Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes.

backgroundAdenosine 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.

methodsMale 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.

resultsTreatment 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.

conclusionsEndogenous 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.