Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various e xtracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expre ssion. In the present study we investigated the effects of increased [Ca2+] (i) on NO production through the iNOS pathway in J774 macrophages. Thapsiga rgin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in [Ca2+](i) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as ac cumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA we re detected by Western blotting and reverse-transcriptase-mediated PCR resp ectively. The activation of nuclear factor kappaB (NF-kappaB) was investiga ted by electrophoretic mobility-shift assay. TG (100 nM) induced a marked s ynthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low conce ntration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS pr otein and NO production by accelerating the degradation of iNOS mRNA. Treat ment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazol ium-5-carboxyaniline ('XTT') method; apoptosis was ruled out by propidium i odide staining and flow cytometry. A23187 (1 muM) also transiently increase d [Ca2+](i) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased [Ca2+](i) induced the stimul ation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor- mediated increase in [Ca2+](i) might be an important factor in the control of the balance between the upregulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflamm atory response.