Scratch Triggers Transient Ca 2+ Entry Through Orai1 in Pulmonary Arterial Endothelial Cells Near the Wound Edge
Year: 2021
Type: conference-abstract
Abstract: Background: During wound healing, ensuring cellular coordination and adhesion involves a variety of intracellular and intercellular signaling processes. Ca 2+ release-activated Ca 2+ channel protein Orai1 contributes to cell transwell migration, number of focal adhesions per cell, cell spreading area, and migration speed. Orai1 favors Ca 2+ permeation and limits Na + permeation. However, Orai1-related cytosolic Ca 2+ and Na + changes in response to scratch and the role of these changes in endothelial wound healing remain unclear, and thus, are the focus of this study. Method: Pulmonary arterial endothelial cells (PAECs) were seeded and grew to confluent monolayers in 4-5 days. Monolayers were loaded with intracellular Ca 2+ dye Cal-590 AM and Na + dye Ion NaTRIUM Green-2 AM. Orai1 inhibitor GSK-7975A (10 M) was added for 15 minutes prior to scratch. Fluorescence images were taken immediately after scratch every 5 seconds for 30 minutes. Fluorescent signals were quantified. Scratch wound healing assay was conducted in the presence and absence of GSK-7975A and Orai1, respectively. Collective migration was triggered by barrier removal in the presence and absence of Orai1. Migration distances were quantified. Results: In the spatial manner, scratch injury immediately increased both cytosolic Ca 2+ and Na + fluorescence intensity at the wound edge. Cytosolic Ca 2+ and Na + quickly decreased within 50 m from the wound edge. However, only cytosolic Ca 2+ but not Na + in cells between 50 and 200 m from the wound edge increased to form a second peak. In the temporal manner, the increases in cytosolic Ca 2+ and Na + at the wound edge reduced in 30 minutes. These reductions were faster within 50 m than those beyond 50 m from the wound edge, respectively. The second Ca 2+ increase was much short-lived and reduced within 6 minutes. Beyond 200 m from the wound edge, both cytosolic Ca 2+ and Na + increased again after 30 minutes. Orai1 inhibition abolished neither the onset nor the decline of scratch-triggered Ca 2+ increase at the wound edge but fully eliminated the second Ca 2+ increase. Orai1 inhibition did not fully abolish the Na + increase or decline at the wound edge either. In the remaining monolayer, Orai1 inhibition appeared to attenuate changes in both cytosolic Ca 2+ and Na + signals. Orai1 expression, but not acute Orai1 activation, contributes to scratch wound closure. Conclusions: Scratch not only evokes acute cytosolic Ca 2+ and Na + increases at the wound edge but also causes transient Ca 2+ entry through Orai1 near the wound edge.
Institution: University of South Alabama
Language: English
Topic: Ion Channels and Receptors
Subfield: Sensory Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4248061809