Kinetics of Endothelial Cell Ectonucleotidases

Summary

Adenosine is a well-recognised vasoactive mediator that is believed to play an important role in reactive hyperaemia. More recently, ATP has also been recognised as a powerful vasoactive agent, with receptor-mediated actions independent of those of adenosine. It is likely that ATP released into the circulation during trauma, platelet aggregation, or possibly as a consequence of hypoxia, significantly modulates vascular tone.

We have characterised in detail the enzymes present on the endothelial cell surface that regulate the circulating levels of adenine nucleotides. ATP or ADP is substantially catabolised on a single passage through the isolated perfused piglet lung or superfused columns of pig aortic endothelial cells cultured on microcarrier beads. By using selective substrates and inhibitors we have concluded that in pig aortic endothelium, catabolism to adenosine is due to separate ATPase, ADPase and 5′-nucleotidase ectoenzymes, and we have determined the kinetic parameters of each. The use of diastereoisomeric pairs of ATP analogues also demonstrated that the ecto-ATPase is distinct from the endothelial P_2Y (ATP-recognising) purinoceptor: both the ecto-ATPase and -ADPase recognise a specific conformer of the Mg²⁺-adenine nucleotide complex as substrate, while the P_2y receptor apparently recognises free nucleotide. Similar enzymes seem to be present in human umbilical vein endothelial cells although their relative activities are different. In contrast, cells cultured from human omental fat microvessels lack significant ecto-ATPase activity, although both these cell types and pig aortic endothelial cells possess ecto-[nucleoside diphosphate kinase].