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

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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