Multiple Calcium Sources Are Required For Intracellular Calcium Mobilization During Gastric Organoid Epithelial Repair

Calcium (Ca2+) is a known accelerator for gastric wound repair. We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1-2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage-gated Ca2+ channels (verapamil, 10 mu M) or store-operated calcium entry (YM58483, 20 mu M) resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C (U73122, 10 mu M) or inositol trisphosphate receptor (2-APB, 50 mu M) likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.