A Role for Nitric Oxide in the Regulated Expression of the 25-Hydroxyvitamin D-1-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 (ie. interferon-gamma, lipopolysaccharide, 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 <10% 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 ≤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.