Molecular biological study on the renal alpha-2 adrenoceptors in genetic hypertension

遗传性高血压肾α-2肾上腺素受体的分子生物学研究

• 批准号: 01044121
• 负责人: ISHII Masao
• 金额: $ 5.06万
• 依托单位: Yokohama City University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01044121/
• 关键词: Dahl salt sensitive rat; Spontaneously hypertensive rat; Restriction fragment length polymorphism; alpha_2 adrenergic receptor; Gene; Essential hypertension; Northern blot; Inner medullary collecting duct; 高血圧自然発症ラット(SHR); Dahl rat; cyclic AMP /

1. Study of alpha_2 adrenergic receptor (alpha_2-AR) gene(1) Restriction fragment length polymorphisms (RFLP) of alpha_2 adrenergic receptor gene in genetically Jlypertensive rats.<I> RFLP between the genome of Dahl Salt sensitive rats (S) and Dahl salt resistant rats (R). We found a RFLP between Dahl S and R rats using a human kidney cDNA alpha_2-AR probe (alpha_2-C_4) and Msp I restriction endonuclease. This probe marked an S allele of 3.0 Kb and an R allele of 2.8 Kb. The frequency of the RFLP distribution was investigated in F_2 rats by crossbreeding R female and S male, and subsequently brother/sister mating of Fi rats. The alpha_2-AR-AR genotype distribution in F_2 population did not follow the Mendelian segregation ratio. After the study of backcrossing of the F_1 rats to their S and R parents, we concluded that the deficiency of SS genotype in F_2 rats may be due to gene recombination. Alternatively, the deficiency may not be due to the alpha_2-AR gene itself, but to the effect … More of other genes closely linked to the _2-AR.<2> RFLP between the genome of spon-tancously hypertensive rats (SHR) and Wister-Kyoto rats (WKY). Dr. Pettinger et. al. found a RFLP between the genome of SHR and WKY using human alpha_2-C_<10> probe. Restriction mapping analysis showed that this RFLP is located in the 5'promoter region of the gene. A genetic cosegregation study showed that this RFLP cosegregates with some of the blood pressure elevation in the SHRXWKY cross.(2) RFLP of alpha_2-AR gene in human. Dr. Pettinger et al. reported a RFLP of human geno. me using human alpha_2-C_<10> probe (J of Hypertension in press). Using same restriction enzyme and probe, we found a same RFLP in Japanese. Now we are under way to evaluate whether any relationship exists between this RFLP and the blood pressure.(3) Characterization of alpha_2-adrenoceptor subtypes in HT29, OK and NG108 cell line ; Northern blot and ribonuclease protection study. Nonhem blot and ribonuclease protection assay were used to identify alpha_2-AR subtypes in human colonic adenocarcinoma (HT29), opossum kidney (OK), and neuroblastoma x glioma rat-mouse hybrid NGlO815 (NG108) cell lines. We concluded that alpha_2-C_4 gene is related to the alpha_<2B> pharmacological alpha_2-adrenoceptor subtype and is actively transcribed in OK and NG108 cells. The alpha_2-C_<10> gene is related to the alpha_<2A> pharmacological subtype and is actively transcribed in HT29 cells.2. Characterization of prazosine-sensitive alpha_<2B>-adrenoceptors expressed by cultured rat IMCD cells. alpha_2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Nonhem blot analysis, and cAMP assay. We demonstrated that IMCD cells express the alpha_<2B> subtype of adrenoceptor and that stimulation of these receptors inhibits vasopressin-stumulated cAMP formation. Less

1.α_2肾上腺素能受体（α_2-AR）基因的研究（1）遗传性高血压大鼠α_2肾上腺素能受体基因的限制性片段长度多态性（RFLP）。<I>Dahl盐敏感大鼠（S）和Dahl盐抗性大鼠（R）基因组之间的RFLP。我们用人肾cDNA α_2-AR探针（α_2-C_4）和Msp Ⅰ限制性内切酶在Dahl S和R大鼠之间发现了一个RFLP。该探针标记3.0Kb的S等位基因和2.8Kb的R等位基因。本研究采用R雌、S雄杂交F_2代大鼠，F_1代大鼠与F_2代大鼠进行兄妹交配，观察F_2代大鼠的RFLP分布频率。α_2-AR-AR基因型在F_2群体中的分布不符合孟德尔分离比。通过对F_1代大鼠与S、R亲本回交的研究，我们认为F_2代大鼠SS基因型缺失可能是由于基因重组所致。或者，这种缺陷可能不是由于α_2-AR基因本身，而是由于 ...更多信息 与_2-AR紧密相关的其他基因。<2>自发性高血压大鼠（SHR）和Wister-Kyoto大鼠（WKY）基因组间的RFLP分析。Dr. Pettinger et.等用人α_2-C_探针在SHR和WKY基因组间发现了一个RFLP<10>。限制性内切酶图谱分析表明，该限制性内切酶位于该基因的5 ′启动子区。遗传共分离研究表明，这种RFLP共分离与一些血压升高的SHRXWKY交叉。(2)人α_2-AR基因的限制性片段长度多态性Pettinger等报道了一个人类基因的RFLP。用人α_2-C_探针测定的方法<10>（高血压杂志出版）。用相同的限制性内切酶和探针，在日本人中发现了相同的RFLP。现在我们正在评估该RFLP与血压之间是否存在任何关系。(3)HT 29、OK和NG 108细胞系中α_2肾上腺素受体亚型的表征;北方印迹和核糖核酸酶保护研究。采用Nonhem印迹和核糖核酸酶保护试验鉴定人结肠腺癌（HT 29）、负鼠肾（OK）和神经母细胞瘤x胶质瘤大鼠-小鼠杂交细胞系NGlO 815（NG 108）中的α_2-AR亚型。结论：α_2-C_4基因与α<2B>_2-肾上腺素能受体亚型有关，在OK和NG 108细胞中转录活跃。α_2-C_<10>基因与α_<2A>药理学亚型相关，在HT 29细胞中转录活跃.培养<2B>的大鼠IMCD细胞表达的哌唑嗪敏感性α-肾上腺素受体的特性用放射配基结合法、Nonhem印迹法和cAMP测定法研究了体外培养的大鼠内髓集合管（IMCD）细胞α_2肾上腺素能受体亚型的表达。我们证明IMCD细胞表达α亚型<2B>肾上腺素受体，刺激这些受体抑制加压素调节的cAMP的形成。少

项目成果

Ishikawa Y,et al.: "Identification of an alpha_2ーadrenoceptor in human coronary arteries by radioligand binding assay" Life Science. 48. 2513-2518 (1991)

Ishikawa Y 等人：“通过放射性配体结合测定鉴定人冠状动脉中的 α_2-肾上腺素受体”生命科学 48. 2513-2518 (1991)。

Sun L.: "Characterization of α_2ーadrenoceptor subtypes in cultured cell lines:Northern blot and ribonuclease protection study" FASEBJ. 5. A700 (1991)

Sun L.：“培养细胞系中 α_2-肾上腺素受体亚型的表征：Northern 印迹和核糖核酸酶保护研究”FASEBJ 5. A700 (1991)。

Umemura S: "Structures and functions of renal alpha adrenoceptors" J Clin Exp Medicine. 151. 199-202 (1989)

Umemura S：“肾α肾上腺素受体的结构和功能”J Clin Exp Medicine。

Umemura S et al.: "Molecular subclassification of a alphal and alpha2 adrenoceptors." J Clin Exp Med.156. 781 (1991)

Umemura S 等人：“α1 和 α2 肾上腺素受体的分子亚分类。”

Jeffries WB: "Receptor regulation of cAMP in initial and terminal inner medullary collecting duct" FASEBJ. 6. (1992)

Jeffries WB：“初始和终末内髓集合管中 cAMP 的受体调节”FASEBJ。