Molecular and functional characterization of a fish inducible-type nitric oxide synthase

Abstract

Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1,127-amino acid protein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transcription factor NF-kappaB was shown to play a role in the induction of iNOS transcription.

Head Kidney-Derived Macrophages of Common Carp (Cyprinus carpio L.) Show Plasticity and Functional Polarization upon Differential Stimulation

“…Recent in vivo experiments suggest that, during infection with T. carassii, macrophages are predominantly alternatively activated during the late phase of infection (J. M. Forlenza, C. M. S. Ribeiro, B. de Vries, H. F. J. Savelkoul, and G. F. Wiegertjes, submitted for publication). In addition, NILT1 gene expression was hardly up-regulated during an infection with T. borreli, where macrophages are primarily innate activated (10,11). Currently we are developing specific Abs against the NILT and CXCR proteins to unequivocally assign these proteins as surface markers of fish macrophages.…”

“…Furthermore, aaMF increase their production of factors involved in tissue remodeling and repair and are able to inhibit type I inflammations (6). Macrophage activation in fish has been well studied with regard to NO production (7)(8)(9)(10)(11) and ROS (12)(13)(14)(15). Recently, we described carp arginase gene expression and activity, which can be used as markers for alternatively activated macrophages, and we proposed an evolutionary conservation of alternatively activated macrophages down to teleost fish (16).…”

Effect of intraperitoneally administered IL-1b-derived peptides on resistance to viral haemorrhagic septicaemia in rainbow trout Oncorhynchus mykiss

“…IL-1 up-regulates TNF-α and iNOS expression in such systems, both of which have potent anti-viral properties (Togashi et al 2000, Lokensgard et al 2001. Recently, iNOS and TNF-α have been fully sequenced in fish (Saeij et al 2000, thus providing a possible pathway for peptide-induced anti-viral activity. Indeed, Tafalla et al (1999) demonstrated that nitric oxide decreases VHSV replication in turbot macrophages in vitro.…”

In Vitro Effects of Lipopolysaccharide and Stress Hormones on Phagocytosis and Nitric Oxide Production by Enriched Head Kidney Macrophage Cultures in the Catfish

“…NO is produced by a wide range of cell types and the reaction is catalyzed by the Ca 2+ -independent enzyme NO synthase (NOS) (Barroso et al, 2000;Saeij et al, 2000;Aktan, 2004). Endothelial (eNOS, membrane bound) and neuronal (nNOS, soluble) NOS do not require any induction for the stimulation as they can be expressed constitutively in any cell type but inducible (iNOS, soluble) requires some induction for its expression, like bacterial products (Aktan, 2004).…”

“…Inducible NOS can be also induced by different cytokines so that NO serves a central role in immune mechanisms (Yang et al, 2013). NO production in macrophages by LPS stimulation has been demonstrated in goldfish (Carassius auratus) and rainbow trout (Oncorhynchus mykiss) but in some fish like common carp (Cyprinus carpio) and goldfish, macrophage-activating factors like interferon-γ (IFN-γ) can synergistically stimulate NO production (Neumann et al, 1995;Laing et al, 1999;Saeij et al, 2000;Arts et al, 2010;Pijanowski et al, 2015). In Heteropneustes fossilis, intraperitoneal injections of LPS increased the activity of iNOS in liver, kidney, heart, gills, muscle and brain and affect homeostasis (Choudhury and Saha, 2016).…”