Na/K pump-induced [Na]i gradients in rat ventricular myocytes measured with two-photon microscopy
Na/K pump-induced [Na]i gradients in rat ventricular myocytes measured with two-photon microscopy
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DOI：
10.1529/biophysj.103.037895
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发表时间：
2004-08-01
影响因子：
3.4
通讯作者：
Bers, DM
中科院分区：
文献类型：
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作者：
Despa, S;Kockskämper, J;Bers, DM
通过Na/Ca和Na/H交换，细胞内Na浓度（[Na]（i））在调节心脏Ca和收缩性中是重要的。功能数据表明，[Na]（i）可能是异质性的，在不稳定状态的心肌细胞，但很少有直接的空间信息。在这里，我们使用SBFI的双光子显微镜空间分辨[Na]（i）在大鼠心室肌细胞。体内校准产生的表观Kd为27 +/-2 mM Na。使用SBFI的双光子或单光子比率测量发现类似的静息[Na]（i）（10.8 +/-0.7 vs. 11.1 +/-0.7mM）。为了评估纵向[Na]（i）梯度，Na/K泵在肌细胞的一端被阻断（局部用移液管施加的无K细胞外溶液）并且在细胞的其余部分中是活跃的。这导致移液管下游部位的[Na]（i）显著增加（Na进入肌细胞，Na/K泵被阻断）。[Na]（i）上游站点的上升较小。这导致持续的[Na]（i）梯度（高达类似于17 mM/120 μ m细胞长度）。这意味着，钠扩散在心肌细胞是缓慢的跨肌膜钠转运率，虽然负责的机制尚不清楚。一个简单的扩散模型表明，这样的梯度需要的Na扩散系数为10 - 12 μ m（2）/s，显着低于在水溶液中。
Via the Na/Ca and Na/H exchange, intracellular Na concentration ([Na](i)) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na](i) might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na](i) in rat ventricular myocytes. In vivo calibration yielded an apparent K-d of 27 +/- 2 mM Na. Similar resting [Na](i) was found using two-photon or single-photon ratiometric measurements with SBFI (10.8 +/- 0.7 vs. 11.1 +/- 0.7mM). To assess longitudinal [Na](i) gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na](i) at sites downstream of the pipette ( where Na enters the myocyte and Na/K pumps are blocked). [Na](i) rise was smaller at upstream sites. This resulted in sustained [Na](i) gradients (up to similar to17 mM/120 mum cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10-12 mum(2)/s, significantly lower than in aqueous solutions.