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Background and Purpose: To investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats., Experimental Approach: Wire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels., Key Results: Superoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate- and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer⁴⁷³ Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling., Conclusion and Implications: The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³ Akt and/or eNOS uncoupling., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Endothelial cells play an important role in the regulation of vascular activity through the release of endothelium derived relaxing factor (EDRF) now believed to be nitric oxide (NO). NO and the NO donor drug nitroglycerin relax vascular smooth muscle by stimulating soluble guanylyl cyclase leading to elevation of intracellular levels of cyclic guanosine 3',5'-monophosphate (cGMP). Halothane has been shown to inhibit the action of NO on blood vessels. This study was designed to further investigate the mechanisms by which halothane attenuates NO-induced vascular relaxations. This was done by examining the effects of halothane on nitroglycerin and NO-induced relaxations in the presence and absence of the inhibitors of soluble guanylyl cyclase, methylene blue and 6-anilino-5,8-quinolinedione (LY 83583). Thoracic aortas from anesthetized male Sprague-Dawley rats were excised and cut into rings and the endothelium was removed. The aortic rings were suspended in organ baths containing Krebs solution and equilibrated at their optimal passive tension. When a stable plateau of contraction was produced by EC60 concentrations of norepinephrine, increasing concentrations of nitroglycerin or NO were added to the baths to relax the rings. This contraction-relaxation procedure was repeated three or four times. In some baths halothane was administered by a calibrated vaporizer 10 min before beginning the second procedure. Either methylene blue or LY 83583 was added to the baths 20 min before the third procedure. The combination of halothane, methylene blue or LY 83583 was added before the fourth procedure. Halothane, methylene blue or LY 83583 significantly inhibited nitroglycerin-induced relaxation individually. Halothane and LY 83583 also significantly inhibited NO-induced relaxations (5 x 10(-9)-3 x 10(-8) M and 5 x 10(-9)-3 x 10(-5) M, respectively) individually. The combination of halothane and methylene blue or halothane and LY 83583 significantly inhibited nitroglycerin-induced relaxation, also, the combination of halothane and LY 83583 significantly inhibited NO-induced relaxations. Halothane, methylene blue and LY 83583 treatment led to rightward shift in the concentration-effect curves. Halothane, in combination with methylene blue or LY 83583, produced inhibition equivalent to the sum of their individual effects. The present study demonstrates that the halothane, methylene blue and LY 83583 attenuate nitroglycerin and NO-induced relaxations of endothelium-denuded rat aortic rings. This suggests that halothane, methylene blue and LY 83583 may act through competitive antagonism at a common site of action on soluble guanylyl cyclase in the EDRF/NO relaxation pathway.

Background: Halothane has been reported to inhibit endothelium-dependent relaxation in a variety of vessels. These studies were done to determine whether this inhibition is caused by interference with synthesis, release, or action of endothelium-derived relaxing factor (EDRF) on cyclic guanosine monophosphate (cGMP) levels within the vascular smooth muscle., Methods: Rat aortic rings were suspended in aerated Krebs solution (37 degrees C) and were contracted to a stable plateau with EC60-70 norepinephrine (NE). Relaxations caused by acetylcholine (ACh; 1 x 10(-8)-1 x 10(-6) M), nitric oxide (NO; 5 x 10(-9)-1 x 10(-6) M), or nitroglycerin (NG; 2 x 10(-9)-3 x 10(-7) M) in rings contracted with NE were compared in the presence and absence of halothane. Tissue cGMP contents were measured using a radioimmunoassay method., Results: In the presence of halothane (0.5, 1.0, and 2.0 MAC), the ACh-induced relaxations were significantly attenuated in a concentration-dependent manner, an effect that was reversible. Halothane (2 MAC) significantly attenuated NO-induced relaxations at all concentrations and NG-induced relaxations at low concentrations (5 x 10(-9)-3 x 10(-8) M) but not at higher concentrations (1 x 10(-9)-3 x 10(-7) M) in denuded vessels. Nitric oxide-stimulated (5 x 10(-8)-5 x 10(-6) M) cGMP content was significantly attenuated by halothane (2 MAC) at NO concentrations between 1 x 10(-7) and 5 x 10(-6) M., Conclusions: Nitric oxide, either endogenous or exogenous, interacts with the enzyme guanylate cyclase to stimulate the production of cGMP. Halothane interfered with the relaxations caused by NO (in rings without endothelium) and decreased the NO-stimulated cGMP content. These results suggest that the site of action of halothane in attenuating endothelium-dependent relaxation in the rat aorta is within the vascular smooth muscle, rather than on the synthesis, release, or transit of the EDRF from the endothelium and that its action may involve an interference with guanylate cyclase activation.