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Am J Physiol Regul Integr Comp Physiol 279: R650-R656, 2000;
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Vol. 279, Issue 2, R650-R656, August 2000

Molecular and kinetic characterization and cell type location of inducible nitric oxide synthase in fish

Juan B. Barroso1, Alfonso Carreras1, Francisco J. Esteban1, María A. Peinado1, Esther Martínez-Lara1, Raquel Valderrama1, Ana Jiménez1, José Rodrigo2, and José A. Lupiáñez3

1 Department of Experimental Biology, Biochemistry and Molecular Biology Area, Faculty of Experimental Sciences, University of Jaén, E-23071 Jaén; 2 Department of Comparative Neuroanatomy, Institute of Neurobiology "Santiago Ramón y Cajal," Consejo Superior Investigaciones Científicas, E-28002 Madrid; and 3 Department of Biochemistry and Molecular Biology, Centre of Biological Sciences, University of Granada, E-18001 Granada, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have found conclusive evidence for inducible nitric oxide synthase (iNOS) activity in rainbow trout (Oncorhynchus mykiss) tissue by means of biochemical, immunohistochemical, and immunoblotting analyses. This Ca2+-independent enzyme uses L-arginine to produce nitric oxide and L-citrulline. It was significantly inhibited by the L-arginine analogs Nomega -monomethyl-L-arginine and NG-nitro-L-arginine methyl ester. Kinetic analyses showed typical Michaelian behavior with no evidence of cooperative effects. The specific activities of the liver and head kidney enzymes were 27 and 106 pmoles · min-1 · mg protein-1, respectively, with similar values for Km (11 µM), all of which correspond well with the values for other previously characterized iNOS. Western blot analyses revealed a single band of MR = 130 kDa tested with an iNOS antiserum. At the ultrastructural level, cells with NADPH-diaphorase activity and iNOS immunoreactivity were identified as being heterophilic granulocytes in head kidney tissue and neutrophils and macrophages in hepatic tissue. The presence of an iNOS isoform in these fish tissues implies that these cells are capable of generating nitric oxide, thus pointing to the potential role of this enzyme in fish defense mechanisms.

cell immunolocalization; fish tissues; kinetic behavior; NADPH diaphorase; rainbow trout


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MECHANISMS FOR THE BIOSYNTHESIS and activity of nitric oxide (NO) in mammalian cells are well established (18, 20). NO is generated from the arginine guanidine group in a reaction catalyzed by NO synthases (NOS). Although it is a reactive free radical and potentially toxic, NO is in fact physiologically useful, being known to participate in such basic processes as vasorelaxation and to act as an intercellular messenger helping to defend against pathogens, and in this way is one of the weapons used by the cell immune system in mammals.

In mammals, the inducible NOS isoform (known as iNOS or NOS 2) is expressed in phagocytes, particularly macrophages, probably in response to proinflammatory cytokines and/or bacterial products such as lipopolysaccharides (LPS). Macrophage-derived NO is an important part of the cytostatic/cytotoxic armament of these cells, participating in the immune response to tumor cells and intracellular parasites, including viruses (22). The role of iNOS-derived NO in other cell types is less clear, but it may represent a primitive immune response to pathogens or inflammatory stimuli, responses presumably mediated by cytokines (21).

Little is known, however, about the mechanism by which fish phagocytes combat pathogens. Furthermore, the generation of reactive oxygen species (13, 21) is clearly not the only method of killing certain pathogens, and alternative mechanisms are still to be clarified (10).

Previous studies have shown that different fish tissues have the capacity to generate NO (6, 10, 23, 25, 28), and a partial cDNA for iNOS has been sequenced in rainbow trout (10) and goldfish macrophages (15). Nevertheless, despite this increasing evidence, to our knowledge, no definitive proof has so far been given for NO production by the action of enzymatic proteins, such as NOSs (EC 1.14.13.39), nor for the cell type responsible for NO production in fish tissues. Neither has the relationship between NO production in fish and their immune defense systems been established (15, 26).

We report here on the presence of an inducible isoform of NOS protein and its activity in fish liver and head kidney and also identify the cell types responsible for NO production. We have also studied several enzymatic, molecular, and immunohistochemical characteristics of this enzyme system and found unequivocal evidence for an NO-producing enzyme system in trout tissues, suggesting the possible participation of NO in fish defense mechanisms.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. L-[3H]arginine (specific activity 68 Ci/mmol) was bought from Amersham (Amersham, UK). Cation-exchange resin, AG 50W-X8, L-arginine, Nomega -monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), cofactors, protease inhibitors, and other biochemicals were bought from Sigma (Sigma Chemical, St. Louis, MO) and Boehringer (Mannheim, Germany). All other chemicals came from Merck (Darmstadt, Germany) and were of the highest purity.

Animals and tissue preparations for analytical procedures. More than sufficient juvenile rainbow trout (Oncorhynchus mykiss) of 250 g body wt were obtained from a local fish farm (Riofrío, Granada, Spain). They were kept in 350-liter fiberglass tanks containing dechlorinated water at 15.0 ± 0.5°C, with continuous aeration at a flow rate of 1.5 liter · min-1 · kg of fish-1. The light period was 12 h. After 2 wk acclimatization to laboratory conditions, we selected fish at random to form five experimental groups of 25 fish each. The fish pathogens Aeromonas salmonicida and Yersinia ruckerii were attenuated either by heating to 100°C for 40 min or by incubation with 0.7% formol for 3 h and then at 0.3% for 4 h. Four groups of fish were anesthetized in water containing 0.3 ml ethylene glycol-mono-phenylether/liter and injected with the four different treatments of the pathogens. Each fish was injected both intraperitoneally (~3 × 106 bacteria) and intravenously (~1.5 × 106 bacteria). The control group was treated similarly but with an equivalent volume of sterile saline solution. At 24 and 48 h after injection, five fish from each of the five groups were killed by cervical dislocation, and their livers and head kidneys were immediately removed and homogenized (1:3 wt/vol) in 30 mM Tris · HCl containing (in µM) 10 EDTA, 15 EGTA, 5 dithiothreitiol (DTT), 0.01 pepstatin-A, 1 phenylmethylsulphonyl fluoride (PMSF), 0.02 leupeptin-A, 0.1 benzamidine, and 0.1 tetrahydrobiopterin (BH4), pH 7.4. The homogenates were centrifuged at 105,000 g for 60 min. All procedures were carried out at 4°C. The fresh supernatant fraction was used for NOS activity and immunoblot assays.

For histochemical studies, the control and treated groups of fish were anesthetized under the same conditions, weighed, and then heparinized through the dorsal aorta (500 IU, Rovi). For head kidney and liver perfusion, the abdominal and heart cavities were exposed and a blunt 20-gauge cannula (Abbot) was inserted either transcardially (for head kidney perfusion) or into the major tributary to the hepatic portal vein (for liver perfusion) and tied securely in place. Perfusion and tissue preparation were done as described elsewhere (1, 8) for NADPH-diaphorase histochemistry and NOS immunohistochemistry.

NOS activity assay and kinetic analysis. NO synthase activity was measured by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline (4). Each sample was assayed for total activity in duplicate for 20 min at 37°C in a reaction medium containing 50 mM HEPES buffer, pH 7.4, 0.1 mM DTT, 1.25 mM CaCl2, 1 mM beta -NADPH, 10 µM FAD, 10 µM flavin mononucleotide, 10 µM BH4, 50 µg protein extract, and variable concentrations of L-arginine supplemented with L-[3H]arginine to a total volume of 200 µl. In similar incubations, 1 mM of the NOS inhibitors L-NMMA or L-NAME were used to establish specific NOS activity. To determine the levels of calcium-dependent and -independent NOS activity, each sample was incubated in both the presence and absence of 1 mM EGTA and 1 mM EGTA plus L-NMMA. When EGTA was used, no calcium was added to the medium. The reactions were stopped by adding 2 ml of chilled 20 mM HEPES buffer (pH 5.5) containing 2 mM EDTA and 2 mM EGTA. To distinguish labeled L-citrulline from L-arginine, the sample was applied to spin columns filled with 0.5 ml of a cation-exchange resin (dowex 50W, 8% cross-linkage, 200-400 mesh, Na+ form), eluted with 2 ml deionized water, and centrifuged at 8,000 g for 2 min. The total column effluent was recovered and counted by liquid scintillation.

We computed the level of L-[3H]citrulline after subtracting the blank value, which represents nonspecific radioactivity in the absence of enzyme. The values obtained in the experiments made in the absence of NADPH, which determine L-[3H]citrulline formation independent of any specific NOS activity, were also subtracted from the total level of L-citrulline. The activities were expressed as pmol L-[3H]citrulline · min-1 · mg protein-1. For kinetic assays, the initial rates of L-[3H]citrulline formation were measured by observing replicate reactions in the presence of arginine (labeled and/or supplemented when necessary) within the range of 0.5-350 µM. Proteins were determined using bovine serum albumin as standard (3).

The kinetic data were obtained with a slight modification of the method described elsewhere (1) and analyzed using a nonlinear regression method based on the rectangular hyperbola described by the Michaelis-Menten equation (7). This nonlinear plot was constructed with the aid of a computer program (GraFit, Microsoft). For illustrative and comparative analyses, the data are also presented as linear double-reciprocal plots. The activity ratio is defined as the relationship between enzyme activity at substrate-subsaturating concentration (Vss) and maximum velocity (Vmax) and is expressed in terms of the quotient Vss/Vmax. This parameter indicates which type of enzyme activity regulation is involved and is used as an index of the capacity of enzyme modulation. Catalytic efficiency, defined as the ratio between enzyme activity and its Km for each substrate, is determined at saturating substrate concentrations (Vmax/Km). This parameter is an indication of the relationship between total enzyme activity and the degree of interaction between the enzyme and its substrate.

Electrophoretic methods and immunoblot analyses. Samples from high-speed liver and head kidney supernatant, containing 30 µg of protein each, were heated to 95°C for 5 min in 62.5 mM Tris · HCl, pH 6.8, containing 2% (wt/vol) SDS, 10% (vol/vol) glycerol, and 10 mM DTT. Polypeptides were separated by 7.5% SDS-PAGE using a Bio-Rad Mini-Protean II slab cell and were transferred onto 0.2-µm polyvinylidene difluoride membrane (Immobilon P, Millipore, Bedford, MA) using a semidry transfer apparatus (Bio-Rad Laboratories) with 10 mM 3-(cyclohexylamino)-1-propanesulphonic (CAPS) buffer, 10% methanol, pH 11.0, at 1.5 mA/cm2 for 2.5 h. The membranes were blocked with 10 mM Tris · HCl, 100 mM NaCl, pH 7.5 buffer (TBS) containing 1.5% nonfat dry milk and 0.05% Tween 20. For immunodetection, the blots were then incubated overnight at 4°C with rabbit anti-iNOS antiserum (27) diluted to 1:2,500 in blocking solution. The blots were then washed with TBS buffer containing 0.1% Tween 20. Immunodetection was performed using an enhanced chemiluminescence kit (ECL-PLUS, Amersham). The blots were scanned with a computer-assisted video densitometer and photographed.

NADPH-diaphorase histochemistry and iNOS immunohistochemistry. We used histochemical staining with NADPH-diaphorase for the indirect visualization of NOS, both by light and electron microscopy (2). Some sections were incubated in the dark in a solution containing 1 mM beta -NADPH, 0.2 mM nitroblue tetrazolium (NBT), and 0.2% Triton X-100 in 0.1 M Tris · HCl (pH 7.4) for 45 min at 37°C. The sections were then rinsed in PBS, dehydrated in a graded ethanol series, cleared, and placed under a DPX (Fluka). Histochemical control experiments, in which beta -NADPH or NBT were excluded from the incubated medium, gave no positive staining.

At the ultrastructural level, NADPH-diaphorase activity was detected in the head kidney after a slight modification of the protocol described elsewhere (9). Briefly, after the NADPH-diaphorase staining mentioned above, but excluding Triton X-100 from the incubation solution, the sections were rinsed in PBS and postfixed in 2.5% glutaraldehyde in PBS for 2 h, followed by 1% osmium tetroxide and 1.5% potassium ferrocyanide in PBS for 2 h at room temperature. The sections were dehydrated through a graded series of ethanol and embedded flat in Durcupan (Fluka) resin on a microslide and, before polymerization, coverslipped with transparent films for overhead projection (Stabilo). The sections were then examined by light microscopy. The areas including NADPH-diaphorase-positive cell bodies were selected, remounted on a polymerized resin block, and serially sectioned with an ultramicrotome (Reicher-Jung). Semithin sections (2 µm) were stained with toluidine blue and examined under an optical microscope (BH2-Olympus). Ultrathin sections (50-70 nm) were then examined with a Zeiss EM 902 electron microscope without counterstaining with lead citrate and uranyl acetate.

For the immunodetection of iNOS, head kidney and free-floating liver sections were incubated with a polyclonal antibody against iNOS (1:1,000), as described elsewhere (27), and examined by light microscopy.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NOS activity and immunoblot analyses. We studied the presence and the kinetic behavior of iNOS in high-speed supernatant fractions from the liver and head kidney of fish infected with either A. salmonicida or Y. ruckerii by measuring the formation of L-[3H]citrulline.

NOS activity was detected in both the liver and head kidney of the treated fish. This activity was inhibited by arginine analogs already known to inhibit selectively mammalian NOS systems (Table 1). Whereas NOS activity was significantly inhibited (~75%) by 1 mM L-NMMA in both tissues, incubation with 1 mM of the Ca2+-chelating agent EGTA without calcium did not modify the initial values of NOS activity to any significant extent. L-NAME, another arginine analog inhibitor of NOS, was also assayed and reduced total activity in a similar way to L-NMMA (results not shown). No activity was detected in these tissues from untreated fish (Table 1). Treatment with A. salmonicida and Y. ruckerii induced iNOS activity in both tissues, although the values were higher in the head kidney than in the liver. This activity reached a maximum in both tissues after 24 h and remained constant when measured again after 48 h (Table 1).

                              
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  		Table 1.   The specific activity of iNOS in the head kidney and liver of both untreated and immunostimulated trout. The effects of NOS inhibitor (L-NMMA) and EGTA on L-citrulline formation in both these tissues of immunostimulated trout

To determine the kinetic behavior of NOS, initial rates were measured from 0.5 to 350 µM substrate. Both hepatic and renal activity followed typical Michaelis-Menten saturation curves, and the double-reciprocal plot of the data revealed close linear relationships, which also exclude any significant cooperative effects (Fig. 1). The kinetic parameters of this NO-forming enzyme in both tissues are shown in Table 2. The specific activity, Vmax, and catalytic efficiency of hepatic NOS were almost fourfold lower than those of renal NOS, whereas no significant changes were observed in Km for L-arginine or in the activity ratio between the two NOS activities. This is clearly reflected by the constancy of the proportionality of the activity rate throughout the saturation curves. To check that these tendencies are valid for the natural conditions in which fish live, we also measured the specific activity values in both tissues at 15°C (these values are given in the legend to Table 2.). As might be expected, enzyme activity in both tissues at 15°C was ~3.3- to 3.8-fold less than at 37°C.


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  		Fig. 1.   Effect of L-arginine concentration on inducible nitric oxide synthase (iNOS) activity from the head kidney and liver of immunostimulated trout. A: the saturation curves of iNOS from head kidney () and liver (open circle ). B: the Lineweaver-Burk plot of the kinetic data. iNOS catalytic activity is saturable by L-arginine with a Km of 11 µM.


                              
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  		Table 2.   Kinetic parameters of iNOS from the head kidney and liver of immunostimulated trout

To characterize the NOS isoform, we analyzed cross-reactivity by Western blotting with a polyclonal antibody against iNOS. This revealed a single polypeptide with an apparent MR of 130 kDa for renal and hepatic tissues with the same mobility as hepatic iNOS from LPS-induced rat and murine macrophage lysate (Transduction Laboratories, Lexington, KY) used as positive controls. No immunoreactive band was detected in either the hepatic or renal tissues of control fish or in the cerebellum of LPS-induced rats that were used as negative controls (Fig. 2). Densitometric scan analysis showed that the liver contained only 45% of the immunoreactive protein found in the head kidney (results not shown).


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  		Fig. 2.   Immunoblot analyses of iNOS protein levels from the head kidney and liver of immunostimulated trout. Samples containing 30 µg protein were subject to SDS-PAGE and then electroblotted onto polyvinylidene difluoride membranes and incubated with a polyclonal antibody against iNOS (1/2,500 dilution). Positions of molecular mass markers are shown on the abcissa. Lane 1: lipopolysaccharide-pretreated rat liver; lane 2: murine macrophage control lysate (Transduction Laboratories); lane 3: liver of immunostimulated trout; lane 4: head kidney of immunostimulated trout; lane 5: rat cerebellum; lane 6: liver of untreated trout; lane 7: head kidney of untreated trout. A single polypeptide with an apparent molecular mass of 130 kDa is recognized in lanes 1-4.

Immunolocalization of NOS. On studying the head kidney of the immunostimulated fish under a light microscope, we detected a significant number of cell groups showing either NADPH-diaphorase activity or iNOS immunoreactivity. These positive cells were located mainly at the periarterial level (Fig. 3, A and B). To ascertain the cell type(s), we made semithin and ultrathin sections. In the semithin sections, all the NADPH-diaphorase positive cells were located periarterially and seemed to have identical morphological features, with a characteristic granular appearance (not shown). At the ultrastructural level (Fig. 3, C and D), the formazan product could be detected inside the granules and, according to Meseguer et al. (19), these cells were identified as being heterophilic granulocytes. In the liver, on the other hand, iNOS immunoreactivity was detected mainly in neutrophils and macrophages located in the hepatic sinusoids (Fig. 3E).


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  		Fig. 3.   Photomicrographs of NADPH-diaphorase positive and iNOS-immunoreactive (IR) cells from the head kidney and liver of rainbow trout. A: light microscope micrograph of NADPH-diaphorase positive cells (*) of the head kidney showing a periarterial location (a). B: light microscope micrograph of iNOS-IR cells (*) of the head kidney showing the same distribution pattern as that of the NADPH-diaphorase positive cells in A. C: electron micrograph of the NADPH-diaphorase positive cells (arrows) of the head kidney, which were identified as heterophilic granulocytes. D: detail of C, in which the formazan product inside the granules (arrowheads) of a heterophilic granulocyte can be seen. E: light microscope micrograph of iNOS-IR cells rainbow trout liver (arrowheads, neutrophils; arrows, Kupffer cells). Scale bars: A and B, 200 µm; C, 6.36 µm; D, 1.6 µm; E, 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The possible presence of NOS activity in fish tissues was reported for the first time by Schoor and Plumb (25) in 1994. Since then, however, little more has been done to characterize this NO-forming enzyme system (5, 23), except for the report of a partial sequence for iNOS in rainbow trout using cDNA stimulated from head kidney macrophages (10, 15), and, to our knowledge, no information is available on the characterization of this enzyme system. Therefore, we have used biochemical and immunochemical approaches to find out more about the molecular properties and cell types involved in this NOS system and thus establish the ability of renal and hepatic fish tissues to express NOS activity. Our results show for the first time the characterization at both cell and molecular level of iNOS protein from the head kidney and liver in fish tissues.

Under our experimental conditions, treatment with fish bacterial pathogens markedly induced NOS activity in the head kidney and liver of immunostimulated fish. L-[3H]citrulline was synthesized in both tissues in an L-arginine-dependent manner. In addition, the traditional NOS inhibitors, L-NMMA and L-NAME, effectively inhibited the formation of L-[3H]citrulline and similarly suppressed NOS activity in these tissues. This specific inhibition would seem to confirm that the conversion of L-arginine to L-citrulline was due to NOS activity. Furthermore, by using EGTA, we have demonstrated that this NOS isoform is not calcium sensitive, indicating that an iNOS may well be the predominant isoform in head kidney and hepatic tissues.

The presence of inducible Ca2+-independent NOS has been described in several mammalian cells on activation with cytokines or LPSs (24). The results set out in Table 1 show that in the presence of a calcium-chelating agent, the activity of iNOS remains unchanged, suggesting that we are dealing with the isoform of the iNOS synthase that is insensitive to calcium. Nevertheless, insensitivity of NOS activity in these tissues toward Ca2+ should not be regarded as the sole criterion to decide which of the various different NOS isoforms is responsible for the activity in question, and so we assayed head kidney and liver proteins by Western blotting to determine the degree of inducible isoform expression. With the use of an antibody against iNOS, we found an immunoreactive polypeptide of an apparent molecular mass of 130 kDa and the same mobility as the hepatic iNOS in the LPS-induced rat and murine macrophages used as controls. This molecular mass is similar to that reported for iNOS protein from other animal sources (14). Furthermore, our Western-blotting results support the idea that the iNOS protein is highly conserved (16), reflecting the importance of this NO-mediated mechanism in its broad appearance throughout evolution (15). Furthermore, an analysis of its kinetic properties reveals that this activity presents an affinity similar to that of other inducible isoforms (11), suggesting that the NO-producing enzyme systems may play a part in the regulation of the concentration of reactive oxygen species in the extracellular microenvironment (12, 30) and act as a backup antimicrobial system (17, 29).

As far as temperature is concerned, the results set out in Table 2 show that the specific activity of iNOS in both types of tissue is some threefold lower at 15°C than it is at 37°C, which indicates that under normal physiological conditions, trout produce a lesser quantity of NO than our experiments show at 37°C. Nevertheless, the production of NO at 15°C, by this isoform, may well be perfectly sufficient to participate effectively in the fish's defense against pathogens, among other functions. This physiological implication opens an interesting field of study into the regulatory aspects of NO production via this enzyme system.

In contrast to the constitutive isoforms of NOS, under our assay conditions, the inducible isoform of this enzyme is capable of producing greater quantities of NO for longer periods of time, and under such conditions, a higher concentration of NO is available to react with O2, thus increasing the production of species that react with NO (RNOS) and generating the so-called indirect toxic effects of this compound (29). In their normal environmental conditions, for healthy development, trout require high levels of oxygen in the water. Nevertheless, the lower production of NO at 15°C would imply a concomitantly lower production of RNOS toxic species. Without these indirect toxic effects, the fish can only benefit from the direct effects of NO that occur at low concentrations of the compound (1-5 µM), i.e., controlling the concentration of free radicals and helping in the fish's defense against pathogens (5, 29).

Furthermore, the specific protein content shows that renal activity is greater than hepatic activity, thus revealing that the kidney is highly involved in NO-production. In fact, the cell site of NO production appears to be critical, although until now, no precise evidence has been found as to its location. Thus Schoor and Plumb (25) have demonstrated that the head kidney of the channel catfish generates NO but without identifying the specific cell type involved.

In fact, iNOS mRNA has been detected previously in macrophage cell lines both in trout (10) and goldfish (15), but in our research, we have detected in situ NADPH-diaphorase activity, which is widely used to locate NOS-containing cells (2), together with the immunohistochemical presence of iNOS in hepatic macrophages and neutrophils and renal heterophilic granulocytes in rainbow trout and have been able to describe the cell type responsible for NO production under immunostimulation. We believe that the production of NO in heterophilic granulocytes of the head kidney is related to the great increase in this type of cell within 24-48 h of injecting the immunostimulatory agent (26). Even though all of these cell types have been described as being involved in the immune response by producing reactive oxygen species (26), we have shown that the generation of iNOS-dependent NO by heterophilic granulocytes in the head kidney and in macrophages and neutrophils in the liver may be a method of fighting external pathogens such as bacteria, viruses, and tumor cells (31). Our results provide clear and conclusive evidence for the production of NO in rainbow trout and thus establish the potential role of iNOS expression in fish defense mechanisms.

Perspectives

A very serious problem in fish farming is one of high mortality rates that occur from time to time with all the consequent economic losses and social repercussions that this implies. Our work is aimed at reducing the risk of disease in intensive fish culture. With this in mind, it has been observed that variations in the expression of NOS can be associated with different disease states, suggesting that the synthesis of NO from L-arginine performs a regulatory role and acts as an important host defense mechanism (14), given that one of the main defense systems in any organism is closely related to the enzyme activity of iNOS (22). A greater understanding of the chemical biology of NO should provide us with a clearer insight into how this molecule can have apparently contradictory toxic, regulatory, and protective effects in biological systems and even in therapeutic applications. For this reason, it is important to arrive at a more complete understanding of the kinetic behavior of this enzyme system, together with its molecular features and cell-type localization, to open the way to controlling the development of numerous illnesses in fish cultures by manipulating the production of this reactive oxygen species. The physiological implications of this enzyme system, which intervenes in the fish's defense against pathogens, open up an interesting avenue of research into the regulatory aspects of NO production.


    ACKNOWLEDGEMENTS

We are indebted to Dr. L. O. Uttenthal (Instituto de Neurobiología Santiago Ramón y Cajal, Madrid) for generous gift of antibodies against iNOS and to Drs. J. L. Barja and A. Toranzo of the University of Santiago de Compostela (Spain) for supplying the bacterial fish pathogens. We thank Dr. A. Gálvez del Postigo of the University of Jaén (Spain) for interest and support in the microbiological techniques. Likewise, we are grateful to Dr. J. Meseguer of the University of Murcia (Spain) for discussions concerning the immunohistochemical results. We also thank our colleague Dr. J. Trout for revision and comments on the text.


    FOOTNOTES

This study has been supported by grants from the Plan Andaluz de Investigación, project-group No. CVI-157 (Junta de Andalucía, Spain) and the Comisión Interministerial de Ciencia y Tecnología, project No. PB95-0752-C03-02 from Ministerio de Educación y Ciencia (Madrid, Spain). Publication #195, from "Drugs, Environmental Toxics and Cell Metabolism" Research Group, Department of Biochemistry and Molecular Biology, Center of Biological Sciences, University of Granada, 18001 Granada, Spain.

Address for reprint requests and other correspondence: J. A. Lupiáñez, Departamento de Bioquímica y Biología Molecular, Centro de Ciencias Biológicas, Universidad de Granada, Avenida Fuentenueva s/n, E-18001 Granada, Spain (E-mail: jlcara{at}goliat.ugr.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 17 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 279(2):R650-R656
0363-6119/00 $5.00 Copyright © 2000 the American Physiological Society



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