title: Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure.
abstract: METHODS
  This in vitro study clarifies the role of nitric oxide (NO) in human lumbar intervertebral disc metabolism.
  
  
  OBJECTIVE
  To investigate the effects of NO on proteoglycan synthesis in human lumbar discs and to test the hypothesis that NO is a mediator of the changes in proteoglycan synthesis in response to hydrostatic pressure.
  
  
  BACKGROUND
  The authors have clarified that hydrostatic pressure has an apparent effect on proteoglycan synthesis as well as matrix metalloproteinase production in the intervertebral disc. The cellular mechanisms 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 proteoglycan synthesis by IL-1 or shear stress.
  
  
  METHODS
  Fifty-eight lumbar intervertebral disc specimens were obtained from patients who had undergone posterior discectomy. The specimens were chopped into 1-2-mm cubes and were incubated in a plastic syringe with 1 mL Dulbecco'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 determined from (35)S-sulfate incorporation rates. Nitrite (the stable oxidation product of NO) concentration in DMEM was determined by a spectrophotometric method based on the Griess reaction. As a competitive inhibitor of NO synthases, N(G)-methyl-l-arginine (l-NMA, 10-1000 micromol) and as an organic donor of NO, S-nitroso-N-acetylpenicillamine (SNAP, 1-200 micromol) were used.
  
  
  RESULTS
  Addition of l-NMA suppressed NO production and increased proteoglycan synthesis rates in the intervertebral disc specimens in a dose-dependent fashion. Addition of SNAP increased exogenous NO content in the medium significantly and suppressed proteoglycan synthesis rates in a dose-dependent fashion. Three-atmosphere hydrostatic pressure stimulated the proteoglycan synthesis rates. Rates were approximately 1.3-fold greater than at 1 atm, whereas 30-atm pressure inhibited proteoglycan synthesis rates. However, the hydrostaticpressure had inverse effect on NO production. At 3 atm, NO production decreased slightly relative to 1 atm, whereas at a pressure of 30 atm, NO production was increased and was approximately 1.32-fold greater than at 1 atm. L-NMA enhanced the 3-atm pressure-induced increase in proteoglycan synthesis and also relieved the suppression of proteoglycan synthesis at a pressure of 30 atm.
  
  
  CONCLUSIONS
  The current study confirmed the previous finding that human herniated lumbar disc cultures spontaneously produce NO. Endogenously generated and exogenously supplied NO inhibited proteoglycan synthesis in the intervertebral disc. Hydrostatic pressure influenced NO production by disc cells, and NO is one of the mediators that changes proteoglycan synthesis in response to hydrostatic pressure. These results may show that autocrine and paracrine mechanisms of NO play an important role in the regulation of disc cell metabolism under mechanical stress and in the pathophysiology of intervertebral disc degeneration.
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.
authors.forename: G Z
authors.initials: GZ
authors.lastname: Liu
authors.forename: H
authors.initials: H
authors.lastname: Ishihara
authors.forename: R
authors.initials: R
authors.lastname: Osada
authors.forename: T
authors.initials: T
authors.lastname: Kimura
authors.forename: H
authors.initials: H
authors.lastname: Tsuji
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D005260:Female; D006801:Humans; D006874:Hydrostatic Pressure; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D008159:Lumbar Vertebrae / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis* / Q000187:drug effects
chemical_list: D011509:Proteoglycans; D009569:Nitric Oxide