内源性一氧化碳调节肺动脉平滑肌细胞增殖的信号转导

Signal transduction of endogenous carbon monoxide-mediated rat pulmonary artery smooth muscle cells proliferation by hypoxia

目的 探讨内源性一氧化碳 (CO)抑制肺动脉平滑肌细胞 (PASMC)增殖的细胞内信号转导机制。方法 培养的大鼠PASMC低氧 2 4h ,采用3 H 胸腺嘧啶 (3 H TdR)参入法检测DNA合成率 ;Western Blot检测细胞外信号调节激酶(ERK)的蛋白表达。结果 低氧使PASMC的3 H TdR参入增加 137 2 8% (P <0 0 1) ;血红素氧合酶 (HO)诱导剂氯化血红素 (Hemin)以浓度依赖的方式抑制低氧PASMC的3 H TdR参入 ,分别降低 19 14 %、2 9 94 %、4 3 97% (P均 <0 0 1) ;HO抑制剂锌原卟啉 (ZnPP)促使低氧PASMC的3 H TdR参入增加 37 89% (P <0 0 1) ;MEK1抑制剂PD980 5 9降低了低氧和低氧 +ZnPP组PASMC的 3 H TdR参入34 88%和 2 3 94 % (P均 <0 0 1)。低氧使PASMC的ERK蛋白表达增加 4 0 9% ;Hemin以浓度依赖的方式抑制低氧PASMC的ERK表达 ,分别降低 33 99%、4 5 97%、6 6 0 1%(P均 <0 0 1) ;ZnPP促使低氧PASMC的ERK表达增加5 7 76 % ,(P <0 0 1) ,PD980 5 9降低了低氧和低氧 +ZnPP组PASMC的ERK表达 34 88%和 2 4 78% (P均 <0 0 1)。

AIM To investigate effect of endogenous carbon monoxide on proliferation of pulmonary artery smooth muscle cells (PASMCs) of rats under hypoxia and study its signal transduction pathway. METHODS In cultured PASMCs of rat, the proliferation of these cells were stimulated by hypoxic treatment for 24 hours. The levels of 3H TdR incorporation were measured to evaluate the speed of DNA synthesis. Western Blot was applied to assay the expression of extracellular signal regulated protein kinase (ERK). RESULTS The levels of 3H TdR incorporation in hypoxic rats PASMCs elevated markedly compared to control ( P <0 01). Hemin, an inducer of activity of heme oxygenase (HO) caused a concentration dependent decrease in 3H TdR incorporation in hypoxic rat PASMCs. 3H TdR incorporation of 10, 20, 40 μmol·L -1 Hemin were 19 14%( P< 0 01), 29 94%( P< 0 01) and 43 97%( P< 0 01) lower than that of hypoxic PASMCs. ZnPP, which is a potent inhibitor of HO activity, increased 37 89%( P< 0 01) 3H TdR incorporation in hypoxic rat PASMCs. PD98059, a selective inhibitor of MEK1, inhibited 3H TdR incorporation in hypoxic rats and ZnPP treated hypoxic rat PASMCs by 34 88%( P< 0 01)and 23 94%( P< 0 01), respectively. The expression of ERK protein in hypoxic PASMCs was 409% higher than control. Hemin caused a concentration dependent inhibition of expression of ERK in hypoxic rat PASMCs. The expression of ERK protein of 10, 20, 40 μmol·L -1 Hemin were 33 99%( P< 0 01), 45 97%( P< 0 01) and 66 01%( P< 0 01) lower than hypoxia PASMCs. ZnPP increased 57 76%( P< 0 01)expression of ERK protein in hypoxic rat PASMCs. PD98059 inhibited expression of ERK protein in hypoxic rats and ZnPP treated hypoxic rat PASMCs by 34 88%( P< 0 01) and 24 78%( P< 0 01), respectively. CONCLUSION Endogenous CO may mediate proliferation of PASMC via MAPK pathway.