Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

The crustacean hepatopancreas is a multifunctional organ with digestive and absorptive properties toward a variety of organic molecules and, in addition,is a site of heavy metal sequestration and detoxification. Detoxification of dietary metals by hepatopancreatic epithelial cells is a result of the simultaneous operation of several intracellular processes working in concert to maintain a low cytoplasmic concentration of these ions while extracellular concentrations may be highly variable(Ahearn et al., 2004a; Roesijadi and Robinson, 1994; Viarengo, 1989). Metallothioneins are low molecular mass proteins in cells that preferentially bind metals and are upregulated when external concentrations of metals rise(Al-Mohanna and Nott, 1985; Brouwer et al., 1989; Brouwer et al., 1992). Besides metallothioneins that reduce the concentrations of soluble cytoplasmic metals,mitochondria (Chavez-Crooker et al.,2002), endoplasmic reticulum(Mandal et al., 2005) and lysosomes (Mandal et al.,2006) are organelle centers of heavy metal sequestration that use membrane transport proteins to accumulate cytoplasmic cationic metals.

Previous studies with lobster (Homarus americanus)heptaopancreatic lysosomes have shown that both copper(Chavez-Crooker et al., 2003)and zinc (Mandal et al., 2006)are transported from hepatopancreatic epithelial cytoplasm into organelle interior by ATP-dependent carrier-mediated transporters that are sensitive to both vanadate and thapsigargin. In addition, both metals were cross-inhibited,suggesting the presence of a metal transporter with a relatively broad substrate specificity. Detoxification of heavy metals in organelles such as lysosomes requires the presence of a mechanism that sequesters the cation in a form that is relatively immobile and cannot be transferred back into the cytoplasm. Electron microprobe analyses of a variety of invertebrate epithelial cells indicate that metals are sequestered together with sulfate and phosphate in solid concretions within membrane-bound organelles(Al-Mohanna and Nott, 1985). While the composition of these concretions has been evaluated in a wide variety of invertebrate species, the mechanisms whereby complexing anions are transferred into these sites of detoxification are unknown. This study examines the nature of anion transport by lobster hepatopancreatic lysosomal membranes and suggests a mechanism by which metal cations and polyvalent anions can be brought together to form detoxifying concretions in these cells.

Live Atlantic lobsters Homarus americanus Milne-Edwards 1837, with a body mass of 500–700 g, were purchased from local commercial dealers in Jacksonville, FL, USA and maintained in a seawater holding tank at 15°C until needed for experimentation. Intermolt (molt stage estimated by gastrolith mass/carapace length ratio) lobsters were used for all experiments. Lobsters were provided with mussel meat for up to 15–20 days and all experiments were conducted only on animals that had been fasted for approximately 24 h to ensure an evacuated hepatopancreas.

Hepatopancreatic lysosomal membrane vesicles (LMV) were prepared from fresh organs of individual lobsters. Hepatopancreatic tissue was quickly placed in chilled Buffer A (in mmol l^–1: 250 sucrose, 20 Hepes, 1 EDTA,0.1 PMSF, adjusted to pH 7.0 with Tris base). The tissue was homogenized with a glass hand homogenizer and diluted tenfold in chilled Buffer A. The homogenate was centrifuged at 800 g for 10 min and the resulting supernatant was centrifuged at 20 000 g for 10 min. The pellet was re-suspended in Buffer B (in mmol l^–1: 250 mmol l^–1 sucrose, 20 Hepes, adjusted to pH 7.0 with Tris base). The suspension was mixed with isotonic Percoll™ in the ratio of 9:11 (pellet suspended in Buffer B:isotonic Percoll™). The Percoll™ mixture was centrifuged at 40 000 g for 90 min. The brownish dense lysosomal band near the bottom of the gradient was removed,diluted with Buffer B and centrifuged at 20 000 g for 10 min. The pellet was then incubated in freshly prepared Buffer B containing 5 mmol l^–1 methionine methyl ester, 2 mg ml^–1bovine serum albumin, and 2 mmol l^–1 MgCl₂ for 20 min at 18°C. An equal volume of ice-cold isotonic Percoll™ was added to the incubation mixture and centrifuged at 35 000 g for 30 min. The purified LMV (located on the top of the gradient as a brownish band)was re-suspended in Preloading Buffer (varied experiment to experiment).

An enzyme characterization of lobster hepatopancreatic LMV produced by the Percoll™ centrifugation method described above has been previously published (Mandal et al.,2006). In this study, the enrichments of three enzymes of known disparate cellular localization were used to show the purification of the LMV samples used in the present investigation. While the brush border enzyme marker, alkaline phosphatase, and the ER marker enzyme, NADPH-cytochrome c reductase, were not significantly enriched (P>0.05),the lysosomal enzyme, acid phosphatase, was purified by more than a factor of 12 in the final vesicle suspension compared to the original tissue homogenate. These data suggest that the LMV fractions used in this previous investigation,and also in the present study, were relatively pure lysosomal membranes and had minimal membrane contamination from other parts of the cell such as the plasma membrane or the endoplasmic reticulum.

Characteristics of ⁶⁵Zn²⁺ transport by isolated vesicles from hepatopancreatic lysosomal membranes were studied at room temperature (23°C). Experiments were initiated by diluting a small volume of vesicle suspension into a medium containing trace amounts of ⁶⁵ZnCl₂ (+ unlabelled zinc sulfate),K³⁶Cl^– (+ unlabelled KCl),K₂³⁵SO₄^2– (+ unlabelled K₂SO₄^2–) or ¹⁴C-oxalic acid(+ unlabelled oxalic acid). The composition of the final vesicle suspension solutions (inside vesicles) and incubation media (outside vesicles) are described separately for each experiment. Uptake of the radiolabelled substrate was initiated by rapidly mixing 20 ml of membrane suspension (150 mg of protein), preloaded with buffer (pH 7.0), with 180 ml of transport medium(described separately for each experiment) and incubating for appropriate time periods. Transport was terminated by addition of 2 ml (tenfold dilution)ice-cold buffer (stop solution) and the suspension was immediately collected under vacuum on a Millipore™ filter (HAWP, Billerica, MA, USA; 45 mm pore size), utilizing the Millipore™ filtration technique developed by Hopfer et al. (Hopfer et al.,1973). Filters were then dissolved in liquid scintillation cocktail (Ecolume™) and the radioactivity counted in a Beckman Coulter LS 6500 multi-purpose scintillation counter. Isotope uptake was expressed as pmol mg^–1 protein s^–1 or as nmol mg^–1 protein s^–1. The protein content of the vesicle suspension was determined according to the Bradford proceedure(BioRad, Hercules, CA, USA), using bovine serum albumin as a standard. Displayed zinc activities were achieved using appropriate concentrations of zinc, NTA (nitriloacetic acid; N,N-bis[carboxymethyl]glycine), and ATP (0.2 mmol l^–1), using Winmax Chelator 2.0 software(Bers et al., 1994).

Isotope uptake into lysosomal vesicles was corrected for non-specific isotope binding (bound activity to exterior of vesicles and not transported to the vesicular interior) by injecting a sample of lysosomal vesicles and isotope directly into ice-cold stop solution without prior mixing. The resulting lysosomal vesicle suspension was then filtered, rinsed and counted as described previously. Resulting values for non-specific isotope binding were subtracted from total isotope uptake in each experiment, providing an index of transmembrane transport of the respective radiolabelled cation or anion. Time points are presented as means of 3–5 replicates and their associated standard errors (s.e.m.). Experiments were repeated at least twice with different animals. Statistical comparisons were made using Student's t-test where a value of P<0.05 was considered significant. Curve-fitting procedures were accomplished using Sigma Plot 9.0 software (Jandel, San Rafael, CA, USA), which provided an iterative best fit to experimental values.

⁶⁵ZnCl₂ was purchased from Oak Ridge National Laboratory, Oak Ridge, TN, USA and ³⁶Cl^–, ³⁵SO₄^2– and ¹⁴C-oxalic acid were obtained from DuPont New England Nuclear Corp., Boston, MA, USA. Valinomycin, Zinc sulfate, Tris, d-mannitol, and other reagent grade chemicals were purchased from Sigma Chemicals (St Louis, MO, USA),Fisher (Pittsburgh, PA, USA), or Bio-Rad (Hercules, CA, USA).

In order to see if the nature of intravesicular anions affected the uptake and equilibrium of ⁶⁵Zn²⁺ by hepatopancreatic lysosomal vesicles, vesicle preparations were filled with a selection of monovalent and polyvalent anions or mannitol and the rate of ⁶⁵Zn²⁺uptake and final metal equilibrium level achieved in these vesicles were compared. As displayed in Fig. 1A, vesicles preloaded with mannitol at pH 7.0 exhibited a slow uptake rate of 25 μmol l^–165Zn²⁺and an equilibrium attained at 30 s of about 2 pmol mg^–1protein. In contrast, when vesicles were preloaded with either 25 μmol l^–1 SO₄^2– or 25 μmol l^–1 PO₄^3– at pH 7.0, the uptake rate of 25 μmol l^–165Zn²⁺ was twice as fast as in mannitol-loaded vesicles and the 20–30 s equilibrium for the metal was also twice that of the vesicles without preloaded anions. There was no significant difference (P>0.05) between the rates of metal uptake or the eventual metal equilibrium attained in vesicles preloaded with the two polyvalent anions.

As shown in Fig. 1B,preloading lysosomal vesicles with either the monovalent anion,Cl^– (25 μmol l^–1) at pH 7.0, or divalent organic anion, oxalate^2– (25 μmol l^–1) at pH 7.0, resulted in 25 μmol l^–165Zn²⁺ uptake rates and metal equilibrium values that were not significantly different (P>0.05) than those achieved by preloading the vesicles with mannitol. These results suggest that the inorganic polyvalent anions, SO₄^2– and PO₄^3–, at pH 7.0 were able to form an intravesicular association with ⁶⁵Zn²⁺ that allowed twice as much metal to accumulate within these compartments than occurred when mannitol, inorganic monovalent anions (Cl^–) or organic polyvalent anions (oxalate^2–) were present.

Because 25 μmol l^–1 SO₄^2–was one of the polyvalent inorganic anions that led to enhanced vesicle accumulation of 25 μmol l^–165Zn²⁺,an experiment was conducted to see what effect a wide range of intravesicular SO₄^2– concentrations would have on the metal uptake by these membrane preparations. Fig. 2 shows that increasing intravesicular SO₄^2– from 25 to 1000 μmol l^–1resulted in a stepwise increase in uptake rate and equilibrium of 25 μmol l^–165Zn²⁺ by hepatopancreatic lysosomal vesicles. Metal uptakes at 500 and 1000 μmol l^–1SO₄^2– were not significantly different(P>0.05) from one another, but were significantly different(P<0.01) from those at lower SO₄^2–concentrations, suggesting that the mechanism resulting in the enhancement of metal uptake reached a maximum around an intravesicular SO₄^2– concentration of approximately 500 μmol l^–1.

Previous studies examining the nature of ⁶⁵Zn²⁺transport by hepatopancreatic lysosomal membrane vesicles showed that metal uptake by these vesicles was stimulated by the presence of ATP in the incubation medium and this nucleotide stimulation was inhibited by vanadate and thapsigargin (Mandal et al.,2006). In addition, ⁶⁵Zn²⁺ uptake was also stimulated by an acidic vesicle interior (pH_i=5.0;pH_o=7.0). These results suggested that zinc transport occurred by a Zn²⁺/H⁺ antiport ATPase, but the exchange stoichiometry of the transporter was unclear. In order to clarify the stoichiometric nature of this exchanger an experiment was conducted using valinomycin (potassium ionophore) and different concentrations of K⁺ across the vesicle membrane. Under these conditions, a transmembrane potential was generated from one side of the membrane to the other. If the Zn²⁺/H⁺antiporter was electroneutral (e.g. 1 Zn²⁺/2H⁺) a transmembrane electrical potential difference would not influence the rate of this exchange, while an electrogenic exchange (e.g. 1 Zn²⁺/1H⁺ or 1 Zn²⁺/3H⁺) would be significantly affected by the imposition of a transmembrane potential.

The influx of ⁶⁵Zn²⁺ was stimulated by a decrease in intravesicular pH and by the presence of external ATP(Fig. 3). Furthermore, in the presence of ATP, metal transport was enhanced by an intravesicular positive electrical potential difference (e.g. K⁺_i<K⁺_o) compared to when the vesicles were either short-circuited (e.g. K⁺_i=K⁺_o) or contained an intravesicular negative electrical potential difference (e.g. K⁺_i>K⁺_o). These results suggest that because an electrically positive intravesicular potential difference increased the uptake of ⁶⁵Zn²⁺, more positive charge must have been transferred out of the vesicle than into the vesicle during the exchange of zinc and protons. The minimum transport stoichiometry that could account for this exchange would be 1Zn²⁺/3H⁺ and this proposed ratio is consistent with the data displayed in Fig. 3.

Because intravesicular polyvalent inorganic anions like SO₄^2– and PO₄^3–enhanced the equilibrium accumulation of ⁶⁵Zn²⁺ within hepatopancreatic lysosomal vesicles, the nature of the uptake process facilitating the transfer of these anions from the cytoplasm was investigated. Two groups of vesicles were prepared. One group was loaded with mannitol and Hepes/Tris at pH 7.0 only and the other had mannitol and intravesicular chloride at pH 7.0. These two vesicle groups were then incubated in media containing ³⁵SO₄^2– or ¹⁴C-oxalate^2– and the time course of isotope uptake into both membrane groups was followed. Fig. 4A shows that ³⁵SO₄^2– uptake was stimulated in vesicles containing intravesicular Cl^– compared to that shown by mannitol-loaded vesicles. Fig. 4B shows a similar response to intravesicular Cl^–by ¹⁴C-oxalate^2– uptake. These results suggest the presence of an anion countertransport process in hepatopancreatic LMV, but do not clarify any of its properties except that it exchanges anions.

The data reported in this figure indicate that there were no significant differences (P>0.05) between the apparent binding affinities (e.g. K_mⁿ) or the relative Hill coefficients(n) at the two pH conditions, but there was a significant (P<0.01)increase in J_max as the internal pH was raised from pH 7.0 to 9.0. In addition, the sigmoidal nature of the influx curves and a Hill coefficient of approximately 2.0 in each instance, suggested that two Cl^– ions were transported across the vesicular membrane during each transport event. Because OH^– ions were the only anion substrate within the vesicles at each of these conditions, the data indicate that these membranes possess a monovalent anion exchanger that exhibited either an antiport stoichiometry of 2Cl^–/1OH^– or 2Cl^–/2OH^–.

While small changes in apparent Cl^– influx K_m ([OH^–] resulting in one-half maximal ³⁶Cl^–) occurred over the range of Cl^– concentrations used, a 12.5-fold increase in Cl^– influx J_max took place from 2.5 to 35 mmol l^–1 Cl^–1. These results implied a far greater effect on maximal ³⁶Cl^– influx rate of intravesicular [OH^–] than on the apparent binding of hydroxyl ions to the exchanger (e.g. K_m). Because the ³⁶Cl^– influx K_m values in all instances were in the acidic pH range, it can be argued that under normal physiological conditions where lysosomes are acidic, the anion antiporter would be saturated with intravesicular OH^– ions and anion exchange would be rate-limited by the cytoplasmic concentration of exchangeable anion substrates. Furthermore, the hyperbolic nature of the Cl^– influx curve over the [OH^–] used implies an electrogenic exchange transport stoichiometry of 2Cl^–/1OH^–.

A series of experiments examining the nature of ³⁵SO₄^2– influx into lysosomal vesicles was performed following the protocol outlined for the investigation of ³⁶Cl^– influx into these preparations as described in Figs 6 and 7. In this instance vesicles were loaded at pH 7.0, 8.0 and 9.0 and were incubated in media containing ³⁵SO₄^2– at concentrations from 2.5 to 50 mmol l^–1. As in the previous series of experiments,OH^– ions were the only exchangeable anion substrate inside the vesicles. Sigmoidal influx kinetics, as described in Eqn 1, were obtained for SO₄^2– influx as a function of external[SO₄^2–] at each of the chosen internal pH conditions (Fig. 8). Results indicated that both sulfate^2– influx K_mⁿ and J_maxincreased significantly (P<0.01) from pH 7.0 to 9.0, but a greater change was observed in the J_max parameters than in the apparent affinity values. A small increase (P<0.02) was also observed in the magnitude of the Hill coefficient n over the pH range examined, but all values approximated 2.0, as was found for Cl^– influx under similar conditions(Fig. 6).

To clarify the exchange stoichiometry between ³⁵SO₄^2– influx into and OH^– efflux from lysosomal vesicles, an experiment was conducted similar to that described in Fig. 7 for ³⁶Cl^–/OH^–exchange. As before, ³⁵SO₄^2– influx was measured into vesicles loaded at several internal pH values and were incubated in media containing either 5 or 10 mmol l^–135SO₄^2–. ³⁵SO₄^2– influx is expressed as a function of internal [OH^–] (nmol l^–1) in Fig. 9. SO₄^2– influx was a hyperbolic function of intravesicular [OH^–] and followed the modified Michaelis–Menten equation (Eqn 4) above except that the respective kinetic parameters related to SO₄^2–, rather than Cl^–, transfer. In this instance increasing external [SO₄^2–] from 5 to 10 mmol l^–1 resulted in apparent K_mvalues for OH^– binding that were not significantly different(P>0.05). In contrast, SO₄^2– influx J_max values were significantly (P<0.01)greater at 10 mmol l^–1 SO₄^2–. The hyperbolic nature of the influx curve in this experiment suggests an electrogenic exchange stoichiometry between the polyvalent and monovalent anions of 2 SO₄^2–/1 OH^–.

Lysosomes are multi-functional intracellular organelles. It is widely accepted that a major role of these organelles in a wide variety of cell types is the disassembly of macromolecules through the use of enzymatic digestion in an acidic microenvironment (Chou et al.,1992; Pisoni and Thoene,1991). In some cells such as renal proximal tubular cells,filtered plasma proteins are reabsorbed by endocytosis and are transferred to lysosomes for degradation (Gekle,1998). This breakdown is taken to tri- and dipeptides, which are exported from lysosomes to cytoplasm where final hydrolysis to amino acids occurs using a lysosomal membrane oligopeptide transporter(Zhou et al., 2000). Similar hydrolysis of other macromolecules such as nucleotides, sugars and protein-associated vitamins (e.g. vitamin B₁₂) occurs in these organelles with monomeric subunits being transferred to the cytoplasm using a variety of membrane-bound lysosomal transport proteins(Pisoni and Thoene, 1991).

In addition to its macromolecular degradation role, lysosomal membranes possess a number of ion transport proteins for transferring both cations and anions between the intralysosomal compartment and the cytoplasm(Pisoni and Thoene, 1991; Chou et al., 1992; Dell'Angelica et al., 2000). Both proton and calcium ATPases have been described for lysosomal membranes(Pisoni and Thoene, 1991; Chou et al., 1992). The V-ATPase was considered responsible for lysosomal acidification, while the Ca-ATPase may help regulate cytoplasmic calcium activity. Calcium transport by human lysosomal membranes was inhibited by other divalent cations(Cd²⁺>Hg²⁺>Zn²⁺>Mg²⁺>Ba²⁺>Sr²⁺),but appeared insensitive to monovalent and trivalent cations(Lemons and Thoene, 1991). Sulfate^2–, phosphate^3–,molybdate^4– and Cl^– are transported across vertebrate lysosomal membranes (Pisoni and Thoene, 1991; Chou et al.,1992). These transport systems are affected by pH and the presence of other anions on the trans-side of a membrane preparation. Jonas and Jobe(Jonas and Jobe, 1990)described sulfate^2– transport across rat liver lysosomal membrane in exchange for Cl^– and suggest the presence of an anion exchanger in this organelle that is regulated by pH or membrane potential. Phosphate^3– transport in human fibroblast lysosomes is strongly affected by pH, but appears highly specific as certain other anions (e.g. SO₄^2–,HCO₃^–, Cl^– or DIDS) have no effect on its transfer (Pisoni and Thoene,1991). However, arsenate^3– was a strong competitive inhibitor of phosphate^3– transport in this system with both substrate and inhibitor having similar binding constants(phosphate^2–K_m=5 μmol l^–1; arsenate^2–K_i=7μmol l^–1). None of the studies that characterized anion transport by lysosomal membranes considered the interactions within the organelle that may result from the simultaneous transport of divalent ions from the cytosol.

Another function of lysosomes is the sequestration of heavy metals such as zinc and copper. Vertebrate lysosomes are known to store zinc by transporting the metal from cytosol to organelle interior by a Znt2 transport protein(McMahon and Cousins, 1998; Liuzzi and Cousins, 2004). In the case of copper, this metal is accumulated in lysosomes as a result of the activities of copper ATPases (ATP7A and ATP7B) or facilitated diffusion systems of the Ctr2 or Ctr6 copper transporter isoforms(Bellemare et al., 2002).

In invertebrate cells, lysosomes are known to sequester a number of cations of both biological relevance (e.g. calcium, copper, zinc, iron) and non-relevance (e.g. cadmium, mercury, lead) in association with anions such as sulfate^2– and phosphate^3– in solid concretions that effectively remove them from cytoplasmic or plasma functionality. These concretions may serve as temporary storage facilities for cations involved in frequent physiological operations such as exoskeletal molting in crustaceans, where lysosomal calcium may be periodically mobilized or stored in cells of various tissues as needed to harden the newly synthesized, and still soft, exoskeletal components. Lysosomal storage of biologically important metals such as copper, zinc and iron may occur in similar endosomes side by side with calcium-containing organelles, but metal reclamation from the depots may occur at rates which are dictated by other physiological needs of the animal such as copper requirements in hemocyanin synthesis or zinc needs in efficient enzymatic activities. Lastly, some metals that have no known biological relevance, such as lead, cadmium and mercury,may be accumulated in certain lysosomes and retained there for the life of the cell, eventually being excreted from the animal through the gastrointestinal tract or some other evacuation site, effectively detoxifying the metals through long-term sequestration.

The experiments reported in this study extend those previously reported for zinc and copper transport by lobster hepatopancreatic lysosomes(Ahearn et al., 2004a; Ahearn et al., 2004b; Chavez-Crooker et al., 2003; Mandal et al., 2006) and for zinc transport by lobster hepatopancreatic endoplasmic reticulum (ER)(Mandal et al., 2005). In these previous studies membranes of both organelles were reported to possess ATP-dependent, thapsigargin- and vanadate-sensitive calcium and metal transport systems that were suggested to play a role in regulating cytosolic concentrations of these divalent cations. The suggestion was made that apparently different isoforms of the ER SERCA (sarco-endoplasmic reticulum calcium ATPase) may be localized at the membranes of both organelles and serve similar regulatory properties in the two sites(Ahearn et al., 2004b). However, the molecular identity of neither transporter was disclosed in these studies and only their relative pharmacological responses were noted.

Data presented in Figs 1 and 2 suggest that the polyvalent anions SO₄^2– and PO₄^3–were more effective in metal accumulation within hepatopancreatic lysosomes than were uncharged solutes (mannitol), monovalent anions(Cl^–), or divalent organic cations(oxalate^2–). In addition, Fig. 3 provides, for the first time, a proposed exchange stoichiometry of the zinc transporter in these membranes. As a result of the action of an ATP-dependent V-ATPase(Chavez-Crooker et al., 2003)on lysosomal membranes, the interior of these organelles is likely acidic and the charge across the membrane would be oriented positive inside. The apparent exchange stoichiometry for the zinc uptake process, as suggested by the results in Fig. 3, and by the proposed orientation of membrane potential, would be 3H⁺/1Zn²⁺ and could be powered by both ATP and the membrane potential. Alternatively, it is possible that the asymmetric cation exchanger is not directly activated by ATP, but is instead only indirectly linked to the ATP-dependent, V-ATPase through the membrane potential it generates.

Generation of an inside positive membrane potential by the V-ATPase serves as a driving force for electrogenic anion exchange whereby cytosolic divalent or trivalent anions exchange with intralysosomal monovalent anions. Results in Figs 5, 6 and 9 suggest that two cytosolic anions (either 2SO₄^2– or 2Cl^–)are able to exchange for single intralysosomal anions (1Cl^–or 1OH^–). In contrast, only a single divalent organic cation(1oxalate^2–) exchanges with a single intralysosomal monovalent anion (1Cl^– or 1OH^–). These stoichiometry differences between inorganic and organic anions may be a function of their relative hydrated radii and the fit they have at the exchanger cytoplasmic binding site. The hydrated radii of Cl^–, SO₄^2– and oxalate^2– are 0.30, 0.40 and 0.45 nm, respectively(Kielland, 1937), suggesting that the exchange stoichiometry of the transporter may convert from 2:1 to 1:1 for cytoplasmic ions possessing a hydrated radius in excess of 0.40 nm.

Once inside the lysosomal acidic interior both metals and polyvalent anions are able to increase in concentration to values in excess of those in the cytosol. Because the intralysosomal binding sites for both the 3H⁺/1Zn²⁺ and 2SO₄^2–/1OH^– exchangers prefer monovalent ions, the transfer of either divalent metals or polyvalent anions back to the cytoplasm would be limited and would effectively trap the metals inside lysosomes in a soluble state at acidic pH. Reduction in V-ATPase activity with coincident elevation of lysosomal pH would likely precipitate metal–anion complexes and lead to the formation of a solid concretion that would detoxify the metal by lowering its availability for transfer from the lysosome. The nature of the control process(es) for variation in V-ATPase activity with resultant changes in intralysosomal pH and concretion formation is not known, but would be a fascinating extention of the present study.

The findings of this study are summarized in Fig. 10, which suggests how the hepatopancreatic lysosomal V-ATPase may power two electrogenic ion exchangers through the use of ATP and the induced membrane potential and bring about the accumulation of metals and polyvalent anions within these organelles. As shown in the figure, the asymmetric exchange stoichiometries for metal and SO₄^2– uptake lead to the accumulation of both ions within the organelle. It is likely that PO₄^3– may also enter lysosomes by the same exchanger and be available for concretion formation under the appropriate conditions (Fig. 1). Previously published experiments have shown that zinc, copper and cadmium are competitive inhibitors for transport into hepatopancreatic lysosomal vesicles(Mandal et al., 2006) and it is likely that other divalent metals and calcium are transported out of the cytosol by this mechanism. The model further suggests that high lysosomal concentrations of metals and polyvalent anions may condense into a concretion with a change in pH brought about by alterations in V-ATPase activity.

Several unanswered questions remain about the nature of concretion formation and heavy metal detoxification by the processes shown in Fig. 10. The three most significant are: (1) what is the molecular nature of the ATP-dependent,electrogenic, metal transporter that exchanges with intralysosomal protons? Is it a lysosomal isoform of the ER thapsigargin- and vanadate-sensitive calcium ATPase (e.g. SERCA) (Mandal et al.,2005)? Or is it a member of the metal transporting proteins identified for vertebrate cells such as the Znt2 or ATP7A or ATP7B(McMahon and Cousins, 1998; Liuzzi and Cousins, 2004; Vulpe and Packman, 1995; Suzuki and Gitlin, 1999)? (2)What is the physiological nature of the regulatory process(es) that control(s)lysosomal pH and therefore regulate(s) whether enclosed metals are soluble or insoluble? (3) Are there multiple populations of lysosomes within the hepatopancreas that individually regulate the sequestration of different cations that are needed for different physiological requirements, as electron microscopic studies coupled with microprobe analysis of lysosomal contents have previously suggested (Al-Mohanna and Nott, 1985; Al-Mohanna and Nott, 1989; Hopkin,1989; Nott, 1991)?Future experiments will be directed at elucidating some of the answers to these critical aspects of heavy metal detoxification.

This study was supported through a grant from the National Science Foundation (IBN04-21986).