title: Post-translational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis.
abstract: BACKGROUND
  Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known.
  
  
  OBJECTIVE
  Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS post-translational phosphorylation and the enzyme's interactions with its regulatory proteins.
  
  
  METHODS
  Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild-type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat-shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium.
  
  
  METHODS
  Molecular mechanisms of eNOS dysfunction in the sickle mouse penis.
  
  
  RESULTS
  eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared with WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared with WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD.
  
  
  CONCLUSIONS
  These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction.
authors.affiliation: The Johns Hopkins University, Department of Urology, Baltimore, MD 21287, USA. bmusicki@jhmi.edu
authors.forename: Biljana
authors.initials: B
authors.lastname: Musicki
authors.forename: Hunter C
authors.initials: HC
authors.lastname: Champion
authors.forename: Lewis L
authors.initials: LL
authors.lastname: Hsu
authors.forename: Trinity J
authors.initials: TJ
authors.lastname: Bivalacqua
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Burnett
mesh_terms: D000755:Anemia, Sickle Cell / Q000150:complications / Q000201:enzymology*; D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010410:Penile Erection; D010413:Penis / Q000201:enzymology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D053833:Wetlands
chemical_list: D051242:Caveolin 1; D018841:HSP90 Heat-Shock Proteins; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse