Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland.
Am J Physiol Cell Physiol. 2013 Sep 15;305(6):C609-22. doi: 10.1152/ajpcell.00025.2013. Epub 2013 Jun 26.
We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.
我们使用穿孔膜片钳技术在 37°C 下研究了兔尿道平滑肌细胞中瞬态大电导钾 (BK) 电流激活的机制。BK 电流需要细胞内 Ca2+的升高，这是通过 Ryanodine 受体 (RyR) 的激活引起的，而 RyR 的激活是由通过 L 型 Ca2+ (CaV) 通道的 Ca2+内流引起的 Ca2+释放触发的。卡巴胆碱通过减少 Ca2+内流和 Ca2+释放来抑制 BK 电流，并改变在电流钳条件下记录的尖峰复合体的形状。BK 电流被iberiotoxin 和 penitrem A（分别为 300 和 100 nM）阻断，当去除外 Ca2+或应用 CaV 通道抑制剂硝苯地平（10 μM）和 Cd2+（100 μM）时，BK 电流也被抑制。BK 电流被咖啡因（10 mM）、Ryanodine（30 μM）和 Tetracaine（100 μM）抑制，这表明 RyR 介导的 Ca2+释放有助于 BK 电流的激活。当用 2-氨基乙氧基二苯硼酸盐（2-APB，100 μM）阻断 IP3 受体（IP3Rs）时，BK 电流的幅度没有降低。然而，当用苯肾上腺素（1 μM）或卡巴胆碱（1 μM）引起 IP3R 介导的 Ca2+释放时，BK 电流被抑制。当用 2-APB 阻断 IP3Rs 或用 M3 受体拮抗剂 4-二苯乙酰氧基-N-甲基哌啶甲碘化物（1 μM）抑制毒蕈碱受体时，卡巴胆碱的作用被消除。在电流钳条件下，用去极化电流注射可以引发动作电位爆发。卡巴胆碱减少每个爆发中的动作电位的数量和幅度，并且在存在 2-APB 时，这些效应会降低。在 Ryanodine 存在下，动作电位的数量和幅度也会降低，卡巴胆碱也没有进一步的作用。这些数据表明，产生 IP3 的激动剂可以调节兔尿道平滑肌细胞的电活动，并可能有助于神经递质对尿道张力的影响。
