Inhibition of acetylcholine-induced EDHF response by elevated glucose in rat mesenteric artery Melike Hacer O ¨ zkan, Serdar Uma * Department of Pharmacology, Faculty of Pharmacy, University of Hacettepe, 06100, Sihhiye, Ankara, Turkey Received 10 December 2004; accepted 23 February 2005 Abstract The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated. After precontraction with phenylephrine (3 Â 10 À 8 – 10 À 7 M), acetylcholine (10 À 8 –3 Â 10 À 6 M) and A 23187 (10 À 8 –3 Â 10 À 6 M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N W -nitro-l-arginine methyl ester (L-NAME) (10 À 4 M) and indomethacin (10 À 5 M). These relaxations were abolished in the presence of charybdotoxin (2 Â 10 À 7 M) plus apamin (10 À 7 M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs – Henseleit solution containing 22.2 mM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 h in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose. Similarly, relaxations to pinacidil (10 À 10 –10 À 5 M) and to sodium nitroprusside (SNP) (10 À 10 –3 Â 10 À 7 M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor- independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide (IO 2 À ) and hydrogen peroxide (H 2 O 2 ) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose. D 2005 Elsevier Inc. All rights reserved. Keywords: Endothelium-derived hyperpolarizing factor; Elevated glucose; Rat mesenteric artery; Reactive oxygen species Introduction Endothelium regulates vascular tone by releasing at least three relaxing factors including nitric oxide (NO) (Palmer et al., 1987), prostacyclin (PGI 2 )(Gryglewski et al., 1986) and an endothelium-derived hyperpolarizing factor (EDHF) that varies among different types of blood vessels (Chen et al., 1988). Although the identity and cellular mechanism of action of EDHF remains uncertain, it causes NO synthase- and cyclooxygenase-independent relaxation which is accompanied by an endothelium-dependent hyperpolarization of vascular smooth muscle (Garland and McPherson, 1992; Adeagbo and Triggle, 1993). While EDHF may provide a compensatory response to decreased NO activity in large arteries under certain pathological conditions, its role in endothelium- dependent relaxation is greater in small resistance arteries (Garland et al., 1995). Mesenteric arteries contribute substan- tially to regulation of vascular resistance and systemic circulation (Christensen and Mulvany, 1993) and EDHF is responsible for the major part of the endothelium-dependent relaxation in this vascular bed (Zygmunt et al., 1995). Diabetes mellitus is associated with a variety of vascular complications. A number of studies have demonstrated that impaired endothelium-dependent relaxations occur in arteries isolated from streptozotocin (STZ)-induced diabetic rats (Pieper and Gross, 1988; Kamata et al., 1989). There may be a primary interaction between diabetes-induced endothelial dysfunction and hyperglycemic conditions. Indeed, hypergly- 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.02.036 * Corresponding author. Tel./fax: +90 312 3052026. E-mail address: seuma@hacettepe.edu.tr (S. Uma). Life Sciences 78 (2005) 14 – 21 www.elsevier.com/locate/lifescie