Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure

Study Design. This in vitro study clarifies the role of nitric oxide (NO) i n human lumbar intervertebral disc metabolism. Objective. To investigate the effects of NO on proteoglycan synthesis in hu man lumbar discs and to test the hypothesis that NO is a mediator of the ch anges in proteoglycan synthesis in response to hydrostatic pressure. Summary of Background Data. The authors have clarified that hydrostatic pre ssure has an apparent effect on proteoglycan synthesis as well as matrix me talloproteinase production in the intervertebral disc. The cellular mechani sms underlying the response of disc cells to hydrostatic pressure remain to be clarified. Herniated lumbar discs produce NO in response to interleukin (IL)-1 beta. In articular cartilage, NO mediates the change of proteoglyca n synthesis by IL-l or shear stress. Methods. Fifty-eight lumbar intervertebral disc specimens were obtained fro m patients who had undergone posterior discectomy. The specimens were chopp ed into 1-2-mm cubes and were incubated in a plastic syringe with 1 mt Dulb ecco's modified Eagle's medium (DMEM). The syringes were placed in a water- filled pressure vessel kept at 37 C. Hydrostatic pressures of 1 (control), 3, and 30 atmospheres (atm) were applied. Proteoglycan synthesis was determ ined from S-36-sulfate incorporation rates. Nitrite (the stable oxidation p roduct of NO) concentration in DMEM was determined by a spectrophotometric method based on the Griess reaction. As a competitive inhibitor of NO synth ases, N-G-methyl-L-arginine (L-NMA, 10-1000 mu mol) and as an organic donor of NO, S-nitroso-N-acetylpenicillamine (SNAP, 1-200 mu mol) were used. Results. Addition of L-NMA suppressed NO production and increased proteogly can synthesis rates in the intervertebral disc specimens in a dose-dependen t fashion. Addition of SNAP increased exogenous NO content in the medium si gnificantly and suppressed proteoglycan synthesis rates in a dose-dependent fashion. Th ree-atmosphere hydrostatic pressure stimulated the proteoglyca n synthesis rates. Rates were approximately 1.3-fold greater than at 1 atm, whereas 30-atm pressure inhibited proteoglycan synthesis rates. However, t he hydrostaticpressure had inverse effect on NO production. AT 3 atm, NO pr oduction decreased slightly relative to 1 atm, whereas at a pressure of 30 atm, NO production was increased and was approximately 1.32-fold greater th an at 1 arm. L-NMA enhanced the 3-atm pressure-induced increase in proteogl ycan synthesis and also relieved the suppression of proteoglycan synthesis at a pressure of 30 atm. Conclusion. The current study confirmed the previous finding that human her niated lumbar disc cultures spontaneously produce NO. Endogenously generate d and exogenously supplied NO inhibited proteoglycan synthesis in the inter vertebral disc. Hydrostatic pressure influenced NO production by disc cells , and NO is one of the mediators that changes proteoglycan synthesis in res ponse to hydrostatic pressure. These results may show that autocrine and pa racrine mechanisms of NO play an important role in the regulation of disc c ell metabolism under mechanical stress and in the pathophysiology of interv ertebral disc degeneration.