Expression and functional role of nitric oxide synthase in osteoblast‐like cells

Nitric oxide synthases (NOS) are enzymes that produce nitric oxide (NO) from L‐arginine in a reaction yielding citrulline as a coproduct. Nitric oxide modulates the activity of a wide variety of cells, but little is known about its effects on bone cells. In the present study we report that the NOS inhibitor NG‐monomethyl‐L‐arginine (NMMA) induced a dose‐dependent inhibitory effect on the proliferation of the osteoblast‐like cell lines MG63 and ROS 17/2.8. The inhibitory effect was prevented by increasing L‐arginine concentrations in the medium and by the NO donor sodium nitroprusside. Likewise, NMMA inhibited interleukin‐6 secretion, independently of its effect on cell number. NOS expression by MG63 cells was confirmed by measuring their ability to metabolize radiolabeled L‐arginine to citrulline. NOS bioactivity was detected in unstimulated cells, but was markedly increased by stimulating the cells with cytokines, lipopolysaccharide, or 1,25‐dihydroxyvitamin D3. NOS activity was partially dependent upon the presence of calcium in the medium. Furthermore, constitutive‐type NOS (c‐NOS) and inducible‐type NOS (i‐NOS) mRNA expression was detected in ROS 17/2.8 cells after reverse transcription and polymerase chain reaction amplification. In conclusion, osteoblast‐like cells express c‐NOS and i‐NOS, and NOS activity seems to play an important role in the regulation of cell proliferation and function.