Miller D J, Smith G L
J Muscle Res Cell Motil. 1985 Oct;6(5):541-67. doi: 10.1007/BF00711914.
Tension responses of rat ventricular trabeculae subjected to successive 'treatment' with EGTA and Triton X-100 are described in order to investigate the effects of chemical 'skinning' techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the 'EGTA-treatment' fails to render cells 'hyperpermeable', i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the 'EGTA-treated' tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of 'hyperpermeability'. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after 'EGTA-treatment' alone. This applies to contractures evoked direct from Ca2+ concentration congruent to 10(-9) M to the test Ca2+ concentration at constant total buffer concentration. 'EGTA-treated' fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24 h produce preparations showing similar behaviour: whatever the explanation for the EGTA-'skinning' phenomenon it is not dependent upon low bathing Ca2+ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after 'EGTA-treatment': this treatment does not produce a model of a selectively 'skinned' cardiac cell.
为了研究化学“去皮”技术的效果，本文描述了大鼠心室小梁在先后用乙二醇双四乙酸（EGTA）和曲拉通X-100“处理”后的张力反应。在一些标本中，在使用曲拉通之前还使用了生物碱皂苷。引用了超微结构证据表明，“EGTA处理”未能使细胞“超通透”，即对小离子自由通透，而皂苷和曲拉通都能做到。在本文中，我们表明可以获得与先前描述的“EGTA处理”组织类似的收缩反应。然而，更详细的检查表明，这种行为在数量上与传统去皮纤维不同，其方式与“超通透”的概念不相容。在我们的实验中，用EGTA、皂苷或曲拉通处理后的钙敏感性是相同的。然而，这并不能通过EGTA处理的标本中钙（和EGTA）自由进入细胞内空间来解释：挛缩的发展具有非常不同的时间进程，在曲拉通处理后最快，首次暴露于皂苷时仅略慢，但仅用“EGTA处理”后慢五倍。这适用于在恒定总缓冲液浓度下，从与10^(-9)M的Ca2+浓度一致的测试Ca2+浓度直接诱发的挛缩。当从浴槽中去除三磷酸腺苷（ATP）或磷酸肌酸（CrP）时，“EGTA处理”的纤维会产生张力。然而，在存在毫摩尔水平的ATP时，对二磷酸腺苷（ADP）和CrP变化的反应仍然存在，这与曲拉通去皮的肌肉完全不同。将各种溶液中的每一种暴露24小时会产生表现出类似行为的标本：无论对EGTA“去皮”现象的解释是什么，它都不依赖于低浴钙浓度。基于这里描述的功能特性以及引用的结构结果，我们得出结论，在“EGTA处理”后细胞膜继续作为选择性通透屏障发挥作用：这种处理不会产生选择性“去皮”心肌细胞的模型。
