title: Genetic disruption of all NO synthase isoforms enhances BMD and bone turnover in mice in vivo: involvement of the renin-angiotensin system.
abstract: BACKGROUND
  NO is synthesized by three different NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The roles of NO in bone metabolism have been extensively investigated in pharmacological studies and in studies with NOS isoform-deficient mice. However, because of the nonspecificity of agents and compensation among the NOS isoforms, the ultimate roles of endogenous NO are still poorly understood. To address this point, we successfully generated mice in which all three NOS genes are completely disrupted. In this study, we examined whether bone metabolism is abnormal in those mice.
  
  
  METHODS
  Experiments were performed in 12-wk-old male wildtype, singly nNOS(-/-), iNOS(-/-), and eNOS(-/-) and triply n/i/eNOS(-/-) mice. BMD was assessed by DXA. The kinetics of osteoblastic bone formation and those of osteoclastic bone resorption were evaluated by measurements of morphological and biochemical markers.
  
  
  RESULTS
  BMD was significantly higher only in the triply NOS(-/-) mice but not in any singly NOS(-/-) mice compared with the wildtype mice. Markers of osteoblastic bone formation, including bone formation rate, mineral apposition rate, and serum alkaline phosphatase concentration, were also significantly larger only in the triply NOS(-/-) mice compared with wildtype mice. Furthermore, markers of osteoclastic bone resorption, including osteoclast number, osteoclast surface, and urinary deoxypyridinoline excretion, were again significantly greater only in the triply NOS(-/-) mice. Importantly, the renin-angiotensin system in bone was significantly activated in the triply NOS(-/-) mice, and long-term oral treatment with an angiotensin II type 1 (AT(1)) receptor blocker normalized this pathological bone remodeling in those mice.
  
  
  CONCLUSIONS
  These results provide the first direct evidence that genetic disruption of the whole NOS system enhances BMD and bone turnover in mice in vivo through the AT(1) receptor pathway, showing the critical role of the endogenous NO/NOS system in maintaining bone homeostasis.
authors.affiliation: Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Sabanai
authors.forename: Masato
authors.initials: M
authors.lastname: Tsutsui
authors.forename: Akinori
authors.initials: A
authors.lastname: Sakai
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Hirasawa
authors.forename: Shinya
authors.initials: S
authors.lastname: Tanaka
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Nakamura
authors.forename: Akihide
authors.initials: A
authors.lastname: Tanimoto
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Sasaguri
authors.forename: Masako
authors.initials: M
authors.lastname: Ito
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shimokawa
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Nakamura
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Yanagihara
mesh_terms: D000818:Animals; D015519:Bone Density; D016723:Bone Remodeling; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019001:Nitric Oxide Synthase / Q000235:genetics*; D012084:Renin-Angiotensin System
chemical_list: D007527:Isoenzymes; D019001:Nitric Oxide Synthase