In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.

The natriuretic peptide (NP) family consists of at least seven members; cardiac ANP, BNP and VNP and brain CNPs (CNP1-4). Phylogenetic and comparative genomic analyses showed that CNP4 is the ancestral molecule of the family, from which CNP3 and CNP1/2 were duplicated in this order, and that the three cardiac NPs were generated from CNP3 by tandem duplication. Seven members existed at the divergence of ray-finned fishes and lobe-finned fishes (tetrapods), but some of the NP genes have disappeared during the course of evolution. In ray-finned fishes, all three cardiac NPs exist in chondrostei and some migratory teleost species, but VNP is generally absent and ANP is absent in a group of teleosts (Beloniformes). In tetrapods, ANP and BNP are present in mammals and amphibians, but ANP is usually absent in reptiles and birds. Thus, BNP is a ubiquitous cardiac NP in bony fishes and tetrapods though elasmobranchs and cyclostomes have only CNP3/4 as a cardiac NP.Functional studies indicate that cardiac NPs are essential Na+-extruding hormones throughout vertebrates; they play critical roles in seawater (SW) adaptation in teleosts, while they are important volume-depleting hormones in mammals as water and Na+ are regulated in parallel in terrestrial animals. In mammals, cardiac NPs become prominent in pathological conditions such as heart failure where they are used in diagnosis and treatment. Although the functional role of BNP has not yet been fully elucidated compared with ANP in non-mammalian vertebrates, it appears that BNP plays pivotal roles in the cardiovascular and body fluid regulation as shown in mammals. ANP has previously been recognized as the principal cardiac NP in mammals and teleosts, but comparative studies have revealed that BNP is the only cardiac NP that exists in all tetrapods and teleosts. This is an excellent example showing that comparative studies have created new insights into the molecular and functional evolution of a hormone family. (C) 2011 Elsevier Inc. All rights reserved.

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.