In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca signaling through mechanotransduction pathways. Because Ca regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca mobilization in human mesenchymal stem cells using 47 MHz focused ultrasound to stimulate single cells at low intensities (~ 110 mW/cm). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled PY purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca from intracellular stores.

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Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is noninvasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not noninvasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1 however plays a different role in mediating the spread of intercellular calcium waves via ATP release.Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy. FIGURE 3. Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUSinduced Ca 2+ oscillation response but uncovers single Ca 2+ transients. (A)Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 M scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 M 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 M Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n=3 (XC), or n=6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet's correction, exact p values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by F/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).

In glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells, the rise of free cytosolic Ca2+ concentration through voltage-gated calcium channels (VGCCs) triggers the exocytosis of insulin-containing granules. Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic β-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.