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First published online September 14, 2007
Journal of Experimental Biology 210, 3484-3493 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008300

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

Kenneth M. Sterling¹, Prabir K. Mandal¹, Barbara A. Roggenbeck¹, Sean E. Ahearn¹, George A. Gerencser² and Gregory A. Ahearn^1,*

¹ Department of Biology, University of North Florida, 4567 St Johns Bluff Road, S., Jacksonville, FL 32224, USA
² Department of Physiology, University of Florida, Gainesville, FL, 32610, USA

^* Author for correspondence (e-mail: gahearn{at}unf.edu)

	Summary

TOP Summary Introduction Materials and methods Results Discussion References

 
Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and ⁶⁵Zn²⁺, ³⁶Cl^–, ³⁵SO₄^2– and ¹⁴C-oxalate^2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO₄^2– or PO₄^3– led to a threefold greater steady-state accumulation of Zn²⁺ than similar vesicles loaded with mannitol, Cl^– or oxalate^2–. The stimulation of ⁶⁵Zn²⁺ uptake by intravesicular sulfate was SO₄^2– concentration dependent with a maximal enhancement at 500 µmol l^–1. Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn⁺/3H⁺. ³⁵SO₄^2– and ¹⁴C-oxalate^2– uptakes were both enhanced in vesicles loaded with intravesicular Cl^– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. ³⁵SO₄^2– influx was a sigmoidal function of external [SO₄^2–] with 25 mmol l^–1 internal [Cl^–], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl^– or OH^– ions. ³⁶Cl^– influx was a sigmoidal function of external [Cl^–] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl^– for 1 OH^–. ¹⁴C-oxalate influx was a hyperbolic function of external [oxalate^2–] with 25 mmol l^–1 internal [Cl^–], suggesting a 1:1 exchange of oxalate^2– for Cl^–. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO₄^2– and PO₄^3–) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Key words: zinc transport, lysosomes, detoxification, sulfate, oxalate, chloride, anion exchange, heavy metals, lobster, Homarus americanus, V-ATPase, electrogenic transport

	Introduction

TOP Summary Introduction Materials and methods Results Discussion References

 
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.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion References

 
Animals
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.

Isolation of hepatopancreatic lysosomal membrane vesicles (LMV)
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^TM in the ratio of 9:11 (pellet suspended in Buffer B:isotonic Percoll^TM). The Percoll^TM 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^–1 bovine serum albumin, and 2 mmol l^–1 MgCl₂ for 20 min at 18°C. An equal volume of ice-cold isotonic Percoll^TM 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^TM 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.

⁶⁵Zn²⁺, ³⁶Cl^–, ³⁵SO₄^2– and ¹⁴C-oxalate^2– transport measurements
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^TM filter (HAWP, Billerica, MA, USA; 45 mm pore size), utilizing the Millipore^TM filtration technique developed by Hopfer et al. (Hopfer et al., 1973). Filters were then dissolved in liquid scintillation cocktail (Ecolume^TM) 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.

Chemicals
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).

	Results

TOP Summary Introduction Materials and methods Results Discussion References

 
Effect of intravesicular anions on ⁶⁵Zn²⁺ uptake
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^{–1 65}Zn²⁺ and an equilibrium attained at 30 s of about 2 pmol mg^–1 protein. 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^{–1 65}Zn²⁺ 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.

View larger version (11K): [in this window] [in a new window]   Fig. 1. (A) Effects of intravesicular polyvalent inorganic anions and mannitol on the time course of 25 µmol l–1 65Zn2+ uptake by hepatopancreatic lysosomal membrane vesicles (LMV). Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, and 25 µmol l–1 K2SO4, K3PO4 or mannitol, and were then incubated in a medium containing 200 mmol l–1 mannitol, 25 µmol l–1 65Zinc-sulfate, 0.5 mmol l–1 NTA, 0.2 mmol l–1 ATP and 20 mmol l–1 Hepes/Tris, pH 7.0. (B) Effects of intravesicular monovalent inorganic anions (Cl–1), polyvalent organic anions (oxalate2–) and mannitol at pH 7.0 on the time course of 25 µmol l–1 65Zn2+ uptake by LMV. Uptake conditions as in A except that in this instance vesicles were loaded with 25 µmol l–1 oxalic acid, NaCl or mannitol. Experiments were conducted in triplicate; values are means ± 1 s.e.m. at each time point.

Because 25 µmol l^–1 SO₄^2– was one of the polyvalent inorganic anions that led to enhanced vesicle accumulation of 25 µmol l^{–1 65}Zn²⁺, 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^–1 resulted in a stepwise increase in uptake rate and equilibrium of 25 µmol l^{–1 65}Zn²⁺ by hepatopancreatic lysosomal vesicles. Metal uptakes at 500 and 1000 µmol l^–1 SO₄^2– 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.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Effect of varying intravesicular SO42– concentration on the time course of 25 µmol l–1 65Zn2+ uptake by LMV. Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0 and K2SO4 concentrations of 25, 50, 100, 250, 500 or 1000 µmol l–1, and were then incubated in a medium containing 200 mmol l–1 mannitol, 25 µmol l–1 65Zinc-sulfate, 0.5 mmol l–1 NTA, 0.2 mmol l–1 ATP, and 20 mmol l–1 Hepes/Tris, pH 7.0. Experiments were conducted in triplicate; values are means ± 1 s.e.m. at each time point.

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.

View larger version (7K): [in this window] [in a new window]   Fig. 3. Effects of intravesicular pH (pH 7.5–5.0), extravesicular ATP (1 mmol l–1), and induced membrane potential on the influx (10 s uptake) of 25 µmol l–1 65Zn2+. A transmembrane electrical potential was induced by equilibrating the vesicles with 50 µmol l–1 valinomycin, varying the extravesicular K+ concentration [0 mmol l–1 (Ki>Ko), 100 mmol l–1 (Ki=Ko) or 200 mmol l–1 (Ki<Ko)], and maintaining a constant intravesicular K+ concentration (100 mmol l–1). Vesicles were loaded with 100 mmol l–1 K2SO4, either 20 mmol l–1 Hepes/Tris (pH 6.5–7.5) or Mes/Tris (pH 5.0–6.0), 50 µmol l–1 valinomycin, and appropriate mannitol to maintain osmolarity. Loaded vesicles were then incubated in media containing 25 µmol l–1 65Zinc-sulfate, 0, 100 or 200 mmol l–1 K2SO4 0.2 or 1.0 mmol l–1 ATP (+ATP), 0.5 mmol l–1 NTA, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was conducted in triplicate; values are means ± 1 s.e.m. at each time point.

Countertransport of anions by lysosomal vesicles
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.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of preloaded Cl– on the time course of 35SO42– and 14C-oxalate2– uptake by hepatopancreatic lysosomal membrane vesicles. (A) Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 KCl, and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 potassium gluconate and 5 mmol l–1 K235SO4. (B) Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 KCl, and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 potassium gluconate, and 5 mmol l–1 14C-oxalate2–. Experiments were in triplicate; values are means ± 1 s.e.m. at each time point.

Polyvalent anion exchange kinetics with intravesicular chloride
In order to more fully describe the features of LMV anion exchange, additional experiments were conducted describing the characteristics of anion influx kinetics into Cl^–-loaded vesicles. Fig. 5A shows ³⁵SO₄^2– influx kinetics into lysosomal vesicles preloaded with 25 mmol l^–1 Cl^–. As displayed in this figure, the influx of the divalent anion was a sigmoidal function of its cytoplasmic concentration when 25 mmol l^–1 intravesicular Cl^– was present inside the preparation. Under these conditions, ³⁵SO₄^2– influx followed the Hill equation of multi-site cooperativity shown below:

(1)

where J_SO4 is sulfate^2– influx at any given [SO₄^2–], J_max is apparent maximum sulfate^2– influx rate (mmol mg^–1 protein s^–1), K_mⁿ (mmol l^–1) is apparent binding affinity of the transporter for the anion corrected for multi-site cooperative interactions, [SO₄^2–]ⁿ is sulfate^2– concentration with multi-site corrections (mmol l^–1), and n is the apparent Hill coefficient, which is an estimate of the number of SO₄^2– ions transported during each transport cycle. The kinetic constants for J_SO4 were K_mⁿ=9.09±0.64 mmol l^–1, J_max=342±18 nmol mg^–1 protein s^–1, and n=2.71±0.51 (Fig. 5A). The results of this experiment suggest the presence of an anion exchange process involved in the simultaneous transfer of monovalent and divalent anions across the lysosomal membrane. It is unclear whether this exchange process is electrogenic or electroneutral, but in either instance it results in the uptake of sulfate from the cytoplasm.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Influx kinetics (10 s uptake) of polyvalent inorganic (35SO42–) and organic (14C-oxalate2–) anions into Cl–-loaded (25 mmol l–1 Cl–) LMV. (A) Vesicles were loaded with 200 mmol l–1 mannitol, 25 mmol l–1 KCl, 20 mmol l–1 Hepes/Tris, pH 7.0 and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, and K235SO4 concentrations from 1 to 50 mmol l–1. (B) Vesicles were loaded with 200 mmol l–1 mannitol, 25 mmol l–1 KCl, 20 mmol l–1 Hepes/Tris, pH 7.0 and incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0 and 14C-oxalic acid concentrations from 0.1 to 10 mmol l–1. Experiments were in triplicate; values are means ± 1 s.e.m. Lines drawn through the curves were computed using Sigma Plot 10.0 Software (Jandal).

Fig. 5B displays a similar exchange process between the organic divalent anion, ¹⁴C-oxalate^2–, and intravesicular Cl^–. In contrast with the results disclosed for sulfate^2– (Fig. 5A), influx kinetics for oxalate^2– followed the hyperbolic Michaelis–Menten relationship described below:

(2)

where J_Ox is oxalate influx at any given [oxalate^2–], K_m is the apparent binding affinity of the transporter for the divalent anion, J_max is apparent maximal oxalate^2– influx rate (nmol mg^–1 protein s^–1), and [Ox^2–] is the oxalate^2– concentration (mmol l^–1). The kinetic constants for J_Ox were K_m=1.81±0.20 mmol l^–1 and J_max=21.1±0.6 nmol mg^–1 protein s^–1. As with sulfate^2– influx, these data suggest that ¹⁴C-oxalate^2– was transported into these vesicles in exchange for intravesicular Cl^–, but the stoichiometric nature of this exchange is unclear. It is likely that the same antiporter was responsible for exchanging both sulfate^2– and oxalate^2– for Cl^–.

Lysosomal ³⁶Cl^– uptake occurs by exchange with OH^–
Because Cl^– ion acted as a `common currency' for exchange with different divalent anions (e.g. Fig. 5), the question arose as to how this monovalent anion was taken up by lysosomal vesicles. Fig. 6 displays the results of an experiment examining the nature of ³⁶Cl^– influx into lysosomal membrane vesicles that were loaded at pH 7.0 or 9.0 (external pH 7.0 at both times), thereby providing only hydroxyl ions as a potential anion substrate for exchange with external ³⁶Cl^–. As indicated in this figure, Cl^– influx was a sigmoidal function of external chloride concentration at each intravesicular pH condition and followed the Hill equation for multisite cooperativity between binding ligands as described previously for ³⁵SO₄^2– influx into these vesicles:

(3)

where J_Cl is Cl^– influx (nmol mg^–1 protein s^–1), J_max is maximal Cl^– influx (nmol mg^–1 protein s^–1), K_mⁿ is an apparent binding affinity constant (mmol l^–1), [Cl^–]ⁿ is the external Cl^– concentration (mmol l^–1) and n is the Hill coefficient.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effect of intravesicular pH on 36Cl– influx kinetics in hepatopancreatic LMV. In both instances extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol and 20 mmol l–1 Hepes/Tris, pH 7.0 (A) or pH 9.0 (B). Loaded vesicles were then incubated in media containing 1, 5, 10, 15, 20 or 75 mmol l–1 K36Cl–, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The sigmoidal curves were drawn through the data using Sigma Plot software (Jandal).

In order to clarify the transport exchange stoichiometry between Cl^– ions and OH^– ions during ³⁶Cl^– influx into lysosomal membrane vesicles, ³⁶Cl^– influx was measured at four different extravesicular [Cl^–] (2.5, 5, 15 and 35 mmol l^–1 Cl^–) over a range of different intravesicular pH values and the ³⁶Cl^– influx values are expressed as a function of intravesicular [OH^–] (nmol l^–1). As the data presented in Fig. 7 indicate, ³⁶Cl^– influx was a hyperbolic function of intravesicular [OH^–] at each extravesicular [Cl^–] from 2.5 to 35 mmol l^–1 Cl^–. These results suggest that ³⁶Cl^–/OH^– exchange followed a modified Michaelis–Menten equation for carrier-mediated transport as shown below:

(4)

where in this instance J_Cl is Cl^– influx by exchange with intravesicular OH^– ions (nmol mg^–1 protein s^–1), J_max is maximal Cl^– influx (nmol mg^–1 protein s^–1), K_m is an apparent binding affinity constant of the exchanger for OH^– ions (nmol l^–1), and [OH^–] is the intravesicular concentration of hydroxyl ions (nmol l^–1).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of intravesicular OH– concentration on 36Cl– influx (2.5, 5, 15, and 35 mmol l–1 Cl–; 5 s uptakes) in hepatopancreatic LMV. In all instances the extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol, and either 20 mmol l–1 Hepes/Tris or Mes/Tris, pH 6.0, 7.0, 8.0 and 9.0. Loaded vesicles were then incubated in media containing 2.5, 5, 15 and 35 mmol l–1 K36Cl–, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The hyperbolic curves were drawn through the data using Sigma Plot software (Jandal).

Lysosomal ³⁵SO₄^2– uptake occurs by exchange with OH^–
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_max increased 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).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effect of intravesicular pH on 35SO42– influx kinetics in hepatopancreatic LMV. In all cases extravesicular pH was held at pH 7.0. Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris at pH 7.0 (A), 8.0 (B) and 9.0 (C). Loaded vesicles were then incubated in media containing 2.5, 5, 10, 25 and 50 mmol l–1 K235SO4, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The sigmoidal curves were drawn through the data using Sigma Plot software (Jandel).

View larger version (11K): [in this window] [in a new window]   Fig. 9. Effect of intravesicular OH– concentration on 35SO42– influx at 5 mmol l–1 (A) and 10 mmol l–1 SO42– (B) (5 s uptakes) in hepatopancreatic LMV. In both cases the extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol and either 20 mmol l–1 Hepes/Tris or Mes/Tris, pH 7.0, 8.0 and 9.0. Loaded vesicles were then incubated in media containing either 5 or 10 mmol l–1 K235SO4, 20 mmol l–1 Hepes/Tris, pH 7.0 and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The hyperbolic curves drawn through the data using Sigma Plot software (Jandal).

	Discussion

TOP Summary Introduction Materials and methods Results Discussion References

 
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=7 µmol 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.

View larger version (69K): [in this window] [in a new window]   Fig. 10. Working model of the role of polyvalent anions in hepatopancreatic lysosomal heavy metal sequestration and detoxification. Membrane-bound, ATP-dependent, V-ATPase (Protein 1) transfers protons into the vesicle interior, creating a decrease in pH, an accumulation of hydrogen ions, and an inside-positive membrane potential. The outwardly directed proton gradient and positive vesicular interior provide the driving force for the asymmetric exchange of cytosolic divalent metals for intravesicular hydrogen ions by an ATP-dependent Zn2+-ATPase, or a 3H+/1Zn2+ exchanger (Protein 2). Polyvalent cytosolic anions such as sulfate2– or phosphate3– exchange with intravesicular monovalent anions such as Cl– or OH– by a second asymmetric antiporter (Protein 3), which uses the membrane potential as a driving force for exchange. Both divalent metals and polyvalent anions increase in concentration inside vesicles at acidic pH and are retained because they cannot be accommodated on the intravesicular binding sites of the exchangers. Divalent metals and polyvalent anions form precipitates (concretions) as the V-ATPase decreases in activity and the intravesicular pH rises.

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.

	Acknowledgments

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

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