Lead hampers gill cell volume regulation in marine crabs: Stronger effect in a weak osmoregulator than in an osmoconformer

Aquatic Toxicology 106–107 (2012) 95–103

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Lead hampers gill cell volume regulation in marine crabs: Stronger effect in a weak osmoregulator than in an osmoconformer Enelise M. Amado b , Carolina A. Freire b , Marco T. Grassi c , Marta M. Souza a,∗ a b c

Departamento de Ciências Fisiológicas, Universidade Estadual de Londrina, Londrina, Paraná, Brazil Departamento de Fisiologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil Departamento de Química, Setor de Ciências Exatas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil

a r t i c l e

i n f o

Article history: Received 30 June 2011 Received in revised form 14 October 2011 Accepted 23 October 2011 Keywords: Callinectes ornatus Crab Gill cells Hepatus pudibundus In vitro Pb2+

a b s t r a c t Hepatus pudibundus is a strictly marine osmoconformer crab, while Callinectes ornatus inhabits estuarine areas, behaving as a weak hyper-osmoregulator in diluted seawater. Osmoconformers are expected to have higher capacity for cell volume regulation, but gill cells of a regulator are expected to display ion transporters to a higher degree. The influence of lead nitrate (10 ␮M) on the ability of isolated gill cells from both species to volume regulate under isosmotic and hyposmotic conditions were here evaluated. Without lead, under a 25% hyposmotic shock, the gill cells of both species were quite capable of cell volume maintenance. Cells of C. ornatus, however, had a little swelling (5%) during the hyposmotic shock of greater intensity (50%), while cells of H. pudibundus were still capable of volume regulation. In the presence of lead, even under isosmoticity, the gill cells of both species showed about 10% volume reduction, indicating that lead promotes the loss of water by the cells. When lead was associated with 25% and 50% hyposmotic shock, C. ornatus cells lost more volume (15%), when compared to isosmotic conditions, while H. pudibundus cells showed volume regulation. We then analyzed the possible ways of action of lead on the mechanisms of cell volume regulation in the two species. Verapamil (100 ␮M) was used to inhibit Ca2+ channels, ouabain (100 ␮M) to inhibit Na+ /K+ -ATPase, and HgCl2 (100 ␮M) to inhibit aquaporins. Our results suggest that: (1) Ca2+ channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus, being the target of lead action in these cells; (2) aquaporins are much more relevant for water flux in H. pudibundus; and (3) the Na+ /K+ -ATPase is much more relevant for volume regulation in C. ornatus. Osmoregulators may be more susceptible to metal contamination than osmoconformers, especially in situations of reduced salinity, for two basic reasons: (1) lower capacity of volume regulation and (2) putative higher uptake of Pb2+ through ionic pathways that operate in salt absorption, such as, for example, the Na+ /K+ -ATPase. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cell volume regulation is an essential process for maintaining cellular homeostasis. The maintenance of a constant volume in face of osmotic disturbances, both extra- and intracellular, is a critical problem faced by all animal cells. Volume changes are usually grouped into two broad categories: anisosmotic and isosmotic. Anisosmotic volume changes are induced by changes in extracellular osmolality and isosmotic volume changes occur through changes in intracellular solute content (Strange, 2004; Hoffmann et al., 2009).

∗ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil, Tel.: +55 53 3233 6852; fax: +55 53 3233 6848. E-mail address: [email protected] (M.M. Souza). 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.10.012

The function and location of gills leads to the fact that their cells are exposed to changes in the external environment, such as changes in salinity and the presence of contaminants (which most often do not affect the osmolality of the water). Furthermore, besides constitutive metabolic processes common to all cells, the function of transepithelial salt transport will also contribute to volume disturbances of gill cells, even in the absence of changes in the external environment. The volume of gill cells can thus be challenged by these two types of osmotic disturbances. Moreover, the external medium of gills cells, given their polarity and condition of interface epithelium, is represented by two possibly different environments, especially in osmoregulators that keep a gradient between their extracellular medium and the water (Freire et al., 2008a,b). To maintain their vital functions, gill cells must have ability to volume regulate after such changes. Under normal conditions, the osmolality of the cytoplasm is equal to the osmolality of the extracellular fluid. Changes in solute

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content in any of these compartments (extracellular or intracellular) generate a trans-membrane osmotic gradient (Strange, 2004). Because the plasma membrane is freely permeable to water, any osmotic gradient results in an immediate flow of water into or out of the cell until a new osmotic equilibrium is reached. This flow causes an increase or decrease in cell volume, which means that for the maintenance of cell volume, the effective intracellular osmolality must be equal to the effective extracellular osmolality (Russell, 2000). After a change in volume, the cell must return to its previous “normal” volume, in order to keep performing its various functions. When the cell, after swelling, uses mechanisms to reduce its volume, this process is called regulatory volume decrease (RVD). Alternatively, mechanisms used to restore the lost volume are called regulatory volume increase (RVI) (Hoffmann and Dunham, 1995; Hoffmann et al., 2009). Both RVD and RVI may be the outcome of the activation of one or more membrane transport systems for inorganic and/or organic solutes (Hallows and Knauf, 1994; Lang et al., 1998; Wehner et al., 2003), solute transport being followed by transport of osmotically obliged water. Cations such as Na+ , K+ and Ca2+ play a role in cell volume regulatory mechanisms, as well as in signaling involved in cell volume regulation (Lang et al., 1998; Wehner et al., 2003; Strange, 2004). When Pb2+ is present in the water or in the extracellular fluid, it may enter the cell because of its similarity to Ca2+ , using calcium ion transport pathways (Rainbow, 1995, 1997; Bridges and Zalups, 2005). Pb2+ can also reduce unidirectional influx of Na+ (Wright, 1995; Ahern and Morris, 1998), often correlated with inhibition of the Na+ /K+ -ATPase (Ahern and Morris, 1998), an essential enzyme for the maintenance of cell volume. Thus, inside the cell, Pb2+ may affect cell volume (as well as other physiological processes), through its resemblance of calcium, or through its other effects on, e.g., sodium fluxes or enzyme activities. Gill cells from marine/estuarine species of decapod crabs with different osmoregulatory strategies are expected to display (1) different volume regulatory behavior (Foster et al., 2010) and possibly (2) a different response to the presence of lead. Hepatus pudibundus is a marine osmoconformer crab, and Callinectes ornatus is a marine/estuarine species with some hyper-regulatory capacity (weak osmoregulator) (Freire et al., 2008a, 2011; Foster et al., 2010). This study thus aimed at comparing the interference by lead of gill cell volume regulation in these two species. More specifically, we asked the questions: (1) is there a difference in gill cell volume regulation under hyposmotic shock (i.e., RVD capacity) between the two species? (2) What is the influence of lead on the maintenance of gill cell volume under isosmotic and hyposmotic conditions in the two species? (3) How does lead interact with some membrane transport systems involved in cell volume regulation?

2.2. Solutions and analytical determinations The control isosmotic saline osmotically corresponded to both crabs’ hemolymph in seawater (in mM): NaCl 470, KCl 8, CaCl2 15, MgSO4 10, Hepes 10, and glucose 5, pH 7.6, measured osmolality of 948 mOsm/kg H2 O, Wescor VAPRO 5520. This control saline had lower magnesium levels than those found in the osmoconformer crab H. pudibundus (Foster et al., 2010). However, we chose to use a same solution for both species, in order to directly compare the ability of their gill cells to perform cell volume regulation. The hyposmotic shocks of 25 and 50% osmolality reduction were accomplished by simple dilution of the isosmotic saline using distilled water. Measured osmolalities were 706 and 484 mOsm/kg H2 O, respectively. Lead nitrate (10 mM, PbNO3 2 ) stock solution was prepared in distilled water. This stock solution was diluted in the isosmotic control saline to reach the final nominal concentration of 10 ␮M lead nitrate. Lead solutions, including the stock solution, were prepared in previously acidified falcon tubes. Lead concentrations in the solutions (total and dissolved – filtered through 0.20 ␮m membranes) were determined through ICP OES – inductively coupled plasma optical emission spectroscopy (PE-LE032/R01), performed by a certified Laboratory at Federal University of Paraná (CEPPA, http://www.ceppa.ufpr.br/). Lead speciation was determined through the chemical equilibrium model CHEAQS-Pro (CHemical Equilibria AQuatic Systems), version P2010.3 (Verweij, 2010). Concentrations of the major ions (Na+ , Mg2+ , K+ , Ca2+ , Cl− , NO3 − , SO4 2− , H+ ) and of lead were introduced in the modeling program and the simulated concentrations of free lead ion as well as lead bound to inorganic ligands were obtained. The concentration of lead employed here fell within the range of lead levels measured in the hemolymph of other species of crabs (the freshwater Barytelphusa guerini and Cherax destructor) exposed for up to 30 days to 0.5 and 100 mg L−1 of lead nitrate (Tulasi et al., 1987; Ahern and Morris, 1998). This high concentration employed was indeed way higher than environmentally relevant estuarine concentrations reported for dissolved lead (e.g., Severini et al., 2011). However, lead is a potential estuarine pollutant in the Brazilian coast, resulting from mining activities and discharge into rivers that run into the ocean (e.g., Farias et al., 2007). In fact, significant lead contamination was detected in the sediment of Paranaguá Bay – Pb was the third most important toxic metal – an estuarine complex inhabited by the swimming crab C. ornatus used in this study (Choueri et al., 2009; Freire et al., 2011). In addition, our purpose here was a mechanistic investigation, rather than environmental assessment of the effects of lead contamination.

2.3. Cell dissociation 2. Materials and methods 2.1. Animals The crabs H. pudibundus and C. ornatus were purchased from fishermen on Ipanema beach, Pontal do Sul, Paraná, Brazil (25◦ 41 S; 48◦ 27 W). Species are sympatric in coastal subtidal marine trawling areas used by shrimp fishermen. They come as a bycatch in shrimp fisheries. The animals collected were transported (∼1.5 h drive) in polystyrene boxes to the Laboratory in the Department of Physiology, Federal University of Paraná, in Curitiba. In the laboratory the crabs were kept in a glass stock aquarium (180 L), containing seawater of salinity 33‰ (∼1000 mOsm/kg H2 O), under constant aeration, biological filtration, natural photoperiod and room temperature (∼20 ◦ C). Crabs were fed with small pieces of fish or shrimp, 2 or 3 times a week.

Crabs were cryoanesthesized and had their carapace quickly manually opened. The 3 pairs of posterior gills were removed and, using a syringe inserted into the efferent hemolymph vessel, gills were perfused with Ca2+ -free PBS (in mM: NaCl 400, Na2 HPO4 25, KH2 PO4 3.5, KCl 20, measured osmolality of 940 mOsm/kg H2 O) for the removal of circulating hemocytes. After being perfused, the area around the efferent vessel was removed using scissors, to allow for proper isolation of gill cells. Gills were then transferred to a Petri dish containing PBS plus EDTA (5 mM) and were sliced into small pieces. Mechanical cell dissociation was then facilitated by repetitive movement of the solution with tissue fragments up and down a Pasteur pipette. Once dissociated, cells were filtered through a nylon mesh (pore size of 30 ␮m) to remove tissue debris; the filtrate was centrifuged at 290 × g for 5 min at 20 ◦ C in a refrigerated centrifuge (Eppendorf Centrifuge 5810R, Germany), and cells were then resuspended in PBS.

E.M. Amado et al. / Aquatic Toxicology 106–107 (2012) 95–103 Table 1 Viability of gill cells of Hepatus pudibundus and Callinectes ornatus under control conditions without lead or after 1, 3, or 6 h of exposure to 10 ␮M lead. Time

Lead concentration (␮M)

Cell viability (%) Hepatus pudibundus

Callinectes ornatus

1h

Control (0) 10

96.7 ± 0.9d 82.8 ± 2.8c

98.7 ± 3.0c 79.7 ± 5.7bc

3h

Control (0) 10

85.9 ± 1.4c 81.8 ± 1.1c

79.4 ± 1.9bca 66.7 ± 5.2aba

6h

Control (0) 10

73.3 ± 2.4b 63.7 ± 1.6a

81.0 ± 8.1b 53.7 ± 4.2a

Different letters indicate groups which are statistically different within the same species. Data are mean ± SEM, n = 4–5 crabs. a Difference between species for the same treatment.

2.4. Cell viability: lead toxicity to the gill cells Freshly dissociated cells were centrifuged, and PBS was replaced by isosmotic saline (controls), or control saline with 10 ␮M lead nitrate for 1, 3 and 6 h. The goal was to assess whether lead would exert, under the concentration chosen, a toxic effect on the gill cells, which could then prevent us from interpreting its putative effects on cell volume regulation. The viability test used was the trypan blue exclusion method. The percentages of viable cells during the 6 h of the experiment are displayed in Table 1. Cells displayed reduced viability with time, being less viable after 3 h, even in the absence of lead. C. ornatus cells were more affected by lead than H. pudibundus cells, showing lower viability for a same experimental procedure (Table 1). Thus, 20 min was a safe maximal experimental time, chosen in order to follow cell volume regulation in the absence or presence of lead.

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response elicited by hyposmotic shocks of two magnitudes: 25 and 50% reduction in saline osmolality, with the addition of 10 ␮M Pb. In order to assure that lead was not quenching the calcein signal, we have loaded gill tissue of a C. ornatus with calcein AM (1 h in isosmotic saline), and washed the tissue with saline to remove external calcein. The tissue was then homogenized to mechanically break up cells and free the now fluorescent calcein. The free fluorescent calcein was then exposed to Pb. There was no decrease in the signal, as would have been expected by lead quenching of calcein; there was even a slight increase. So, the method employed is a reliable indicator of cell volume changes.

2.7. Analysis of the Pb2+ action on possible pathways involved in cell volume regulation Solute routes known to operate for volume regulation were selectively inhibited in order to investigate how Pb2+ exerts its inhibitory effects on cell volume regulation (verapamil 100 ␮M – calcium channel blocker and ouabain 100 ␮M – Na+ /K+ -ATPase inhibitor). The inhibitors were added individually to either (1) isosmotic control saline, (2) isosmotic saline + 10 ␮M Pb, and (3) 25% hyposmotic solution + 10 ␮M Pb, and cell volume was followed for 20 min, as already described. The role played by water flux through aquaporins was also investigated, through the use of HgCl2 (100 ␮M, Marinelli et al., 1997; Meinild et al., 1998; Belyantseva et al., 2000; Watanabe et al., 2005) on these three experimental situations. The influence of lead on ion pathways was evaluated only under 25% hyposmotic shock, as this milder shock is actually more likely to occur in the lives of these crabs.

2.8. Statistical analysis 2.5. Cell volume regulation A fluorescence self-quenching technique using Calcein-AM (Sigma–Aldrich) was employed to detect changes in cell volume, as described by Hamann et al. (2002) and Capó-Aponte et al. (2006). Freshly dissociated cells were counted in a Neubauer chamber and were then placed in a black 96-well plate (Optiplate) at a density of about 104 cells per well in 200 ␮l of control isosmotic saline. The plate was then centrifuged at 290 × g for 5 min to spin down cells, and the supernatant (control saline) was replaced by the calcein-AM solution (10 ␮M calcein in control saline). Plates were incubated in the dark for 1 h with fluorescence readings every 3 min, with excitation at 485 nm and emission at 530 nm, to follow calcein entry into the cells (Tecan Infinity M200, Austria). After incubation, the plate was again centrifuged and the calcein solution removed and replaced by the control saline. Isosmotic fluorescence was then read every 30 s for 5 min to assess the stability of the fluorescence emission. The plate was centrifuged again and the cells were exposed to either control saline, 25% or 50% hyposmotic shock. This procedure was repeated until the final “n” indicated in the legends was reached. Fluorescence was then read every 30 s for 20 min. Fluorescence data were expressed relative to the initial volume reading of each well used. 2.6. The effect of lead on cell volume regulation We first analyzed whether lead would affect gill cell volume regulation in isosmotic conditions, as it had a time-dependent effect reducing viability of cells in isosmotic conditions (Table 1). According to the protocol described in the previous section, cells of both species were exposed to either control isosmotic saline, or to isosmotic saline + 10 ␮M Pb. We also probed its effect on the RVD

Data are presented as mean ± standard error of mean. For cell viability data, one-way ANOVAs were applied for each species separately. Data from the two species, for the same experimental treatment were compared using Student’s t-tests (Table 1). Cell volume data were treated statistically only in times 0.5, 5, 10, 15 and 20 min through one-way ANOVAs for each of these time points (3 treatments, Figs. 1, 3 and 4). Inhibitor data (cell volume after 20 min) were also treated statistically using one-way ANOVAs (Fig. 5). The ANOVA was followed by the Holm–Sidak post hoc test in the case of normally distributed data. Kruskal–Wallis one-way ANOVA on ranks followed by Dunn’s test was used in the case of non-normally distributed data. When two treatments were compared (control × experimental), unpaired Student’s t-tests were applied (Fig. 2). The level of significance considered was p < 0.05. All statistical analyses were performed by Sigma Plot® software.

3. Results 3.1. Evaluation of the capacity for regulatory volume decrease in crab gill cells H. pudibundus gill cells, when exposed to hyposmotic shocks of two different magnitudes – 25 and 50% reduction with respect to the isosmotic control – showed no change in cell volume. The cells maintained their volume constant throughout the whole experimental period of 20 min (Fig. 1A). However, gill cells of C. ornatus exposed to the 50% hyposmotic shock showed swelling (∼4%) after 10 min of exposure. Their maximum swelling (5%) was reached after 10 min, and after 20 min the cells still displayed a swelling of 3%, thus not returning to their original volume (Fig. 1B).

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H. pudibundus - hyposmoc shock

1.1

A volume variaon

volume variaon

1.1

1.0

0.9 isosmoc 25% hyposmoc 50% hyposmoc

0.8

0

5

H. pudibundus - isosmoc + Pb

A

1.0 * 0.9

15

20

25

0

5

me (min)

b

volume variaon

a 1.0

a

a a

a a

a a

1.1

b

b

a a

a

volume variaon

B

a

0.9

0.8

isosmoc 25% hyposmoc 50% hyposmoc 0

5

10

15

20

25

15

20

25

me (min) Fig. 1. Evaluation of the capacity for regulatory volume decrease in crab gill cells. Time course of volume variation in gill cells of Hepatus pudibundus (A) and Callinectes ornatus (B) exposed either to isosmotic (control) or hyposmotic conditions (25% and 50% reduction with respect to the control saline). Different letters indicate statistical differences between these 3 treatments, at: 0.5, 5, 10, 15 and 20 min. Data are mean ± SEM, n = 5–13 crabs.

3.2. Lead levels in experimental salines 3.2.1. Measured lead There were no differences between total and dissolved lead concentrations measured in the isosmotic solution and the hyposmotic solutions used to evaluate the influence of lead on cell volume regulation. The respective values of total and dissolved (filtered in 0.20 ␮m) lead concentrations were (n = 3 determinations): 8.35 ± 0.73 ␮M and 8.20 ± 0.73 ␮M in the isosmotic saline; 8.25 ± 0.73 ␮M and 8.20 ± 0.19 ␮M in the 25% hyposmotic saline; 8.20 ± 0.24 ␮M and 8.40 ± 0.10 ␮M in the 50% hyposmotic saline. This range of values corresponds to ∼1.70 mg L−1 of lead. 3.2.2. Lead calculated using CHEAQS The calculations carried out via the chemical equilibrium model showed that lead is entirely present in the dissolved phase in the solutions under study. For the isosmotic solution the most important species indicated by the model were PbCl+ (46.80%), PbCl2 (aq) (24.78%), the free ionic species Pb2+ (9.90%), PbCl3 − (8.86%), Pb(OH)+ (3.94%), PbCl4 2− (2.95%), and PbSO4 (aq) (2.06%). Calculations were also performed for the hyposmotic solutions. The main species containing Pb specified by the model for the 25% hyposmotic solution were PbCl+ (50.11%), PbCl2 (aq) (20.33%), Pb2+ (13.91%), PbCl3 − (5.60%), Pb(OH)+ (3.94%), and PbSO4 (aq) (2.39%). Finally, for the 50% hyposmotic solution, the major species revealed were PbCl+ (51.02%), Pb2+ (20.12%), PbCl2 (aq) (14.34%), Pb(OH)+ (8.12%), PbCl3 − (2.69%), and PbSO4 (aq) (2.63%). As expected, an

C. ornatus - isosmoc + Pb

B

1.0 *

*

*

0.9

0.8

10

*

me (min)

C. ornatus - hyposmoc shock 1.1

*

isosmoc 10μM Pb

0.8 10

*

*

isosmoc 10μM Pb 0

5

10

15

20

25

me (min) Fig. 2. Lead effect in isosmotic conditions. Time course of volume variation in gill cells of Hepatus pudibundus (A) and Callinectes ornatus (B) exposed either to isosmotic (control) or isosmotic with the addition of 10 ␮M lead nitrate. *Pb2+ caused volume reduction at 0.5, 5, 10, 15, and 20 min. Data are mean ± SEM, n = 5–13 crabs. Isosmotic (control) data repeated from Fig. 1.

increase of the free ion concentration was observed as a result of the dilution of the isosmotic saline stock solution. However, given that these species are labile, all this lead is considered as dissolved and bioavailable. 3.3. Lead effect in isosmotic conditions Lead nitrate (10 ␮M) caused cell volume decrease in gill cells of both species. Cell volume decrease in gill cells of H. pudibundus was detected after 5 min, and cells continued to shrink continuously, with 12% volume decrease at the end of the experimental time (Fig. 2A). In gill cells of C. ornatus, volume reduction (3%) started at ∼5 min and the cells continued to lose volume until the end of the experimental time, reaching 10% of volume loss (Fig. 2B). 3.4. Lead effect in 25% and 50% hyposmotic conditions Exposure of gill cells of H. pudibundus to the concentration of 10 ␮M lead nitrate associated with 25% hyposmotic shock resulted in cell volume decrease. Cells began to lose volume at 5 min of exposure (∼8%), maintained until 15 min of exposure, when cells start to return to their original volume, which is reached by the end of the experimental time, 20 min (Fig. 3A). In constrast, cells of C. ornatus did not recover from the volume lost upon 25% hypoosmotic shock plus lead, reaching 17% volume reduction at the end of the 20 min experimental time (Fig. 3B). When lead was applied together with the hyposmotic shock of greater magnitude (50%), cells of H. pudibundus displayed no

E.M. Amado et al. / Aquatic Toxicology 106–107 (2012) 95–103

H. pudibundus - 25% hyposmoc + Pb

1.1

A a a

1.0

a

0.9

a a b

a

a

a

a

b

b

volume variaon

volume variaon

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a a a

isosmoc 25% hyposmoc 25% hyposmoc + 10μM Pb

0.8

0

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10

H. pudibundus - 50% hyposmoc + Pb

A

1.0

0.9 isosmoc 50% hyposmoc 50% hyposmoc + 10μM Pb

0.8

15

20

99

0

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C. ornatus - 25% hyposmoc + Pb

B

volume variaon

a 1.0

1.1

a a

a a

a

a

a

a

a

a

0.9 b 0.8

isosmoc b 25% hyposmoc 25% hyposmoc + 10μM Pb 0

5

10

15

20

25

C. ornatus - 50% hyposmoc + Pb

B

b a

volume variaon

1.1

10

me (min)

me (min)

b

15

a

ab a 0.9

0.8

b 20

1.0

volume change (Fig. 4A). However, again, cells of C. ornatus behaved differently, showing significant volume loss (7%) after 5 min, and 16% after 20 min (Fig. 4B). 3.5. Analysis of the Pb2+ action on possible pathways involved in cell volume regulation

a

isosmoc c 50% hyposmoc 50% hyposmoc + 10μM Pb 5

a

a

c

c

10

15

20

25

me (min)

me (min) Fig. 3. Lead effect in 25% hyposmotic conditions. Time course of volume variation in gill cells of Hepatus pudibundus (A) and Callinectes ornatus (B) exposed either to isosmotic (control), 25% hyposmotic condition, or 25% hyposmotic condition with 10 ␮M lead nitrate. Different letters indicate statistical differences between these 3 treatments at 0.5, 5, 10, 15 and 20 min. Data are mean ± SEM, n = 5–13 crabs. Isosmotic (control) and 25% hyposmotic data repeated from Fig. 1.

b

b

0

25

b

a

Fig. 4. Lead effect in 50% hyposmotic conditions. Time course of volume variation in gill cells of Hepatus pudibundus (A) and Callinectes ornatus (B) exposed either to isosmotic (control), 50% hyposmotic condition, or 50% hyposmotic condition with 10 ␮M lead nitrate. Different letters indicate statistical differences between these 3 treatments at 0.5, 5, 10, 15 and 20 min. Data are mean ± SEM, n = 5–13 crabs. Isosmotic (control) and 50% hyposmotic data repeated from Fig. 1.

20 min), now in the presence of ouabain the cells displayed 20% volume decrease after 20 min (Fig. 5B). Gill cells of C. ornatus did not show volume loss in the presence of verapamil and ouabain, as they had shown in their absence under 25% hyposmotic shock in the presence of lead (18%). Thus, these inhibitors reverse the lead effect on hyposmotic condition (Fig. 5D). 4. Discussion

Only the final volume changes (experimental time of 20 min) were compared to assess the effects of inhibitors (Fig. 5). The addition of verapamil or ouabain in isosmotic conditions resulted in no change in the volume of H. pudibundus cells, and cells behaved as they did in plain isosmotic saline. However, HgCl2 caused a volume increase of ∼23% in isosmotic conditions (Fig. 5A). Under the same experimental conditions, cells of C. ornatus showed volume increase when either verapamil (11%) or ouabain (9%) were added, but remained unchanged upon addition of HgCl2 (Fig. 5C). In the presence of lead under isosmotic conditions cells of H. pudibundus submitted to verapamil and HgCl2 did not show volume decrease, as they did in the presence of lead alone (Fig. 5A). C. ornatus cells lost volume equally, with or without verapamil and HgCl2 , in the presence of lead in isosmotic conditions (Fig. 5C). For both species, the addition of ouabain did not modify the loss in cell volume observed in the presence of lead. When the blockers were associated to the 25% hyposmotic shock with lead, the only difference in H. pudibundus cells was noted with ouabain. If previously, in hyposmotic conditions with lead, cells did not show the volume reduction caused only by lead (12% after the

4.1. Cell volume regulation H. pudibundus and C. ornatus gill cells exposed to a 25% hyposmotic shock displayed a marked capacity of cell volume regulation, keeping their volume unchanged. Cells from both species activate mechanisms to prevent water influx to the cell. However, a hyposmotic shock of greater magnitude (50% osmolality reduction) revealed differences in the responses of cells from these species. While H. pudibundus cells managed to maintain their volume stable, C. ornatus cells swell by 5%, and despite displaying a trend for recovery, after 20 min the cells were still showing a significant swelling of 3%. Still, this volume change (maximum of 5%) is 10fold less than the intensity of the applied shock (50%). This means that, although at a slower pace, C. ornatus gill cells activate cell volume regulatory mechanisms in order to minimize cell swelling (Neufeld and Wright, 1996; Souza and Scemes, 2000; Amado et al., 2006; Ruiz and Souza, 2008; Cruz and Souza, 2008). The result of better capacity for RVD in the osmoconformer crab (H. pudibundus) than in the weak osmoregulator (C. ornatus) was entirely

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H. pudibundus

A 1.3

isosmotic

d

1.2 1.1

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1.0

c

c

CT + V

CT + O

c

c

bc

ab

Pb + V

Pb + O

a

0.9 0.8

B

1.3

CT

Pb

CT + Hg

hyposmotic

1.2 1.1

b

b

1.0

b

b

b

0.9

a

0.8

CT

C

1.2

25%hypo + Pb

25%hypo

1.0

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25%hypo + Pb + V

25%hypo + Pb + Hg

C. ornatus isosmotic

d

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Pb + Hg

cd

bc

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c

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CT

Pb

CT + V

CT + O

CT + Hg

Pb + V

Pb + O

Pb + Hg

hyposmotic

1.1 1.0

c

bc

bc

bc ab

0.9

a

0.8

CT

25%hypo

25%hypo + Pb

25%hypo + Pb + V

25%hypo + Pb + O

25%hypo + Pb + Hg

experimental condions Fig. 5. Analysis of possible target pathways of lead involved in cell volume regulation. Volume variation after 20 min of exposure of gill cells of Hepatus pudibundus (A – isosmotic; B – 25% hyposmotic) and Callinectes ornatus (C – isosmotic; D – 25% hyposmotic) to Pb2+ and transport inhibitors: verapamil (V), ouabain (O), and mercury chloride (Hg). Different letters above the bars indicate statistical differences. Data are mean ± SEM, n = 4–6 crabs. Control, Pb2+ (isosmotic), and 25% hyposmotic data at 20 min were taken from Figs. 1 and 2. The dashed line indicates absence of volume variation with respect to initial values.

compatible with our previous findings reported in Foster et al. (2010). Osmoconformers do not keep their hemolymph osmolality stable when facing water salinity changes, while weak osmoregulators such as estuarine crabs tend to hyper-regulate when salinity decreases (Freire et al., 2008a,b; Foster et al., 2010). Thus, while the osmoconformer H. pudibundus invests more in volume regulation of cells, regulators such as C. ornatus invest in anisosmotic extracellular regulation and therefore prevent ample fluctuations in hemolymph osmolality. In consequence, their cells are spared of intense need for volume regulation (Freire et al., 2008b; Foster et al., 2010). In Foster et al. (2010), muscle tissue slices of H. pudibundus were submitted to a 30% hyposmotic shock. In that situation, as well as in the isolated gill cells here (submitted to 25% hyposmotic shock), the tissue/cells were fully capable of maintaining their water content. Also using muscle slices, tissue of H. pudibundus

was exposed to a 50% dilution of saline, gained volume initially, but was also able to recover (Freire et al., 2008b). In contrast, here, isolated gill cells exposed to the same shock (50%) did not even swell transiently. The difference is most probably explained by the fact that volume control is more evident in isolated cells, as would be expected, and that initial swelling found in muscle tissue could be due to some interstitial water (also resulting from cellular RVD), buffering the response. In addition, different tissues have been used, here gill cells, and muscle tissue in the previous study (Freire et al., 2008b). In summary, as cell volume regulation is such a vital function for cells, the difference in behavior between species with these different osmoregulatory patterns was noted only when the more intense shock was applied (50%), both here and in the previous studies (Freire et al., 2008b; Foster et al., 2010).

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4.2. Lead effect on cell volume regulation, and possible pathways involved 4.2.1. Lead effect under isosmotic conditions The exposure of gill cells of both species to Pb (10 ␮M) under isosmotic conditions led to a significant and similar loss of cell volume. As no osmotic shock was applied, the result indicates that lead somehow caused an osmotic and/or ionic imbalance causing gill cells to lose water. Cell volume decreases upon exposure to lead was also found in human erythrocytes. The erythrocytes lost volume in even lower concentrations of lead nitrate (0.3, 1.3 ␮M) than that used here; the explanation was that Pb2+ acts on a calcium dependent-K+ channel, promoting efflux of K+ and consequent cell volume decrease (Kempe et al., 2005). We did not assess K+ fluxes here, but this could also be the reason for the volume loss upon Pb2+ exposure in our crab gill cells. Pb2+ has been shown to interact (Beeby, 1978; Simons, 1993; Rogers et al., 2003; Kempe et al., 2005; Li et al., 2008) or even mimic Ca2+ (Bridges and Zalups, 2005) in several cell models, both in vivo and in vitro. Thus, putatively increased Pb2+ inside the cells could mimic an increase in intracellular Ca2+ concentration. It is known that increases in intracellular Ca2+ concentration results, in many cellular systems, in the activation of regulatory mechanisms for volume decrease – RVD (McCarty and O’Neil, 1992; Hoffmann and Dunham, 1995; Wehner et al., 2003). RVD mechanisms involve the release of organic and inorganic osmolytes with consequent loss of water and reduction in cell volume. Several transport systems involved in RVD have been reported as calcium-dependent, such as conductive pathways for K+ and Cl− (Pasantes-Morales and Morales-Mulia, 2000; Wehner et al., 2003) and osmosensitive pathways of taurine release (Cardin et al., 2003). Notably, cells of H. pudibundus did not lose volume in the presence of Pb2+ and verapamil, a calcium channel blocker. Without lead in isosmotic saline, gill cells of C. ornatus swelled with the inhibition of Ca2+ channels and of the Na+ /K+ -ATPase, indicating that these transporters are important for the maintenance of cell volume in isosmotic conditions in the osmoregulator crab. Cell volume increase in isosmotic conditions after inhibition of Na+ /K+ ATPase by ouabain has been described in other cell types (Lang et al., 1998; Wehner et al., 2003). Accumulation of sodium in the cell due to inhibition of Na+ /K+ -ATPase results in water entry and increase in cell volume (Lang et al., 1998). Further, the inhibition of Ca2+ entry (verapamil) into the cell may compromise the leakage of potassium through calcium dependent-K+ channels (Wehner et al., 2003), as discussed above, resulting in accumulation of K+ into the cell, with consequent volume increase. This hypothesis could be tested using K+ channel blockers, which would then be expected to reduce or stop volume loss by the cells. Consistently, verapamil, in the presence of Pb, causes cell volume to return to normal values, when compared to volume reduction caused by Pb alone, supporting the idea of Pb entry through calcium channels. Verapamil thus blocks both calcium and Pb entry through calcium channels in both species. The intense water efflux putatively caused by activation of calcium-dependent K+ channels by intracellular Pb2+ probably happens due to a strong binding of Pb2+ to the intracellular side of the K+ channel. This binding is probably much less reversible than the binding of the physiological cation, calcium, as reported for the troponin C superfamily of calcium-binding proteins (Fullmer et al., 1985). One can expect that this strong binding will increase the open state probability of these channels, and in consequence, K+ leakage and water efflux, in agreement with the findings that 1–100 ␮M lead activates the conductance of calcium-dependent K+ channels in erythrocytes and neuroblastoma cells, more potently than calcium itself (Vijverberg et al., 1994). Aquaporins are also known to be regulated by Ca2+ . AQP6 expressed in intercalate cells of the collecting duct of the mammalian kidney has a binding site for calmodulin (Rabaud et al.,

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2009). Moreover, lead induces increased permeability of AQP4 in astrocytes, and the effect of lead was attenuated in the presence of an inhibitor of protein kinase II Ca2+ /calmodulin-dependent (Gunnarson et al., 2005). At least with H. pudibundus cells, the general aquaporin inhibitor mercury chloride led to decreased cell volume loss in the presence of lead. However, this result is compatible both with a direct effect of Pb2+ on aquaporins (again mimicking Ca2+ ), or with its putative indirect effect: mercury simply blocking water efflux through aquaporins, indirectly caused by lead. In support of our contention of lead causing volume loss in gill cells, it is important to add that, if Pb2+ would be quenching the calcein signal – thereby producing an artifact of reduced signal – aquaporin inhibition using HgCl2 in H. pudibundus (Fig. 5A) would not prevent the decreased signal, returning the fluorescence to control levels (group Pb + HgCl2 ). It is thus apparent that Pb2+ makes water leave the cells through aquaporins (by activating RVD mechanisms, as discussed). When this exit is blocked by mercury, water does not leave the cells. In addition, inhibition of aquaporins in isosmotic conditions without lead affected cell volume only in H. pudibundus, consistent with the interpretation that this species is more dependent on aquaporins for cell volume regulation than C. ornatus. Thus, the higher efficiency of H. pudibundus cells in RVD could be due, at least partially, to a higher expression of aquaporins. 4.2.2. Lead effect under hyposmotic conditions Under isosmotic conditions, lead had the same effect (i.e., volume decrease) in both species. However, when gill cells were exposed to lead during the 25% hyposmotic shock, there was a marked difference in the response of the cells of the two species. Lead associated with the hyposmotic saline not only made C. ornatus cells to lose the ability to regulate volume which it displays for this magnitude of shock, but caused an even larger decrease in cell volume, when compared to cells exposed to lead under isosmoticity. However, cells of H. pudibundus remained with the ability to regulate volume. They did not show volume reduction as found during exposure to lead alone (under isosmoticity). The reason is probably that in the diluted salines, there were favourable inward water gradients for water influx (in 50% shock even more than in the 25% shock), thus compensating for the activation of the RVD mechanisms. Again, the osmoconformer shows higher capacity to regulate cell volume. The pathways and solutes involved, and how are they different from the osmoregulators, remain to be investigated. It can be proposed that this higher capacity is not dependent on the Na+ /K+ -ATPase. Conversely, in C. ornatus, verapamil and ouabain induced volume increase in isosmotic conditions without lead, and in hyposmotic conditions with lead, both situations when compared to respective controls, results compatible with the above discussion on the role of calcium channels and the Na+ /K+ -ATPase on volume regulation in the osmoregulator crab. Another issue is relevant to the discussion of lead effects under hyposmotic conditions. Given that Pb availability was demonstrated to be approximately the same (labile and dissolved complexes) in the isosmotic and both hyposmotic solutions, it is important to point out that the most intense deleterious effects (i.e., resulting in volume loss) of Pb observed in C. ornatus gill cells exposed to hyposmotic solutions was probably not due to a significantly higher chemical availability of Pb2+ (as this would apply equally to both species) but most importantly, due to physiological differences between the gill cells of the two crabs. This is entirely compatible with the conclusion of Blanchard and Grosell (2006), when evaluating copper effects on the estuarine killifish, in different salinities. Besides a general difference in the capacity for cell volume regulation that may apply to all cells and tissues of osmoconformers and osmoregulators, as discussed above, gill cells present a particularity. Gill cells of an osmoregulator (C. ornatus) are expected to display membrane transport systems for salt uptake,

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which may not be present at all in gill cells of osmoconformers (Freire et al., 2008a). Thus, a higher Pb2+ uptake into the cells of C. ornatus would be expected, resulting in the more intense effects of the metal in this species, especially under hyposmotic conditions. In agreement, the inhibitors verapamil and ouabain affected volume in C. ornatus gill cells more than in H. pudibundus cells, under both isosmotic and hyposmotic conditions. These last results are compatible with a much higher Na+ /K+ -ATPase activity in the gills of osmoregulators, when compared to osmoconformers (Freire et al., 2008a). Furthermore, in the diluted solutions there is a decrease in the Ca2+ concentration. Less Ca2+ implies less competition between Ca2+ and Pb2+ for the binding site at the membrane calcium transporters, further facilitating the entry of lead (Grosell et al., 2006). A greater amount of lead enters the cells and enhances the efflux of water, especially in C. ornatus. 5. Conclusion Our results suggest that Ca2+ channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus and targets of lead action in these cells. Results have disclosed that aquaporins are much more relevant for water flux in H. pudibundus, and that calcium channels and the Na+ /K+ -ATPase are much more relevant for volume regulation in C. ornatus. Thus, lead effects (direct and indirect) on gill cells are different, depending on the osmoregulatory strategy displayed by the crabs, and accordingly, on their degree of cell volume regulatory capacity. Under decreasing salinity, when hemolymph dilution is expected, gill cell volume regulation of the weak osmoregulator species, which is already less efficient than that of the osmoconformer, is also more affected by lead than that of the osmoconformer species, possibly because of the higher expression of ion transporters, e.g., the Na+ /K+ -ATPase. Acknowledgements Authors would like to thank Dr. R.T.H. Fogac¸a (Department of Physiology, UFPR) for kindly donating the transport blocker verapamil. Dr. Enelise M. Amado was supported through a PhD fellowship from CNPq, Brazil. Experiments were conducted in accordance with Brazilian laws of Ethics in Animal Experimentation (CEEA – UFPR Certificate number 23075.068428/2009-28, issued on May 12, 2009). Authors wish to express their gratitude to all anonymous referees, but very especially to Referee #3. References Ahern, M.D., Morris, S., 1998. Accumulation of lead and its effects on Na+ balance in the freshwater crayfish Cherax destructor. J. Exp. Zool. 281, 270–279. Amado, E.M., Freire, C.A., Souza, M.M., 2006. Osmoregulation and tissue water regulation in the freshwater red crab Dilocarcinus pagei (Crustacea, Decapoda), and the effect of waterborne inorganic lead. Aquat. Toxicol. 79, 1–8. Beeby, A., 1978. Interaction of lead and calcium uptake by the woodlouse, Porcellio scaber (Isopoda, Porcellionidae). Oecologia 32, 255–262. Belyantseva, I.A., Frolenkov, G.I., Wade, J.B., Mammamo, F., Kachar, B., 2000. Water permeability of cochlear outer hair cells: characterization and relationship to electromotility. J. Neurosci. 20, 8996–9003. Blanchard, J., Grosell, M., 2006. Copper toxicity across salinities from freshwater to seawater in the euryhaline fish Fundulus heteroclitus: is copper an ionoregulatory toxicant in high salinities? Aquat. Toxicol. 80, 131–139. Bridges, C.C., Zalups, R.K., 2005. Molecular and ionic mimicry and the transport of toxic metals. Toxicol. Appl. Farmacol. 204, 274–308. Capó-Aponte, J.E., Iserovich, P., Reinach, P.S., 2006. Characterization of regulatory volume behavior by fluorescence quenching in human corneal epithelial cells. J. Membr. Biol. 207, 11–22. Cardin, V., Lezama, R., Torres-Márquez, M.E., Pasantes-Morales, H., 2003. Potentiation of the osmosensitive taurine release and cell volume regulation by cytosolic Ca2+ rise in cultured cerebellar astrocytes. Glia 44, 119–128. Choueri, R.B., Cesar, A., Torres, R.J., Abessa, D.M.S., Morais, R.D., Pereira, C.D.S., Nascimento, M.R.L., Mozeto, A.A., Riba, I., DelValls, T.A., 2009. Integrated sediment quality assessment in Paranaguá Estuarine System, Southern Brazil. Ecotox. Environ. Saf. 72, 1824–1831.

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