Investigation of G protein-initiated, Ca2+-dependent release of ATP from endothelial cells.
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.
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Jason A
JA
Gruenhagen
Edward S
ES
Yeung
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005905:Glyburide / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D009569:Nitric Oxide; D000001:Calcimycin; D003189:p-Methoxy-N-methylphenethylamine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D020962:Heterotrimeric GTP-Binding Proteins; D005905:Glyburide; D002118:Calcium