Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic ß-Cell Line.

In glucose-stimulated insulin secretion (GSIS) of pancreatic ß-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 ß-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.

Anti-inflammatory effect of Lycium barbarum on polarized human intestinal epithelial cells.

BACKGROUND/OBJECTIVES: Inflammatory Bowel Disease (IBD) has rapidly escalated in Asia (including Korea) due to increasing westernized diet patterns subsequent to industrialization. Factors associated with endoplasmic reticulum (ER) stress are demonstrated to be one of the major causes of IBD. This study was conducted to investigate the effect of Lycium barbarum (L. barbarum) on ER stress. MATERIALS/METHODS: Mouse embryonic fibroblast (MEF) cell line and polarized Caco-2 human intestinal epithelial cells were treated with crude extract of the L. chinense fruit (LF). Paracellular permeability was measured to examine the effect of tight junction (TJ) integrity. The regulatory pathways of ER stress were evaluated in MEF knockout (KO) cell lines by qPCR for interleukin (IL) 6, IL8 and XBP1 spliced form (XBP1s). Immunoglobulin binding protein (BiP), XBP1s and CCAAT/enhancer-binding homologous protein (CHOP) expressions were measured by RT-PCR. Scanning Ion Conductance Microscopy (SICM) at high resolution was applied to observe morphological changes after treatments. RESULTS: Exposure to LF extract strengthened the TJ, both in the presence and absence of inflammation. In polarized Caco-2 pretreated with LF, induction in the expression of proinflammatory marker IL8 was not significant, whereas ER stress marker XBP1s expression was significantly increased. In wild type (wt) MEF cells, IL6, CHOP and XBP1 spliced form were dose-dependently induced when exposed to 12.5-50 µg/mL extract. However, absence of XBP1 or IRE1α in MEF cells abolished this effect. CONCLUSION: Results of this study show that LF treatment enhances the barrier function and reduces inflammation and ER stress in an IRE1α-XBP1-dependent manner. These results suggest the preventive effect of LF on healthy intestine, and the possibility of reducing the degree of inflammatory symptoms in IBD patients.

Low-Intensity Ultrasound Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel.

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 Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47 MHz focused ultrasound to stimulate single cells at low intensities (~ 110 mW/cm2). 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 P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.