## 4 Discussion
Furthermore, the membrane potential, which indicates the electrical potential difference between the intracellular and extracellular space resulting from the transport of ions through channels and transporters [44; 45; 46], could not be changed due to ES, as the experiments with DiBAC\({}_{4}\)(3) showed. Otherwise, [PERSON] et al. were able to prove in their study that ES has an influence on the change in the ion gradient [47]. To further analyze membrane-related components under ES the expression of aquaporin channels were observed. We found that AQP 1 is strongly expressed in human MG-63 osteoblastic cells. The AQP 1 expression pattern is similar with cells from the _Nucleus Pulposus_ in the central region of the intervertebral disc, which is rich in proteoglycans [48]. [PERSON] et al. showed that AQP 1 and 5 expressions decreased when the intervertebraldisc was degenerated, indicating a role of AQPs in hyperosmotic regulation [48]. In our experiments, after electrical stimulation with 1 and 5 V at 20 Hz, most AQPs showed a trend towards increased expression. Since the AQPs 1, 3, 5, 7, 8, and 9 are involved in the transport of hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) [16], the assumption of a connection between the expression changes in the AQPs and the H\({}_{2}\)O\({}_{2}\) exposure is reasonable. Intracellular H\({}_{2}\)O\({}_{2}\) concentrations that are too high exceed the capacity of antioxidant enzymes and can have a damaging effect [49]. On the other hand, after low-to-moderate H\({}_{2}\)O\({}_{2}\) exposure, the expression of most AQPs increases to cope with the concentration balance. This conclusion is based on the fact that AQPs 3 and 5 have been shown to have a proliferation advantage under oxidative stress [50] and that physiological concentrations of hydrogen peroxide play an essential role in cellular signaling cascades. Therefore, physiological intracellular concentrations of H\({}_{2}\)O\({}_{2}\) are necessary for cellular balance [49; 50]. The influence of ES on internal cell structures was also demonstrated by flow cytometry and the evaluation of the FSC/SSC dot plot [51]. The forward scatter (FSC) is proportional to the size, and the side scatter (SSC) is proportional to the granularity of the cells [51]. Very few groups focused on ES and cell granularity [52; 53; 54]. In our short-time AC stimulation experiments, the granularity of MG-63 cells was increased after 10 min with 5 V and 20 Hz. Also, the authors [PERSON] et al. recognized increased cell granularity in diverse cell lines such as from epithelia, pancreas, and glioblastoma after applying tumor-treating fields (TTField) [55]. TTField is an antitumor treatment modality that applies 1-3 V/cm with kilohertz-frequency stimulation (100-300 kHz), which is nearly 10,000 times higher than we used. The cell granularity was not only shown by FACS, as in our experiments, but was also visible in images from ultra-structural scanning transmission electron microscopy (STEM). Autophagosomes and autolysosomes were seen in U-87 MG and A172 cells after TTField-application for 48 h [55]. As a conclusion to our findings, it is worth observing the expression of MG-63 cells' transmembrane adhesion receptors in defined time frames after the short electrical stimulus, i.e., integrins with \(\beta\)1-, \(\beta\)3-, and \(\alpha\)-subtypes as well as CD44 hyaluron and syndecan receptors, which all are typically expressed in these osteoblastic cells [11].