[HTML][HTML] Investigation of G protein-initiated, Ca2+-dependent release of ATP from endothelial cells

JA Gruenhagen, ES Yeung - … et Biophysica Acta (BBA)-Molecular Cell …, 2004 - Elsevier
JA Gruenhagen, ES Yeung
Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2004Elsevier
We investigated G protein-stimulated release of ATP from human umbilical vein endothelial
cells (HUVECs) using the G protein stimulant compound 48/80. Application of compound
48/80 resulted in dose-dependent ATP evolution from cultured HUVECs. This release was
not cytotoxic as demonstrated by a lactate dehydrogenase assay and the ability of the cells
to load and retain the viability dye calcein following stimulation. Mastoparan also stimulated
release of ATP, further suggesting the process was G-protein initiated. This G protein was …
We investigated G protein-stimulated release of ATP from human umbilical vein endothelial cells (HUVECs) using the G protein stimulant compound 48/80. Application of compound 48/80 resulted in dose-dependent ATP evolution from cultured HUVECs. This release was not cytotoxic as demonstrated by a lactate dehydrogenase assay and the ability of the cells to load and retain the viability dye calcein following stimulation. Mastoparan also stimulated release of ATP, further suggesting the process was G-protein initiated. This G protein was insensitive to pertussis toxin and appeared to be of the Gq-subtype. The ATP efflux was completely abolished in the presence of EGTA and thapsigargin signifying a strict Ca2+ dependence. Furthermore, compound 48/80-induced release was significantly decreased in cells pretreated with the phospholipase C inhibitor U73122. Thus, the release pathway appears to proceed through an increase in intracellular Ca2+ via PLC activation. Additionally, the G protein-initiated release was attenuated by pretreatment of the cells with either phorbol ester or indolactam V, both activators of protein kinase C. Finally, ATP release was not affected by treating HUVECs with nitric oxide synthase (NOS) inhibitors or glybenclamide.
Elsevier