Neuronal nitric oxide synthase can mediate no production in mechanically stimulated mouse osteoblasts

The performance of the skeleton is secured by the constant adaptation of bone to its mechanical loading environment. Nitric oxide (NO) has long been implicated in the cellular process of mechanical adaptation in bone and NO donors are trialled as treatment for osteoporosis. The precise mechanism whereby NO is produced and acts in bone, however, remains poorly understood. In particular, it has not been conclusively demonstrated which NO synthase (NOS) is activated by mechanical forces in bone cells. We addressed this question using, for the first time, bone cells from NOS knockout mice. Since we did not have access to double or triple NOS knockout mice, given the difficulties in breeding and crossing single NOS deleted strains, we used single NOS gene-deleted strains in combination with a range of pharmacological NOS inhibitors. Mechanical force was applied in the form of pulsatile fluid flow (PFF), since this is a reproducible and accepted method to mechanically stimulate bone cells in vitro. We measured the response to PFF in osteocytes (MLO-Y4) and in primary osteoblasts from both long bones and calvarial bones of wildtype mice and mice with a deletion in endothelial NOS (eNOS), inducible NOS (iNOS), or neuronal NOS (nNOS). Osteoblasts were kept in static culture or subjected to PFF in the presence or absence of the NOS inhibitors l-canavanine, 1400W, or L-NAME. NO production was measured using Griess reagent (to measure cumulative NO production) or DAR4M-AM (to measure NO production in real time). NOS expression was quantified using taqman PCR. PFF stimulated NO production to a similar extent in MLO-Y4 osteocytes and WT, eNOS −/− and iNOS−/− osteoblasts, even though iNOS−/− osteoblasts neither expressed functional iNOS nor eNOS mRNA. l-Canavanine and 1400W reduced PFF-induced NO production in iNOS−/− cells, suggesting that nNOS was responsible. L-NAME, an inhibitor of eNOS and nNOS, reduced PFF-induced NO production in wildtype and eNOS−/− osteoblasts, cell types that did not express eNOS, again pointing to nNOS as the enzyme responsible. Interestingly, NO production was not reduced in PFF-treated nNOS−/− osteoblasts, suggesting that other enzymes, especially iNOS, may play a role when nNOS is absent. Our results challenge the notion that fluid flow-induced NO production in bone cells is only dependent on eNOS and suggest a more important role for nNOS in bone maintenance through mechanical stimuli than has previously been considered.