INDUCTION OF THE P2Z P2X(7) NUCLEOTIDE RECEPTOR AND ASSOCIATED PHOSPHOLIPASE-D ACTIVITY BY LIPOPOLYSACCHARIDE AND IFN-GAMMA IN THE HUMAN THP-1 MONOCYTIC CELL-LINE/

The activation of phospholipase D (PLD) was used as a marker for P2z n ucleotide receptor (P2zR) activity in the human THP-1 monocytic cell l ine. While untreated THP-1 cells displayed little PLD response to the P2zR agonist 3'-O-(4-benzoyl)benzoyl (Bz)ATP, treatment with either IF N-gamma or bacterial LPS induced a BzATP-mediated stimulation of PLD a ctivity, Treatment of cells with combinations of IFN-gamma and LPS res ulted in synergistic induction, P2z receptors mediated these effects b ecause: 1) reduction of extracellular divalent cation concentration in creased agonist potency; 2) only BzATP or ATP acted as agonist nucleot ides; 3) oxidized ATP, an inhibitor of the P2z receptor, abolished the response. The P2zR-stimulated PLD was rapidly activated (t(1/2) = 1.5 min), completely inhibited by KN-62, a calcium-calmodulin kinase II i nhibitor, and only partially repressed by bisindolylmaleimide, a prote in kinase C inhibitor, The P2zR-mediated PLD activity was distinguishe d from phorbol ester-stimulated PLD activity because the latter was sl owly activated (t(1/2) > 15 min), unaffected by oxidized ATP or KN-62, and completely inhibited by bisindolylmaleimide. IFN-gamma and LPS tr eatment also synergistically induced P2zR-dependent changes in membran e permeability and cytolysis, as indicated by BzATP-mediated Ca2+ infl ux, ethidium bromide uptake, and lactate dehydrogenase release. Finall y, IFN-gamma and LPS synergistically up-regulated mRNA encoding the P2 X(7) receptor, a recently cloned ATP-gated channel that exhibits a P2z R phenotype.