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-associat ed, mitochondrial-based 25-hydroxyvitamin D (25-OHD)-1-hydroxylation r eaction 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 express ion of the macrophage nitric oxide snythase (mac NOS), we investigated the possibility that endogenous nitric oxide (NO) production may be l inked to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) synthesis by HD-11 cel ls in vitro. To test this hypothesis we investigated the effects exclu ding 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. Depletio n of L-arginine from the extracellular medium for as little as 6 h res ulted in a significant decrease (p<0.02) in basal 1,25-(OH)2D synthesi s; after 15 h in an L-arginine-free environment hormone production was reduced to <1O% of basal levels without any adverse affect on cell vi ability Reintroduction of L-arginine, but not D-arginine, into the ext racellular medium restored 1,25-(OH)2D3 synthetic capacity fully if do ne after less than or equal to 6 h of incubation in the absence of L-a rginine. Competitive inhibition of NOS with N-W-nitro-L-arginine methy l ester (p<0.002) and N-W-nitro-L-arginine (p<0.02) significantly inhi bited 1,25-(OH)2D synthesis, indicating that macrophage NO generating capacity is functionally Linked to endogenous synthesis of the active vitamin D metabolite.