Abstract
BackgroundDiabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. Peroxynitrite can induce formation of 3-nitrotyrosine-modified proteins. RhoA/ROCK signaling is also involved in diabetes-induced vascular dysfunction. The study aimed to investigate possible links between Rho/ROCK signaling, hyperglycemia, and peroxynitrite in small coronary arteries.MethodsRat small coronary arteries were exposed to normal (NG; 5.5 mM) or high (HG; 23 mM) D-glucose. Vascular ring constriction to 3 mM 4-aminopyridine and dilation to 1 μM forskolin were measured. Protein expression (immunohistochemistry and western blot), mRNA expression (real-time PCR), and protein activity (luminescence-based G-LISA and kinase activity spectroscopy assays) of RhoA, ROCK1, and ROCK2 were determined.ResultsVascular ring constriction and dilation were smaller in the HG group than in the NG group (P < 0.05); inhibition of RhoA or ROCK partially reversed the effects of HG. Peroxynitrite impaired vascular ring constriction/dilation; this was partially reversed by inhibition of RhoA or ROCK. Protein and mRNA expressions of RhoA, ROCK1, and ROCK2 were higher under HG than NG (P < 0.05). This HG-induced upregulation was attenuated by inhibition of RhoA or ROCK (P < 0.05). HG increased RhoA, ROCK1, and ROCK2 activity (P < 0.05). Peroxynitrite also enhanced RhoA, ROCK1, and ROCK2 activity; these actions were partially inhibited by 100 μM urate (peroxynitrite scavenger). Exogenous peroxynitrite had no effect on the expression of the voltage-dependent K+ channels 1.2 and 1.5.ConclusionsPeroxynitrite-induced coronary vascular dysfunction may be mediated, at least in part, through increased expressions and activities of RhoA, ROCK1, and ROCK2.
Highlights
Diabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite
Impairment of vascular ring contraction and dilation by high glucose or peroxynitrite may involve RhoA/RhoA-associated protein kinase (ROCK) signaling Figure 1a presents concentration-response curves showing the contractile responses of vascular rings in the various experimental groups treated with 4-AP
Inhibition of RhoA or ROCK partially reversed this effect of high glucose: the maximal contractile response of rings treated with 4-AP was significantly larger in the HG + C3 (2.13 ± 0.09 mN) and HG + Y-27632 (2.02 ± 0.16 mN) groups than in the HG group (P < 0.05; n = 6)
Summary
Diabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. RhoA/ROCK signaling is involved in diabetes-induced vascular dysfunction. DM has been reported to cause dysfunction of endothelial-dependent vasodilation of small coronary arteries [1,2,3], even during the early stages of the disease [4]. This impairment in the relaxation of coronary vessels may involve, at least in part, reduced availability of nitric oxide (NO) [5, 6]. There is strong evidence that DM increases the expressions and activities of RhoA and ROCK in various tissues, and that the resulting phosphorylation of downstream targets enhances the contraction of vascular smooth muscle cells [27,28,29,30,31,32,33]
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