肺动脉平滑肌细胞钾电流记录方法的建立

Establishment of recording potassium channel currents in pulmonary arterial smooth muscle cells

目的在肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)上建立钙离子激活钾通道(calcium-activated potassium channel,KCa)电流、电压门控钾通道(voltage-gated potassium channel,KV)电流和内向整流钾通道(Inward rectifier channel,Kir)电流的记录方法。方法用急性酶解的方法分离出单个大鼠PASMC,利用全细胞膜片钳方法记录钾电流。结果①急性酶解分离得到高质量的单个大鼠PASMC,呈梭形,边界清楚,胞浆均匀透亮。②KV电流可以被5mmol/L4-AP明显抑制,使电流-电压关系曲线明显下移,在55mV时,KV电流密度从(133.86±7.36)pA/pF减少到(59.09±3.35)pA/pF(n=6,P<0.05)。③1mmol/L TEA对KCa电流有明显抑制作用,使电流-电压关系曲线明显下移,在55mV时,KCa电流密度从(9.03±1.42)pA/pF减少到(2.12±0.52)pA/pF(n=7,P<0.05)。④KATP电流可以被10μmol/L尼可地尔激活,使电流-电压关系曲线明显上移,在50mV时,Kv电流密度从(29.08±5.90)pA/pF增加到(88.90±7.98)pA/pF(n=10,P<0.05)。结论在肺动脉平滑肌细胞上成功地建立了KCa、KV和Kir电流的记录方法,可以为肺动脉相关疾病的发病机制和治疗方案的探索,提供有效的细胞模型基础。

Objectives To establish the methods for recording calcium-activated potassium channel ( KCa) , voltage-gated potassium channel (KV) and inward rectifier channel (Kir) in pulmonary arterial smooth muscle cells (PASMC). Methods PASMC was acutely isolated by enzyme. Potassium currents were recorded by whole-cell patch- clamp technique. Results (1) High-quality single PASMCs were obtained. A typical single cell was fusiform shape and its cytoplasm was uniform and lucent. (2) KV currents were inhibited by 5 mmol / L 4-AP, Ⅰ-Ⅴ relation curve was shifted downward, KV current density were reduced from ( 133.86 ±7.36) pA / pF to ( 59.09 ±3.35) pA / pF (n=6, P<0.05) at 55 mV. (3) KCa currents were inhibited by 1 mmol/ L TEA. Ⅰ-Ⅴ relation curve was shifted downward. KV current density were reduced from (9.03±1.42) pA / pF to (2.12±0.52) pA / pF (n=7, P<0.05) at 55 mV. (4) KATP currents were activated by 10 μmol / L nicorandil. I-V relation curve was shifted upward. KV current density were increased from (29.08±5.90) pA / pF to (88.90±7.98) pA / pF (n=10, P<0.05) at 50 mV. Conclusions KCa, KV and Kir currents were recorded successfully in rat single PASMC, which offers an effective cell model to explore the pathogenesis of pulmonary artery diseases.