Role of nitric oxide and endothelin in endothelial maintenance of vasoconstrictor responses in aortas of diabetic female rats.

BACKGROUND: Diabetes differentially affects the vascular system in males and females. Although various results have been reported, very few studies have focused on responses in females. In the present study, we investigated contractile responses to norepinephrine in aortas of alloxan-diabetic female rats and evaluated endothelial modulation of these responses. METHODS: Concentration-response curves were constructed to norepinephrine in the absence or presence of N(G) -nitro-l-arginine methyl ester (l-NAME), indomethacin, losartan, tezosentan, and calphostin C; pre-pro-endothelin mRNA expression was evaluated; and norepinephrine-stimulated expression of phosphorylated (p-) Akt Ser(473) , p-endothelial nitric oxide synthase (eNOS) Ser(1177) , and p-eNOS Ser(633) was determined in endothelial cells incubated in the presence of low (5 mmol/L) or high (25 mmol/L) glucose concentrations. RESULTS: Similar maximal responses (Rmax ) to norepinephrine were seen in control and diabetic endothelium-intact aortas; however, Rmax was reduced in diabetic endothelium-denuded aortas. Incubation of endothelium-intact aortas with 100 µmol/L l-NAME increased Rmax in the control group only. Inhibition of cyclo-oxygenase (10 µmol/L indomethacin) and blockade of angiotensin II receptors (10 µmol/L losartan) reduced Rmax in endothelium-intact aortas in both the control and diabetic groups. Blockade of endothelin receptors (0.1 µmol/L tezosentan) and inhibition of protein kinase C (PKC; 0.1 µmol/L calphostin C) reduced Rmax only in endothelium-intact aortas from diabetic rats. Pre-pro-endothelin mRNA expression was increased in aortas from diabetic female rats. Finally, p-Akt Ser(473) , p-eNOS Ser(1177) , and p-eNOS Ser(633) levels were enhanced after norepinephrine stimulation only in low glucose-treated endothelial cells. CONCLUSIONS: In aortas of diabetic female rats, reductions in smooth muscle contractile responses to norepinephrine are counterbalanced by the endothelium via reduced eNOS activation and increased endothelin release and PKC activation.

Decreased endothelium-dependent vasodilation in diabetic female rats: role of prostanoids.

Overproduction of vasoconstrictor prostanoids and reduced prostacyclin levels have been related to the male diabetic-linked vascular dysfunction. However, it is not clear yet if these changes also occur in diabetic females. The aim of this study was to verify the role of prostanoids in the vascular dysfunction of diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy and isolated perfused arteriolar bed), prostanoid measurement (enzyme immunoassay), superoxide generation (intravital fluorescence microscopy), and the presence of peroxynitrite (Western blot for nitrotyrosine-containing proteins). The response to acetylcholine was decreased in arterioles of diabetic female rats and diclofenac, but not ridogrel, corrected the altered response. The unstimulated (basal) release of thromboxane B2 (TXB2), but not prostaglandin F2alpha (PGF2alpha) or 6-keto-PGF1alpha, was increased in the mesenteric perfusate from diabetic female rats. Increased production of PGF2alpha and 6-keto-PGF1alpha, but not TXB2, was induced by acetylcholine in diabetic arterioles. The superoxide generation was increased in diabetic female rats and diclofenac corrected it. Diabetes increased nitrotyrosine-containing proteins in mesenteric microvessels. In conclusion, our data show that the increase of constrictor prostanoid release, most likely PGF2alpha, could be involved in the reduced endothelium-dependent vasodilation of diabetic female rats. In addition, the enhanced activation of cyclooxygenase may be a source of superoxide anion generation in this model.

Correction of endothelial dysfunction in diabetic female rats by tetrahydrobiopterin and chronic insulin.

Diabetes-induced vascular dysfunction has mainly been studied in males. However, the mechanisms involved may not correspond to those in females. Here we analyzed the effects of tetrahydrobiopterin (BH(4)) and chronic insulin on the physiology of mesenteric arterioles of alloxan-diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy), nitric oxide synthase (NOS) activity (conversion of L-arginine to L-citrulline), eNOS gene expression (RT-PCR), NO production (diaminofluorescein), reactive oxygen species (ROS) generation (intravital fluorescence microscopy) and Cu/Zn superoxide dismutase (SOD) activity (spectrophotometry) and gene expression (RT-PCR). The reduced endothelium-dependent vasodilation of diabetic females was corrected by both BH(4) and insulin. NOS activity was decreased by diabetes, but insulin did not correct it. However, NOS expression was not modified by either diabetes or insulin. Arterioles of diabetic rats exhibited lower NO production, which was fully corrected by BH(4) and only partially by insulin. ROS generation was increased in diabetic rats, and both BH(4) and insulin normalized it. Diabetes did not change SOD activity and gene expression. However, insulin increased SOD activity but not its expression. Our data suggest that, similarly to males, endothelial dysfunction in female diabetic rats involves an altered ROS/NO imbalance. In contrast to males, however, insulin does not regulate NOS in the microcirculation of diabetic females.