We have investigated responses of human monocyte/macrophage cells to extracellular ATP (ATPe). Freshly isolated peripheral blood monocytes showed responses linked to P2Y but not P2Z purinergic receptors; however, during in vitro macrophage differentiation, these cells also exhibited responses suggestive of the presence of the membrane-permeabilizing P2z receptor. In fact, in human macrophages a brief (15-min) exposure to ATPe, but not other nucleotides, caused (1) a rapid and long-lasting plasma membrane depolarization; (2) a large increase in intracellular Ca2+ concentration followed by efflux of the Ca2+ indicator; (3) uptake of low molecular weight hydrophilic molecules such as Lucifer yellow and ethidium bromide; and (4) cell rounding, swelling, and eventual release of the cytoplasmic enzyme lactate dehydrogenase. rIFN-y enhanced both membranepermeabilizing and cytotoxic ATPe effects. Membrane permeabilization and cytotoxicity were fully blocked by pretreatment of the cells with oxidized ATP, a compound recently shown to block P2z receptors covalently in macrophages. Blocking of the P2z receptor by oxidized ATP also inhibited multinucleated giant cell generation stimulated by concanavalin A or rIFN-y without decreasing monocyte migration or membrane adhesion molecule expression. These data suggest that human macrophages express rIFN-ymodulated purinergic P2z receptors in vitro and hint at a role for these plasma membrane molecules in the generation of macrophage polykarions. (J. Clin. Invest. 1995Invest. . 95:1207Invest. -1216

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1 The present study describes the e ect of low frequency, low energy, pulsing electromagnetic ®elds (PEMFs) on A 2A adenosine receptors in human neutrophils.2 Saturation experiments performed using a high a nity adenosine antagonist [ 3 H]-ZM 241385 revealed a single class of binding sites in control and in PEMF-treated human neutrophils with similar a nity (K D =1.05+0.10 and 1.08+0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P50.01) (B max =126+10 and 215+15 fmol mg 71 protein, respectively).3 The e ect of PEMFs was speci®c to the A 2A adenosine receptors. This e ect was also intensity, time and temperature dependent. 4 In the adenylyl cyclase assays the A 2A receptor agonists, HE-NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC 50 value of 43 (40 ± 47) and 255 (228 ± 284) nM, respectively. The capability of HE-NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P50.01) after exposure to PEMFs with an EC 50 value of 10(8 ± 13) and 61(52 ± 71) nM, respectively. 5 In the superoxide anion (O 2 7 ) production assays HE-NECA and NECA inhibited the generation of O 2 7 in untreated human neutrophils, with an EC 50 value of 3.6(3.1 ± 4.2) and of 23(20 ± 27) nM, respectively. Moreover, in PEMF-treated human neutrophils, the same compounds show an EC 50 value of 1.6(1.2 ± 2.1) and of 6.0(4.7 ± 7.5) nM respectively. 6 These results indicate the presence of signi®cant alterations in the expression and in the functionality of adenosine A 2A receptors in human neutrophils treated with PEMFs.

The highly hydrophobic C60 (buckminsterfullerene) was water solubilized by covalently linking the synthon 1,2-dihydro-1,2-methanofullerene [60]-61-carboxylic acid to the alpha-amino group of the hydrophilic 4-8 sequence of peptide T, known to display potent human monocyte chemotaxis. The resulting compound, characterized by a variety of analytical techniques, including a UV spectrum in aqueous solution, exhibits remarkable chemotactic potency, comparable to that of the parent pentapeptide. Furthermore, this fullerene-peptide conjugate inhibits, albeit weakly, HIV-1 protease.

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