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肾上腺素受体基因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。我们使用人肾 cDNA alpha_2-AR 探针 (alpha_2-C_4) 和 Msp I 限制性内切酶在 Dahl S 和 R 大鼠之间发现了 RFLP。该探针标记了 3.0 Kb 的 S 等位基因和 2.8 Kb 的 R 等位基因。通过将 R 雌性和 S 雄性杂交，以及随后 Fi 大鼠的兄弟/姐妹交配，研究了 F_2 大鼠中 RFLP 分布的频率。 F_2群体中α_2-AR-AR基因型分布不遵循孟德尔分离比例。通过对F_1大鼠与其S和R亲本的回交研究，我们得出结论：F_2大鼠SS基因型的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 alpha_2-AR 基因本身，而是由于与 _2-AR 密切相关的其他基因的影响。<2>自发性高血压大鼠 (SHR) 和 Wister-Kyoto 大鼠 (WKY) 基因组之间的 RFLP。佩廷格博士等。等人。使用人类α_2-C_<10>探针发现SHR和WKY基因组之间的RFLP。限制性图谱分析显示该RFLP位于该基因的5'启动子区域。基因共分离研究表明，该RFLP与SHRXWKY杂交中的部分血压升高共分离。(2)人α_2-AR基因的RFLP。佩廷格博士等人。报道了人类基因的 RFLP。我使用人类 alpha_2-C_<10> 探针（J of Hypertension）。使用相同的限制性内切酶和探针，我们在日语中发现了相同的 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)HT29、OK和NG108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。使用Nonhem印迹和核糖核酸酶保护测定来鉴定人结肠腺癌(HT29)、负鼠肾(OK)和神经母细胞瘤x神经胶质瘤大鼠-小鼠杂交NG10815(NG108)细胞系中的α_2-AR亚型。我们得出结论，α_2-C_4基因与α_<2B>药理学α_2-肾上腺素受体亚型相关，并且在OK和NG108细胞中活跃转录。 α_2-C_<10>基因与α_2A>药理学亚型相关，并在HT29细胞中活跃转录。2．由培养的大鼠IMCD细胞表达的哌唑嗪敏感的α 2B -肾上腺素受体的表征。使用放射性配体结合研究、Nonhem 印迹分析和 cAMP 测定，研究了培养的大鼠内髓集合管 (IMCD) 细胞中的 alpha_2-肾上腺素受体亚型表达。我们证明IMCD细胞表达肾上腺素受体的α_2B亚型并且这些受体的刺激抑制加压素刺激的cAMP形成。较少的

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。