Levi A J, Lee C O, Brooksby P
Department of Physiology, University of Bristol, United Kingdom.
J Cardiovasc Electrophysiol. 1994 Mar;5(3):241-57. doi: 10.1111/j.1540-8167.1994.tb01161.x.
Although some properties of the fluorescent sodium indicator "SBFI" are known, there is no accepted method by which the SBFI signal might be calibrated for intracellular Na (Nai) in cardiac cells. The first aim of this study was to characterize the loading and compartmentalization of this indicator in single cardiac myocytes. The second aim was, from experimental observation, to develop a rational calibration method for SBFI. The third aim was to use this Na indicator to study the dependence of tonic contraction on Nai in voltage-clamped ventricular myocytes.
SBFI was incorporated into myocytes by incubating with the acetoxymethyl ester (10 microM) for 2 hours. This led to an intracellular concentration of SBFI free acid of 122 +/- 17 microM. We considered a number of issues with respect to compartmentalization of indicator and, under our conditions, we found the majority (71%) of indicator was situated in the cytoplasm. Therefore, SBFI indicates mainly changes of cytoplasmic Na. Calibration of the indicator for Nai was performed by equilibrating internal and external Na. We investigated the conditions required for optimum transmembrane Na equilibration and found it necessary to use a calibration solution free of both Ca and magnesium (Mg). The Na ionophores gramicidin D and monensin were both required, and it was also necessary to inhibit the Na/K pump for optimum Na equilibration. Using these conditions, the Nai concentration in quiescent rat ventricular myocytes was 10.9 +/- 0.74 mM (mean +/- SEM, n = 40; equivalent to an Na activity of 8.3 mM). The concentration of Nai was significantly lower in quiescent rabbit myocytes: 3.8 +/- 0.23 mM (n = 24; equivalent to an Na activity of 2.9 mM). In voltage-clamped rabbit myocytes, inhibition of the Na/K pump caused a rise of Nai; there were also marked effects on the tonic shortening elicited by ramp depolarizations. As Nai rose, the magnitude of tonic shortening increased and its voltage dependence also changed.
These results confirm the suitability of SBFI for measuring Nai in cardiac cells. Provided that steps are taken to optimize transmembrane Na equilibration, the indicator can be calibrated for Nai. The different Nai of rat and rabbit myocytes has implications for the function of sarcolemmal Na/Ca exchange in each cell type. An Nai of 10.9 mM in rat myocytes gives a calculated reversal potential for the exchange of -35 mV. In comparison, an Nai of 3.8 mM in rabbit myocytes leads to a reversal potential for the exchange +45 mV. Therefore, relatively small changes of Nai can shift the reversal potential of the exchange to values that are substantially more positive or negative than zero. The behavior of voltage-dependent tonic contraction in rabbit myocytes was consistent with the Na/Ca exchange reversal potential being more positive than zero in these cells.
尽管荧光钠指示剂“SBFI”的一些特性已为人所知，但尚无公认的方法可对心肌细胞内的钠（Nai）进行SBFI信号校准。本研究的首要目的是表征该指示剂在单个心肌细胞中的负载和区室化情况。第二个目的是根据实验观察结果，开发一种合理的SBFI校准方法。第三个目的是使用这种钠指示剂研究电压钳制的心室肌细胞中张力收缩对Nai的依赖性。
通过与乙酰氧基甲酯（10 microM）孵育2小时，将SBFI掺入心肌细胞。这导致细胞内游离酸形式的SBFI浓度为122±17 microM。我们考虑了与指示剂区室化相关的一些问题，在我们的实验条件下，发现大部分（71%）指示剂位于细胞质中。因此，SBFI主要指示细胞质钠的变化。通过平衡细胞内和细胞外的钠来对指示剂进行Nai校准。我们研究了实现最佳跨膜钠平衡所需的条件，发现有必要使用不含钙和镁（Mg）的校准溶液。钠离子载体短杆菌肽D和莫能菌素均为必需，并且为了实现最佳钠平衡，抑制钠钾泵也很有必要。使用这些条件，静息大鼠心室肌细胞中的Nai浓度为10.9±0.74 mM（平均值±标准误，n = 40；相当于钠活性为8.3 mM）。静息兔心肌细胞中的Nai浓度显著较低：3.8±0.23 mM（n = 24；相当于钠活性为2.9 mM）。在电压钳制的兔心肌细胞中，抑制钠钾泵导致Nai升高；对斜坡去极化引发的张力缩短也有显著影响。随着Nai升高，张力缩短的幅度增加，其电压依赖性也发生变化。
这些结果证实了SBFI适用于测量心肌细胞中的Nai。只要采取措施优化跨膜钠平衡，就可以对该指示剂进行Nai校准。大鼠和兔心肌细胞中不同的Nai水平对每种细胞类型的肌膜钠钙交换功能有影响。大鼠心肌细胞中10.9 mM的Nai计算得出交换的反转电位为-35 mV。相比之下，兔心肌细胞中3.8 mM的Nai导致交换的反转电位为+45 mV。因此，Nai的相对较小变化可能会使交换的反转电位向比零更正向或更负向的值偏移。兔心肌细胞中电压依赖性张力收缩的行为与这些细胞中钠钙交换反转电位大于零一致。
