Plasma Levels of Nitrite and Nitrate in Early and Recent Classes of Fish

The stable metabolite of nitric oxide in plasma is NO_x, the sum of nitrite plus nitrate. Measures of plasma NO_x may provide information about the nitric oxide tonus of the entire endothelium including capillary microvessels. Although data are available for mammalian species, plasma NO_x measurements in early vertebrate species are scarce. The purpose of this study was to test the hypothesis that plasma NO_x would be similar to the NO_x in the water environment for fish in early classes (Agnatha and Chondrichthye) and would exceed water NO_x levels in the known nitrite-sensitive fish (Osteichthye). Plasma samples were obtained from 18 species of adult fish (n = 167) and from their housing or natural water environment. NO_x was measured by using chemiluminescence. Plasma NO_x was detected in all species and ranged from 0.5 nmol/ml (skate) to 453.9 nmol/ml (shortnose gar). Average plasma NO_x was significantly higher in sea lamprey than in Atlantic hagfish whereas that of little skate was 3-fold lower than in spiny dogfish shark. Plasma NO_x differed significantly among early bony fish (paddlefish, pallid sturgeon, gar) yet was similar among modern bony fish, with the exception of rainbow trout. Plasma NO_x reflected water NO_x in only 2 species (hagfish and shark), and levels did not coincide with nitrite sensitivity. This study provides an expanded comparative view of plasma NO_x levels across 3 groups of early fish. The data obtained suggest a nitric oxide system in early and modern fish.Abbreviations: NOS, nitric oxide synthaseNitric oxide is generated from oxygen and L-arginine by nitric oxide synthase (NOS), an enzyme with 3 isoforms: constitutive (or endothelial cell), neuronal, and inducible. In mammals, nitric oxide is an important signaling molecule that is responsible for functions in the cardiovascular, nervous, and immune systems.¹⁹ The role that nitric oxide plays as a vasodilator molecule in the peripheral circulation is of particular importance because it serves to regulate vascular tone and total peripheral resistance.⁹Comparative studies focused on nitric oxide provide valuable information about the conserved nature of this ubiquitous molecule. Staining for NADPH-diaphorase (an enzyme equivalent to NOS) or testing for reactivity of vessels isolated from the species of interest have been used to investigate the phylogenetic roots of NOS. Insects^13,20 as well as marine invertebrates, including the horseshoe crab with its copper-containing erythrocytes,²⁶ display NOS activity and produce measurable amounts of nitric oxide. Nitric oxide appears to be responsible for a wide variety of physiology including immune function, growth, development, and neural responses.Nitric oxide is released continuously into surrounding tissues as well as into circulation. From biochemistry studies nitric oxide apparently interacts with platelets and leukocytes²⁸ as well as with hemoglobin inside red blood cells, albeit more slowly than had been thought.³⁴ In addition to their interactions with cells, nitric oxide molecules bind to various proteins in plasma, including albumin, the most abundant protein in circulation.^28,29In vivo, nitric oxide also may exist in its unbound form.²⁷ The region along the vascular wall that remains free of erythrocytes has been suggested as 1 location of unbound nitric oxide with a biologic lifetime in the range of 100 to 500 s.⁶ This length of time allows nitric oxide to affect vessels downstream from its release point, thus performing a hormone-like function.²⁸The stable metabolite of nitric oxide in the plasma portion of blood is referred to as NO_x and is the sum of the oxidative products of nitric oxide, nitrite plus nitrate. Measures of plasma NO_x provide information about chronic basal NOS activity for the entire endothelium including capillary microvessels (accounting for the largest surface area of endothelial cells). Although nitrite appears to reflect acute changes in endothelial cell NOS activity in humans,¹⁴ reports indicate uniformity in both nitrite and NO_x levels across a range of mammalian species,^12,35 likely reflecting similarities in chronic basal NOS activity, that is, nitric oxide tonus.Few studies report measures of NO_x in the plasma of early vertebrate species. Previous work indicated differences in NO_x levels of 8 mammalian species compared with the other vertebrates that were tested.³⁵ One observation from that study was that NO_x was higher and more variable in plasma sampled from fish in the classes Agnatha, Chondrichthye, and Osteichthye. In the present study, the observations were expanded to include plasma NO_x levels for a range of fishes sampled from 3 groups of fish with a spectrum of nitrite sensitivity and from a variety of natural and research housing habitats. Water NO_x levels were measured also. Our hypothesis was that levels of NO_x circulating in plasma would be similar to NO_x in the water environment for fish in the early classes (Agnatha and Chondrichthye) and above water NO_x levels only in the nitrite-sensitive fish of the Osteichthye class. Average NO_x for lamprey, skate, and trout have been published previously.³⁵