Nitric oxide (NO) signalling pathways were examined in the lateral aortae and dorsal aorta of the cane toad Bufo marinus. NADPH diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry found no evidence for endothelial NOS in the endothelium of toad

of a large number of mediators that regulate vascular tone, including nitric oxide (NO). First identified as endotheliumderived relaxing factor (EDRF) by Furchgott and Zawadzki (1980), it was not until 1987 that Palmer et al. (1987) demonstrated that NO and EDRF were the same molecule. Nitric oxide is a potent vasodilator that is synthesised by nitric oxide synthase (NOS), an enzyme that occurs in three major isoforms, endothelial NOS (eNOS), neural NOS (nNOS) and inducible NOS (iNOS) (Förstermann et al., 1994). Both eNOS and nNOS are calcium-dependent and NO is released via the activation of NOS by a variety of signalling molecules, including acetylcholine (Furchgott, 1996). Once synthesised, NO diffuses out of the endothelial cell or nerve fibre and into the adjacent smooth muscle cells (Ignarro, 1989; Moncada et al., 1991; Lowenstein et al., 1994). In the mammalian vasculature, NO derived from the vascular endothelium is primarily responsible for vasorelaxation; however, some blood vessels of the cerebral, pelvic and gut regions have perivascular nerves that contain neural NOS, and neurally released NO can induce relaxation (Young et al., 2000). In mammals, the vasodilatory effect of NO is mediated via an intracellular soluble guanylyl cyclase (soluble GC) located in the cytoplasm of the vascular smooth muscle cells (Chinkers and Garbers, 1991; Schmidt et al., 1993; Lucas et al., 2000). Once activated, the soluble GC catalyses the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). The subsequent increase in cGMP concentration activates cGMP-dependent protein kinase, which lowers the intracellular calcium in vascular smooth muscle cells to mediate a vasodilatory response (Lucas et al., 2000). Compared with mammals, there have only been a few studies in non-mammalian vertebrates that have suggested that an endothelial NO system exists in the vasculature of birds (Hasegawa and Nishimura, 1991), reptiles (Knight and Burnstock, 1993), and amphibians (Rumbaut et al., 1995; Knight and Burnstock, 1996). The presence of an endothelial NO system in blood vessels of fish remains controversial because of studies proposing both its existence (Mustafa and Agnisola, 1998; Fritsche et al., 2000) and non-existence (Olson and Villa, 1991; Kågström and Holmgren, 1997; Evans and Gunderson, 1998). Miller and Vanhoutte (1986) were the first to demonstrate, in amphibians, that the vasodilatory effect of applied acetylcholine in the vasculature of the American bullfrog Rana catesbeiana is endothelium-dependent. In addition, the effect of acetylcholine was reversed by methylene blue, which suggested that the vasodilation was occurring via activation of a soluble GC. However, it was the study by Knight and Burnstock (1996) that indicated that an endothelial 3093 The Journal of Experimental Biology 205, 3093–3100 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 JEB4479