REGULATION OF NA,K ATPASE BY THE AH RECEPTOR

人类在怀孕期间暴露于持久性环境污染物，如2,3,7,8-四氯二苯并-对二恶英（TCDD）及相关化学物质，会导致出生体重下降、神经功能缺陷、甲状腺激素改变、肺部听诊和色素沉着。TCDD介导发育毒性的机制尚未阐明。基本的螺旋-环-螺旋- pas转录因子，芳烃受体（AhR），是tcdd诱导小鼠致畸所必需的，并可能介导其他物种的致畸。TCDD毒性可能是由AhR基因转录的改变引起的。一个可能解释TCDD致畸作用的AhR基因靶点是Na+/K+ atp酶alpha1。假定的二恶英反应元件保守于哺乳动物和鸟类Na+/K+ atp酶α 1基因的5'增强子区。在鸡胚中，TCDD降低心肌Na+/K+ atp酶α 1蛋白表达，诱导扩张性心肌病，改变心电图，这些都与Na+/K+ atp酶活性降低一致。在缺乏AhR的小鼠中，胚胎会发展为肥厚性心肌病和心脏纤维化，并随着年龄的增长而恶化，这与Na+/K+ atp酶α 1的潜在过度表达和高血压的发展相一致。我将使用鸡胚和AhR缺失小鼠来验证AhR调节心肌Na+/K+ atp酶α 1基因表达，改变心血管发育的假设。本研究的目的是：(1)利用TCDD、RT-PCR和禽类基因启动子分析，阐明AhR缺失小鼠和发育中的鸡胚心肌Na+/K+ atp酶α 1基因的调控作用；(2)通过定量测定AhR缺失小鼠和tcdd暴露鸡胚心肌中乌阿巴因结合位点和Na+/K+ atp酶活性，确定这一调控的组织意义；(3)通过ECG测量AhR缺失小鼠血压和tcdd暴露鸡胚心肌对瓦巴因的敏感性，确定其功能意义；(4)通过pcr选择减法杂交方法和基因微阵列筛选，分析由亚利桑那大学Selmin博士鉴定的TCDD在大鼠胚胎心脏和肺中表达改变的候选基因的小鼠和鸟类同源物的表达。

Human exposure during pregnancy to persistent environmental pollutants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals, results in decreased birth weights, neurological deficits, thyroid hormone alterations, lung auscultation, and hyperpigmentation. The mechanism by which TCDD mediates developmental toxicity has not been elucidated. The basic helix-loop-helix-PAS transcription factor, aryl hydrocarbon receptor (AhR), is required for TCDD-induced teratogenicity in mice and likely mediates teratogenicity in other species. TCDD toxicity may result from alterations in gene transcription by the AhR. One potential AhR gene target that could account for some of TCDD's teratogenic effects is the Na+/K+ ATPase alpha1. Putative dioxin response elements are conserved in the 5' enhancer region of the mammalian and avian Na+/K+ ATPase alpha1 gene. In the chick embryo, TCDD reduces myocardial Na+/K+ ATPase alpha1 protein expression, induces a dilated cardiomyopathy, and alters ECGs, all consistent with reduced Na+/K+ ATPase activity. In mice lacking the AhR, embryos develop a hypertrophic cardiomyopathy and cardiac fibrosis which worsens with age, consistent with the potential overexpression of Na+/K+ ATPase alpha1 and development of hypertension. I will use the chick embryo and AhR null mice to test the hypothesis that the AhR regulates myocardial expression of the Na+/K+ ATPase alpha1 gene, altering cardiovascular development. The aims of this proposal are to (1) elucidate the regulation of myocardial Na+/K+ ATPase alpha1 gene in AhR null mice and in the developing chick embryo by TCDD using RT-PCR, and in vitro by promoter analysis of the avian gene; (2) determine the tissue significance of this regulation by quantitating myocardial ouabain binding sites and Na+/K+ ATPase enzyme activity in AhR null mice and TCDD-exposed chick embryos; (3) determine the functional significance by measuring blood pressure in AhR null mice and myocardial sensitivity to ouabain in TCDD-exposed chick embryos by ECG; and (4) analyze the expression of murine and avian homologues of candidates genes, whose expression is altered by TCDD in the rat embryo heart and lung as identified by Dr. Selmin, University of Arizona, by PCR-selected subtractive hybridization method and screening of gene microarrays.