响，缺氧对SH-PTP-1、MKP-1和MKP-3活性和表达的影响，缺氧对MAP激酶ERK和JNK活性和表达的影响;（3）缺氧对SH-PTP-1、MKP-1和MKP-3细胞半胱氨酸亚硝酰化的影响：（4）NO介导的半胱氨酸亚硝酰化与蛋白酪氨酸磷酸酶和MAP激酶磷酸酶MKP-1和MKP-3活性抑制的关系;（5）NO供体对神经元胞核Ca ~（++）内流的影响：（6）缺氧对神经元胞核Ca ~（++）激酶活性及凋亡促进蛋白Bax表达的影响;和（7）在缺氧期间施用一氧化氮合酶（NOS）抑制剂对磷酸酶活性、Bcl-2和CREB的磷酸化的影响，Bax的表达和神经元死亡的免疫细胞化学和形态学指标。这些研究将提供一种新的机制，NO介导的特定蛋白磷酸化导致缺氧神经元死亡的转录非依赖性和转录依赖性途径。NO介导的细胞死亡阻遏蛋白Bcl-2的磷酸化和细胞死亡促进蛋白Bax在缺氧反应中的表达的分子机制的阐明，将有助于开发新的预防策略缺氧诱导的新生儿脑功能障碍。

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.