Metabolism of inositol phosphates in ATP-stimulated vascular endothelial cells

Abstract

The accumulation of InsP1, InsP2, InsP3 and InsP4 isomers was investigated in bovine aortic endothelial cells labelled with [3H]inositol and stimulated with ATP. The separation of these isomers was performed by ion-pairing reverse-phase h.p.l.c. on a mu Bondapack C18 column for the InsP3 and InsP4 isomers and by ion-exchange h.p.l.c. on a Partisil SAX column for the InsP1 and InsP2 isomers. In unstimulated endothelial cells, a large amount of material was co-eluted with InsP5 and InsP6, whereas amounts of InsP3 and InsP4 were small. The addition of ATP (100 microM) induced a striking (35-fold stimulation) and transient increase of Ins(1,4,5)P3 that was maximal around 15 s. This peak was followed by a more sustained accumulation of Ins(1,3,4,5)P4 and Ins(1,3,4)P3, but the amounts of these two metabolites accumulated in response to ATP were much smaller than that of Ins(1,4,5)P3. The increase in InsP2 isomers in response to ATP had similar characteristics: a rapid and transient accumulation of Ins(1,4)P2, followed by an increase of Ins(3,4)P2 and Ins(1,3)P2, which was more sustained but had a smaller magnitude. ATP also induced the accumulation of both Ins1P and Ins4P, but with different time courses: the level of Ins4P was maximal at 1 min (60 times the control value) and returned to baseline after 5 min, whereas the increase in Ins1P was undetectable at 1 min and reached a maximum after 5 min, which represented 240% of the basal level. These data indicate that Ins(1,4,5)P3, which is rapidly formed in aortic endothelial cells as a result of activation of P2Y receptors, is preferentially metabolized at early times (less than 1 min) by a 5-phosphatase, with the sequential formation of Ins(1,4)P2 and Ins4P. Afterwards, a small but sustained increase in the content of Ins(1,3,4)P3, Ins(1,3)P2, Ins(3,4)P2 and Ins1P was observed, reflecting the activation of the Ins(1,4,5)P3 3-kinase.