PYRIMIDINOCEPTOR-MEDIATED POTENTIATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE INDUCTION IN J774 MACROPHAGES - ROLE OF INTRACELLULAR CALCIUM

We have shown that, in murine J774 macrophages, binding of UTP to pyri midinoceptors stimulates phosphoinositide (PI) breakdown and an increa se in [Ca2+](i). In this study, UTP modulation of the expression of in ducible nitric-oxide synthase (iNOS) was investigated. Although UTP al one had no effect, stimulation of J774 cells with a combination of UTP (10-300 mu M) and LPS (0.1-3 mu g/ml) resulted in a potentiated incre ase in nitrite levels. In parallel, the amount of iNOS protein induced by LPS was also potentiated by UTP treatment. The UTP potentiating ef fect was attenuated by U73122, suggesting involvement of the downstrea m signaling pathways of phosphatidylinositide turnover. The tyrosine k inase inhibitor genistein inhibited both the LPS-induced nitrite respo nse and the UTP potentiation. Conversely, two protein kinase C inhibit ors, Ro 31-8220 and Go 6976, and a phosphatidylcholine-specific phosph olipase C inhibitor, D609, inhibited LPS-stimulated nitrite induction, but did not affect the potentiating effect of UTP, which was also una ffected by pretreatment with phorbol 12-myristate 13-acetate for 8 h. Furthermore, the UTP-induced potentiation was abolished by BAPTA/AM or KN-93 (a selective inhibitor of Ca2+/calmodulin-dependent protein kin ase (CaMK)). Nitrite potentiation and iNOS induction were prominent wh en UTP was added simultaneously with LPS, with the potentiating effect being lost when UTP was added 3 h after treatment with LPS. Pyrrolidi nedithiocarbamate (3-30 mu M), an inhibitor of NF-kappa B, caused a co ncentration-dependent reduction in the nitrite response to LPS and UTP . In electrophoretic mobility shift assays, LPS produced marked activa tion of NF-kappa B and AP-1, which was potentiated by UTP. LPS-induced degradation of I kappa B-alpha as well as the phosphorylation of I ka ppa B-alpha were also increased by UTP. Moreover, the UTP-potentiated activation of NF-kappa B and AP-1 and the degradation and phosphorylat ion of I kappa B-alpha were inhibited by KN-93. Taken together, these data demonstrate that nucleotides, especially UTP, can potentiate the LPS-induced activation of NF-kappa B and AP-1 and of iNOS induction vi a a CaMK-dependent pathway and suggest that the UTP-dependent up-regul ation of iNOS may constitute a novel element in the inflammatory proce ss.