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Altered aortic reactivity by diabetes mellitus in ovariectomized rats: role of epoxyeicosatrienoic acids (EETs)

Background: Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are vasodilators believed to be the endothelium-derived hyperpolarizing factor in a number of vascular beds. EETs may play a role in the secretion and action of insulin and the metabolism of carbohydrates and lipids. In our previous study we have demonstrated that oestrogen may have protective effect on acetylcholine induced relaxation (AChIR) of aortic rings in diabetic rats (1). Thus, the aim of our study was to investigate if EETs are involved in the mechanisms of AChIR and hypoxia-induced relaxation (HIR) of aortic rings in ovariectomized (OVX) diabetic rats.Matherials and Methods: 26 healthy, OVX Sprague-Dawley rats were divided in control (14 weeks old, N=13) and DM group (8 weeks of DM, N=13). Bilateral ovariectomy (ventral approach) was performed under anesthesia (75 mg/kg ketamine + 2.5 mg/kg midazolam i.p.) at the 5th week of age (2). After the surgery and the next day, the rats received analgesia (15 mg/kg metamizole sodium i.m.). Diabetes mellitus (DM) was induced by streptozotocin (60mg/kg i.p.) at the 6th week of age, and duration of DM was 8 weeks. Prior to decapitation, rats were anesthetized (75 mg/kg ketamine + 2.5 mg/kg midazolam i.p.). Thoracic aortic rings were used to test ACh response (10-9- 10-5 M) and response to reduced pO2 (bath gas mixture: N2 95%, CO2 5%) after precontraction with noradrenaline (NA) for 5 minutes, in the absence/presence of the NOS inhibitor L-NAME, COX- 1, 2 inhibitor indomethacin (INDO) and inhibitor of the CYP4A2 and CYP4A3 epoxygenation reactions (MS-PPOH) in the tissue bath. To test differences among groups Two-way ANOVA (AChIR protocol) and One-way ANOVA (HIR protocol) were used ; p<0.05 considered significant (SigmaPlot v11.2, Chicago, USA). The Ethical Committee of Faculty of Medicine University of Osijek approved the study.Results: OVX DM rats had significantly reduced both AChIR (P<0.001) and HIR (P=0.0017) compared to OVX controls. While both L-NAME (P<0.001) and MS-PPOH (P<0.05) significantly blocked AChIR in OVX controls, only L-NAME blocked AchIR in DM group (P<0.001). While in OVX controls HIR was significantly reduced with L-NAME (P<0.05, 19.64%), INDO (P<0.001, 47.78%) and MS-PPOH (P<0.01, 29.98%), in DM rats only INDO significantly reduced HIR (P<0.05, 38, 94%) with no significant effect of L-NAME (15.9%) and MS-PPOH (13.39%) administration on HIR. Conclusion: The results of this study confirmed our previos finding that DM impairs aortic reactivity to both ACh and hypoxia in OVX rats. Since MS-PPOH administration significantly blocked AChIR and HIR in OVX controls, and the same effect wasn't observed in DM rats, this finding suggests that diabetes may alter vascular aortic response in OVX rats by: 1) ovariectomy per se changes the mechanisms of vasorelaxation to ACh and hypoxia ; 2) diabetes alters either production of EETs and/or 3) bioavailability and/or sensitivity to EETs.

diabetes mellitus; ovariectomy; aorta; epoxyeicosatrienoic acids

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