Purine nucleotides induce regulated secretion of von Willebrand factor: involvement of cytosolic Ca2+ and cyclic adenosine monophosphate-dependent signaling in endothelial exocytosis.
von Willebrand factor (vWF) is stored and released from endothelial secretory granules called Weibel-Palade (WP) bodies. Acute release can be induced by thrombin, histamine, and other mediators of thrombosis or inflammation. Their effect is thought to be mediated by an increase in intracellular free calcium ([Ca2+]i). Purine nucleotides such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are released from platelet dense granules and from ischemic tissues and are important regulators of platelet function and vascular tone. In the present study, we investigated whether they could also induce exocytosis from cultured endothelial cells. ATP (1 to 100 micromol/L) induced a dose-related increase in vWF release, with a 2.3-fold maximal increase after 30 minutes. Similar responses were observed with ADP. ATP induced calcium mobilization from intracellular stores, an effect mimicked by 2-methylthio-ATP, a selective agonist for P2y receptors. However, 2-methylthio-ATP-induced vWF release was only 43% of the ATP response. ATP-induced vWF release was also associated with a twofold increase in cellular cyclic adenosine monophosphate (cAMP) content, and was potentiated by 3-isobutyl-1-methylxanthine ([IBMX] added to increase cAMP levels by blocking cellular phosphodiesterases) and 8-bromo-cAMP and inhibited by more than 50% by Rp-8-CPT-cAMPS, a competitive protein kinase A inhibitor. Adenosine but not 2-methylthio-ATP mimicked the ATP-induced increase in cAMP. ATP-induced vWF release was partly inhibited by adenosine deaminase, which degrades adenosine generated from ATP in the incubation medium. Adenosine (1 to 100 micromol/L) failed to induce vWF release, but potentiated the secretory response to 2-methylthio-ATP and thrombin without modifying the calcium response to these agents. Our results suggest that ATP/ADP can induce vWF release from endothelial cells via dual activation of P2y and adenosine A2 receptors. ATP/ADP-induced exocytosis could be involved in the regulation of thrombus formation and ischemia-reperfusion injuries. Further, we provide evidence that a receptor-mediated increase in cellular cAMP can potentiate the secretory response to calcium-mobilizing agents.
Vischer
U M
UM
Division de Biochimie Clinique, Department of Medicine, Centre Médical Universitaire, Geneva, Switzerland.
Wollheim
C B
CB
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000243:Adenosine Deaminase / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D003594:Cytoplasmic Granules / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D018047:Receptors, Purinergic P1 / Q000187:drug effects* / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology*; D012636:Secretory Rate / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000378:metabolism*
D000227:Adenine Nucleotides; D004791:Enzyme Inhibitors; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; D014841:von Willebrand Factor; D015124:8-Bromo Cyclic Adenosine Monophosphate; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin; D000243:Adenosine Deaminase; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine; C008501:2-methylthio-ATP