During Ca ²⁺ ‐dependent gastric epithelial repair, Ca ²⁺ is sourced from both Ca ²⁺ uptake and intracellular Ca ²⁺ release

Background Calcium (Ca 2+ ) is a known accelerator for gastric repair. However the mechanism by which Ca 2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo . Using gastric organoids derived from transgenic mice expressing a fluorescent Ca 2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca 2+ increases in cells directly adjacent to a damaged cell. Using this Ca 2+ sensor, we investigate the potential sources of intracellular Ca 2+ essential for repair. Methods Gastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca 2+ . Change in intracellular Ca 2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca 2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca 2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB). Results Unperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca 2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca 2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca 2+ by BAPTA/AM resulted in significant dampening of Ca 2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca 2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca 2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca 2+ response. Conclusion These results suggest both extracellular and intracellular Ca 2+ sources are essential for supplying the Ca 2+ that stimulates repair. The findings implicate an intracellular Ca 2+ raise mediated via Ca 2+ uptake via plasma membrane Ca 2+ channels and intracellular Ca 2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca 2+ dynamics and further investigate the signaling cascade behind Ca 2+ ‐mediated repair. Support or Funding Information This work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .