ATP Release from Cultured Endothelial Cells and Intercellular Calcium Signaling during Shear Stress Exposure

Abstract

Intracellular calcium ([Ca2+]i) is a second messenger molecule critical for numerous intracellular signaling pathways in endothelial cells (ECs). Direct mechanical stimulation imposed on single ECs by a microprobe has been demonstrated to increase [Ca2+]i levels in ECs. After an initial delay time, this Ca2+-signal propagates from the mechanically stimulated cell to its neighboring cells in the form of an intercellular Ca2+-wave, a process termed intercellular communication. Although intercellular communication is a fundamental property of many multicellular systems, it remains unclear as to whether intercellular communication following shear stress of ECs actually occurs, and if so, whether this communication occurs via gap junctions or the release of extracellular mediators including ATP. In the current study, we investigated ATP release from ECs during periods of shear stress and measured intercellular Ca2+-waves using adenosine 5'-triphosphate, P3-(1-(2-nitrophenyl)ethyl)ester and disodium salt (NPE-caged ATP) stimulation. In addition, we investigated intercellular communication in ECs during shear stress using chemical inhibitors of both gap junctions and various components of the ATP paracrine signaling pathway. ECs subjected to shear stress loading released ATP. Using NPE-caged ATP, local increase of extracellular ATP induced a Ca2+-wave. Furthermore, [Ca2+]i responses in ECs under shear stress loading was inhibited by the purinergic receptor blocker, ATPase, and several metabolic inhibitors including FCCP and rotenone. These results suggest that [Ca2+]i communication mediated by ATP exists in ECs under shear stress loading in vitro.