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

1. α _2肾上腺素能受体（alpha_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限制性内切片段长度多态性（RFLP）。达尔盐敏感大鼠(S)和达尔盐抗性大鼠(R)基因组间RFLP < 1 >。我们利用人肾cDNA alpha_2-AR探针（alpha_2-C_4）和Msp I限制性内切酶发现了Dahl S和R大鼠之间的RFLP。该探针标记出一个3.0 Kb的S等位基因和一个2.8 Kb的R等位基因。通过对F_2大鼠进行R雌性与S雄性杂交，并对F_2大鼠进行兄弟姐妹交配，研究RFLP分布频率。F_2群体的alpha_2-AR-AR基因型分布不符合孟德尔分离比。通过对F_1大鼠与S、R亲本回交的研究，我们认为F_2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，而是由于与α _2-AR密切相关的其他基因的影响。自旋性高血压大鼠（SHR）和Wister-Kyoto大鼠（WKY）基因组间RFLP <2 b>。Dr. Pettinger等使用human alpha_2-C_<10>探针发现SHR和WKY基因组之间存在RFLP。限制性位图分析表明，该RFLP位于该基因的5'启动子区。遗传共分离研究表明，该RFLP与SHRXWKY杂交中部分血压升高存在共分离。(2)人α _2- ar基因的RFLP。Dr. Pettinger等人报道了人类基因的RFLP。我使用人类alpha_2-C_<10>探针（高血压新闻J）。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RFLP和血压之间是否存在任何关系。(3) HT29、OK和NG108细胞系中α _2-肾上腺素能受体亚型的特征；Northern blot和核糖核酸酶保护研究。采用Nonhem blot和核糖核酸酶保护实验鉴定人结肠腺癌（HT29）、负鼠肾（OK）和神经母细胞瘤×胶质瘤大鼠-小鼠杂交NGlO815 （NG108）细胞系中α _2- ar亚型。我们得出结论，alpha_2-C_4基因与α _<2B>药理alpha_2-肾上腺素受体亚型相关，并在OK和NG108细胞中活跃转录。alpha_2-C_<10>基因与alpha_<2A>药理亚型相关，在HT29细胞中活跃转录。培养的大鼠IMCD细胞表达普拉唑嗪敏感的α _<2B>-肾上腺素受体的特性。采用放射配体结合研究、Nonhem印迹分析和cAMP法研究α _2-肾上腺素能受体亚型在培养大鼠髓内集管（IMCD）细胞中的表达。我们证明了IMCD细胞表达α _<2B>亚型的肾上腺素能受体，刺激这些受体可抑制抗利雅得激素介导的cAMP形成。少

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