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

Donna A Williams,1,* Mary H Flood,1 Debra A Lewis,2 Virginia M Miller,3 and William J Krause4

The stable metabolite of nitric oxide in plasma is NOx, the sum of nitrite plus nitrate. Measures of plasma NOx may provide information about the nitric oxide tonus of the entire endothelium including capillary microvessels. Although data are available for mammalian species, plasma NOx measurements in early vertebrate species are scarce. The purpose of this study was to test the hypothesis that plasma NOx would be similar to the NOx in the water environment for fish in early classes (Agnatha and Chondrich- thye) and would exceed water NOx 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. NOx was measured by using chemiluminescence.

Plasma NOx was detected in all species and ranged from 0.5 nmol/ml (skate) to 453.9 nmol/ml (shortnose gar). Average plasma NOx 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 NOx differed significantly among early bony fish (paddlefish, pallid sturgeon, gar) yet was similar among modern bony fish, with the exception of rainbow trout. Plasma NOx reflected water NOx in only 2 species (hagfish and shark), and levels did not coincide with nitrite sensitivity. This study provides an expanded comparative view of plasma NOx levels across 3 groups of early fish. The data obtained suggest a nitric oxide system in early and modern fish.

Abbreviation: NOS, nitric oxide synthase

Nitric oxide is generated from oxygen and L-arginine by nitric tric oxide molecules bind to various proteins in plasma, including oxide synthase (NOS), an enzyme with 3 isoforms: constitutive albumin, the most abundant protein in circulation.28,29 (or endothelial cell), neuronal, and inducible. In mammals, nitric In vivo, nitric oxide also may exist in its unbound form.27 The oxide is an important signaling molecule that is responsible for region along the vascular wall that remains free of erythrocytes functions in the cardiovascular, nervous, and immune systems.19 has been suggested as 1 location of unbound nitric oxide with a The role that nitric oxide plays as a vasodilator molecule in the biologic lifetime in the range of 100 to 500 s.6 This length of time peripheral circulation is of particular importance because it serves allows nitric oxide to affect vessels downstream from its release to regulate vascular tone and total peripheral resistance.9 point, thus performing a hormone-like function.28 Comparative studies focused on nitric oxide provide valuable The stable metabolite of nitric oxide in the plasma portion of information about the conserved nature of this ubiquitous mol- blood is referred to as NOx and is the sum of the oxidative prod- ecule. Staining for NADPH-diaphorase (an enzyme equivalent to ucts of nitric oxide, nitrite plus nitrate. Measures of plasma NOx NOS) or testing for reactivity of vessels isolated from the species provide information about chronic basal NOS activity for the en- of interest have been used to investigate the phylogenetic roots tire endothelium including capillary microvessels (accounting of NOS. Insects13,20 as well as marine invertebrates, including the for the largest surface area of endothelial cells). Although nitrite horseshoe crab with its copper-containing erythrocytes,26 display appears to reflect acute changes in endothelial cell NOS activity in 14 NOS activity and produce measurable amounts of nitric oxide. humans, reports indicate uniformity in both nitrite and NOx lev- Nitric oxide appears to be responsible for a wide variety of physi- els across a range of mammalian species,12,35 likely reflecting simi- ology including immune function, growth, development, and larities in chronic basal NOS activity, that is, nitric oxide tonus. neural responses. Few studies report measures of NOx in the plasma of early

Nitric oxide is released continuously into surrounding tissues vertebrate species. Previous work indicated differences in NOx as well as into circulation. From biochemistry studies nitric oxide levels of 8 mammalian species compared with the other verte- apparently interacts with platelets and leukocytes28 as well as brates that were tested.35 One observation from that study was with hemoglobin inside red blood cells, albeit more slowly than that NOx was higher and more variable in plasma sampled from had been thought.34 In addition to their interactions with cells, ni- fish in the classes Agnatha, Chondrichthye, and Osteichthye. In the present study, the observations were expanded to include

plasma NOx levels for a range of fishes sampled from 3 groups Received: 29 April 2008. Revision requested: 23 Feb 2008. Accepted: 7 March 2008. of fish with a spectrum of nitrite sensitivity and from a variety 1Capillary Physiology and Microcirculation Research Laboratory, Montana State of natural and research housing habitats. Water NO levels were 2 3 x University, Bozeman, MT; Applied Neuroradiology Research Laboratory, Departments measured also. Our hypothesis was that levels of NO circulating of Surgery, Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, MN; x 4Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, in plasma would be similar to NOx in the water environment for Columbia, MO fish in the early classes (Agnatha and Chondrichthye) and above *Corresponding author. Email: [email protected]

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Table 1. Class, order, and common and Latin names for each species water NOx levels only in the nitrite-sensitive fish of the Osteich- thye class. Average NOx for lamprey, skate, and trout have been Class Species n 35 published previously. 1 Agnatha Sea lamprey (Petromyzon marinus) 10a Atlantic hagfish (Myxine glutinosa) 5 Materials and Methods 2 Chondrichthye Animal subjects. Samples were obtained from 18 species of 1 Order: Selachii adult fish (n = 167) selected from the 3 groups of fish, Agnatha Spiny dogfish shark (Squalus acan- 5 (2 species), Chondrichthye (2 species), and Osteichthye (Chon- thias) drostei, 3 species; Teleostei, 11 species; Table 1) All fish appeared 2 Order: Rajiformes healthy and free of disease by physical examination. Procedures Little skate (Raja erinacea) 10a were approved by the Institutional Animal Care and Use Com- mittees at the University of Missouri–Columbia. 3 Osteichthye Housing and natural habitats. Table 2 lists the original source of Chondrostei 1 Order: Acipenseriformes each fish species as well as location of the housing tank if the ani- Pallid sturgeon (Scaphirhynchus albus) 8 mal was held in captivity before sampling; the sampling labora- Paddlefish (Polyodon spathula) 14 tory or field location also is provided. Within the overall sample, 2 Order: Lepisosteiformes 14 species were wild-caught, and 4 were bred in captivity. Four Shortnose gar (Lepisosteus platysto- 7 of the 14 wild-caught species were saltwater and the remaining mus) inhabited fresh water. Convenience sampling was used for 8 of the 11 teleosts. One unique source of teleosts was a large scour pit Teleostei 2 Order: Clupeiformes that formed naturally along the Missouri River during the 500-y Gizzard shad (Dorosoma cepedianum) 12 flood of 1993. The scour pit contained a number of different spe- 3 Order: Salmoniformes cies that had existed for 3 y in the pit and were isolated from the a river with no known inlet or outlet. Rainbow trout (Oncorhynchus 14 mykiss)/Montana Light cycles differed among the captive species. The lampreys were exposed to 9:15 light:dark (US Fish and Wildlife Service, 4 Order: Siluriformes Marquette, MI). For the hagfish, skate, shark, and flounder, light Brown bullhead catfish (Ameiurus 3 cycles were 12:12-h light:dark at both the Marine Biological Labo- nebulosus) ratory (Woods Hole, MA) and Mt Desert Island Biological Lab- Black bullhead catfish (Ameiurus 3 oratories (Salisbury Cove, ME). The Columbia Environmental melas) Research Center (United State Department of Interior, Columbia, Channel catfish (Ictalurus punctatus) 23 MO) maintained their light:dark cycles at 16:8 for the sturgeon, 7 Order: Perciformes paddlefish, trout, and brown bullhead catfish. Largemouth bass (Micropterus 10 Temperature. Each species was maintained at a temperature salmoides) compatible with their natural habitat. Temperature of the flow- White crappie (Pomoxis annularis) 9 through and holding tanks was monitored continuously (digital, Bluegill (Lepomis macrochirus) 11 mercury, or red-line thermometers). Average temperature for the 9 Order: Pleuronectiformes tanks was maintained within 2 °C of the temperature value indi- Yellowtail flounder (Limanda fer- 3 cated for the captive species listed in Table 2. ruginea) Dietary status. A fasting period of at least 12 h occurred be- 10 Order: Cypriniformes fore sampling blood from captive species. Prior to their fasting Common carp (Cyprinus carpio) 11 period, those fish that were bred in captivity received diets that Bigmouth buffalo (Ictiobus cyprinel- 9 were standard for each and nitrate-free. At the US Department lus) of the Interior facility, trout and brown bullhead catfish were fed a 35 standard pellets (Zeigler Brothers, Gardners, PA). Sturgeon and Published previously paddlefish ate adult brine shrimp. Some wild-caught species also were housed and maintained in tanks. The lampreys were not fed for 90 d. Hagfish, shark, and Some species were used subsequently for experiments. Four spe- flounder ate chopped squid and shrimp (Mt Desert Island Bio- cies (paddlefish, channel catfish, largemouth bass, and bluegill) logical Laboratories). The skates housed at the Marine Biological were caught and sampled in 2 different geographic locations Laboratory ate both vertebrates and invertebrates caught in high- (Table 2). In the second location for largemouth bass and bluegill quality water with a fast current, which was assumed to be free (Little Dixie Lake), samples were obtained after the fish recovered of algae accumulation and thus relatively nitrate free. The gars from census data collection procedures performed by the Mis- were fed live goldfish. souri Department of Conservation. Blood collection. Blood was collected into syringes by using 35 Measurement of NOx. Plasma samples. As with previous work, accessible sampling sites and sedation or anesthesia that was ap- samples of whole blood were placed immediately into tubes con- propriate for each species. Table 3 provides blood sampling sites, taining EDTA, transferred to wet ice, and then centrifuged (Het- sampling month, dietary status, and condition during and after tich MIKRO 22 R, Proscientific, Oxford, CT) within 1 h at 3200× g blood sampling for each species. None of the fish had surgery or for 15 min (4 to 5 °C). The plasma was removed and injected into an experimental procedure before the samples were obtained. siliconized vacuum tubes for storage at –70 °C.

432 Basal plasma NOx in early and recent fish

Table 2. Original source, housing, and geographic sampling location for each species Housing type (water Class and species Original source (no. of fish if more than 1 source) temperature) Sampling laboratory or location 1 Agnatha Sea lamprey wild-caught: Lake Michigan, MI outdoor holding tank (8–9°C) US Fish and Wildlife Service, Marquette, MI Atlantic hagfish wild-caught: Bay of Fundy, offshore New flow-through tank (seawater, MDIBL, Eastern Bay, ME Brunswick, CA 13 °C)

2 Chondrichthyes Spiny dogfish shark wild-caught: Frenchman’s Bay, ME flow-through tank (seawater, MDIBL, Eastern Bay, ME 13 °C) Little skate wild-caught: SW end of Martha’s Vineyard indoor holding tank (12–14 °C) MBL, Aquatic Resources Division; Sound, MA Woods Hole, MA

3 Chondrostei Pallid sturgeon captive-bred: egg source: USDI, Columbia, MO flow-through tank (17° C) USDI, Columbia, MO Paddlefisha captive-bred: egg source: Blind Pony Fish outdoor holding tank (17°C) Blind Pony Fish Hatchery, Sweet Hatchery (10) Springs, MO captive-bred: egg source: USDI, Columbia, MO (4) flow-through tank (17° C) USDI, Columbia, MO Shortnose gar wild-caught: Lake of the Ozarks, MO outdoor holding tank Boone County, MO 3 Teleostei Gizzard shad wild-caught – 3-y scour pit natural Missouri River, Boone County, MO Rainbow trout captive-bred: egg source, Ennis National Fish flow-through tank (17° C) USDI, Columbia, MO Hatchery, Ennis, MT Brown bullhead catfish captive-bred: egg source, USDI, Columbia, MO flow-through tank (17° C) USDI, Columbia, MO Black bullhead catfish wild-caught: 3-y scour pit natural Missouri River, Boone County, MO Channel catfisha wild-caught: private pond (7) natural Boone County, MO wild-caught: Locust Creek off Grand R. (16) natural Chariton County, MO Largemouth bassa wild-caught: 3-y scour pit (2) natural Missouri River, Boone County, MO wild-caught: Little Dixie Lake (8) natural Callaway County, MO White crappie wild-caught: 3-y scour pit natural Missouri River, Boone County, MO Bluegilla wild-caught: 3-y scour pit (3) natural Missouri River, Boone County, MO wild-caught: Little Dixie Lake (8) natural Callaway County, MO Yellowtail flounder wild-caught: Bay of Fundy, offshore New flow-through tank (seawater, MDIBL, Eastern Bay, ME Brunswick, CA 13 °C) Common carp wild-caught: 3-y scour pit natural Missouri River, Boone County, MO Bigmouth buffalo wild-caught: 3-y scour pit natural Missouri River, Boone County, MO MBL, Marine Biological Laboratories; MDIBL, Mt Desert Island Biological Laboratories; USDI, US Department of the Interior a2 locations

1,16 Water samples. Water was collected from each housing or natu- °C) and reduces NOx to nitric oxide at 85 °C. The area under the ral environment for all species at the time of blood sampling. The curve obtained from the nitric oxide analyzer was integrated and water samples were injected into siliconized vacuum tubes, kept recorded to a computer (Pentium 4, GX260 Optiplex, Dell, Round

cool in wet ice, and stored at –70 °C. Levels of NOx for 3 of the Rock, TX). The nitric oxide analyzer was calibrated daily with natural environments were sampled twice to compare NOx over KNO3 standards (range, 0.5 to 5 nM). NOx was measured within time; the scour pit could not be sampled twice because it no lon- 1 mo of sample collection.

ger exists due to changes in the riverbed. Data analysis and statistics. Levels of NOx measured in plasma

Chemiluminescence. Samples were heated to 96 °C and NOx are presented according to class and superorder of each fish spe- was measured by using chemiluminescence (Sievers Nitric Ox- cies. A wide range of maximal abscissa values that are not con- ide Analyzer, model 280i, Boulder, CO; sensitivity, 1 pmol/ml). sistent between figures was necessary due to broad differences Plasma (5 to 200 µl per sample) was injected into a receptacle that between species. For all analyses, n equals the number in each contained 5 ml 0.1 M vanadium III chloride (Aldrich Chemical, group. Plasma NOx data for each species were tested for normal- Milwaukee, WI) in 3.0 M HCl (Ricca Chemical, Arlington, TX). ity (Shapiro–Wilk W test) and are presented as individual data The vanadium III–HCl solution was made fresh weekly. Vana- points, along with an indicator for each mean. Data were ana- dium III reduces nitrite to nitric oxide at room temperature (20 lyzed by using Student t tests or 1-way ANOVA for comparisons

433 Vol 58, No 5 Comparative Medicine October 2008

Table 3. Blood collection conditions for each species Species Sampling site Month Dietary status Condition during sampling Condition after sampling 1 Agnatha Lamprey heart Jun 90-d fast sedated, sodium pentobarbital terminally anesthetized, sodium pento- (50 mg/kg IP) barbital (100 mg/kg) Hagfish caudal vein Oct 10-d fast sedated, 1-phenoxy-2-propanol terminally anesthetized ( 1-phenoxy-2- in 0.4% seawater propanol)

2 Chondrichthye Shark caudal artery Oct 2-d fast conscious euthanized (spinal cord pith) Skate caudal vein Jun 24-h fast conscious terminally anesthetized (tricane) terminally anesthetized (MS222)

3 Osteichthye Chondrostei Sturgeon caudal artery Aug unknown conscious nonterminal Paddlefish caudal artery Aug–Sep unknown conscious nonterminal Gar heart Jul 1-wk fast conscious nonterminal Teleostei Shad heart Oct unknown conscious nonterminal Trout heart Jan, Jun 24-h fast conscious euthanized (spinal transaction) Catfish heart Aug–Oct unknown conscious nonterminal Largemouth bass heart Sept unknown conscious nonterminal White crappie heart Sept unknown conscious nonterminal Bluegill heart Sept unknown conscious nonterminal Flounder caudal artery Oct 4-d fast conscious terminally anesthetized (MS222) Carp heart Sept unknown conscious nonterminal Bigmouth buffalo heart Sept unknown conscious nonterminal

within each class and superorder (JMP, SAS Institute, Cary, NC). Among Chondrichthyes, plasma NOx for shark and skate

In the case of unequal variance (Bartlett test), a Welch ANOVA ranged from 0.5 to 4.3 nmol/ml with an average plasma NOx for was used. Tukey–Kramer post hoc tests were used to compare skate almost 3-fold lower (P < 0.0001) than that for shark (Figure between species; t tests were used to compare data within species. 3). For shark, average plasma and water NOx values were indis-

Statistical significance was set at P value of less than 0.05. tinguishable. In contrast, average NOx in plasma obtained from

skate (1.1 ± 0.1 nmol/ml) was lower (P < 0.0001) than the NOx Results measured in the water of their holding tank (33.4 nmol/ml). Among Osteichthyes Superorder Chondrostei, plasma NO Water quality. Figure 1 provides levels of NO in the water that x x differed (P < 0.0001) among all 3 of the early bony fish species was sampled from the immediate surroundings of the fish in this tested (Figure 4). Levels in gars were 3 times higher than those study. NO in the water varied depending on sampling site. Flow- x in sturgeon and at least 15 times higher than those in paddle- through tanks appeared to be more effective at maintaining water fish. Average plasma NO in paddlefish was lower than the lev- quality (low NO ) than were holding tanks. Water NO levels in x x x els measured in both gar and sturgeon. All plasma levels of NO 4 flow-through tanks and the 4 natural water environments were x were higher than those of the water environments (P < 0.005). lower and more consistent than those of holding tanks with the Among Osteichthyes Superorder Teleosti, plasma NO for 11 exception of the first sampling of water from Locust Creek, MO. x fish species representing 6 of 10 orders of teleosts are presented NO in the water of Locust Creek, MO changed dramatically be- x in Figure 5. Average plasma NO ranged from 6.1 nmol/ml for tween the first and second sampling time points, most likely due x largemouth bass to 15.3 nmol/ml for bigmouth buffalo. Average to a change in run-off water quality. The second sampling of Lo- plasma NO did not differ among the 3 species of catfish (brown cust Creek was more consistent with levels in the 3 other natural x bullhead, black bullhead, and channel) that were caught and environments. sampled at a private pond in Boone County, MO. Consequently, NO in plasma of fishes. Broad differences in plasma NO ex- x x the catfish data were pooled. Significant differences in average isted between classes and species within classes. plasma NO were not detected among teleosts with the exception Among Agnatha, plasma NO was higher in lamprey than hag- x x of trout. Trout plasma NO was 2- to 3-fold higher (P < 0.0001) fish (Figure 2). For hagfish, average NO values for plasma and x x compared with all other teleosts tested. All samples of plasma water were numerically identical (3.0 nmol/ml), whereas for lam- had NO levels that exceeded (P < 0.0001) that measured in the prey plasma NO exceeded the water level. x x surrounding water.

434 Basal plasma NOx in early and recent fish

Figure 3. Nitrite and nitrate (NOx) measured in 2 species of cartilagi- Figure 1. Water nitrite and nitrate (NO ; mean ± SE) for the housing nous fish: Vertebrate Class 2, Chondrichthye. Data are presented as indi- x vidual points along with the plasma average. Water NO data are from environments (Table 2) of all species tested. Light gray bars, water sam- x pled at the same time that plasma samples were obtained; open bars, Figure 1. Note break in abscissa. *, P < 0.0001. repeat sampling of natural water environments (plasma not obtained with this set of samples); USDI, United States Department of the Inte- rior. The 3-y Missouri (MO) River scour pit did not exist at the time of the second sampling.

Figure 4. Nitrite and nitrate (NOx) measured in the plasma of 3 species

Figure 2. Nitrite and nitrate (NOx) measured in 2 species of jawless fish: of Chondrostei bony fish: Vertebrate Class 3, Osteichthye. Data are pre- Vertebrate Class 1, Agnatha. Data are presented as individual points sented as individual points along with the plasma average. Water NOx

along with the plasma average. Water NOx data are from Figure 1. Note data are from Figure 1. Location for paddlefish was Blind Pony Fish break in abscissa. *, P < 0.0001 Hatchery (Table 2). Note break in abscissa. *, P < 0.0001.

Plasma NOx in fish sampled from different locations. Figure 6 sampled from Locust Creek, MO, plasma NOx was 12 times high-

shows data for plasma NOx measured in species that existed in 2 er (P < 0.0001) than the values obtained for channel catfish ob-

different geographic locations. Average plasma NOx was higher tained from a private pond in Boone County, MO. Water NOx in (P = 0.01) for the 4 paddlefish housed in the flow-through hold- Locust Creek was 55.4 nmol/ml, at least 11 times greater than that ing tank at the US Department of the Interior facility compared at the private pond (4.8 nmol/ml). Largemouth bass from Little with the 10 paddlefish that were housed in the outdoor holding Dixie Lake, MO, and the 3-y scour pit formed by the Missouri tank at Blind Pony Fish Hatchery. Levels of NOx in the tank water River had similar average plasma NOx levels. In contrast, bluegill for these 2 locations were similar. For channel catfish caught and 435 Vol 58, No 5 Comparative Medicine October 2008

sampled from Little Dixie Lake had higher (P = 0.02) plasma NOx than did the bluegill sampled from the 3-y scour pit.

Discussion Four key points can be derived from the present study. First,

the baseline plasma NOx data confirm the presence of NOx in all

categories of fish and increase our knowledge of NOx in various species including the earliest forms. Second, 1 jawless fish (lam-

prey) and 1 cartilaginous fish (skate) displayed NOx in plasma

that differed from NOx in the water environment, whereas plasma

NOx was indistinguishable from water NOx in the other 2 spe-

cies in these groups (hagfish and shark). Third, plasma NOx was detected and displayed considerable variation among the early bony fish (Chondrostei). Fourth, with the exception of trout, strik-

ing similarities existed for plasma NOx levels among the Teleostei despite their wide range of sensitivities for accumulating nitrite. Portions of the central nervous system in lamprey stain intense- ly for NADPH-diaphorase,30 thus providing a histochemical cor- Figure 5. Nitrite and nitrate (NOx) measured in the plasma of 11 species of Teleostei bony fish: Vertebrate Class 3, Osteichthye. Data are present- relate for the high levels of circulating NOx in lamprey reported ed as individual points along with the plasma average. Water NOx data in the current study (Figure 2). In addition, nitric oxide produces are from Figure 1. Data for brown bullhead, black bullhead, and channel a biphasic (constriction then dilation) response in lamprey aortic catfish were pooled as catfish. Location for channel catfish was a pri- rings.3 These data support the existence of a nitric oxide system vate pond. Location for largemouth bass and bluegill was a 3-y Missouri in lamprey. River scour pit. One outlier for catfish (20.6 nmol/ml) and one outlier for carp (32.0 nmol/ml) were not included in the analysis. *, P < 0.0001. In contrast, the concentration of NOx in the plasma of hagfish could not be distinguished from that in the water environment. This observation is consistent with other data10 indicating no response of islet organ blood flow to nitric oxide in hagfish. In addition, nitric oxide constricted, rather than dilated, hagfish aortic rings.3 Therefore, nitric oxide is unlikely to play a prominent role in regulating vascular tone in hagfish.

In skate, plasma NOx was the lowest of all species tested in this

study and was detected at a level that was 30-fold lower than NOx in the water surrounding these fish (Figure 3). These data suggest

that the skate can isolate itself quite effectively from NOx in its immediate environment. The physiologic purpose of the small

amount of NOx in circulation of skates is unknown at this time. Histochemical studies of NOS have not been performed in this species; however, the NOS enzyme may be active in skate. Some evidence4 indicates that acetylcholine (10−5 M and greater) relaxed coronary artery rings obtained from rough skate (Raja nasuta), consistent with NOS activity.

Data in the present study for plasma NOx in spiny dogfish shark are consistent with published reports. For example, previous investigators2 did not detect a nitric oxide system in the shark ventral aorta. Similarly, vasoactivity in response to acetylcholine was not present in the dogfish coronary artery.5 Use of the whole fish to detect changes in resistance due to ni- Figure 6. Nitrite and nitrate (NO ) measured in plasma for fish species x tric oxide is a methodologic approach that reflects NOS activ- sampled in 2 geographic locations. Data are presented as individual ity more broadly than by testing rings harvested from a specific points along with the plasma average. Water NOx data are from Figure 1. Black solid markers are data provided for comparison from Figure blood vessel. Neither systemic infusion of L-arginine (a substrate 4 for paddlefish (Blind Pony Fish Hatchery) and Figure 5 for channel for NOS) nor hemoglobin (a scavenger of nitric oxide in mam- 32 catfish, largemouth bass, and bluegill. Two outlier data points for large- mals) had any hemodynamic effect in Squalus acanthias. NOx mouth bass (12.3 and 15.8 nmol/ml) and 1 outlier for bluegill (245.6 levels reported in the present study were measured in plasma nmol/ml) were not included in the analysis. #, P = 0.01; *, P < 0.0001; that was sampled systemically, could not be distinguished from &, P = 0.02. those measured in the aquatic environment, and, therefore are consistent with the earlier data.32 Consequently, it appears that

NOx is not released and does not accumulate in the circulation of normoxic shark. Nitric oxide vasoregulation however did oc- cur when sharks were exposed to hypoxia.32 In systemic plasma

samples drawn from hypoxic shark, NOx would be expected to 436 Basal plasma NOx in early and recent fish

exceed that in the surrounding water, most likely reflecting rapid predicted that largemouth bass,24 bluegill,33 flounder,11 and carp36 upregulation of vascular NOS under these conditions. Elevat- would be least susceptible to nitrite accumulation, in light of low ed levels in plasma from hypoxic shark would further validate chloride transport capabilities (largemouth bass, bluegill, carp) plasma NOx as a systemic indicator of NOS activity, potentially or an abundance of chloride in the environment to compete with reflecting endothelial cell function. nitrite (saltwater flounder). Along the same line of reasoning, we 24,33 15 To our knowledge, nitric oxide has not been studied in stur- anticipated that plasma NOx from channel catfish and trout geon, paddlefish, or gar. Consequently, the data reported here (the 2 species in this study with known nitrite sensitivity) would

(Figure 4) provide the first information on detectable levels of NOx be higher than that of the other teleosts studied. Instead, no dif- in plasma sampled from 3 early bony fish species. All plasma NOx ferences in plasma NOx levels were found among all teleosts levels exceeded NOx in the surrounding water environments. Gar studied, with the exception of trout, despite the fact that plasma displayed the highest values for NOx measured in fish to date. NOx exceeded the water levels for all of the teleosts. This obser-

The distinct differences in NOx among these fish may reflect dif- vation suggests that the use of chemiluminescence, a very sensi- ferences in nitric oxide systems, gill transport, or NOx elimination. tive technique to detect NOx, and sampling from fish species that The broad range between species coupled with relatively small were maintained in or lived in clean conditions, have revealed variance within, for example, paddlefish, may suggest the pres- the potential for basal endothelial cell NOS activity in the teleosts ence of a nitric oxide system in the circulatory systems of these tested. These data support the possibility that endogenous NOx is fish. Further study of nitric oxide and NOS enzyme activity in separate from nitrite accumulation. Chondrostei could yield important information about evolution Data for shad, crappie, and bigmouth buffalo broaden the scope of the nitric oxide system. for NOx in teleosts and are the first for these species. Again, these

The comparatively modern Teleostei have received consider- data indicate that baseline plasma NOx was higher than that in able attention from investigators who are interested in determin- the surrounding water environment, yet were not different from ing the evolutionary course of NOS, and endothelial cell NOS the other species tested, with the exception of trout. in particular. Early investigators presented evidence against en- The higher levels of NOx in water from Locust Creek, MO did dothelial cell NOS activity in trout (Oncorhynchus mykiss5,17,18,23,31) appear to be reflected in the catfish plasma samples (Figure 6) and carp (Cyprinus carpio25). Evidence of blood vessel responsive- when compared with the channel catfish from the Missouri River ness to acetylcholine in teleosts was presented first in 199531 by 3-y scour pit. This result could be predicted by the chloride trans- investigators working with the gas bladder of jeju (Hoplerythri- port hypothesis for nitrite accumulation. However, the higher 8 nus unitaneniatus) and by other scientists who studied cerebral levels of plasma NOx in bluegill (known to have low chloride blood flow in crucian carp (Carassius carassius). Other investiga- transport33) was inconsistent with a chloride transport mecha- tors, using a different approach, then provided data indicating the nism for concentrating nitrite because levels of water NOx were presence of endothelial cell NOS in the trout coronary system.21,22 similar between Little Dixie Lake and the Missouri River 3-y Further, sodium nitroprusside was shown to dilate body vascula- scour pit. Another possible explanation for the higher plasma 7 ture of a second salmonid, Atlantic salmon (Salmo salar aleuins ). NOx in bluegill from the Little Dixie Lake could be due to the

Data presented here for NOx in plasma (Figure 5) suggest that procedures used for census taking in the lake (stunning fish with the trout is unique among the teleosts tested, an interesting re- electricity). If so, the data for bluegill may reflect a unique and sult in light of the positive and negative conclusions for the pres- unanticipated response to the census procedures compared with ence of endothelial cell NOS in teleosts drawn previously from those from largemouth bass, which also experienced the census trout. The higher and more variable level of plasma NOx in trout procedure prior to sampling but did not have increased plasma relative to the other 8 teleost species suggests either a more ac- NOx. Further testing on environmental influences of water NOx tive endothelial cell NOS in trout or that trout concentrate nitrite levels and stress on plasma NOx is recommended. from their environment more effectively than do other teleost Limits of this study correspond to those associated with whole- species. Trout accumulate as much as 60 times the environmental animal studies. The data presented here are not informative with concentration of nitrite in their plasma.15 The LC50 for nitrite36 regard to cause and effect or cellular mechanisms. However, in- is 0.7 mmol/l, which is a level 140 times greater than the NOx formation obtained from cellular preparations can and must be measured in the flow-through tanks at the United States Depart- verified by studies on intact animals. Therefore, the present study ment of Interior. If all NOx in the tank water was nitrite (which is verifies the findings of some cellular reports by testing the whole- unlikely because it was well aerated), then the trout plasma NOx animal species and identifies the need for expanded studies of likely would be quite high. Instead, plasma NOx was only 6 times NOx in some early species. The data presented here have implica- the environmental level, suggesting that the water was not the tions for comparative aspects of endothelial cell biology, cardio- source of NOx. Further, the positive endothelial cell NOS results vascular physiology, whole-organ function, and environmental in whole-heart preparations of trout22 reflect the large surface area health for fish and those who eat them. of endothelium in the microcirculation (arterioles, venules, and This study was planned to present and record baseline data of capillaries), similar to our interpretation of systemically drawn NOx in the plasma of early fish. Baseline NOx information is use- plasma samples. Taking into account the plasma NOx data for ful to veterinarians and persons associated with animal husband- trout reported here along with data from whole-organ studies ry who advise researchers who work with fish. These data may be of trout, an endogenous source of NOx likely is present in this useful with regard to evaluating animal housing units and aquar- teleost. ia in a university setting. For example, 1 important species used Chloride transport in the gill appears to be associated with currently in developmental biology is the zebrafish. Data reported whether teleosts accumulate nitrite from their environment.11,36 in Figure 5 for carp and bigmouth buffalo would provide a useful From studies of chloride transport and nitrite accumulation, we comparison to the zebrafish, which also is categorized in Order

437 Vol 58, No 5 Comparative Medicine October 2008

10, Cypriniforme (Table 1). Further, Fish and Wildlife divisions at 10. Jansson L, Falkmer S. 1998. Blood flow to the pancreatic islet paren- universities and various government environmental groups as- chyma of the Atlantic hagfish (Myxine glutinosa). Horm Metab Res signed to monitor water quality and the impact of water runoff on 30:182–187. fish may find the NO data useful for future comparisons. Finally, 11. Jensen FB. 2003. Nitrite disrupts multiple physiological functions x in aquatic animals. Comp Biochem Physiol A Mol Integr Physiol the comparative baseline data for NO in plasma may be useful x 135:9–24. for study of the function of circulating nitric oxide. 12. Kleinbongard P, Dejam A, Lauer T, Rassaf T, Schindler A, Picker In conclusion, data in this study provide an expanded com- O, Scheeren T, Godecke A, Schrader J, Schulz R, Heusch G, Schaub parative view of baseline plasma NOx levels across 3 groups of GA, Bryan NS, Feelisch M, Kelm M. 2003. Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radic early fish. NOx was detected in the plasma of all species tested. In the context of the aquatic environment, lamprey and skate from Biol Med 35:790–796. classes Agnatha and Chondrichthye show evidence of a nitric ox- 13. Kuzin B, Roberts I, Peunova N, Enikolopov G. 1996. Nitric oxide ide system. This conclusion is consistent with available literature regulates cell proliferation during Drosophila development. Cell 87:639–649. on these ancient vertebrates. Class Osteichthye demonstrated 14. Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, variability in plasma NOx among early bony fish and consistency Kelm M. 2001. Plasma nitrite rather than nitrate reflects regional in levels of circulating NOx in plasma of the more modern bony endothelial nitric oxide synthase activity but lacks intrinsic vasodila- fish, with the exception of trout. The trout was distinctive among tor action. Proc Natl Acad Sci USA 98:12814–12819. teleosts tested with regard to nitric oxide. 15. Margiocco C, Arillo A, Mensi P, Schenone G. 1983. Nitrite bioac- cumulation in Salmo gairdneri Rich. and hematological consequences. Aquat Toxicol 3:261–270. Acknowledgments 16. Menon NK, Pataricza J, Binder T, Bing RJ. 1991. Reduction of The authors offer a special thank you to the following individuals and biological effluents in purge and trap micro reaction vessels and institutions involved in providing blood samples for many different detection of endothelium-derived nitric oxide (EDNO) by chemilu- species: Greg Baldwin and Geraldine Larson (US Fish and Wildlife minescence. J Mol Cell Cardiol 23:389–393. Service, Marquette, MI), Martha Radner (Mt Desert Island Biological 17. Miller VM, Vanhoutte PM. 1992. Endothelium-dependent vascular Laboratories, Salisbury Cove, ME), Dr Susan Jones (US Department of responsiveness: evolutionary aspects. New York: Marcel Decker. the Interior, Columbia, MO), Andy Sexton (Aquatic Resources Division, 18. Miller VM, Vanhoutte PM. 2000. Prostaglandins by not nitric oxide Marine Biological Laboratories, Woods Hole, MA), Dr George R Kracke are endothelium-derived relaxing factors in the trout aorta. Acta (Department of Anesthesiology, School of Medicine, University of Pharmacol Sin 21:871–876. Missouri–Columbia, MO), Dr Leonard R Forte (Department of Medical 19. Moncada S, Palmer RM, Higgs EA. 1991. Nitric oxide: physiology, Pharmacology and Physiology, University of Missouri–Columbia, MO), pathophysiology, and pharmacology. Pharmacol Rev 43:109–142. and Dr Charles F Rabeni (Department of Fisheries and Wildlife Sciences, 20. Muller U. 1997. The nitric oxide system in insects. Prog Neurobiol University of Missouri–Columbia, MO), Department of Conservation at 51:363–381. Blind Pony Fish Hatchery (Sweet Springs, MO), and the Missouri River 21. Mustafa T, Agnisola C. 1998. 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