酪氨酸激酶抑制剂对hTrp3蛋白介导的α_(1B)-AR引起的Ca~(2+)内流的影响

目的　探讨Trp3(transientreceptorpotential 3)蛋白是否参与α1B AR引起的Ca2 + 内流以及酪氨酸激酶对其调控作用。方法　采用脂质体转染 ,将hTrp3cDNA分别转染到HEK2 93细胞和已有α1B受体稳定表达的HEK2 93细胞 ;Westernblot方法检测Trp3蛋白表达情况 ;Fura 2 /AM荧光分光光度法 ,测定胞浆游离Ca2 + 浓度。结果　HEK2 93细胞上可检测到hTrp3的内源性表达 ,转染后其表达增加。α1B HEK2 93细胞转染hTrp3cDNA后 ,α1B AR引起的Ca2 +内流显著增加 (P <0 0 1) ;转染hTrp3cDNA对thapsigargin诱导的Ca2 + 内流无作用。 5～ 30 μmol·L-1genistein对转染细胞α1B AR诱发的Ca2 + 内流有抑制作用 ,最大抑制率达(75 2± 12 6 ) %。结论　Trp3cDNA转染可能主要通过非CRAC(calciumreleaseactivatedcalcium )途径增加α1B AR引起的Ca2 + 内流 ,这一过程很大程度上依赖酪氨酸激酶的调控

Effects of tyrosine kinase inhibitor on α1B-AR-induced Ca2+ influx involved in hTrp3 protein

AIM To investigate the role of Trp3 in the Ca 2+ influx induced by α 1B AR in HEK293 cells and the effect of tyrosine kinase on it. METHODS With lipofect AMINE2000 reagent, hTrp3 cDNA was transfected to HEK293 cells and α 1B HEK293 cells respectively. The expression of Trp3 was examined by Western blot. With Fura 2/AM spectrophoto fluorometry, Ca 2+ influx was determined. RESULTS HTrp3 was expressed endogenously in HEK293 cells, and the expression increased in hTrp3 transfected cells. Compared with untransfected cells, transfection of hTrp3 cDNA increased Ca 2+ influx induced by α 1B AR ( P< 0 01) without any change in thapsigargin induced Ca 2+ influx ( P> 0 05). 5～30 μmol·L -1 genistein inhibited Ca 2+ influx induced by α 1B AR in hTrp3 cDNA transfected cells and the maximum inhibitory rate was (75 2±12 6)% . CONCLUSION Transfection of hTrp3 cDNA increased Ca 2+ influx induced by α 1B AR in HEK293 cells. This process was regulated by tyrosine kinase.