Characterization of Cyprinus carpio brain nitric oxide synthase

Neuroscience Letters 242 (1998) 155–158

Characterization of Cyprinus carpio brain nitric oxide synthase A. Conte*, E. Ottaviani Department of Animal Biology, University of Modena, via Berengario 14, 41100 Modena, Italy Received 12 October 1997; received in revised form 12 December 1997; accepted 9 January 1998

Abstract Nitric oxide synthase (NOS) activity is found both in soluble and in particulate fractions of the carp brain. The Km values for arginine are 2.8 ± 0.5 and 3.3 ± 0.4 mM for the soluble and particulate fractions, respectively. Ki for NG-monomethyl-L-arginine inhibitor are 2.6 ± 0.5 and 2.9 ± 0.6 mM, and activation energy for the breakdown of the substrate-enzyme complex 8120 ± 710 and 4620 ± 450 cal per mole. Carp enzyme shows higher affinity than rat NOS for Ca2+ and for the competitive inhibitor 7nitroindazole.  1998 Elsevier Science Ireland Ltd.

Keywords: Nitric oxide synthase; Teleost; Brain

The free radical nitric oxide (NO) plays an important role as a physiological mediator, performing a number of different functions, including regulation of vascular tone, brain cellular signalling, and elimination of pathogens [13]. Three isoforms of NO synthase (NOS) have been characterized in mammals. Two of these are constitutive and Ca2+-calmodulin dependent: one is soluble and present in neurons, while the other is bound to the membrane in endothelial cells. The third isoform is inducible and Ca2+-independent, and is expressed in activated macrophages [13]. Using histochemical and immunocytochemical methods, putative NO-producing systems have been described in the brain of different non-mammalian vertebrates including fish [4,11,16], amphibians [5], reptiles [6] and birds [3]. Holmqvist et al. [10] demonstrated that in the brain of Atlantic salmon neuronal, NOS-like molecules are associated with vesicles in neural processes and are accumulated at different sites along dendritic profiles, as well as at synaptic and nonsynaptic sites. In the brain of this fish, NO may play an important role in modulating neural signals involved in the visual system and the hypothalamo-hypophysial axis. Moreover, in teleosts, NO may participate in the functions of specialized neural circuits such as the cerebrospinal fluid (CSF)-contacting system [10,16]. NO involvement in cere-

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bral flow regulation in the carp has also been demonstrated [12]. No data are presently available on the quantitative expression or the kinetic and regulatory properties of cerebral isoenzymes in non-mammals. We have investigated the properties of NOS in the carp brain, since this fish tolerates large thermal variations and survives for several months in very low oxygen tension conditions. For comparison some properties of rat brain NOS are also reported. 3 L-[2,3,4,5– H]Arginine monohydrochloride (58 Ci/ mmol), purified as reported in [7], was obtained from Amersham, (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrocloride (BH4) from Schircks Laboratories and all other reagents from Sigma. Soluble and pellet fractions from the brain of male Wistar rat and carp (Cyprinus carpio) were prepared according to Conte and Ottaviani [7]. Pellets were re-suspended twice with PBS, centrifuged and re-suspended with the homogenization buffer. NOS activity in soluble and particulate fractions was determined by citrulline formation. Standard reaction mixtures contained 50 mM HEPES pH 7.4, 0.5 mM ethylene diamine tetracetic acid, 1.4 mM CaCl2, 1 mM MgCl2, 1 mM NADPH, 1 mM dithiothreitol, 10 mM flavin adenine dinucleotide (FAD), 10 mM flavin mononucleotide (FMN), 100 mM BH4, 1 mM calmodulin, 12 mM L-valine, 1 mM citrulline, a variable amount (0.5–10 mM) of L-arginine, 80 000–

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00063- 9

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100 000 ct/min of purified tritiated arginine and 2–10 ml of enzyme fraction in a final volume of 100 ml. In some cases, incubations also contained variable concentrations of 7nitroindazole (7-NI) S-methylisothiurea sulfate (SMT) and diphenyleneiodonium (DPI) or trifluoperazine (TFP). After 5–20 min incubation at 37°C, [3H]citrulline was quantified by liquid-scintillation counting after separation from [3H]arginine [7]. The NOS activity was quantified by calculating the difference between [3H]citrulline produced in the presence and in the absence of 10 mM NG-monomethyl-Larginine (L-NMMA). Enzyme activity is given as nmol of [3H]citrulline formed per min for each gram of tissue. In all experimental conditions, the initial velocity was determined. To validate the NOS activity of the carp brain preparations, the stoichiometry of nitrite + nitrate and citrulline was also determined [7]. The free calcium concentrations were calculated following Fabiato [9]. The results are the mean ± SD of at least four independent experiments in triplicate. The Km values for L-arginine were 2.8 ± 0.5 and 3.3 ± 0.4 mM for soluble and particulate carp NOS, respectively. The Ki values for L-NMMA were 2.6 ± 0.5 and 2.9 ± 0.6 mM. The Km values for soluble and particulate rat brain were 1.6 ± 0.3 and 1.2 ± 0.4 mM, and the LNMMA Ki 1.3 ± 0.2 and 1.1 ± 0.2 mM, respectively. The NOS activity in the soluble and particulate fractions of the carp brain determined as [3H]citrulline formation at 37°C and extrapolated to the saturating arginine concentration, was 2.08 ± 0.65 and 0.73 ± 0.21 nmol/min per g tissue. The NOS activity in soluble and particulate fractions determined by the nitrate + nitrite formation at 25 mM arginine was 2.41 ± 0.86 and 0.78 ± 0.27 nmol/min per g tissue respectively. NOS activity shows the same distribution in rat brain (3.64 ± 0.89 and 1.10 ± 0.29 nmol/min per g tissue in soluble and particulate, respectively). Table 1 shows the co-factor requirement. NOS activity is almost completely inhibited in the soluble and particulate

fractions by the omission of NADPH. When FAD, FMN, BH4 or calmodulin are omitted, there is less inhibition. The NOS activity of both carp supernatant and particulate isoforms are Ca2+-dependent and show greater affinity for Ca2+ than rat NOS. At low Ca2+ concentrations (20 nM), carp NOS is still active, while rat NOS is almost completely inactive. We also studied the effect of 7-NI (a selective inhibitor of brain NOS [14]), SMT (a selective inhibitor of inducible NOS [18]), DPI (which interferes with both NADPH and FAD [17]), and TFP (a calmodulin antagonist [8]). The IC50 values are shown in Table 2. 7-NI is a more effective inhibitor of carp NOS. Soluble and particulate NOS show opposing sensitivity to 7-NI in carp and rat brain. Similar inhibition of soluble and pellet carp activities is observed with DPI and SMT. These also inhibit rat NOS. Soluble NOS is more sensitive to TFP than particulate NOS in both carp and rat fractions. We tested the NOS activity of soluble and particulate fractions at different temperatures (5, 10, 15, 19, 23, 28 and 37°C). Fig. 1 shows the Arrenhius plot of NOS activity at saturating arginine concentration. A straight line is obtained for the four enzyme preparations. The soluble and particulate carp NOS preparations show an activation energy of 8120 ± 710 and 4620 ± 450 cal/mol, respectively. Higher values are obtained for rat enzyme, i.e. 11480 ± 860 cal/mol for soluble NOS and 9980 ± 790 cal/mol for the particulate fraction. Histochemical and immunocytochemical investigations using NADPH-diaphorase detection and antisera against mammalian neuronal NOS have shown that putative NOS molecules are found in various areas of the brain in teleost fish (salmon, trout, goldfish) [4,10,11,16]. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) completely blocks the increase in cerebral flow velocity induced by acetylcholine in crucian carp [12]. We first demontrated that NOS activity is present in the carp brain in both soluble

Table 1 Effect of various co-factors Co-factors omitted

Residual activity (%) Carp

None NADPH BH4 FAD FMN BH4, FAD, FMN Calmodulin Ca2+ (100 nM) Ca2+ (20 nM) Ca2+ (3 nM)

Rat

Soluble

Pellet

Soluble

Pellet

100 5 ± 0.4 18 ± 1.3 93 ± 7.4 60 ± 4.2 7 ± 0.6 87 ± 7.8 85 ± 6.2 39 ± 2.7 4 ± 1.1

100 0 20 ± 1.6 92 ± 8.2 55 ± 6.0 6 ± 0.6 72 ± 7.5 68 ± 6.0 16 ± 1.5 3 ± 0.4

100 6 ± 0.4 19 ± 1.5 95 ± 10 89 ± 8.1 13 ± 1.0 96 ± 7.6 32 ± 4.0 2 ± 0.2 0

100 0 21 ± 1.7 91 ± 6.3 74 ± 8.1 8 ± 0.7 79 ± 7.1 44 ± 5.3 4 ± 0.3 0

Standard reaction mixtures containing 3 and 1.5 mM L-arginine for carp and rat, respectively, were prepared as detailed in the text. Each co-factor was omitted from the reaction mixture and the NOS activity was determined and expressed as a percentage of the value obtained in the presence of all co-factors. Data are the mean ± SD of four independent experiments performed in triplicate.

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A. Conte, E. Ottaviani / Neuroscience Letters 242 (1998) 155–158 Table 2 IC50 values of various inhibitors on NOS activity Inhibitor added Carp Soluble Pellet Rat Soluble Pellet

7-NI (nM)

DPI (nM)

SMT (nM)

TFP (mM)

390 ± 25 590 ± 43

350 ± 21 210 ± 16

437 ± 31 395 ± 27

95 ± 7 505 ± 33

7800 ± 546 2300 ± 168

320 ± 29 437 ± 32

596 ± 45 1000 ± 93

90 ± 8 420 ± 34

The inhibitors were included in standard reaction mixtures containing 3 and 1.5 mM L-arginine for carp and rat, respectively, and prepared as detailed in the text. Data are the mean ± SD of four independent experiments performed in triplicate.

and particulate fractions. Seventy-five percent of the total activity is soluble. Mammalian neuronal isoenzyme is in the soluble fraction, while endothelial isoenzyme is associated to the plasma membrane by a N-terminal myristate [13]. In the brain of Atlantic salmon, NOS activity is localized in neuronal processes associated with the plasma membrane of dendrites, at terminal formations within synaptic vesicles, and in neuronal somata, mainly in the cristae of some large mitochondria [10]. On the basis of these observations, the NOS present in the carp particulate fraction could be constituted by both neuronal and endothelial isoenzymes. The behaviour of soluble and particulate activities with respect to the so called ‘specific’ inhibitors gives no additional information. 7-NI, an inhibitor of neuronal mammalian isoform, inhibits the soluble and corpuscolate NOS of carp and rat brain. However, the anti-psychotic drug TFP, a calmodulin antagonist, is less effective on carp and rat membranebound NOS. This is particularly significant, because in vivo

the action of TFP may be less effective on the calmodulindependent enzymes when they are membrane-bound. From a biological point of view, NO is an old signalling molecule. In invertebrates it is also involved in the longterm potentiation of synaptic transmission as in mammals [15]. In fish, several neural circuits involved in sensory, hypophysiotrophic brain functions, as well as the CSF-contacting system, probably utilize NO as a signal molecule. Neuronal NOS isoenzyme is widespread in the rat brain, with particularly large amounts in the cerebellum [2]. The neural circuits that utilize NO as a messenger may differ in different groups of vertebrates. Although NO synthesis is a biochemically conserved mechanism, the interspecies variability might correspond to differences in the relative activity of the different brain areas in each class of vertebrates [1]. The total brain NOS measured in carp at 37°C is about 59% of rat NOS. However, the carp generally lives at 18°C in the aquarium, and at this temperature NOS activity falls

Fig. 1. Effect of temperature on initial velocity of the soluble and particulate NOS in carp and rat brains. Standard reaction mixtures containing saturating arginine concentration were prepared as detailed in the text. Each point is the mean of four independent experiments.

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to 25% of the level in rat. The lower NOS activity in the carp brain could be due to fewer NOS neurons, or to less enzyme activity in these neurons or both. The hypothesis of less activity in NOS neurons is supported by the longer incubation time required by the histochemical demonstration of NOS activity in rainbow trout [16]. At low concentration (,5 mM), NO performs various activities, including defence and free radical scavenging [19]. However, at high concentrations (.20 mM), it may also be toxic as a result mainly of reactive nitrogen oxide species (RNOS) formed from NO and O2. It is known that neuronal and endothelial NOS generate low levels of NO controlled by Ca2+ activation [19]. Furthermore direct reactions of NO with oxyhemoglobine, superoxide and components of the mitochondria prevent RNOS formation, while endogenous antioxidants (e.g. glutathione, ascorbate) play a critical role in scavenging RNOS [19]. However it must be remembered that NO chemistry has mainly been studied at 37°C and in normoxic conditions. Even if between 20 and 37°C the reaction between NO and O2 is little affected by pH and temperature, at lower temperature differences in NO reactivity and toxicity may be expected. Carp is able to sustain large variations in temperature and relative oxygen pressure, and the control of NO concentration appears to be more difficult than in mammals. For this reason, we surmise that the lower NOS activity and the smaller quantity of NO produced by neurons and endothelial cells may be more advantageous for this animal. We have observed that the hemocytes of the freshwater snail Viviparus ater, which share the same habitat as the carp, produce, after stimulation, less NO than the corresponding mammalian cells (macrophages) [7]. In the carp brain, NO intervenes in the regulation of cerebral flow in normoxic conditions, while in anoxia or during hibernation, adenosine is the main regulator of cerebral flow [12]. As seen in many poikilotherms, the decrease in activity as a function of temperature is less than in homeotherms. The curves of the Arrenhius plot up to 5°C show that particulate and soluble NOSs are still active at this temperature, producing 73 and 49%, respectively, of the activity seen at 18°C. This observation suggests that the lack of NO production at low temperature during hypoxia and hibernation may be due either to calcium regulation of neuronal and endothelial NOS activity or to the fact that oxygen may become the limiting factor in the reaction. In this condition, the regulation of cerebral blood flow depends on the unbalance of energy metabolism and not on Ca2+-dependent cellular NOS activities. This may be advantageous, since no oxygen is consumed in the reaction and no potentially toxic free radicals are formed under these conditions. This work was supported by a MURST Grant to E.O. [1] Arevalo, R., Alonso, J.R., Garcı`a-Ojeda, E., Brinon, J.G., Crespo, C. and Aijon, J., NADPH-diaphorase in the central

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