Mitcheson J S, Hancox J C, Levi A J
Department of Physiology University of Bristol, School of Medical Sciences, UK.
Pflugers Arch. 1997 Apr;433(6):817-26. doi: 10.1007/s004240050350.
We used the whole-cell patch-clamp technique and monitoring of Fura-2 fluorescence to investigate the voltage dependence of the L-type Ca current (ICa,L) and intracellular Ca (Cai) transient in rabbit atrial myocytes at 37 degrees C. Imaging the atrial cell membrane with Di-4-ANNEPS showed (in contrast to ventricular cells) that atrial cells had very few transverse tubules. We measured ICa,L using a Cs-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak ICa,L amplitude was bell-shaped: ICa,L was maximal at +10 mV and declined at more negative and positive potentials. For measuring the Fura-2 (Cai) transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. Ryanodine (20 microM) plus thapsigargin (2 microM) (blockers of the sarcoplasmic reticulum, SR) abolished the phasic component of the Fura-2 transient (n = 5), demonstrating that the phasic Fura-2 transient provided an index of the magnitude of SR release. The Fura-2 transient also showed bell-shaped voltage dependence, but this was different from that for ICa,L. The Fura-2 transient peaked at +30 mV and partially declined at more positive potentials; but at potentials where inward ICa,L was small (if not absent), the phasic Fura-2 transient still attained a significant amplitude. We used a rapid application of nifedipine (32 microM), and of nifedipine plus 5 mM Ni, to assess the ability of ICa,L and reverse-mode Na-Ca exchange to trigger SR Ca release. With test pulses to +10 mV and +60 mV, a rapid switch to nifedipine (which blocked ICa,L) produced no significant reduction in phasic Fura-2 transient amplitude. This suggests that in the absence of ICa,L, another mechanism was able to trigger SR release. With pulses to +10 and +60 mV, a single beat switch to nifedipine plus 5 mM Ni almost completely abolished the phasic transient. Since 5 mM Ni inhibits Na-Ca exchange, this suggests that, in the absence of ICa,L, trigger Ca entry via reverse Na-Ca exchange was able to activate SR Ca release in atrial cells at 37 degrees C. The mechanisms underlying the Fura-2 transient in atrial cells, and differences with pre-existing data from rabbit ventricular cells, are discussed.
我们采用全细胞膜片钳技术并监测Fura-2荧光，以研究37℃下兔心房肌细胞中L型钙电流（ICa,L）和细胞内钙（Cai）瞬变的电压依赖性。用Di-4-ANNEPS对心房细胞膜成像显示（与心室细胞不同），心房细胞的横管很少。我们使用基于铯的内部透析液测量ICa,L，以消除干扰性钾电流。峰值ICa,L幅度的电压依赖性呈钟形：ICa,L在+10 mV时最大，在更负和更正的电位时下降。为了测量Fura-2（Cai）瞬变，我们使用基于钾的内部透析液来维持正常的兴奋-收缩偶联。Ryanodine（20 microM）加毒胡萝卜素（2 microM）（肌浆网，SR的阻滞剂）消除了Fura-2瞬变的相位成分（n = 5），表明相位Fura-2瞬变提供了SR释放量的指标。Fura-2瞬变也显示出钟形电压依赖性，但这与ICa,L的不同。Fura-2瞬变在+30 mV时达到峰值，在更正的电位时部分下降；但在内向ICa,L较小（如果不是不存在）的电位下，相位Fura-2瞬变仍达到显著幅度。我们快速应用硝苯地平（3 microM）以及硝苯地平加5 mM镍，以评估ICa,L和反向模式钠-钙交换触发SR钙释放的能力。用测试脉冲至+10 mV和+60 mV，快速切换至硝苯地平（阻断ICa,L）不会使相位Fura-2瞬变幅度显著降低。这表明在没有ICa,L的情况下，另一种机制能够触发SR释放。用脉冲至+10和+60 mV，单次心跳切换至硝苯地平加5 mM镍几乎完全消除了相位瞬变。由于5 mM镍抑制钠-钙交换，这表明在没有ICa,L的情况下，通过反向钠-钙交换的触发钙内流能够在37℃下激活心房细胞中的SR钙释放。讨论了心房细胞中Fura-2瞬变的潜在机制以及与兔心室细胞现有数据的差异。
