We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain of Staphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.

1 Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X 7 nucleotide receptor (P2X 7 R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X 7 R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X 7 R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. 2 oATP decreased by 40 -70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-a (TNF-a) in all three cell types, by IL-1b in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-a-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X 7 R. 3 oATP also attenuated cytokine-stimulated expression of nuclear factor-kB-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IkB. 4 oATP had no effect on uridine triphosphate-induced activation of native P2Y 2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y 1 receptors. However, inhibition of P2Y 1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-a-stimulated IL-8 secretion. 5 Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. 6 In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-κB and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of ∼1 nM when assayed as monolayers of 106 adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.