I_1咪唑啉受体对α_(2A)肾上腺素能受体表达和功能的影响

Effect of I_1 imidazoline receptor on the expression and function of α_(2A) adrenergic receptor

目的在细胞水平研究I_1咪唑啉受体(I_1R)对α_(2A)肾上腺素能受体(α_(2A)AR)表达及功能的影响。方法在完成小鼠I_1R基因和α_(2A)AR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I_1R和α_(2A)AR的CHO细胞系,以及共同稳定表达I_1R与α_(2A)AR的CHO细胞系。采用放射性配体-受体结合实验确定α_(2A)AR的亲和力及表达量,用Western印迹法研究I_1R对α_(2A)AR表达量的影响和对α_(2A)AR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I_1R和α_(2A)AR基因质粒后,CHO细胞生长正常。在为稳定表达小鼠α_(2A)AR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I_1R的CHO细胞以及稳定表达小鼠α_(2A)AR的CHO细胞中再分别转染I_1R基因后,I_1R蛋白表达水平均明显上升(P<0.05,P<0.01)。在共同稳定转染I_1R和α_(2A)AR的CHO细胞系中,I_1R的表达上调了CHO细胞中α_(2A)AR的表达水平(P<0.01),同时也进一步上调了右美托咪定激活α_(2A)AR引起的ERK磷酸化水平(P<0.01)。结论在外源表达的细胞系中,I_1R上调α_(2A)AR蛋白的表达和右美托咪定激活α_(2A)AR后的ERK磷酸化水平。本研究为后续分子水平深入探索I_1R和α_(2A)AR之间的相互作用奠定了基础。

Objective To investigate the effect of I1 imidazoline receptor（I1 R）on the expression and function of α2Aadrenergic receptor（α2AAR）at the cellular level. Methods After sequencing and enzymatic identification,the mouse I1 R and α2AAR plasmids were transfected into CHO cells,respectively. The radioligand receptor binding assay and flow cytometry were used to select single cell clones,and the CHO cell lines stably expressing the mouse I1 R or α2AAR were established. The CHO cell line that stably expresses both the mouse I1 R and α2AAR were also established by the same technology and strategy. Then,the radioligand receptor binding assay was used to determine the affinity and expression of α2AAR. Further,the effects of I1 R on the α2AAR expression and theα2AAR agonist dexmedetomidine（DEX）-induced extracellular regulated protein kinase（ERK）phosphorylation were evaluated by the Western blotting. Results After transfection of mouse I1 R 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 of3 H-RX821002 were（0.96± 0.24）nmol/L and（0.29 ± 0.03）nmol/g protein,respectively. The expression levels of I1 R were significantly increased in both the CHO cells expressing I1 R and the CHO cells co-expressing α2AAR and I1 R（P〈0.05,P〈0.01）,when the cells that express exogenous I1 R or α2AAR alone were trasfected again with the I1 R plasmids. Moreover,in the CHO cells that transfected both I1 R and α2AAR stably,the I1 R expression upregulated the α2AAR expression（P〈0.01）,and further increased the ERK phosphorylation induced by DEX through activating α2AAR（P〈0.01）. Conclusion I1 R could upregulate the α2AAR expression and the ERK phosphorylation induced by DEX through activating α2AAR in the CHO cells that express exogenous I1 R and α2AAR. This study presents a groundwork for further exploration of the relationship between I1 R and α2AAR at the molecular level in future.