Effect of asymmetric dimethylarginine on osteoblastic differentiation

Background. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor o f nitric oxide synthase (NOS) that accumulates in renal insufficiency and m ay be a uremic toxin. To determine whether ADMA inhibits bone metabolism, w e investigated the in vitro effect of ADMA on osteoblastic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). Methods. The effect of ADMA on nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium u sing commercial NO kit. The temporal sequence of osteoblastic differentiati on in BMSCs was assessed in the presence and absence of ADMA by measuring a lkaline phosphatase (ALP) activity, mineralization, and osteoblast gene exp ression at 0, 4, 8, 12 days of culture. Results. ADMA (5, 50, 500 mu mol.(-1)) resulted in a dose-dependent decreas e in nitrite formation in conditioned media of BMCS cultures, consistent wi th inhibition of NOS. ADMA treatment was associated with reduced ALP activi ty, calcium deposition and osteoblast-related gene expression in BMSCs cult ures. Concurrent treatment with v-arginine (3600 mu mol.L-1) reversed the A DMA (500 mu mol.L-1)-mediated decrease in NO production, restored the diffe rentiation potential of BMSCs, and significantly attenuated the down-regula tion of Cbfa1 and ostcocalcin gene expression by ADMA. Conclusions. ADMA inhibition of the NO-NOS pathway in BMSCs impairs osteobl astic differentiation of mouse BMSC cultures. These studies further support a role of NO in the local regulation of bone metabolism and the possibilit y that ADMA may act as uremic toxin on bone through its effect to inhibit N O actions in osteoblasts.