(Rhamdia Quelen) Exposed to Different Salinities

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Biochemical Systematics and Ecology 46 (2013) 44–49

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Biochemical Systematics and Ecology

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Ectonucleotidase and acetylcholinesterase activities in silver catﬁsh (Rhamdia quelen) exposed to different salinities

Alexssandro Geferson Becker a,*, Thaylise Vey Parodi a, Jamile Fabbrin Gonçalves c, Margarete Dulce Bagatini d, Roselia Maria Spanevello e, Vera Maria Morsch b, Maria Rosa Chitolina Schetinger b,c, Bernardo Baldisserotto a

a Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Av. Roraima n 1000 – Cidade Univ, 97105-900 Santa Maria, RS, Brazil b Química, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil c Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil d Colegiado do Curso de Enfermagem, Universidade Federal da Fronteira Sul, 89812-000 Chapecó, SC, Brazil e Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Setor de Bioquímica, Universidade Federal de Pelotas, Campus Universitário Capão do Leão, 96010-900 Pelotas, RS, Brazil

article info abstract

Article history: The purpose of this study was to investigate whether salinity adaptation can alter the Received 8 April 2012 purinergic (ecto-nucleoside triphosphate diphosphohydrolase; NTPDase and, 50-nucleo- Accepted 8 September 2012 tidase) and cholinergic (acetylcholinesterase; AChE) systems in whole brain and blood Available online 4 October 2012 tissue of the silver catfish, Rhamdia quelen. Silver catfish were gradually adapted to salinities of 0, 4 or 8 ppt and maintained at the experimental salinity for 10 days before Keywords: brain and blood samples were collected. Blood AChE activity decreased significantly at AChE 8 ppt and significant decreases in AChE activity were observed in the brain with salinity Blood NTPDase increases. ATP hydrolysis did not change between the groups. In contrast, ADP and AMP fi fi Salinity hydrolysis in silver cat sh maintained at salinities of 4 and 8 ppt were signi cantly higher Stenohaline fish than those kept at 0 ppt. In conclusion, this study showed that there is an enhancement in 0 50-nucleotidase the NTPDase (ADP hydrolysis) and 5 -nucleotidase activities in the brains of silver catfish exposed to increased salinity. Therefore, the activities of these enzymes can act as markers of salinity changes. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Transition from fresh- to seawater can be tolerated by some fish species, called euryhaline fish, which are efficient osmoregulators (Prodócimo and Freire, 2001). Most studies regarding fish adaptation to different salinities were performed with euryhaline fish species. For example, Mozambique tilapia (Oreochromis mossambicus)(Baldisserotto et al., 1994), pufferfish (Sphoeroides testudineus and Sphoeroides greeleyi)(Prodócimo and Freire, 2001) and flounder (Paralichthys orbignyanus)(Sampaio and Bianchini, 2002) regulate plasma ion levels efficiently in salinities between 0-35 ppt. In contrast, freshwater stenohaline fishes, such as the Indian catfish (Heteropneustes fossilis)(Parwez et al., 1979) and silver catfish (Rhamdia quelen)(Marchioro and Baldisserotto, 1999), can only survive in salinities up to 10–12 ppt.

* Corresponding author. Tel.: þ55 55 3220 9382; fax: þ55 55 3220 8241. E-mail address: [email protected] (A.G. Becker).

Responses to changes in salinity may need to be rapid, such as during tidal cycles or rapid movements through estuaries, or slow, such as during the seasonal or ontogenetic acquisition of salinity tolerance in anadromous fish (McCormick, 2001). Osmotic imbalance commonly occurs during salinity adaptation, which leads to an increased energy expenditure that affects fish growth and reproduction (Franklin et al., 1992; Borski et al., 1994; Morgan and Iwama, 1996; Sampaio and Bianchini, 2002). The primary intracellular energy source is ATP, which also acts as an extracellular signaling molecule (Fields and Burnstock, 2006). Because ATP has many functions, several studies have been performed that investigate modifications in the activity of this molecule regardless of the organism in question (Sarkis and Salto, 1991; Battastini et al., 1991; Schetinger et al., 2001; Kaizer et al., 2009). ATP and the neurotransmitter acetylcholine (ACh) are both co-released and inactivated enzymatically in the synaptic cleft of ectonucleotidases and cholinesterases, respectively (Gonçalves and Silva, 2007). ATP acts as a co-transmitter and modulator of cholinergic transmission by regulating ACh release via signaling through the P2X and P2Y receptors (Cunha and Ribeiro, 2000; Dahm et al., 2006). Alterations in acetylcholinesterase (AChE) activity can affect locomotion and equilib- rium in fish exposed to pollutants, for example, and may also impair feeding, escape, and reproductive behavior (Saglio and Trijasse, 1998; Bretaud et al., 2000; Miron et al., 2005). Nucleotidases constitute a highly refined system for regulating nucleotide-mediated signaling through ATP and/or ADP hydrolysis at the surface of many cell types (Robson et al., 2006; Rico et al., 2008). The ability of ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) to hydrolyze ATP and ADP nucleotides establishes it as a fundamental player in cellular metabolism (Schetinger et al., 2001). Additionally, following hydrolysis of ATP to AMP, AMP is dephosphorylated by 50- nucleotidase to produce adenosine, which has a neuromodulatory role in the nervous system of some fish, such as Carassius auratus (Zimmermann, 1992; Rosati et al., 1995). The purpose of this study was to investigate whether salinity adaptation can alter the purinergic and cholinergic systems of a stenohaline fish: the silver catfish. To achieve this purpose, blood AChE and NTPDase as well as brain AChE and 50-nucleotidase activities in silver catfish adapted to different salinities were evaluated. As the first study of its kind, these results will contribute to the current knowledge of the roles of the central and peripheral systems of stenohaline fish during salinity adaptation.

2. Materials and methods

2.1. Chemicals and reagents

ATP, ADP and AMP substrates as well as Trizma base, sodium azide, HEPES, acetylthiocholine iodide, 5,50dithiobis-2- nitrobenzoic acid (DTNB), and Coomassie brilliant blue G were obtained from Sigma Chemical Co (St. Louis, MO, USA). Bovine serum albumin and K2HPO4 were obtained from Reagen (Colombo, PR, Brazil). All other chemicals used in this study were of the highest purity.

2.2. Experimental procedure

Silver catfish (328.7 21.4 g; 28.9 1.3 cm) were obtained from a fish farm near Santa Maria City in southern Brazil. Fish were maintained in continuously aerated 250 L tanks for at least one week prior to the start of an experiment. Eight fish per tank were progressively acclimated for seven days to the experimental salinities (0, 4 or 8 ppt; three replicates per salinity). The experimental salinities were obtained by the addition of ions according to the ion levels reported in similar salinities in an estuary in southern Brazil (Becker et al., 2011)(Table 1). Fish were maintained in these salinities for 10 days. During this period, they were fed to satiety with a commercial diet (Supra, 32% Crude Protein). Feces were siphoned out once daily, and at least 50% of the water was renewed each day as well. After the 10-day experimental period, blood (3 mL) was collected from the caudal vein with syringes using EDTA as an anticoagulant and then stored in its receptacle on ice. Fish were then euthanized by separation of the spinal cord to remove the brain, which was weighed and rapidly placed in a solution of 320 mM sucrose, 0.1 mM EDTA, 5 mM Tris–HCl, pH 7.5 (medium I) at 4 C. Brains were homogenized in 10 volumes of medium I in a motor-driven Teflon-glass homogenizer and centrifuged for 10 min at 1000 g to isolate the supernatant.

Table 1 Ion levels of the different experimental salinities used in each experiment.

Salinity (ppt)

Ion levels (mmol L 1)0 4 8 þ Na 0.68 0.17 63.95 2.12 99.41 2.23 Cl 0.87 0.15 77.25 1.98 120.54 2.48 Kþ 0.10 0.02 2.09 0.24 3.28 0.31 þ Ca2 1.11 0.06 2.29 0.28 3.62 0.26

Means SEM. 46 A.G. Becker et al. / Biochemical Systematics and Ecology 46 (2013) 44–49

2.3. Acetylcholinesterase (AChE; E.C. 3.1.1.7) activity assay for whole blood

An aliquot (40 mL) of blood was hemolyzed with phosphate buffer 0.1 M, pH 7.4 containing Triton X-100 (0.03%) and stored at 30 C for two weeks. Whole blood AChE activity was determined by the method previously described by Ellman et al. (1961) and modiﬁed by Worek et al. (1999). To achieve temperature equilibration and complete reaction of the sample matrix sulf- hydryl groups with DTNB, the mixture was incubated at 37 C for 10 min prior to the addition of 2.3 mM ACh. Enzyme activity was corrected for spontaneous hydrolysis of the substrate and DTNB degradation. Butyrylcholinesterase was inhibited by ethopropazine. AChE activity was measured at 436 nm and calculated based on the quotient of AChE activity and hemoglobin content (Hb). Hb was determined using a Zylstra-modiﬁed solution. The results were expressed as mU mmol Hb 1.

2.4. Acetylcholinesterase (AChE; E.C. 3.1.1.7) activity assay for whole brain

The supernatants of whole brain extracts were used in the AChE enzymatic assay. AChE activity was determined according þ to Ellman et al. (1961) and modiﬁed as described by Rocha et al. (1993). The reaction mixture contained 100 mM K -phosphate buffer, pH 7.5 and 1 mM 5,50-dithio-bis-nitrobenzoic acid (DTNB). This method is based on the formation of the yellow anion, 5,50-dithio-bis-nitrobenzoic acid, as measured by absorbance at 412 nm during a 2-min incubation at 25 C. The enzyme (40–50 mg of protein) was pre-incubated for 2 min. The reaction was initiated by adding 0.8 mM acetylthiocholine iodide. All samples were run in duplicate or triplicate, and enzyme activity was expressed as mmol AcSCh h 1 mg of protein 1.

2.5. NTPDase and 50-nucleotidase activity assays for whole brain

The NTPDase enzymatic assay of whole brain was performed in a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM Tris–HCl buffer, pH 8.0, in a final volume of 200 mL as described by Schetinger et al. (2000). Twenty microliters of enzyme preparation (8–12 mg of protein) was added to the reaction mixture and pre-incubated at 37 C for 10 min. The reaction was initiated by the addition of ATP or ADP as substrate to obtain a final concentration of 1.0 mM, followed by incubation for 20 min. The activity of 50-nucleotidase was determined as described by Heymann et al. (1984) in a reaction medium containing 10 mM MgSO4 and 100 mM Tris–HCl buffer, pH 7.5, in a final volume of 200 mL. Twenty microliters of enzyme preparation (8–12 mg of protein) was added to the reaction mixture and pre-incubated at 37 C for 10 min. The reaction was initiated by the addition of AMP as substrate to a final concentration of 2.0 mM, followed by incubation for 20 min. In all cases, the reaction was stopped by the addition of 200 mL of 10% trichloroacetic acid (TCA) to obtain a final concentration of 5%. Samples were then chilled on ice for 10 min. The released inorganic phosphate (Pi) was assayed as described by Chan et al. (1986) using malachite green as the colorimetric reagent and KH2PO4 as a standard. All samples were analyzed in triplicate. Enzyme activities were reported as nmol Pi released min 1 mg of protein 1.

2.6. Protein determination

Protein was measured by the Coomassie blue method according to Bradford (1976) using serum albumin as a standard.

2.7. Statistical analysis

All data were expressed as the means SEM. Homogeneity of variances among treatments was tested with the Levene test. The data presented homogeneous variances; therefore, comparisons among different groups were made using a one- way ANOVA and Tukey’s test. Analysis was performed using the software Statistica 7.0, and the minimum signiﬁcance level was set at P < 0.05.

3. Results

No mortality was observed in the silver catfish specimens adapted to the different salinities. There was a decrease of 15.36% in blood AChE activity in fish exposed to a salinity of 8 ppt compared to fish exposed to 0 ppt (Fig. 1A). Brain AChE activity decreased 37.34% and 19.28% in fish kept at 4 and 8 ppt, respectively, compared to fish maintained at 0 ppt (Fig. 1B). In addition, different salinities altered the NTPDase and 50-nucleotidase activities. ATP hydrolysis was not significantly different between the groups. Nevertheless, the ADP and AMP hydrolysis increased significantly at 4 ppt (71.37% and 47.73%, respectively) and 8 ppt (111.58% and 69.05%, respectively) when compared to the fish exposed to 0 ppt (Fig. 2).

4. Discussion

Silver catﬁsh (mean weight 1.68 g) tolerate up to 9 g L 1 of common marine salt- and seawater up to a salinity of 10 ppt for at least 96 h without mortality (Marchioro and Baldisserotto, 1999). In our study, the salinity concentrations (0, 4 or 8 ppt) were lower than that reported as the maximum concentration tolerable by Marchioro and Baldisserotto (1999) for this A.G. Becker et al. / Biochemical Systematics and Ecology 46 (2013) 44–49 47

Fig. 1. AChE activities in whole blood (A) and brain (B) extracts from silver catﬁsh adapted to different salinities. Means SEM. Different lowercase letters indicate signiﬁcant differences between salinities (P < 0.05).

species. In addition, the mean weight in this study was approximately 195-fold higher than that used by the authors reported above, indicating that at least in silver catfish, size does not influence salinity tolerance. The increase in salinity resulted in decreased brain and blood AChE activity in silver catfish. Acetylcholine is not degraded when AChE activity decreases; therefore, it accumulates within synapses, which prevents them from functioning normally (Dutta and Arends, 2003). Consequently, it is possible that an increase in salinity leads to an accumulation of ACh, which stimulates the receptors in silver catfish. A decrease of AChE activity in whole blood or plasma is characteristic of exposure to several agrichemicals: methidathion and deltamethrin in carp (Cyprinus carpio)(Balint et al., 1995), chlorpyrifos in channel catfish (Ictalurus punctatus)(Straus and Chambers, 1995) and fenitrothion in European eel (Anguilla anguilla)(Sancho et al., 1998). Thus, this parameter could be used to evaluate the adaptative “status” to salinity of fish. NTPDase enzyme hydrolyzes the nucleotides ATP and ADP (Robson et al., 2006; Rico et al., 2008). In the present study, ATP hydrolysis was not affected by salinity. However, ADP and AMP hydrolyzes, the latter of which consists of dephosphorylation of AMP to produce adenosine by the 50-nucleotidase enzyme (Zimmermann, 1992), were altered by salinity. This finding suggests an enhanced adenosine production, which is confirmed by the observed increase in AMP hydrolysis. The NTPDase þ þ þ þ and 50-nucleotidase enzymes are activated by Ca2 or Mg2 ; therefore, alterations in the ionic availability of Ca2 or Mg2 might affect the activities of these enzymes (Kaizer et al., 2007). Moreover, the increase in NTPDase and 50-nucleotidase þ þ activities could be related to a compensatory mechanism due to an increase in intraneuronal Ca2 or Mg2 levels caused by þ þ exposure to higher salinities. Higher Ca2 and Mg2 levels might facilitate formation of nucleotide-cation complexes as similarly reported in rats exposed to heavy metals, such as aluminum (Kaizer et al., 2007).

Fig. 2. NTPDase and 50-nucleotidase activities using ATP (A), ADP (B) and AMP (C) as substrates in whole brains from silver catﬁsh adapted to different salinities. Means SEM. Different lowercase letters indicate signiﬁcant differences between salinities (P < 0.05). 48 A.G. Becker et al. / Biochemical Systematics and Ecology 46 (2013) 44–49

The increase in AMP hydrolysis results in a higher level of adenosine production, which indicates that this nucleoside could act as a neuroprotector of the central nervous system (CNS) in silver catfish under stress induced by an increase in salinity. In addition, this salinity change can enhance the production of free radicals or reactive oxygen species (ROS), which damage the integrity of cellular membranes due to lipid peroxidation and protein oxidation (Miyata et al., 1993). In conclusion, AChE activity in the brain and blood decreased (the latter decreased only at a salinity of 8 ppt) in silver catfish exposed to increased salinity. Nevertheless, an increase in NTPDase (only ADP hydrolysis) and 50-nucleotidase activities was observed in the brains of silver catfish exposed to a salinity increase. Most likely, purinergic and cholinergic systems are affected by the salinity change; therefore, the activities of these enzymes can act as markers of the response to stress caused by salinity changes.

Conﬂict of interest statement

No conﬂict of interest to be stated for any of the authors.

Acknowledgments

The authors thank Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowships granted to T.V. Parodi, J.F. Gonçalves and M.D. Bagatini and also thank the Conselho Nacional de Pesquisa e Desenvolvimento Cientíﬁco (CNPq) for fellowships granted to B. Baldisserotto, V.M. Morsch, M.R.C. Schetinger, R.M. Spanevello and A.G. Becker.

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