A ROLE FOR NITRIC-OXIDE IN THE REGULATED EXPRESSION OF THE 25-HYDROXY-VITAMIN D-L-HYDROXYLATION REACTION IN THE CHICK MYELOMONOCYTIC CELL-LINE HD-11

We have recently described the existence of a cytochrome P450-associated, mitochondrial-based 25-hydroxyvitamin D (25-OHD)-1-hydroxylation reaction in the chick macrophage-like cell Line HD-11. Considering that this reaction is regulated by the same set of factors tie. interferon-gamma, lipoporysaccharide, and glucocorticoids) that modulate expression of the macrophage nitric oxide snythase (mac NOS), we investigated the possibility that endogenous nitric oxide (NO) production may be linked to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) synthesis by HD-11 cells in vitro. To test this hypothesis we investigated the effects excluding from the extracellular medium the essential amino acid L-arginine, substrate for endogenous NO production, on the basal and stimulated expression of the HD-11 cell 25-OHD-1-hydroxylation reaction. Depletion of L-arginine from the extracellular medium for as little as 6 h resulted in a significant decrease (p<0.02) in basal 1,25-(OH)2D synthesis; after 15 h in an L-arginine-free environment hormone production was reduced to <1O% of basal levels without any adverse affect on cell viability Reintroduction of L-arginine, but not D-arginine, into the extracellular medium restored 1,25-(OH)2D3 synthetic capacity fully if done after less than or equal to 6 h of incubation in the absence of L-arginine. Competitive inhibition of NOS with N-W-nitro-L-arginine methyl ester (p<0.002) and N-W-nitro-L-arginine (p<0.02) significantly inhibited 1,25-(OH)2D synthesis, indicating that macrophage NO generating capacity is functionally Linked to endogenous synthesis of the active vitamin D metabolite.