title: Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes.
abstract: BACKGROUND
  Adenosine has been shown to induce nitric oxide (NO) production via inducible NO synthase (iNOS) activation in vascular smooth muscle cells (VSMCs). Although this is interpreted as a beneficial vasodilating pathway in vaso-occlusive disorders, iNOS is also involved in diabetic vascular dysfunction. Because the turnover of and the potential to modulate iNOS by adenosine in experimental diabetes have not been explored, we hypothesized that both the adenosine system and control of iNOS function are impaired in VSMCs from streptozotocin-diabetic rats.
  
  
  METHODS
  Male Sprague-Dawley rats were injected with streptozotocin once to induce diabetes. Aortic VSMCs from diabetic and nondiabetic rats were isolated, cultured and exposed to lipopolysaccharide (LPS) plus a cytokine mix for 24 h in the presence or absence of (1) exogenous adenosine and related compounds, and/or (2) pharmacological agents affecting adenosine turnover. iNOS functional expression was determined by immunoblotting and NO metabolite assays. Concentrations of adenosine, related compounds and metabolites thereof were assayed by HPLC. Vasomotor responses to adenosine were determined in endothelium-deprived aortic rings.
  
  
  RESULTS
  Treatment with adenosine-degrading enzymes or receptor antagonists increased iNOS formation in activated VSMCs from nondiabetic and diabetic rats. Following treatment with the adenosine transport inhibitor NBTI, iNOS levels increased in nondiabetic but decreased in diabetic VSMCs. The amount of secreted NO metabolites was uncoupled from iNOS levels in diabetic VSMCs. Addition of high concentrations of adenosine and its precursors or analogues enhanced iNOS formation solely in diabetic VSMCs. Exogenous adenosine and AMP were completely removed from the culture medium and converted into metabolites. A tendency towards elevated inosine generation was observed in diabetic VSMCs, which were also less sensitive to CD73 inhibition, but inosine supplementation did not affect iNOS levels. Pharmacological inhibition of NOS abolished adenosine-induced vasorelaxation in aortic tissues from diabetic but not nondiabetic animals.
  
  
  CONCLUSIONS
  Endogenous adenosine prevented cytokine- and LPS-induced iNOS activation in VSMCs. By contrast, supplementation with adenosine and its precursors or analogues enhanced iNOS levels in diabetic VSMCs. This effect was associated with alterations in exogenous adenosine turnover. Thus, overactivation of the adenosine system may foster iNOS-mediated diabetic vascular dysfunction.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy. nassialberto@gmail.com.|Transplant Immunology Unit, Padua University Hospital, Padova, Italy. nassialberto@gmail.com.
authors.forename: Alberto
authors.initials: A
authors.lastname: Nassi
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy. francesca.malorgio@unipd.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Malorgio
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy. serena.tedesco1988@gmail.com.
authors.forename: Serena
authors.initials: S
authors.lastname: Tedesco
authors.affiliation: Department of Medicine, University of Padova, Padova, Italy. andrea.cignarella@unipd.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cignarella
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy. rosamaria.gaion@unipd.it.
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Gaion
mesh_terms: D000241:Adenosine / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology / Q000503:physiopathology; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology* / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D058915:Purinergic P1 Receptor Antagonists / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D058915:Purinergic P1 Receptor Antagonists; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000241:Adenosine