Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes

Previous studies have indicated that physiological levels of dynamic mechan ical strain produce rapid increases in nitric oxide (NO) release from rat u lna explants and primary cultures of osteoblast-like cells and embryonic ch ick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric o xide synthase (NOS) isoform mRNA and protein expression, the effect of mech anical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot anal yses, in situ hybridization, and immunocytochemistry we have established th at the predominant NOS isoform expressed in rat long bone periosteal osteob lasts and in a distinct population of cortical bone osteocytes is the endot helial form of NOS (eNOS), with little or no expression of the inducible NO S or neuronal NOS isoforms, In contrast, in non-load-bearing calvariae ther e are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in r esponse to loading in vitro, presumably through the activation of eNOS, whe reas calvarial explants do not The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, whic h showed that purified populations of osteocytes produce significantly grea ter quantities of NO per cell in response to mechanical strain than osteobl ast-like cells derived from the same bones. Using Northern blot hybridizati on, we have also shown that neither a single nor five consecutive daily per iods of in vivo mechanical loading produced any significant effect on diffe rent NOS isoform mRNA expression in rat ulnae. In conclusion, our results i ndicate that eNOS is the prevailing isoform expressed by cells of the osteo blast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.