In contrast, substance P only activates the classical EDHF response through a HEPES-sensitive mechanism which may involve myo-endothelial gap junctions. Acknowledgments The generous gift of iloprost by Schering AG, Berlin is gratefully acknowledged. substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode answer or Krebs made up of 10?mM HEPES the EDHF response to material Fulvestrant (Faslodex) P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode answer was inhibited by iberiotoxin and by 10?M 17-octadecynoic acid. We conclude that material P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Fulvestrant (Faslodex) Incubation in HEPES-buffered Tyrode answer abolishes the EDHF responses to material P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K+ channels, activated only by bradykinin. activation of BKCa. Several studies have provided evidence that bradykinin’s action may be mediated, at least in part, by a cytochrome P450 metabolite, probably the epoxyeicosatrienoic acid, 11,12-EET (Hayabuchi em et al /em ., 1998; Frieden em et al /em ., 1999; Edwards em et al /em ., 2000). This eicosanoid is known to hyperpolarize vascular easy muscle by opening BKCa (Edwards em et al /em ., 2000). Furthermore, 17-ODYA, a suicide substrate inhibitor of cytochrome P450 epoxygenase (Zou em et al /em ., 1994), abolishes the endothelium-dependent opening of smooth muscle mass BKCa channels and partially inhibits the relaxation to bradykinin whereas it has no effect on the relaxant response to material P (Hayabuchi em et al /em ., 1998; Frieden em et al /em ., 1999). In the present study, 17-ODYA abolished the residual endothelium-dependent smooth muscle mass hyperpolarization to bradykinin following incubation in HEPES-buffered Tyrode answer. This finding strongly favours the identity of the additional hyperpolarizing factor released by bradykinin but not by material P (and which has a pharmacology unique from that of EDHF’) as an epoxyeicosatrienoic acid, probably 11,12-EET as proposed by Fisslthaler em et al /em . (2000). Conclusions The results of the present study show that this classical EDHF pathway in porcine coronary artery does not involve a cytochrome P450-derived metabolite as previously claimed (Fisslthaler em et al /em ., 2000; Fleming em et al /em ., 2001). Instead, the data indicate that bradykinin stimulates not only the EDHF pathway but also one which involves cytochrome P450. In contrast, material P only activates the classical EDHF response through a HEPES-sensitive mechanism which may involve myo-endothelial Fulvestrant (Faslodex) space junctions. Acknowledgments The nice gift of iloprost by Schering AG, Berlin is usually gratefully acknowledged. We are very grateful to Dalehead Abattoir, Ashton-under-Lyne for the supply of new pig hearts. This study was supported by grants from your British Heart Foundation (G. Edwards, M.J. Gardener, G.R. Richards, A.H. Weston) and the Medical Research Council (C.D. Glen). Abbreviations BKCalarge conductance calcium-sensitive K+ channel1-EBIO1-ethyl-2-benzimidazolinoneEDHFendothelium-derived hyperpolarizing factorHEPESN-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid)KATPATP-sensitive K+ channelL-NAN-nitro-L-arginineNOR-1()-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamideNS16191-(2-hydroxy-5 trifluoromethylphenyl)-5-trifluoromethyl-2(3 em H TSPAN16 /em )benzimidazolone17-ODYA17-octadecynoic acid.