Clinical and Experimental Pharmacology and Physiology (2005) 32, 728–734 MECHANISMS UNDERLYING RELAXATION OF RABBIT AORTA BY BAY 41-2272, A NITRIC OXIDE-INDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR Fernanda BM Priviero, Juliana S Baracat, Cleber E Teixeira, Mário A Claudino, Gilberto De Nucci and Edson Antunes Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, Brazil SUMMARY 1. The compound BAY 41-2272 (5-cyclopropyl-2-[1-(2- fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4- ylamine) has been described as a potent, nitric oxide (NO)- independent, stimulator of soluble guanylate cyclase. In the present study, the mechanisms underlying the relaxant effect of BAY 41-2272 in endothelium-intact and -denuded precon- tracted rabbit aortic rings were investigated. 2. Male New Zealand white rabbits were anaesthetized with pentobarbital sodium. Aortic rings were transferred to 10 mL organ baths containing oxygenated and warmed Krebs’ solution. Tissues were connected to force-displacement trans- ducers and changes in isometric force were recorded. Aortic rings were precontracted submaximally with phenylephrine (1 mol/L). 3. The addition of BAY 41-2272 (0.01–10 mol/L) to the organ bath produced concentration-dependent relaxations of the aortic rings with a higher potency in endothelium-intact (pEC50 6.59  0.05) compared with endothelium-denuded (pEC50 6.19  0.04; P < 0.05) preparations. No differences in maximal responses were observed in either preparation. The NO synthesis inhibitor N G -nitro-L-arginine methyl ester (100 mol/L) produced a 2.1-fold rightward shift in endothelium-intact (P < 0.01) rings, but had no effect in endothelium-denuded rings. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 mol/L) caused significant rightward shifts of the concentration–response curves to BAY 41-2272 of 4.9- and 2.6-fold in endothelium-intact and -denuded rings, respectively. The phosphodiesterase-5 inhibitor sildenafil (0.1 mol/L) significantly potentiated the relaxant effects of BAY 41-2272 in both endothelium-intact and -denuded rings. 4. At 1 mol/L, BAY 41-2272 significantly elevated the aortic cGMP content above basal levels in both endothelium- intact and -denuded rings. Furthermore, ODQ reduced BAY 41-2272-elicited increases in cGMP content by 17 and 90% in endothelium-intact and -denuded rings, respectively (P < 0.01). 5. In conclusion, BAY 41-2272 potently relaxes endo- thelium-intact and -denuded rabbit aortic rings. The basal release of endothelium-derived NO enhances BAY 41-2272- induced relaxations, suggesting a synergistic effect of BAY 41- 2272 and NO on soluble guanylate cyclase. In addition, the endothelium-independent relaxation involves both GMP- dependent and -independent mechanisms. Key words: aorta, BAY 41-2272, 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one, nitric oxide, sildenafil, soluble guanylate cyclase. INTRODUCTION Nitric oxide (NO) has emerged as an important signalling molecule playing a crucial role in a number of cellular functions, including the regulation of vascular smooth muscle tone. 1,2 The most prominent physiological target of NO is the enzyme soluble guanylate cyclase (sGC), which catalyses the conversion of GTP to the intracellular second messenger cGMP, which mediates NO- induced vasorelaxation. 3 The cellular effects of cGMP are mediated by the activation of different effector proteins, such as the cGMP-dependent protein kinases 4 and the cGMP-regulated phosphodiesterases (PDE). 5 The integrity of the NO/cGMP pathway is important in many physiological processes and the pathogenesis of various disease states, particularly of the cardio- vascular system, has been attributed to inappropriate activation of sGC. 6 Organic nitrates mimic the action of endogenous NO by bioconversion to NO or NO-related compounds 7 and have been used for over a century in the treatment of angina pectoris, although the development of tolerance following prolonged use represents a major drawback of this therapy. A synthetic benzylin- dazole derivative, namely 3-(5'-hydroxymethyl-2'-furyl)-1- benzylindazole (YC-1), has been described to directly stimulate sGC in platelets in an NO-independent manner and to sensitize the enzyme towards its native activator NO. 8–12 Recently, the compound BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)- 1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) has been shown to potently stimulate recombinant sGC. 13 Photoaffinity label studies identified the cysteine 238 and cysteine 243 spanning region in the -subunit of the enzyme as part of the target site for Correspondence: Dr Edson Antunes, Department of Pharmacology, Faculty of Medical Sciences, UNICAMP, PO Box 6111, 13084-971, Campinas (SP), Brazil. Email: edson.antunes@uol.com.br Received 27 January 2005; revision 6 April 2005; accepted 17 April 2005.