Supplementary Material for: The Mechanism of Injury-Induced Intracellular Calcium Concentration Oscillations in the Endothelium of Excised Rat Aorta

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca2+ response in the endothelium of excised rat aorta, which comprises an initial Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive stores followed by a long-lasting decay phase due to Ca2+ entry through uncoupled connexons. The Ca2+ signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca2+ spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca2+ oscillations. The latter reversibly ceased upon removal of extracellular Ca2+ or addition of the gap junction blockers heptanol, 18 α,β-glycyrrhetinic acid, La3+ and Ni2+, but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca2+ entry mode of the Na+/Ca2+ exchanger (NCX). The InsP3-producing agonist ATP resumed Ca2+ oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca2+ transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca2+ entry and InsP3-dependent Ca2+ release contributes to injury-induced intracellular Ca2+ concentration oscillations.