The Role Of Protein Kinase Ca In Hypoxia-induced Rat Pulmonary Artery Smooth Muscle Cell Proliferation

Objective:To investigate the change of PKCa protein expression level in hypoxia-induced rat pulmonary artery smooth muscle cells, the effect of PKCa on the proliferation of rats PASMCs under hypoxic condition, and therefore to explore the role of PKCa in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation.Methods:(1) Rat PASMCs were primarily cultured by tissue-piece inoculation and the identity of PASMCs were verified by immunofluorescence staining using specific mouse monoclonal antibodies against smooth muscle cell alpha-actin.(2) PASMCs were exposed to hypoxia for 24h、48h or 72h respectively, then the proliferation of PASMCs was examined by MTT assay.(3) PASMCs were exposed to hypoxia for 24h、48h or 72h respectively, PKCa protein expression was detected by Western-blotting.(4) PASMCs were pretreated by PKC promoter PMA(0.4μM), PKCa special inhibitor Safingol(0.6μM), MEK-1 inhibitor PD98059(20μM) or MEK inhibitor U0126(5μM) for 30min respectively before exposed to hypoxia for 72h, then the proliferation of PASMCs was examined by MTT assay.(5)PASMCs were exposed to hypoxia for 24h,48h or 72h, or pretreated by PKC promoter PMA(0.4μM) or PKCα special inhibitor Safingol(0.6μM) for 30min respectively before exposed to hypoxia for 72h, ERK1/2 protein expression was detected by Western-blotting.Results:(1) The absorbance value in PASMCs exposed to hypoxia (3%O2) for different time (24h,48h or 72h) were (0.191±0.014,0.250±0.060,0.359±0.032 respectively), the absorbance value in PASMCs exposed to normoxia (21% O2) for different time (24h,48h or 72h) were (0.155±0.009,0.133±0.016,0.163±0.032 respectively). Compared with those of control group exposed to normoxia for different time, the absorbance value of hypoxia groups were significantly increased (p<0.05). Compared with PASMCs exposed to hypoxia for 24h, the absorbance value in PASMCs exposed to hypoxia for 72h was significantly increased (p<0.05).(2) PKCa protein expression in PASMCs exposed to hypoxia (3% O2) for different time (24h,48h or 72h) were (0.336±0.160,0.656±0.085 �'� 0.808±0.098 respectively) and were significantly increased compared with that of control group exposed to normoxia (21% O2) for 72h (0.290±0.060,p<0.05).(3) The absorbance value in PASMCs exposed to hypoxia (3% O2) for 72h (0.529±0.015) was significantly increased compared with that of control group (0.177±0.010) exposed to normoxia (21% O2) for 72h (p<0.05); the absorbance value in PASMCs pretreated by PKC promoter PMA (0.4μM) for 30min before exposed to hypoxia for 72h (0.587±0.044) was significantly increased compared with that of PASMCs exposed to hypoxia for 72h (p<0.05); the absorbance value in PASMCs pretreated by PKCa special inhibitor Safingol (0.6μM) for 30min before exposed to hypoxia for 72h (0.426±0.033) was significantly reduced compared with that of PASMCs exposed to hypoxia for 72h (p<0.05).(4) ERK1/2 protein expression in PASMCs exposed to hypoxia (3% O2) for different time (24h,48h or 72h) were (0.134±0.071,0.246±0.083 �'� 0.291±0.054 respectively). ERK1/2 protein expression in PASMCs exposed to hypoxia (3% O2) for 48h or 72h were significantly increased compared with that of control group exposed to normoxia (21% O2) for 72h (0.156±0.040,p<0.05).(5) The absorbance value in PASMCs exposed to hypoxia (3% O2) for 72h (0.529±0.015) was significantly increased compared with that of control group (0.177±0.010) exposed to normoxia (21% O2) for 72h (p<0.05); the absorbance value in PASMCs pretreated by MEK-1 inhibitor PD98059 (20μM) or MEK inhibitor U0126 (5μM) for 30min respectively before exposed to hypoxia for 72h were (0.489±0.011,0.306±0.004 respectively) and were significantly reduced compared with that of PASMCs exposed to hypoxia for 72h (p<0.05).(6) ERK1/2 protein expression in PASMCs pretreated by PKC promoter PMA (0.4μM) for 30min before exposed to hypoxia (3% O2) for 72h (0.558±0.039) was significantly increased compared with that of PASMCs exposed to hypoxia (3% O2) for 72h (0.432±0.047,p<0.05); ERK1/2 protein expression in PASMCs pretreated by PKCa special inhibitor Safingol (0.6μM) for 30min before exposed to hypoxia (3% O2) for 72h (0.311±0.082) was significantly reduced compared with that of PASMCs exposed to hypoxia (3% O2) for 72h p<0.05).Conclusion:(1) The method of tissue-piece inoculation of PASMCs was simple, and the cell condition was good, the survival rate was high.(2) The protein expression of PKCa was detected in rat PASMCs. PKCa was upregulated in hypoxic rat PASMCs in culture and involved in hypoxia-induced PASMCs proliferation.(3) PKCa participated in hypoxia-induced rat PASMCs proliferation via the ERK1/2 pathway in vitro.