Endotoxin activation of macrophages does not induce ATP release and autocrine stimulation of P2 nucleotide receptors

Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in ma crophages, The most compelling evidence has been the observation that inhib itors of ionotropic nucleotide (P2X) receptors, including periodate-oxidize d ATP (oATP), attenuate a subset of endotoxin-induced effects such as activ ation of NF-kappaB and up-regulation of inducible NO synthase, We investiga ted whether endotoxin induces ATP release from a murine macrophage cell lin e (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These ce lls constitutively released ATP, producing steady-state extracellular conce ntrations of similar to1 nM when assayed as monolayers of 10(6) adherent ce lls bathed in 1 ml of medium. However, the macrophages did not release addi tional ATP during either acute or prolonged endotoxin stimulation. In addit ion, cellular ecto-ATPase activities were measured following prolonged endo toxin activation and were found not to be significantly altered. Although o ATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubat ed with apyrase, a highly effective ATP scavenger. These results indicate t hat activation of macrophages by endotoxin does not induce autocrine stimul ation of P2 nucleotide receptors by endogenous ATP released to extracellula r compartments. Moreover, the data suggest that the ability of oATP to inte rfere with endotoxin signaling is due to its interaction with molecular spe cies other than ATP-binding P2 receptors.