Decreasing the time gap between two identical electric pulses is expected to render bioeffects similar to those of a single pulse of equivalent total duration. In this study, we show that it is not necessarily true, and that the effects vary for different permeabilization markers. We exposed individual CHO or NG108 cells to one 300-ns pulse (3.7–11.6 kV/cm), or a pair of such pulses (0.4–1000 μs interval), or to a single 600-ns pulse of the same amplitude. Electropermeabilization was evaluated (a) by the uptake of YO-PRO-1 (YP) dye; (b) by the amplitude of elicited Ca²⁺ transients, and (c) by the entry of Tl⁺ ions. For YP uptake, applying a 600-ns pulse or a pair of 300-ns pulses doubled the effect of a single 300-ns pulse; this additive effect did not depend on the time interval between pulses or the electric field, indicating that already permeabilized cells are as susceptible to electropermeabilization as naïve cells. In contrast, Ca²⁺ transients and Tl⁺ uptake increased in a supra-additive fashion when two pulses were delivered instead of one. Paired pulses at 3.7 kV/cm with minimal separation (0.4 and 1 μs) elicited 50–100% larger Ca²⁺ transients than either a single 600-ns pulse or paired pulses with longer separation (10–1000 μs). This paradoxically high efficiency of the closest spaced pulses was emphasized when Ca²⁺ transients were elicited in a Ca²⁺-free solution (when the endoplasmic reticulum (ER) was the sole significant source of Ca²⁺), but was eliminated by Ca²⁺ depletion from the ER and was not observed for Tl⁺ entry through the electropermeabilized membrane. We conclude that closely spaced paired pulses specifically target ER, by either permeabilizing it to a greater extent than a single double-duration pulse thus causing more Ca²⁺ leak, or by amplifying Ca²⁺-induced Ca²⁺ release by an unknown mechanism.

减少两个相同电脉冲之间的时间间隔有望使生物效应类似于总持续时间相等的单个脉冲的生物效应。在这项研究中，我们表明它不一定是正确的，并且对于不同的通透性标记物，其效果也有所不同。我们将单个CHO或NG108细胞暴露于一个300 ns的脉冲（3.7-11.6 kV / cm）或一对这样的脉冲（0.4-1000μs的间隔），或暴露于相同幅度的单个600 ns的脉冲。（a）通过摄取YO-PRO-1（YP）染料来评估电通透性；（b）通过诱发的Ca ²⁺瞬变的幅度，以及（c）通过Tl ⁺的进入离子。对于YP吸收，施加600 ns脉冲或一对300 ns脉冲会使单个300 ns脉冲的效果加倍。这种加性效应不取决于脉冲之间的时间间隔或电场，这表明已经透化的细胞对电透化的敏感性与幼稚的细胞一样。相反，当递送两个脉冲而不是一个时，Ca ²⁺瞬态和Tl ⁺吸收以超加性方式增加。在3.7 kV / cm的最小间隔（0.4和1μs）下，成对脉冲比单个600 ns脉冲或间隔更长（10-1000μs）的成对脉冲引起的Ca ²⁺瞬变大50-100％。当Ca ²⁺在不含Ca ^2+的溶液中引发瞬变（当内质网（ER）是Ca ²⁺的唯一重要来源时），但被ER中的Ca ²⁺耗尽所消除，并且没有观察到Tl ⁺进入电透膜。我们的结论是，紧密隔开的成对脉冲专门针对ER，通过比单个双持续时间脉冲更大程度地透化ER，从而引起更多的Ca ²⁺泄漏，或者通过未知机制放大Ca ²⁺诱导的Ca ²⁺释放。 。