I1咪唑啉受体对α2A肾上腺素能受体表达和功能的影响

摘要

Objective To investigate the effect of I1imidazoline receptor(I1R)on the expression and function of α2Aadrener-gic receptor(α2AAR)at the cellular level.Methods After sequencing and enzymatic identification,the mouse I1R and α2AAR plas-mids were transfected into CHO cells,respectively.The radioligand receptor binding assay and flow cytometry were used to select sin-gle cell clones,and the CHO cell lines stably expressing the mouse I1R or α2AAR were established.The CHO cell line that stably ex-presses both the mouse I1R and α2AAR were also established by the same technology and strategy.Then,the radioligand receptor bind-ing assay was used to determine the affinity and expression of α2AAR. Further,the effects of I1R on the α2AAR expression and the α2AAR agonist dexmedetomidine(DEX)-induced extracellular regulated protein kinase(ERK)phosphorylation were evaluated by the Western blotting.Results After transfection of mouse I1R and α2AAR plasmids,CHO cells grew normally.In the saturation binding experiments of membrane proteins from the CHO cells that stably expressed α2AAR,the Kdand Bmaxvalues of 3H-RX821002 were(0.96 ± 0.24)nmol/L and(0.29 ± 0.03)nmol/g protein,respectively.The expression levels of I1R were significantly increased in both the CHO cells expressing I1R and the CHO cells co-expressing α2AAR and I1R(P<0.05,P<0.01),when the cells that express exogenous I1R or α2AAR alone were trasfected again with the I1R plasmids.Moreover,in the CHO cells that transfected both I1R and α2AAR sta-bly,the I1R expression upregulated the α2AAR expression(P<0.01),and further increased the ERK phosphorylation induced by DEX through activating α2AAR(P<0.01).Conclusion I1R could upregulate the α2AAR expression and the ERK phosphorylation in-duced by DEX through activating α2AAR in the CHO cells that express exogenous I1R and α2AAR.This study presents a groundwork for further exploration of the relationship between I1R and α2AAR at the molecular level in future.%目的 在细胞水平研究I1咪唑啉受体(I1R)对α2A肾上腺素能受体(α2AAR)表达及功能的影响.方法 在完成小鼠I1R基因和α2AAR基因的质粒测序及酶切鉴定后,分别转染CHO细胞,采用放射性配体-受体结合实验、流式细胞分选技术进行阳性细胞单克隆筛选,建立分别稳定表达I1R和α2AAR的CHO细胞系,以及共同稳定表达I1R与α2AAR的CHO细胞系.采用放射性配体-受体结合实验确定α2AAR的亲和力及表达量,用Western印迹法研究I1R对α2AAR表达量的影响和对α2AAR激动剂右美托咪定刺激细胞外调节蛋白激酶(ERK)磷酸化作用的影响.结果 在转染小鼠I1R和α2AAR基因质粒后,CHO细胞生长正常.在为稳定表达小鼠α2AAR的CHO细胞膜蛋白所做的放射性配体饱和实验中,3H-RX821002的Kd和Bmax值分别为(0.96± 0.24)nmol/L和(0.29±0.03)nmol/g蛋白.在稳定表达小鼠I1R的CHO细胞以及稳定表达小鼠α2AAR的CHO细胞中再分别转染I1R基因后,I1R蛋白表达水平均明显上升(P<0.05,P<0.01).在共同稳定转染I1R和α2AAR的CHO细胞系中,I1R的表达上调了CHO细胞中α2AAR的表达水平(P<0.01),同时也进一步上调了右美托咪定激活α2AAR引起的ERK磷酸化水平(P<0.01).结论 在外源表达的细胞系中,I1R上调α2AAR蛋白的表达和右美托咪定激活α2AAR后的ERK磷酸化水平.本研究为后续分子水平深入探索I1R和α2AAR之间的相互作用奠定了基础.