Effects of Hg2+ on Ca2+ Dynamics in the Scallop Sarcoplasmic Reticulum (Pecten jacobaeus): Protective Role of Glutathione

Comp. Biochem. Physiol. Vol. 116C, No. 1, pp. 77–83, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00126-0

Effects of Hg21 on Ca21 Dynamics in the Scallop Sarcoplasmic Reticulum (Pecten jacobaeus): Protective Role of Glutathione B. Burlando,* A. Viarengo,* M. Pertica,† G. Mancinelli,† B. Marchi† and M. Orunesu† *Dipartimento di Scienze e Tecnologie Avanzate, Universita` di Torino, Corso Borsalino 54, 15100 Alessandria, Italy, and †Istituto di Fisiologia Generale, Universita` di Genova, Corso Europa 26, 16132 Genova, Italy

ABSTRACT. Scallop (Pecten jacobaeus) sarcoplasmic reticulum (SR) vesicles were functionally characterized and tested for Hg21 effects. An SR-containing 20,000 g supernatant from adductor muscle homogenate was incubated with fluo-3, allowing free Ca21 variations to be spectrofluorimetrically followed. Data showed an ATPdependent thapsigargin-sensitive Ca21 uptake, revealing the activity of the SR Ca21 ATPase. Treatment with ryanodine elicited Ca21 release, showing the presence of ryanodine-sensitive Ca21 channels, whereas InsP3 caused negligible effects. Exposure to different concentrations of Hg21 (1–50 µM) produced a dose-dependent Ca21 release from SR vesicles, which was shown to depend on both Ca21 ATPase inhibition and Ca21 channel opening. Hg21 binding to sulfhydryls was pointed out by incubation with Thiolyte, whereas an involvement of sulfhydryls in Ca21 release was assessed by treatment with the sulfhydryl reagent N-ethylmaleimide (NEM). Yet, conversely to Hg21, NEM seemed unable to open Ca21 channels, suggesting that the latter effect occurs via some specific heavy metal interaction, possibly involving sulfhydryls not available to larger molecules or even components other than sulfhydryls. Pre-incubation of SR with reduced glutathione (GSH) largely prevented Hg21 effects, whereas a certain reduction of metal injury also occurred by adding GSH after Hg21 exposure, thus confirming the role of GSH as a first line of defense against heavy metals. Copyright  1997 Elsevier Science Inc. comp biochem physiol 116C;1:77–83, 1997. KEY WORDS. Scallop, sarcoplasmic reticulum, Ca21 dynamics, Hg21-induced intracellular Ca21 release, Ca21dependent cytotoxicity, reduced glutathione

INTRODUCTION Heavy metals are known to increase the concentration of cytosolic Ca 21 ([Ca21]i) in a variety of cell types (14,15,22– 24,33,36,40). Sustained [Ca21]i increase is often considered a main pathway of cytotoxicity, and in the case of heavy metals, it is generally ascribed to interactions with cell components involved in cytosolic Ca21 modulation, such as the plasma membrane/endoplasmic Ca 21 ATPases (2,13,19,28, 29) and Ca21 channels (9,16,18,40). Marine organisms are frequently exposed to variable concentrations of heavy metals, which can reach particularly high levels in specific coastal areas. Hence, these organisms are of particular interest for studying the effects of heavy metals on Ca21 homeostasis. Marine invertebrates, for instance, can accumulate high amounts of heavy metals in their tissues (3), but in these organisms the mechanisms Correspondence to: A. Viarengo, Istituto di Fisiologia, Universita` di Genova, Corso Europa 26, 16132 Genova, Italy. Received 8 March 1996; accepted 27 June 1996.

involved in Ca21 homeostasis and Ca21-mediated cytotoxicity are still scarcely known. Heavy metals were found to increase [Ca21]i in mussel hemolymph cells (33) and in protozoa (31), and evidence was given that different heavy metals inhibit a Ca21 ATPase of mussel gill cell plasma membranes both in vitro (36,35) and in vivo (37). However, data concerning heavy metal effects on endocellular Ca21 stores in invertebrates were hitherto nearly absent. This study aims to clarify the mechanisms of Ca21 handling and metal-dependent Ca21 cytotoxicity in the sarcoplasmic reticulum (SR) of the marine scallop Pecten jacobaeus. We first characterized the functioning of SR vesicles from the scallop cross-striated adductor muscle and then we tested the SR sensitivity to mercury (Hg). This non-essential heavy metal is one of the most hazardous seawater pollutants (4) and was studied before for its modulatory action on cytosolic Ca21 (30). Considering that reduced glutathione (GSH) may represent an important cell defense against heavy metals (20), we also explored the possibility that GSH reduces Hg21 effects on the scallop SR and thus preserves muscle cells from Ca21-mediated cytotoxicity.

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MATERIALS AND METHODS Materials Ouabain, EGTA, adenosine 5′-triphosphate (ATP), Nethylmaleimide (NEM), reduced GSH and calcium ionophore A23187 were from Sigma Chemical Company (St Louis, MO, U.S.A.). Ryanodine, thapsigargin and d-inositol 1,4,5-tris-phosphate (InsP3) were from Alomone Labs (Jerusalem, Israel). Fluorescent probe fluo-3 was from Molecular Probes Inc. (Eugene, OR, U.S.A.), Thiolyte was from Calbiochem-Novabiochem Corp. (La Jolla, CA, U.S.A.). All other reagents were of analytical grade. SR Isolation SR-containing fractions were prepared from fresh adductor muscle of scallops (P. jacobaeus) obtained from a commercial fishery. The adductor muscle was homogenized with a blendor in a buffer consisting of 0.32 M sucrose, 20 mM HEPES (pH 7.2), 1 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride and 2 µM leupeptine. The homogenate was centrifuged at 20,000 g for 20 min at 4°C, and the resulting SR-containing supernatant was stored at 280°C until use. Aliquots of this supernatant were also centrifuged at 100,000 g for 1 hr at 4°C, and the resulting microsomal pellet was stored at 280°C. Ca21 Transport Assays Ca21 uptake and release were determined on the SR-containing 20,000 g supernatant using a Perkin Elmer LS 50B spectrofluorimeter (Perkin Elmer Ltd., Beaconsfield, England) (Ex 5 510 nm, Em 5 530, slit 2.5 nm). The 100,000 g microsomal pellet was not suitable for studying Ca21 dynamics. Aliquots of the 20,000 g supernatant (approximately 2 mg protein) were incubated with 30 mM HEPES (pH 7.2), 200 mM KCl, 1 mM MgCl 2, and 2.5 µM fluo-3, a fluorescent indicator for free Ca 21 (10). The reaction mixture was adjusted to obtain experimental conditions as close as possible to physiological parameters of marine invertebrates, concerning in particular pH, ionic strength, ATP levels and free Ca21 concentration. Ca21 uptake was started by addition of 4 mM ATP, time was allowed for the reaching of a stable baseline level and thereafter vesicles were loaded with Ca21 by addition of suitable amounts of CaCl2 (50 µmol⋅121). Free Ca21 concentrations in the reaction mixture were estimated by the formula (7) [Ca21] 5

F 2 Fmin 3 Kd, Fmax 2 F

where F is the fluorescence value to be converted into [Ca21], Fmin is minimum fluorescence, Fmax is maximum fluorescence and Kd 5 400 nM is the dissociation constant of the fluo-3/Ca21 complex. The effects of Hg21 on Ca21 modulations were investigated by adding HgCl2 (1–50 µM) to the reaction mixture

during incubation of the 20,000 g supernatant in the presence of ATP. In these experiments, the metal was added during spectrofluorimetric recording after having loaded SR vesicles by Ca21 addition. Thapsigargin (200 nM), ryanodine (25 µM), InsP 3 (10 µM), NEM (500 µM) and GSH (500 µM) were also added in some cases to the reaction mixture during incubation. It was also assessed that under the conditions used for Ca21 transport assays, Hg21 levels up to 80 µM cause a negligible change in the fluo-3 signal. Assay of Hg21 Binding to SH Residues Aliquots of the microsomal preparation (0.3 ml, corresponding to approximately 600 µg protein) were incubated with the reaction mixture used for Ca21 transport assays, which in some cases contained 20–50 µM Hg21. In these experiments, instead of fluo-3 the reaction mixture contained 50 µM Thiolyte (monobromobimane), a compound forming a fluorescent adduct by specifically binding to sulfhydryls (11). Kinetics of probe binding to SH were followed using a spectrofluorimeter (Ex 5 391 nm, Em 5 474 nm). A test for Hg21 (50 µM) interference with Thiolyte showed no detectable change of the probe signal (data not shown). Assay of Ca21 ATPase Activity Ca21 ATPase activities were evaluated on the microsomal pellet through determination of organic phosphate (Pi) release according to the method of Martin and Doty (12) as modified by Viarengo et al. (38). The reaction mixture contained 30 mM HEPES (pH 7), 200 mM KCl, 2 mM MgCl2, 2 mM EGTA when present and 5 µM free Ca 21 when present. Titration of free Ca21 in the reaction mixture was achieved by using the software described by Fabiato and Fabiato (5). Microsomes (about 92 µg protein) were incubated in the mixture, containing in some cases Hg21 (1– 20 µM); the reaction was then immediately started by the addition of 2 mM ATP and after 30 min at 19°C it was stopped with cold trichloroacetic acid 50%. The Ca21-stimulated ATPase activity was evaluated by subtracting the amount of Pi released in the absence of free Ca21 (2 mM EGTA, no Ca 21 added) from that released in the presence of 5 µM Ca21. Assay of Protein Content Protein content was evaluated by the method of Hartree (8) on both the 20,000 g supernatant and the 100,000 g microsomal pellet, using bovine serum albumin as standard. RESULTS In experiments concerning Ca21 uptake and release, repeatable results were obtained with the SR-containing 20,000 g supernatant but not with vesicles obtained from resuspen-

Effects of Hg21 on Ca21 Dynamics in the Scallop SR

FIG. 1. Characterization of Ca21 handling in the scallop SR

vesicles. (a) When ATP (4 mM) was present in the incubation medium, sequential additions of Ca21 (50 mmol⋅l21), inducing a sudden increase of the free Ca21 concentration, were invariably followed by Ca21 sequestration into SR vesicles, restoring Ca21 values down to baseline. Moreover, addition of the calcium ionophore A23187 caused prompt Ca21 efflux from SR vesicles. By contrast, when ATP was absent, there was a slow release of Ca21 from SR vesicles, which also occurred after Ca21 addition until a plateau was reached. (b) Addition of thapsigargin (200 nM) to Ca21-loaded vesicles induced Ca21 efflux, whereas a further addition of Ca21 was not followed by uptake. (c) Treatment of SR vesicles with ryanodine (25 mM) produced a sudden release of Ca21 followed by resequestration. (d) Conversely, treatment with InsP3 (10 mM) caused negligible Ca21 release. In this and the following figures, spectrofluorimetric traces are representative of at least three experiments; pCa 5 2log10 [Ca21].

sion of the 100,000 g microsomal pellet. Therefore, the latter preparation was only used for assays of Ca21-ATPase activity through determination of Pi release and for the evaluation of sulfhydryls availability by using Thiolyte. Incubation of the 20,000 g supernatant with the fluorescent dye fluo-3, coupled to spectrofluorimetric recording, allowed for the following of Ca 21 uptake and release kinetics under different experimental conditions. When incubation was started in the presence of ATP, background free Ca21 was rapidly driven to a baseline level of 70–100 nM (fluorescence decreased), which is very close to normal cytosolic Ca 21 levels. Moreover, the system was able to repeatedly restore the above baseline level within about 10 min of each consecutive Ca21 addition (Fig. 1a). Conversely, when no ATP was present there was a slow release of Ca21 from the SR vesicles to the incubation medium (fluorescence increased), which also continued after a sudden rise of the free Ca21 level induced by addition of CaCl2 (Fig. 1a). The ATP-dependent capability of the system to restore Ca21 levels down to base-

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line, also occurring after repeated Ca21 additions, clearly indicated Ca21 sequestration into SR vesicles. Also, addition of the Ca21 ionophore A23187 (1 µM) after Ca 21 sequestration elicited Ca21 efflux from SR vesicles (Fig. 1a). When SR vesicles, pre-incubated with ATP and loaded with Ca 21, were treated with 200 nM thapsigargin, a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca21 ATPase (25), an efflux of Ca21 from the SR vesicles was detected, whereas a subsequent addition of Ca21 was not followed by uptake (Fig. 1b). In different experiments, addition of 50 µM ryanodine induced a sudden Ca21 release (Fig. 1c) followed by resequestration, whereas treatment with 10 µM InsP3 induced a negligible variation of baseline Ca 21 levels (Fig. 1d). Evaluation of mercury effects involved the exposure of Ca21-loaded SR vesicles to increasing Hg 21 levels. Addition of 1 µM Hg 21 (Fig. 2a) produced a minimal rise of the baseline Ca21 level, whereas a subsequent Ca21 addition was followed by normal uptake. By using 5 µM Hg21 (Fig. 2b), there was a higher Ca21 release followed by a stabilization of the Ca 21 concentration at a steady-state level above the control baseline, whereas after Ca21 addition the Ca21 level was restored down to the metal-induced steady-state level. Treatment with 10 µM Hg21 (Fig. 2c) also led to a sudden Ca21 release that was higher than that recorded at 5 µM

FIG. 2. Effects of Hg21 on Ca21-loaded SR vesicles. (a) Addi-

tion of 1 mM Hg21 to the SR-containing reaction mixture produced a minimal increase of the baseline Ca21 level, whereas the ensuing addition of Ca21 was followed by Ca21 sequestration down to near baseline levels. (b) Addition of 5 mM Hg21 caused a fast Ca21 release followed by a stabilization of the free Ca21 concentration to a level higher than control baseline. Moreover, a subsequent Ca21 addition was followed by Ca21 sequestration restoring the free Ca21 concentration down to the altered level. (c) Treatment with 10 mM Hg21 caused sudden Ca21 release followed by a stabilization and then by a further release occurring at a slower rate. Thereafter, Ca21 addition elicited a partial Ca21 sequestration but Ca21 release eventually prevailed. (d) Treatment with 50 mM Hg21 induced a sudden and massive release of Ca21, rapidly driving the free Ca21 concentration to a plateau.

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TABLE 1. Effects of Hg 21 on the Ca 21-dependent Pi release

in microsomal fractions from scallop adductor muscle Treatment Control 1 µM Hg 21 5 µM Hg 21 20 µM Hg 21 FIG. 3. Effects of NEM on SR vesicles and of NEM and Hg21

after pre-incubation with thapsigargin. (a) Treatments with increasing concentrations of thapsigargin (0.2, 5.0, 10 mM) caused dose-dependent Ca21 efflux from SR vesicles. Addition of 50 mM Hg21 after any thapsigargin treatment caused a further increase of the rate of the thapsigargin-induced Ca21 efflux. In a different experiment, addition of NEM (500 mM) after thapsigargin (0.2 mM) did not modify the rate of Ca21 efflux. (b) Treatment of Ca21-loaded SR vesicles with NEM (500 mM) produced a release of Ca21, whereas a subsequent addition of Ca21 was not followed by Ca21 sequestration.

Hg21, whereas after Ca21 addition, the uptake process was initially manifested but was eventually overwhelmed by massive Ca21 release from SR vesicles. Finally, the use of 50 µM Hg 21 (Fig. 2d) led to an abrupt and massive Ca21 release, whereas Ca21 uptake was no longer detectable. Measurements of the amounts of Ca 21 mobilized from SR vesicles just after metal additions yielded mean values (n 5 4 to 5) of 30 6 2 pmol Ca21 /mg protein at 5 µM Hg21, 50 6 3.5 pmol/mg protein at 10 µM, and 1000 6 200 pmol/ mg protein at 50 µM. The mechanisms of the Hg21-dependent Ca21 modulation were investigated by using treatments with thapsigargin followed by exposure to Hg21. Increasing thapsigargin concentrations (0.2–10 µM) resulted in dose-dependent rates of Ca21 efflux from SR vesicles, with a faster release occurring immediately after thapsigargin addition followed by a slow down of the efflux rate (Fig. 3a). However, at all thapsigargin concentrations, addition of 50 µM Hg21 a few minutes after thapsigargin strongly increased the rate of Ca21 efflux from SR vesicles (Fig. 3a). Treatment of loaded vesicles with NEM (500 µM), a sulfhydryl reagent, also led to Ca21 efflux, whereas no uptake was recorded after Ca21 addition (Fig. 3b). Conversely to what was observed with Hg 21, when NEM was added after exposure to the lowest concentration of thapsigargin, it was not able to increase the rate of the thapsigargin-induced Ca21 efflux (Fig. 3a). A direct indication of the SR Ca 21 ATPase inhibition by Hg21 was obtained through the evaluation of Ca21-dependent Pi release during microsomal incubation under different Hg21 concentrations. Pi release was significantly inhibited even at a minimal concentration of 1 µM Hg21, whereas increasing inhibitions were found for increasing (5–20 µM) Hg21 concentrations (Table 1). The binding of Hg 21 to microsomal sulfhydryl residues

Activity 2.93 1.80 1.60 1.33

6 6 6 6

0.20 0.08* 0.68** 0.23**

n

Inhibition (%)

5 4 4 4

0 38.6 45.2 54.6

Values are expressed as µmol Pi ⋅ h 21 ⋅ mg protein 21. All treatments are significantly different from control, according to Bonferroni t test (*P , 0.05, **P , 0.01).

was evaluated by using kinetics of Thiolyte binding to sulfhydryls, recorded as increasing fluorescence over time. Incubation of microsomes with 20 µM Hg21 led to a drastic reduction in the rate of increase of the Thiolyte fluorescence signal with respect to controls, whereas at 50 µM Hg21 there was no fluorescence increase (Fig. 4). When SR vesicles were pre-incubated with 500 µM GSH, loaded with Ca21 and then exposed to 20 µM Hg21, the metal-induced Ca21 release was drastically reduced with respect to a parallel test where GSH was absent, whereas a subsequent Ca21 addition showed that GSH also preserved the activity of Ca 21 uptake (Fig. 5a). In a different experiment, Ca21-loaded SR vesicles were exposed to 10 µM Hg 21, producing a sudden alteration of the baseline Ca21 level, and thereafter sequential additions of GSH restored the control Ca 21 baseline level in a stepwise manner (Fig. 5b). DISCUSSION The cell-free system obtained from scallop adductor muscle showed in vitro free Ca 21 modulations comparable with those reported for other SR preparations deriving from dif-

FIG. 4. Spectrofluorimetric traces showing kinetics of Thio-

lyte (50 mM) binding to microsomal sulfhydryls (increasing fluorescence). Addition of Hg21 to the incubation medium induced a clear dose-dependent reduction of the rate of Thiolyte binding with respect to the control curve. int. 5 fluorescence intensity.

Effects of Hg21 on Ca21 Dynamics in the Scallop SR

FIG. 5. Protective action of GSH against the effects of Hg21

on SR vesicles. (a) Pre-incubation of SR vesicles with 500 mM GSH led to a sharp reduction in the Ca21 release induced by Hg21 (20 mM), also allowing an almost complete sequestration of sequentially added Ca21. (b) After treatment with 10 mM Hg21, a series of GSH additions (each 500 mmol⋅l21) were able to restore, in a stepwise manner, the metal-induced alteration of Ca21 level, also allowing an almost complete Ca21 sequestration after final Ca21 addition.

ferent organisms (6,18,41). The occurrence of an ATP-dependent Ca21 uptake, which was also sensitive to thapsigargin, clearly revealed the activity of the SR Ca21 ATPase. In the presence of ATP, sequential additions of CaCl2 demonstrated that the capacity of Ca21 sequestration was maintained by SR vesicles for at least one hour (i.e., a time length comparable with the duration of all other tests performed in this study). Also, the Ca21 release induced by ryanodine showed the presence of ryanodine-sensitive Ca21 channels, whereas the negligible variation of free Ca21 obtained with InsP 3 was an indication that this compound does not play an important role as a physiological activator of Ca21 channels in the scallop SR. Taken together, these features of the scallop SR compare with what is known for the mammalian skeletal muscle (17). A variety of heavy metals, such as Ag1 , Cd21, Cu21, Zn21 and Hg 21, are known to stimulate Ca21 release from SR, as well as from other endocellular Ca21 stores, although this does not seem to be a general rule because, for example, Ni 21, Cd 21 and Cu 21, were found to be ineffective on cardiac SR vesicles (18). Such a Ca 21 release has been reported to occur through the opening of Ca21 channels because of metal interaction with sulfhydryl groups closely linked to the channel components (1,18,27,41). In addition, Cd21, Cu 21 and Zn21 have also been found to inhibit the endoplasmic reticulum Ca21 ATPase both in the skeletal muscle (1) and in liver cells (41), suggesting that such an effect could also play a role in the metal-induced Ca21 efflux from endocellular stores. Considering our data, an effect of Hg21 on both Ca21 channels and Ca 21 ATPase seems confirmed also in the scallop SR vesicles. Hg21 treatments triggered a release of Ca21 that varied in intensity and pattern as the metal concentration rose. The lowest metal concentration (1 µM) only resulted in a minimal variation of the free Ca21 level, whereas at 5 µM Hg21 the higher upward shifting of free Ca21, followed by stabilization, suggested a sudden Ca21 release from SR vesicles that was then partially compensated for by resequestration. At still higher concentrations of Hg21, there

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was no stabilization of the Ca21 levels after metal addition, probably due to the release of larger Ca21 amounts occurring together with a progressive decline of the Ca21 uptake efficiency. By examining our data more in detail, evidence of Ca21 channel opening by Hg 21 derived from the fact that the metal was able to increase the rates of Ca21 efflux generated by thapsigargin, even after treatments with high drug concentrations. On the other hand, an effect of Hg21 on the SR Ca21 ATPase activity was also shown by the inhibition of the Ca21-dependent P i release. Such a result is consistent with spectrofluorimetric recordings showing a progressive inability of SR vesicles to compensate for the Hg 21-induced Ca21 release as the metal concentration increased. However, it should be noted that the inhibitory effects of Hg21 on Pi release, evaluated on the 100,000 g microsomal pellet, are not quantitatively comparable with the Hg 21 effects on Ca21 sequestration observed spectrofluorimetrically, as the latter were obtained using the 20,000 g supernatant that contains higher amounts of soluble heavy metal ligands. As reported above, different literature sources ascribe to sulfhydryls a pivotal role in the heavy metal-dependent mobilization of intracellular Ca21. In our study, Hg 21 binding to sulfhydryls was confirmed by the metal ability to strongly reduce the fluorescence of the sulfhydryl probe Thiolyte. In addition, a relationship between sulfhydryls and Ca21 release was also shown by exposure of SR vesicles to the sulfhydryl reagent NEM. Yet, NEM was not able to increase the rate of Ca21 release beyond that induced by thapsigargin, in contrast to the effect observed when Hg21 was added after thapsigargin. The latter result was also found in a study where different heavy metals were used on liver endoplasmic Ca21 stores (41), which led the authors to hypothesize an effect of NEM on the Ca21-pumping ATPase but not on Ca21 channels. The evidence that particular heavy metals are able to open Ca21 channels whereas a sulfhydryl reagent seems ineffective argues for the existence of some specific heavy metal-sulfhydryl interaction. Sulfhydryl oxidation with disulfide formation was, in fact, proposed as a possible mechanism for the opening of ryanodine-sensitive Ca21 channels, at least under physiopathological conditions (1,27). This suggests that an equivalent effect may also derive from the formation of a complex between a heavy metal cation and different SH groups. Alternatively, heavy metal cations could mimic Ca21-induced Ca21 release by interacting with some protein site not accessible to larger molecules. Finally, it should be noted that the available evidence does not exclude the possibility that heavy metal-induced Ca21 release from endocellular stores may also involve metal binding to components other than sulfhydryls, such as, for instance, imidazole groups, which for example are reported as a possible target for the interaction of heavy metals with GABA receptors (21). Compounds such as dithiothreitol (DTT) and reduced GSH have been found to moderate heavy metal-induced

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Ca21 modulations. DTT is reported to be more effective than GSH (14,41), but GSH is a native cell thiol and therefore provides better insight about the possibilities of the cell to detoxify heavy metals in natural situations. The concentrations of GSH used in our experiments fall within the physiological range reported for in different mollusk species, also including the scallop (32,34). This clearly shows that physiological concentrations of GSH are able to counteract the action of Hg 21 on scallop SR vesicles by either preventing or reversing metal effects. Such a result is a confirmation of the supposed role of GSH as a first-line defense mechanism against heavy metals within cells (20,30). However, it should be noted that the quantitative aspects of these in vitro experiments cannot be directly applied to natural situations, as the GSH cell content of marine mollusks is known to undergo marked seasonal variations (34). Moreover, environmental stress can involve depletion of cell defense systems, including GSH (34,37), thereby affecting the possibility of cells to counteract the toxicity of pollutant compounds such as heavy metals. The finding that Hg 21 can affect Ca21 handling in the scallop SR vesicles fairly integrates with previous data about heavy metal injury to plasma membrane Ca21 homeostasis systems in the mussel (35,37), as scallop and mussels are two closely related organisms. The metal concentrations used in these investigations are higher than those to which are commonly exposed marine organisms. However, data of Hg21 bioaccumulation in the Mediterranean (26,39) suggest that in heavily contaminated areas, Hg 21 levels in the muscle of bivalve mollusks can reach values as high as those used in experimental studies. Also, it has been shown in marine mollusks that experimental exposure to heavy metal mixtures results in additive effects on Ca21 homeostasis systems (30), hence suggesting the possible occurrence of Ca21-mediated cytotoxic effects also in areas affected by moderate levels of different pollutants. Such a complex of evidence allows a first outline of heavy metal cytotoxicity pathways in marine mollusks. Heavy metal-induced alterations of cytosolic Ca21 are likely to start from metal interactions with plasma membrane components, probably the first target of metal offence to cells, but the process can be further sustained after heavy metal penetration into the cells through a direct action on endocellular Ca 21 stores.

4. 5.

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This work was supported by grants from the Italian Ministry for University and Scientific Research (MURST).

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