【摘要】 目的:研究α₁-AR三种亚型对细胞增殖和Ca²⁺-钙调蛋白依赖性蛋白激酶（CCDPK）的作用。方法:采用磷酸钙沉淀法进行转染,用放射配基结合实验测定α₁-AR表达量。用[³H]胸腺嘧啶参入量测定细胞增殖,用免疫沉淀和髓鞘蛋白底物法测定CCDPK的活性。结果:三株表达α_1A-,α_1B-和α_1D-AR的细胞株表达受体密度约为0.6 nmol·g^-1。在普萘洛尔存在下,去甲肾上腺素（NE）作用24 h可浓度依赖地刺激HEK293/α_1A-AR和HEK293/α_1B-AR细胞DNA合成。NE 10 μmol·L^-1可促进HEK293/α_1A-AR和HEK293/α_1B-AR细胞DNA合成增加,刺激CCDPK活性的升高。NE不引起HEK293/α_1D-AR细胞DNA合成和CCDPK活性的显著改变。结论:在转染α₁-AR亚型的HEK293细胞中激动α_1A-或α_1B-AR可引起细胞增殖,激动α_1D-AR则无显著改变。更多还原

【Abstract】 AIM: To examine the ability of α1-AR subtypes on proliferation and Ca2 + -calmodulin dependent protein ki-nase(CCDPK, formerly called MAPK) activation in transfected human embryo kidney 293 (HEK293) cells. METHODS: pREP8/α1A-AR, pREP4/α1B-AR, and pREP9/α1D-AR were transfected, respectively, into HEK293 cells by calcium phosphate precipitation. The expression of α1-AR was detected by radioligand binding assays. DNA synthesis was measured by [3H]thymidine incorporation. CCDPK activity was determined by immunoprecipitation method and myelin basic protein was used as substrate. RESULTS: Three clonal HEK293 cell lines stably expressing α1A-or α1B- or α1D-AR were chosen and characterized by radioligand binding assay with receptor densities of about 0.6 nmol·g-1. Treatment with norepinephrine (NE) in the presence of propranolol for 24 h increased DNA synthesis in HEK293/α1A- or HEK293/α1B-AR cells concentration-dependently, with EC50 values of 48.8 nmol·L-1 (95 % confidence limits 9.7-246 nmol·L-1) and 8.4 nmol·L-1 (95 % confidence limits 2.1 - 32.9 nmol·L-1), respectively. The increase of DNA synthesis induced by NE 10 μmol·L-1 was 201 %±28 % and 269 %±44 % of basal, and the activation of CCDPK was 171 %±84 % and 292 %±92 % of basal in HEK293/α1A-AR and HEK293/α1B-AR cells, respectively. Preincubation with prazosin completely abolished NE-induced CCDPK activation in HEK293/α1A- and α1B-AR cells. Those changes were not found in HEK293/α1D-AR cells. CONCLUSION: The activation of α1A- orα1B-AR but not α1D-AR induces cell proliferation.更多还原