COORDINATE REGULATION OF NITRIC-OXIDE AND 1,25-DIHYDROXYVITAMIN-D PRODUCTION IN THE AVIAN MYELOMONOCYTIC CELL-LINE HD-11

Cells of the monocyte/macrophage lineage are capable of both nitric ox ide (NO) and 1,25-dihydroxyvitamin D [1, 25-(OH)(2)D] production throu gh expression of inducible nitric oxide synthase (iNOS) and a putative 25-hydroxyvitamin D (25-OHD)-1-hydroxylase, respectively. We have rec ently reported that 1,25-(OH)(2)D synthesis in the chick myelomonocyti c cell Line HD-11 is restricted by inhibition of iNOS. In the current set of experiments, measuring nitrite, a stable water-soluble secreted metabolite of NO as an index of iNOS activity and 1,25-(OH)(2)D-3 in lipid extracts of cells incubated with 200 nM 25-OHD3 as an index of 1 -hydroxylase activity, we demonstrate that NO and 1,25-(OH)(2)D produc tion by HD-11 cells are temporally related, induced by the same kinds of activating agents, and coordinately regulated. NO and 1,25-(OH)(2)D -3 production by HD-11 cells was stimulated severalfold by the macroph age stimulators interferon-gamma and lipopolysaccharide and by an auto logous, nonlipid, heat-labile factor with an apparent molecular mass a pproximate to 10,000 daltons. As expected NO synthesis was 1) dependen t upon the presence of L-arginine in the extracellular medium, 2) subj ect to significant stimulation by N-W-hydroxy-L-arginine, an L-arginin e-derived intermediate in NO biosynthesis, and by sodium nitroprusside , a non-L-arginine-dependent source of intracellular NO, and 3) inhibi ted by N-W-nitro-L-arginine methyl ester, a competitive inhibitor of i NOS. At high NO production rates, induced either by high-dose lipopoly saccharide or by sodium nitroprusside exposure, there was an apparent downturn in 1,25(OH)(2)D-3 synthesis, suggesting functional dependence of the 1-hydroxylase on NO but ultimate inhibition of 1,25-(OH)(2)D-3 synthetic capacity at high levels of intracellular NO production. On the basis of these results we postulate that the macrophage 25-OHD-1-h ydroxylation reaction may be dependent on iNOS-generated NO as a solub le source of electrons and regulated in an autocrine mode by a macroph age-derived NO stimulatory factor and NO itself.