Arch Environ Contam Toxicol (2008) 55:49–56 DOI 10.1007/s00244-007-9102-5
Response of Na+-dependent ATPase Activities to the Contaminant Ammonia Nitrogen in Tapes philippinarum: Possible ATPase Involvement in Ammonium Transport Alessandra Pagliarani Æ Patrizia Bandiera Æ Vittoria Ventrella Æ Fabiana Trombetti Æ Maria Pia Manuzzi Æ Maurizio Pirini Æ Anna Rosa Borgatti
Received: 22 November 2007 / Accepted: 22 November 2007 / Published online: 4 January 2008 Ó Springer Science+Business Media, LLC 2007
Abstract In vivo and in vitro experiments elicited different responses to ammonia nitrogen (ammonia-N) of gill and mantle Na,K-ATPase and ouabain-insensitive NaATPase activities in the Philippine clam Tapes philippinarum. Short-term (120 h) exposed clams to sublethal ammonia-N (NH3+NH+4 ) concentrations (1.5 and 3.0 mg/L ammonia-N) showed enhanced gill and mantle ouabaininsensitive ATPase activity and decreased mantle Na, K-ATPase activity with respect to unexposed clams, while gill Na,K-ATPase was unaffected. In vitro experiments showed that NH+4 could efficiently replace Na+ in ouabain-insensitive ATPase activation and K+, but not Na+, in Na, K-ATPase activation. Simple saturation kinetics was constantly followed with similar K0.5 values to that of the substituted cation. The same maximal ouabain-insensitive ATPase activation was obtained at 80 mM Na+ or NH+4 in the gills and at 50 mM Na+ or NH+4 in the mantle and that of Na,K-ATPase at 10 mM K+ or NH+4 in the presence of 100 mM Na+ in both tissues. The two coexistent ATPase activities maintained their typical response to ouabain also when stimulated by NH+4 : when activated by Na++K+ or by Na++NH+4 the ATPase activity was completely suppressed by 10-3 M ouabain, whereas the Na+- or NH+4 -stimulated ATPase activity was unaffected by up to 10-2 M ouabain. The whole of the data suggests a possible involvement of the two ATPase activities in NH+4 transmembrane transport.
A. Pagliarani (&) P. Bandiera V. Ventrella F. Trombetti M. P. Manuzzi M. Pirini A. R. Borgatti Department of Biochemistry ‘‘G. Moruzzi’’, Section of Veterinary Biochemistry, University of Bologna, Via Tolara di Sopra, 50, 40064 Ozzano Emilia, BO, Italy e-mail:
[email protected]
In farmed coastal waters with high mollusc density, ammonia nitrogen (ammonia-N), represents the main contaminant (Ali and Nakamura 2000). Ammonia-N especially accumulates at seawater–sediment interfaces (Camargo and Alonso 2006), negatively affects many biological functions and reduces mollusc production (Huchette et al. 2003). The chemical equilibrium between the two forms of ammonia-N, NH3 and NH+4 , is a function of temperature, salinity and, to a large extent, pH (Emerson et al. 1975, Johansson and Wedborg 1980). The mechanisms of ammonia-N toxicity are yet not completely elucidated, but have been mainly ascribed to the lipophilic uncharged form NH3, though it represents the far lower component at seawater and physiological pHs (Emerson et al. 1975, Johansson and Wedborg 1980). Recently, the prevailing NH+4 form has been increasingly considered (Camargo and Alonso 2006, Kater et al. 2006): NH+4 can replace K+ in the Na, K-ATPase activation, leading to membrane ionic gradient and nervous conductance impairment (Randall and Tsui 2002); increases in NH+4 can also reduce internal Na+ to levels incompatible with life (Camargo and Alonso 2006). The transmembrane NH+4 transport by the Na,K-ATPase has been involved in NH3 excretion in invertebrates and vertebrates (Henry and Mangum 1980, Randall and Tsui 2002, Alam and Frankel 2006, Garc¸on et al. 2007, Weiner and Hamm 2007). The NH+4 binding to the Na, K-ATPase on the sites for K+, traditionally ascribed to the similar biophysical characteristics of the two cations (Weiner and Hamm 2007), is probably a widely shared mechanism (Garc¸on et al. 2007). Interestingly, other than the classical sodium pump (E.C.3.6.1.37) (Skou 1957), bivalve mollusc plasma membranes contain a second Mg2+-dependent Na-ATPase which is insensitive to ouabain and is activated by only one monovalent cation. In clams the ouabain-
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insensitive Na-ATPase attains similar enzyme activity to the Na,K-ATPase (Trombetti et al. 2000, Pagliarani et al. 2006) and even overwhelms the latter in mussels (Howland and Faus 1985, Ventrella et al. 1992, Pagliarani et al. 1996), in contrast with vertebrates where it is generally far lower (Proverbio et al. 1991). The two coexistent P-type ATPases (Marin et al. 1999), both actively translocating Na+ (Proverbio et al. 1991), are differently susceptive to a variety of effectors (Proverbio et al. 1991, Pagliarani et al. 1996, 2006, Silva et al. 2005). In mussels the two ATPases responded differently to waterborne ammonia-N (Pagliarani et al. 1996) and NH+4 could replace Na+ in the ouabain-insensitive ATPase activation (Howland and Faus 1985, Ventrella et al. 1992). These findings are suggestive of their possible involvement in internal ammonia-N regulation. Despite their commercial and nutritional value, ammonia-N sublethal effects in this taxonomic group have been poorly considered by literature (Keppler 2007). This goal was pursued by investigating ammonia-N effects on the Na,K-ATPase and Na-ATPase activities in the Philippine clam Tapes philippinarum (Bivalvia, Mollusca) in vivo and in vitro. Of the two ammonia-N forms, we mainly focused on NH+4 , largely prevailing under the adopted experimental conditions (Emerson et al. 1975, Johansson and Wedborg 1980). The allochthonous Philippine clam, widely cultivated in Italian coastal lagoons (Bartoli et al. 2001), is especially suitable for these studies because it is largely ammonia-N tolerant (Wang 1989, Borgatti et al. 1998, Ali and Nakamura 2000) and exhibits both ouabain-sensitive and ouabain-insensitive Na+-ATPase activities (Trombetti et al. 2000, Pagliarani et al. 2006). The main aim of the present work was to cast light on the possible role of the two ATPases in NH+4 transport in marine bivalves. The short-term exposure of clams to ammonia-N aimed at evaluating possible effects on the Na,K-ATPase and Na-ATPase activities by mimicking contaminant peaks in culture plants (Bartoli et al. 2001). The in vitro investigation of the possible replacement by NH+4 of the monovalent cations Na+ and K+ in the activation of the two ATPase activities was designed to provide further information on the kinetic mechanisms underlying enzyme activity responses.
Materials and Methods Chemicals MgATP (vanadium-free) and ouabain were purchased from Sigma-Aldrich (Milan, Italy). All other chemicals were reagent grade. Quartz double-distilled water was used for all reagent solutions.
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Animals Approximately 1000 adult clams (Tapes philippinarum Adams & Reeve) of commercial size (average soft body wet weight 3.8 ± 0.5 g) from different stocks obtained from coastal culture plants in the Northern Adriatic Sea were transported live in aerated seawater plastic tanks to the laboratory and maintained unfed under natural light regime in a thermostatic room at 18–20°C (a temperature range close to that in the natural habitat) in air-stone-aerated synthetic seawater (Coral Life salt mixture plus tap water) tanks at 30 ppt salinity, pH 8.0 until use. Most clams were sacrificed after 24 h acclimation, pooled, and used as tissue source for the in vitro experiments. Approximately 300 clams were used for the in vivo experiment.
Clam Exposure to Ammonia-N Before starting the experiment, clams were carefully checked and dead and shell-damaged individuals were removed. Bivalves were considered dead when the shells gaped open. The experiment was run in acrylic tanks under controlled conditions. Oxygen content, temperature, and pH were assayed daily in tank waters by portable instruments (Aqualytic OX22 Oxygen meter and Knick Portamess type 911). The nominal ammonia-N concentrations of 1.0 (ammonia I) and 2.0 mg/L (ammonia II) were selected and each treatment was run in duplicate. Clams were randomly distributed among six experimental tanks: each tank contained 50 clams spread on plastic racks laid on the bottom and 50 L of air-stone-aerated synthetic seawater (30 ppt salinity, pH 8.0), in a static system. The water was not filtered. Tanks were sheltered with shade plastic to prevent accidental introduction of extraneous matter and development of algal biofilms. The ambient temperature was gradually lowered from 18–20°C to 12–13°C in 24 h and maintained within this range throughout the experiment to minimize ambient stress. Accordingly, preliminary tests indicated that, under the experimental conditions adopted, higher temperatures, independently of contaminant exposure, implied significant mortality, which deeply affected the experimental trial. Low experimental temperatures were also suggested by literature reporting that ammonia excretion increases as temperature increases (Zhu et al. 1999), whereas tolerance to contaminants decreases (Camargo and Alonso 2006). Clams were acclimated for 24 h at 12–13°C before contaminant addition and not fed both during the acclimation and the exposure periods. Adequate amounts of NH4Cl solutions were added to seawater in order to obtain the nominal ammonia-N concentrations of 1.0 (ammonia I) and 2.0 (ammonia II) mg/L.
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Ambient water was renewed once after 48 h exposure. Control clams (C) underwent the same protocol, apart from NH4Cl addition. Before starting the exposure trial (zero time), immediately before and 1 h after NH4Cl addition and after 24, 48, 72, and 120 h exposure, water samples were collected from all tanks, stored at –20°C in the dark, and used later for colorimetric determination of total NNH3 (Koroleff 1983). Added NH4Cl did not significantly modify ambient pH which was 8.0 ± 0.1 in all tanks throughout the experiment. After 120 h exposure clams were dissected and the gills and the mantle quickly removed. Throughout the experiment clam survival was daily checked in all tanks; dead individuals were promptly removed and counted in order to evaluate the cumulative mortality percentages in each tank at the end of the trial.
Preparation of the Microsomal Fractions Mantle and gill excised from dissected clams were dried on blotting paper, repeatedly rinsed in ice-cold medium A, containing 0.25 M sucrose, 5 mM Tris- ethylenediaminetetraacetic acid (EDTA), pH 7.4, pooled, and stored in liquid nitrogen until use. The microsomal fraction was obtained by stepwise centrifugation as reported by Pagliarani et al. (2006). Microsomal preparations were then stored in liquid nitrogen until the evaluation of ATPase activities.
Assay of Na-Dependent ATPase Activities Immediately after thawing, mantle and gill microsomal preparations were used for the Na,K-ATPase and NaATPase activity assays. Enzyme activities, measured as hydrolysis of ATP, were evaluated as reported by Pagliarani et al. (2006). The ATPase reaction was carried out at 30°C under the Na,K- and Na-ATPase assay conditions found to be optimal for the two ATPases (Borgatti et al. 1998). In detail, the Na,K-ATPase reaction medium contained 75 mM Hepes, 23 mM Tris (pH 7.0) plus 3 mM MgATP, 100 mM NaCl, and 10 mM KCl for the gills and 2 mM MgATP, 10 mM NaCl, and 10 mM KCl for the mantle. The reaction mixture for the ouabain-insensitive ATPase assay contained 75 mM Hepes plus 46 mM Tris (pH 7.5), 5 mM MgATP, and 80 mM NaCl for the gills and 23 mM Tris (pH 7.0), 1 mM MgATP, and 50 mM NaCl for the mantle. ATPase activities were determined as lmoles Pimgprotein-1h-1 in triplicate in each assay. The data represent the mean ± standard error (SE) of at least three experiments carried out on different pools of animals.
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In Vitro Tests The in vitro experiments were carried out on microsomal preparations of gill and mantle from unexposed clams. The possible replacement by NH+4 of Na+ or K+ in the activation of the Na,K-ATPase and of Na+ in that of the Na-ATPase was verified by evaluating in parallel the two ATPase activities in the presence of their classical activating monovalent cations and in the presence of equimolar NH+4 concentrations in the absence of Na+ or K+ and of Na+, respectively. The dependence curves of the enzyme activities as a function of K+ or NH+4 concentrations for the Na,K-ATPase and of Na+ or NH+4 for the ouabain-insensitive ATPase were built by adding to the ATPase reaction system an appropriate concentration of chloride salt solutions of one monovalent cation in the absence of the substituted ion and maintaining all other parameters unchanged. The activation kinetics by individual monovalent cations was assayed by computing the Hill coefficient nH from the slope of Hill plots in the activating Na+, K+, and NH+4 concentration range. Once assessed Michaelis–Menten kinetics (nH = 1), apparent K0.5 values were calculated from Lineweaver– Burk plots. In order to check the preservation of the enzyme sensitivity to the glycoside ouabain, specific inhibitor of the classical Na,K-ATPase, ATPase assays were carried out in parallel by adding increasing concentrations of ouabain to the ATPase reaction medium in the presence of Na+ and K+ or Na+ and NH+4 . A similar protocol was followed to assess the response to increasing ouabain concentrations of the so-called ouabain-insensitive ATPase activated by only one monovalent cation, namely Na+ or NH+4 in the gills. The two ATPase activities were expressed as percentages of 100% activities in the absence of inhibitor. One hundred percent enzyme inhibition occurred at the ouabain concentration which reduced the enzyme activity to the basal level determined in the absence of monovalent cations. The IC50 values, corresponding to the ouabain concentration which reduced by 50% the ATPase activity, were obtained by graphical interpolation from the dose– response curves of enzyme activities.
Statistics Differences between the data from ammonia-exposed and unexposed animals as well as between that detected in the presence and in the absence of NH4Cl in the two ATPase reation mixtures were evaluated by one-way analysis of variance (ANOVA) followed by the Student–Newman– Keuls test when F values indicated significance (p B 0.05).
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Table 1 Total ammonia-N and unionized ammonia (NH3) content (mg/L) in the experimental tanks Treatment
Exposure time (h) 0
Control Ammonia I Ammonia II
24
48 Before change
After change
72
120
Ammonia-N
0.25 ± 0.14
0.75 ± 0.10
1.12 ± 0.05
n.d.
0.25 ± 0.14
0.78 ± 0.12
NH3
5.5 9 10-3
16.5 9 10-3
24.6 9 10-3
—
5.5 9 10-3
17.2 9 10-3
Ammonia-N NH3
1.54 ± 0.29 33.9 9 10-3
2.31 ± 0.05 50.8 9 10-3
2.04 ± 0.05 44.9 9 10-3
1.53 ± 0.05 34.0 9 10-3
1.55 ± 0.05 34.1 91 0-3
2.00 ± 0.22 48.4 9 10-3
Ammonia-N
3.00 ± 0.12
3.15 ± 0.1
2.95 ± 0.22
2.61 ± 0.15
2.55 ± 0.10
2.80 ± 0.15
NH3
66.0 9 10-3
69.3 9 10-3
65.2 9 10-3
57.4 9 10-3
56.1 9 10-3
61.6 9 10-3
Ammonia-N data are the mean value ± SE of duplicate tanks. Total ammonia-N indicates the sum of the two forms NH3 + NH+4 . NH3 was calculated according to Emerson et al. (1975). Before and after change refer to samplings carried out immediately before and 1 hr after water renewal. n.d. = not detectable
Mortality data were analyzed by the v2 test. Percentage data were arcsin transformed prior to statistical analysis to ensure normality.
Results Clam Exposure to Ammonia-N Ammonia I and II clams were exposed to approximately 1.5 mg/L and 3.0 mg/L ammonia-N, respectively (Table 1), probably because the physiological excretion by ammonotelic clams raised ammonia-N level in all tanks with respect to the nominal values (1.0 and 2.0 mg/L, respectively). Ammonia-N and unionized ammonia NH3 contents, the latter computed as 2.2% of total ammonia-N (Emerson et al. 1975), followed the expected order: control \ ammonia I \ ammonia II tanks (Table 1). Cumulative mortality percentages at the end of the experimental trial were below 5.0% in all tanks with no significant differences between control and treated tanks. Irrespective of the dose tested, a strikingly different response of the Na-ATPase and Na,K-ATPase activities occurred in exposed clams with respect to control ones: the former was enhanced in both treated groups, while the Na,K-ATPase was unaffected in the gills and depressed in the mantle (Fig. 1).
In Vitro Tests The activating cations, Na+ plus K+ or Na+ plus NH+4 or NH+4 plus K+ or Na+ alone for the Na,K-ATPase and Na+ or NH+4 for Na-ATPase, were added to the reaction media in order to raise the enzyme activity (Tables 2 and 3). As a first step, the capability of NH+4 concentrations equal to the optimal
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Fig. 1 Na,K-ATPase and Na-ATPase activities in gills (A) and mantle (B) from control and ammonia-N-exposed clams. Data represent the mean ± SE (vertical bar) from duplicate tanks. An asterisk indicates significant differences from control ATPase activities (p B 0.05)
concentrations (Borgatti et al. 1998) of the substituted cation to stimulate the two ATPase activity was tested (Table 2). The first row of each block (in bold type) shows the ATPase activity under standard conditions. Within each tissue, the data in the upper four rows of each block refer to the ouabain-sensitive ATPase (the classical Na,K-ATPase) activation, and that in the lower two refer to the ouabaininsensitive ATPase activation. Due to the appreciable Na-ATPase activity both in gill and mantle microsomes, in this first step Na+ replacement tests were performed in both tissues. The results can be summarized as follows: (1) NH+4 replaces K+, but not Na+
Arch Environ Contam Toxicol (2008) 55:49–56 Table 2 Replacement of Na+ and K+ by NH+4 in the stimulation of the microsomal Mg2+ dependent ouabainsensitive and ouabaininsensitive ATPase activities
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Tissue
Monovalent cations
ATPase activities (lmoles Pimg protein-1h-1)
Gills
100 mM Na+ + 10 mM K+
1.7 ± 0.2
100 mM Na+ + 10 mM NH+4
1.6 ± 0.1
100 mM NH+4 + 10 mM K+
Not detectable
100 mM or 110 mM Na
Mantle
Data represent the mean ± SE from different microsomal preparations (N = 3). a Assayed in the presence of 1 mM ouabain. Bold type: control Na,K- and Na-ATPase activities under standard conditions
+
Not detectable
80 mM Na+
a
2.2 ± 0.3
80 mM NH+4
a
2.4 ± 0.6
10 mM Na+ + 10 mM K+
2.2 ± 0.4
10 mM Na+ + 10 mM NH+4
2.2 ± 0.1
10 mM
NH+4
+
+ 10 mM K
Not detectable
10 mM or 20 mM Na+
Not detectable
50 mM Na+ a 50 mM NH+4 a
1.7 ± 0.2 1.7 ± 0.9
Table 3 Optimal concentrations (OC), Hill coefficients (nH), and K0.5 values for the activating monovalent cations Gills
Mantle
OC (mM) +
+
+
Na , K -ATPase
K 10
Na , NH+4 -ATPase + a
NH+4 +
10
Na 80
NH+4 -ATPasea
NH+4 80
+
Na -ATPase
nH
K0.5 (mM)
1.4 ± 0.1
OC (mM) +
3.6 ± 1.0
K 10
1.2 ± 0.1
2.9 ± 0.7
NH+4
1.3 ± 0.1
37.5 ± 1.7
1.2 ± 0.1
35.5 ± 3.5
10
nH
K0.5 (mM)
1.3 ± 0.2
1.2 ± 0.4
1.1 ± 0.2
1.4 ± 0.3
Not assayed Not assayed a
Data represent the mean ± SE from different microsomal preparations (N = 3). Assayed in the presence of 1 mM ouabain
ATPase activity –1 –1 (µmoles Pi · mg protein · hr )
2.0
ATPase activity –1 –1 (µmoles Pi · mg protein · hr )
in the activation of gill and mantle Na,K-ATPases; equal NH+4 and K+ concentrations display the same activation efficiency of the Na,K-ATPase. (2) The ouabain-sensitive enzyme activity obligatorily requires Na+ plus another monovalent cation to be activated, Na+ plus K+ or indifferently Na+ plus NH+4 . (3) NH+4 efficiently replaces Na+ in the activation of the gill and mantle ouabain-insensitive Na-ATPase. In a second series of experiments, the replacement of K+ by NH+4 in the activation of the classical Na,K-ATPase was further analyzed. The dependence curves of the enzyme activities in the absence of K+ and in the presence of increasing concentrations of NH+4 at constant optimal Na+ concentrations (100 mM Na+ in the gills and 10 mM Na+ in the mantle) are shown in Fig. 2. The two curves were nearly overlapping over the whole range of concentration tested both in the gills (Fig. 2A) and in the mantle (Fig. 2B) and the maximal enzyme activation occurred at the same K+ or NH+4 concentration in both tissues. Also for the activation of gill ouabain-insensitive ATPase by Na+ or NH+4 , the two dependence curves nearly overlapped in the range 1–200 mM, both showing a peak at 80 mM Na+ or NH+4 (Fig. 3). In the mantle the low and poorly reproducible Na-ATPase activity prevented us to build the dependence curve.
1.6
A GILLS
1.2 0.8 0.4 0.0 0.0
2.4 2.0
5.0
B
10.0 15.0 monovalent cation (mM)
20.0
MANTLE
1.6 1.2 0.8 0.4 0.0 0.0
5.0
10.0
15.02
20.0
monovalent cation (mM)
Fig. 2 Stimulation of the ouabain-sensitive ATPase activity by Na++K+ (continuous line) or Na++NH+4 (dashed line) in the gill (A) and in the mantle (B) at fixed concentration of Na+, 100 mM in the gills and 10 mM in the mantle, and increasing concentrations of the other cation. Each point represents the mean ± SE (vertical bar) from distinct microsomal preparations (N = 3)
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Arch Environ Contam Toxicol (2008) 55:49–56 140
GILLS
2.5
120
2.0
100 ATPase activity
ATPase activity – – (µmol Pi · mg protein 1 · hr 1)
3.0
1.5 1.0 0.5
+
NH4
80
Na
+
60 40 20
0.0 0.0
50.0
100.0 150.0 monovalent cation (mM)
200.0
0 0
Fig. 3 Stimulation of gill ouabain-insensitive ATPase activity by Na+ (continuous line) or NH+4 (dashed line). Each point represents the mean ± SE (vertical bar) from distinct microsomal preparations (N = 3)
The activation kinetics of both ATPase activities always obeyed Michaelis-Menten kinetics (nH & 1.0) irrespective of the activating cation K+ or NH+4 for the Na,K-ATPase or Na+ or NH+4 for the ouabain-insensitive ATPase (Table 3). The apparent K0.5 values indicate that, within each tissue, the Na,KATPase affinity for K+ or NH+4 was similar, as well as gill ouabain-insensitive ATPase affinity for Na+ or NH+4 (p B 0.05). Both in the gills (Fig. 4A) and in the mantle (Fig. 4B) the microsomal Na++K+- or Na++NH+4 -stimulated ATPase
1
2
3
4 5 6 ouabain (- Log M)
7
8
Fig. 5 Response to ouabain of gill ATPase activated by Na+ (continuous line) or NH+4 (dashed line). ATPase activities are expressed as a percentage of the enzyme activity detected in the absence of inhibitor. Each point represents the mean ± SE (vertical bar) from three distinct microsomal preparations (N = 3)
activities plotted versus ouabain concentration (logarithmic scale) had a sigmoidal shape. In both tissues the enzyme activity was always substantially suppressed by 10-3 M ouabain. The far lower IC50 values show that the enzyme activity was slightly more susceptive to ouabain in the gills than in the mantle. Interestingly, in both tissues the replacement of K+ with NH+4 in the ATPase activation apparently somehow increased the enzyme susceptibility to ouabain, yielding tenfold lower IC50 values when K+ was replaced by NH+4 . Conversely, the ATPase activated only by Na+ or NH+4 was unaffected by ouabain concentrations over the whole range tested up to 10-2 M (Fig. 5), a tenfold higher concentration than that which abolishes the coexistent Na,KATPase (Fig. 4).
Discussion
Fig. 4 Response to ouabain of gill (A) and mantle (B) ouabainsensitive ATPase activated by Na++ K+ (continuous line) or Na++NH+4 (dashed line). ATPase activities are expressed as a percentage of the enzyme activity detected in the absence of inhibitor. Each point represents the mean ± SE (vertical bar) from distinct microsomal preparations (N = 3)
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Both at environmental and physiological pHs, NH+4 is the prevailing ammonia-N form. In the in vivo experiment, approximately 97.8% of total ammonia-N was taken by NH+4 whereas NH3 only amounted to 2.2% (Table 1). Under the adopted in vitro pH (6.5–7.5) and temperature conditions (30°C), the NH3 contribution was 0.2–2.5% (Emerson et al. 1975; Johansson and Wedborg 1980). Therefore all ATPase responses can be mainly related to NH+4 . The ammonia-N levels tested on ammonia I and ammonia II clams, which were in the range registered in aquaculture plants (Bartoli et al. 2001, Huchette et al. 2003, Cherry et al. 2005), were apparently well tolerated, as expected, according to the largely higher 96-h LC50 value in this species (Wang 1989). Even if ammonia-N responses were reported to usually require long-term exposures in
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bivalves (Hickey and Martin 1999), the apparent stimulation of the ouabain-insensitive gill Na-ATPase and the Na,K-ATPase depression confined to the mantle after short-term exposure of clams (Fig. 1) suggest that sublethal ammonia-N concentrations primarily affect the ouabaininsensitive Na-ATPase; higher exposure doses and time are probably required to elicit changes in the Na,K-ATPase activity. Accordingly, previous experiments carried out on the same species employing lower ammonia-N concentrations (0.5 and 1.0 mg/L) and shorter exposure time (96 h) left Na,K-ATPase unaltered and stimulated gill Na-ATPase (unpublished data). The lower contaminant level in treated tanks with respect to that expected on the basis of control values (Table 1), suggests that in clams physiological ammonia-N excretion is probably decreased by high environmental ammonia-N concentrations, as in fish (Randall and Tsui 2002) and crustaceans (Chen and Lin 1992, Chen and Nan 1992). Under the experimental conditions adopted, clams faced environmental ammonia-N concentrations exceeding tissue levels, which may favor contaminant influx and interaction with membranes. The lipophilic form NH3 is the most likely candidate to perturb the membrane-bound enzyme microenvironment and promote enzyme conformational changes yielding enzyme activation or inhibition. Assuming that the contaminant interacts through membrane lipids, the different membrane fatty acid composition (Trigari et al. 2001), differently favoring contaminant incorporation and/or access to the enzyme microenvironment (Pagliarani et al. 2006) may result in a different responsiveness of the two ATPases in different mollusc tissues (Pagliarani et al. 1996). However, since the Na,KATPase interacts with lipids both by electrostactic and nonelectrostactic bonds (Esmann and Marsch 2006), NH+4 could also be involved somehow. In the in vitro activation of the two enzyme activities, NH+4 displays similar efficiency and kinetic features to the substituted cation, namely K+ for the Na, K-ATPase and Na+ for the Na-ATPase, as well as striking similarities in the optimal concentrations, Michaelis–Menten activation kinetics and affinity constants (Tables 2 and 3, Fig. 2–5). All the in vitro results suggest a possible interchange between the activating monovalent cations, as already hinted in teleosts (Ventrella et al. 1987). The different requirement for monovalent cations of the two Na-dependent ATPases is here confirmed. The essentiality of Na+ and the replacement of K+ in the activation of the ouabain-sensitive enzyme activity (Table 2) are both consistent with known Na,K-ATPase features (Skou 1957). The obligated requirement for Na+ shared by teleost (Ventrella et al. 1987, Randall and Tsui 2002) and crustacean (Garc¸on et al. 2007) Na,K-ATPases could be
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tentatively ascribed to steric selectivities of the binding sites (Di Stasio 2004). The aspecific activation by monovalent cations appear to be typical of mollusc (Howland and Faus 1986, Ventrella et al. 1992) and fish (Ventrella et al. 1987) ouabain-insensitive ATPase activities, where it distinguishes it from the coexistent Na,K-ATPase; conversely, mammalian ouabain-insensitive ATPase activities are known to be strictly dependent on Na+ (Proverbio et al. 1991). The different sensitivity to the glycoside ouabain is universally considered as one of the main distinctive features between the two coexistent microsomal Mg2+-dependent Na-ATPase activities (Ventrella et al. 1987, Proverbio et al. 1991, Caruso-Neves et al. 2004). When stimulated by either of the two cations, clam gill ATPase activity maintained its typical ouabain insensitivity which distinguishes it from the coexistent Na,K-ATPase. The enhanced ouabain susceptivity of the Na+ plus NH+4 -activated ATPase with respect to the Na,K-ATPase (Fig. 4) may mirror a favored ouabain access to the enzyme structure, possibly due to conformational changes. Accordingly, the E2 ouabain-sensitive enzyme conformation is known to prevail in the presence of millimolar NH4Cl concentrations (Furriel et al. 2004). The classical sodium pump has already been shown to be involved in the NH+4 extrusion from bivalve cells (Henry and Mangum 1980). According to a widely accepted mechanism, NH+4 is taken up in competition with K+ over the cytoplasmic membrane through the ouabain-sensitive ATPase (Weiner and Hamm 2007), then NH3 passively diffuses outwards. However, in clam cells, the ouabaininsensitive ATPase probably supports the main load of NH+4 transport, though it apparently shows lower affinity for NH+4 than the Na,K-ATPase (Table 3). On these bases, the ouabain-insensitive ATPase stimulation in ammonia-N exposed clams may be interpreted in terms of an adaptive response to counteract contaminant load: the enzyme activity may bind NH+4 as easily as Na+ and then directly expel it from the cell. On the other hand, on considering the enhancement of gill Na,K-ATPase in ammonia-N-exposed teleosts (Alam and Frankel 2006) and crustaceans (Chen and Nan 1992), it seems tempting to speculate that high external ammonia-N levels stimulate the ATPase playing the major role in ionic regulation, namely the ouabainsensitive ATPase in most animals and the ouabain-insensitive ATPase in molluscs. Finally, it cannot be ruled out that under high ammonia-N contamination the replacement of K+ by NH+4 in the activation of the Na,K-ATPase can somehow lead to ionic disturbances. Taken as a whole, the present findings substantiate the hypothesis that the two ATPase activities, and especially the ouabain-insensitive ATPase, may be involved in maintaining mollusc ionic regulation under ammonia-N accumulation conditions.
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56 Acknowledgment This work was financed by am RFO (ex 60%) grant to A.R. Borgatti.
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