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 µM) or store-operated calcium entry (YM58483, 20 µM) resulted in delayed repair and dampened intracellular Ca²⁺ response. Furthermore, inhibition of phospholipase C (U73122, 10 µM) or inositol trisphosphate receptor (2-APB, 50 µM) 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.