Hardy M E L, Lawrence C L, Standen N B, Rodrigo G C
Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
J Pharmacol Toxicol Methods. 2006 Sep-Oct;54(2):173-82. doi: 10.1016/j.vascn.2006.02.013. Epub 2006 Mar 6.
Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology.
We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte.
Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM).
These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.
电位敏感染料主要用于光学记录全心脏组织中的动作电位（AP）。使用这些染料记录药物引起的离体心肌细胞AP形态变化，可为制药行业开发全细胞检测方法提供契机。理想情况下，这要求染料对膜电位具有一致且快速的响应，对细胞运动不敏感，并且本身不影响AP形态。
我们在单激发双发射比率系统中使用di-8-ANEPPS对成年豚鼠离体心室肌细胞的AP进行光学记录，记录方式既可以是在电场刺激的细胞中单独记录，也可以是在全细胞桥接模式下与用膜片电极记录的电AP同时进行记录。使用开关钳将心肌细胞钳制到电压钳状态，根据膜电位对di-8-ANEPPS荧光信号的比率进行校准。
我们的数据表明，在AP的电压范围内，560/620nm处发射的光信号比率与电压呈线性关系，每100mV比率变化7.5%，不受细胞运动影响，并且与用膜片电极同时记录的AP相同。然而，用di-8-ANEPPS加载的心肌细胞中光学记录的APD90明显长于用膜片电极记录的未加载心肌细胞中的APD90（355.6±13.5对296.2±16.2毫秒；p<0.01）。尽管有这种影响，但通过阻断IKr来延长AP的西沙必利的表观IC50，无论是通过光学方法还是用膜片电极测定，均无显著差异（91±46对81±20nM）。
这些数据表明，使用di-8-ANEPPS从单个心室肌细胞以比率方式光学记录的AP能准确跟踪动作电位形态。尽管di-8-ANEPPS本身会延长APD90，但该技术可用于评估化合物对AP的延长作用。光学染料所需的技术技能较少，与传统电生理技术相比侵入性较小，并且与心室肌细胞结合使用时，可减少动物使用量并便于进行更高通量的检测。
