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meso-2,3-Dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells
Toxicology 138 (1999) 81 – 91 www.elsevier.com/locate/toxicol
meso-2,3-Dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells Philip L. Pokorski, Michael J. McCabe Jr., Joel G. Pounds * Department of Pharmaceutical Sciences, Institute of Chemical Toxicology, Wayne State Uni6ersity, 2727 Second A6enue Room 4000 MCHT, Detroit, MI 48201, USA Received 6 May 1999; accepted 7 July 1999
Abstract The maintenance of intracellular Ca2 + homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2 + ]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimercaptosuccinic acid (DMSA) evoked transient increases in [Ca2 + ]i. Changes in [Ca2 + ]i were monitored using the Ca2 + indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2 + , DMSA treatment caused a concentration-dependent (15 – 500 mM) transient increase in [Ca2 + ]i returning to baseline levels within 30–60 s. Pharmacologic concentrations of DMSA (30 mM) stimulated a three-fold increase in [Ca2 + ]i, which was spatiotemporally comparable to Ca2 + transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2 + ]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2 + ]i, suggesting that Ca2 + mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2 + pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5%-trisphosphate (ATP) also elicited transient increases in [Ca2 + ]i similar to those of DMSA. No transient increases in [Ca2 + ]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2 + messenger system. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DMSA; Ca2 + transients; Proximal tubular cells; Lead
1. Introduction Calcium acts as an important second messenger in many cell signaling pathways and cellular pro* Corresponding author. Tel.: +1-313-577-0100; fax: + 1313-577-0082. E-mail address: [email protected] (J.G. Pounds)
cesses. Hormones, electrical signals, antigens, neurotransmitters, toxicants, and therapeutic agents can act extracelluarly to influence free intracellular calcium levels ([Ca2 + ]i), resulting in many varied responses such as muscle contraction, cell growth, metabolism changes, alterations in gene expression, secretion and exocytosis, and even cell
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death. Because of the potential for toxicity due to increases in [Ca2 + ]i, intracellular calcium homeostasis is tightly regulated. An important effect of lead at the cellular level is the perturbation or disruption of cellular calcium homeostasis. Lead inhibits calcium entry through voltage-gated calcium channels in neuronal cells (Simons 1988a,b) where it is thought that lead inhibits this calcium transport by entering the channel and competing with calcium at a single Ca2 + binding site, but not by transversing the channel (Busselberg et al., 1991). In bovine adrenal medullary cells, lead blocks calcium movement across the plasma membrane by blocking calcium channels (Pocock and Simons, 1987). Calcium pumps or Ca2 + -ATPases are present in both the plasma membrane and in the endoplasmic reticulum. Calcium pumps transport calcium ions from the cytoplasm to the extracellular environment or from the cytoplasm to the endoplasmic reticulum lumen. Lead has been shown to affect these pumps in two ways; lead is an effective substrate for these pumps (Simons, 1988a), and lead may inhibit Ca2 + -ATPases, depending on the ratio of calcium present compared to lead (Mas-Oliva, 1989). Lead has been known for some time to disrupt intracellular calcium homeostasis, but this phenomenon remains poorly understood (Pounds, 1984; Pounds et al., 1991). Intracellular calcium homeostasis is a tightly regulated process since any changes from the norm can cause secondary effects on intracellular signaling mechanisms, many of which are calcium dependent. In general, exposure of isolated or cultured cells to lead causes an increase in total cytoplasmic calcium levels (free Ca2 + plus bound Ca2 + ). These elevated calcium levels in turn overstimulate the calcium signaling processes. For example, treatment of neuroblastoma ×glioma cells with 5 mM Pb for 2 h produced a two-fold increase in [Ca2 + ]i (Schanne et al., 1989b). Rat osteosarcoma cells (ROS 1712.8) exposed to 5 and 25 mM Pb, sustained 50 and 120% increases in [Ca2 + ]i when measured by 19F-BAPTA NMR (Schanne et al., 1989a) In contrast, neither acute nor chronic exposure to 5–50 mM Pb caused an increase in basal [Ca2 + ]i levels in LLC-MK2 cells (Pokorski et al., 1999).
The importance of investigations to define the effects of lead on calcium metabolism in renal proximal tubular cells is two-fold. First, if lead perturbs calcium homeostasis in proximal tubular cells, the disruption may lead to inhibition of the calcium-dependent 1a-hydroxylase, which is responsible for the synthesis of l,25-dihydroxyvitamin D3. If 1a-hydroxylase is inhibited by a lead-induced increase in [Ca2 + ]i, this could be used as evidence to explain the clinical observation that serum 1,25-dihydroxyvitamin D3 levels are decreased in lead-poisoned children (Rosen et al., 1980). Second, the kidney and proximal tubular cells are a site of effective chelation of lead by meso-2,3-dimercaptosuccinic acid. In rats and mice, DMSA has been shown to reduce the lead burden of soft organs, including the kidney (Jones et al., 1984; Cory-Slechta, 1988; Smith and Flegal, 1992). In any chelation therapy regime, not only is the physical removal of the metal from the body of greatest importance, but also the restoration of affected organ and cellular processes. However, no clinical studies have been performed to monitor serum 1,25-dihydroxyvitamin D3 levels before and after DMSA therapy. Given the pathophysiological importance of renal proximal tubular cells in vitamin D3 metabolism, LLC-MK2 cells are a rational model for mechanistic studies pertaining to the mode of DMSA action. More evidence supporting proximal tubular cells as a potential site of DMSA-lead chelation comes from the fact that 99mTc-DMSA has been used in renal scintigraphy for over the past 20 years. The mechanism of uptake is unclear, but it is known that 99mTc-DMSA accumulates in renal proximal tubular cells (DeLange et al., 1989). Additionally, 99mTc-DMSA has been shown to have high binding affinity for the proximal convoluted tubules, providing good imaging of the renal parenchyma (Vanlic´-Razumenic´ and Pertovic´, 1982; McLorie et al., 1989). Since both lead and DMSA may share a common cellular target (i.e. renal proximal tubular cells), and since we have already shown that Pb does not increase [Ca2 + ]i in the LLC-MK2 cell line, attention now can be focused on the effects of DMSA, if any, on renal proximal tubular cell calcium homeostasis. It is well known that certain
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pharmaceutical agents (e.g. the antibiotic gentamicin) exhibit organ toxicity specific to kidney cells. Little is known pertaining to the potential toxic effects of DMSA on kidney cells. Specifically, the question asked in this investigation was: does DMSA treatment affect intracellular calcium levels in LLC-MK2 cells. During experimentation, we encountered a novel, previously unknown, and unexpected pharmacodynamic property of DMSA: the ability to elicit transient increases in [Ca2 + ]i. This observation warranted further study and characterization which is described in this paper.
2. Materials and methods
2.1. Cell culture Rhesus Monkey Kidney cells (LLC-MK2, macca mullata) were obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were initially cultured in M199 supplemented with 1% horse serum, penicillin (10 000 U/ml), and streptomycin (10 000 mg/ml). Cells were gradually adapted to Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (Hyclone,) with penicillin and streptomycin. The cell culture medium was replaced at 48-h intervals until cells reached confluency. Upon reaching confluency, cells were detached from T75 culture flasks using a solution of 0.1% trypsin-EDTA in calcium and magnesium free Hank’s Balanced Salt Solution (HBSS, l× ) and plated onto experimental dishes. At all times cells were incubated in a humidified 95% air:5% CO2 environment. All cell culture medium and supplements were from Gibco, BRL (Grand Island, NY).
2.2. Fura-2 loading Approximately 104 LLC-MK2 cells were plated onto 35-mm glass bottom microwells (MatTek Corp. Ashland, MA) and incubated for 3 – 4 days to near confluence. The kidney cells were then incubated with 2 mM of Fura-2-acetoxymethyl ester (Fura-2AM, Molecular Probes, Eugene, OR)
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in DMEM for l h at 37°C. The timing of Fura-2 loading was established by experimentation and observation of maximal fluorescent emission at 380 nm. After l h loading, the microwells were rinsed three times with analysis buffer (5.3 mM KCl, 44 mM KH2PO4, 137 mM NaCl, 4.2 mM NaHCO3, 0.33 mM Na2HPO4, 11 mM D-glucose, 5 mM HEPES, 2 mM CaCl2, 0.1% BSA, from Sigma Chemical Co, St. Louis), and incubated at room temperature for 15 min for dye equilibration (unesterified). The cells were then rinsed twice with analysis buffer and the microwell was examined for fluorescent emissions. For ‘calciumdeficient’ experiments, CaCl2 was omitted from the analysis buffer and excess EGTA (500 mM) was added. Measurement of free Ca2 + in this ‘calcium-deficient’ analysis buffer confirmed levels less than 1 nM.
2.3. Single cell fluorescence imaging Ca2 + measurements were made using Fura-2 ratio fluorescence from several individual cells in the microscope field using digitized images captured by an intensified video camera. Experiments were stopped and discarded if at any time during the monitoring period cells lifted off the coverslip and floated from their original position. Experiments were carried out at room temperature in a darkened environment with a Nikon TMS inverted microscope using an InCa+ +™ dual wavelength illumination system (Intracellular Imaging Inc., Cincinnati, OH). Fluorescent measurements were obtained by recording the intensities of Fura-2’s emission at a single wavelength (510 nm) due to excitation at two wavelengths (340 and 380 nm). The ratio of the two fluorescent emission intensities (340/380) was automatically calculated and converted to calcium concentrations according to Grynkiewicz et al. (1985) by comparison to a within-run calibration curve performed with the hexapotassium salt form of Fura2 and commercial calcium solution standards buffered by EGTA (Molecular Probes, Eugene, OR). Ten–15 single cells were selected for study based upon their intensity of Fura-2 emission at 380 nm. Typical experiments were between 7 and 9 min long, consisting of an initial 90 s period to
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measure basal [Ca2 + ]i followed by addition of specified treatments. The 35-mm dishes held 1 ml of analysis buffer and were open to room air. Most experiments were ended by addition (5 – 6 mM) of the calcium ionophore ionomycin (Sigma) to test the responsive integrity of the cells. Other treatment compounds used in this investigation, i.e. thapsigargin, ATP, were obtained from Sigma and stock solutions were made using analysis buffer. All experiments were replicated in triplicate using separate 35-mm dishes.
Fig. 2. DMSA concentration-response curve of the intensity of calcium transients. Data represent the mean 9SEM of triplicate dishes of the peak height of individual cell responses (n = 6 – 12 cells/dish) from a representative experiment.
2.4. DMSA treatment Pharmaccutical grade meso-2,3-dimercaptosuccinic acid (DMSA) was kindly provided by McNeil Consumer Products, Co. (Skillman, NJ). Fresh stock solutions of DMSA were prepared daily in degassed analysis buffer. Cells were treated by addition of DMSA to a pre-determined final concentration within the test microwell by pipette.
3. Results
Fig. 1. Calcium transients in Fura-2 loaded LLC-MK2 cells elicited by DMSA. Arrows indicate addition of 100 ml of an 150 mM DMSA stock solution (A) or of a 300 mM DMSA stock solution to 1.0 ml of analysis buffer (B). In all experiments described, stock solutions of treatment compounds were made in extracellular analysis buffer.
While performing control experiments to investigate the action of lead, we discovered that addition of DMSA to Fura-2 loaded LLC-MK2 cells elicited immediate intracellular calcium transient spikes which returned to near basal levels within 30 s. Further investigation demonstrated that repeated additions of DMSA elicited additional Ca2 + spikes in a concentration-dependent manner (Fig. 1). Addition of a stock solution of DMSA to give a final concentration of 5 mM DMSA caused a 2099 nM increase in peak [Ca2 + ]i, 30 mM DMSA; 97 9 26 nM; 60 mM DMSA; 1029 41 nM and, 150 mM DMSA caused an average peak calcium increase of 124916 nM calcium (Fig. 2).
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Basal intracellular calcium levels in Fura-2 loaded, untreated, control LLC-MK2 cells were 78.89 8 nM (n= 18 cells per microscope field, data not shown). In comparison to aequorin techniques used by other investigators for measuring [Ca2 + ]i levels in the LLC-MK2 cell line (Borle and Snowdowne, 1982; Snowdowne et al., 1985), Fura-2 appears to be an accurate and reliable indicator for intracellular calcium measurement. Depletion of intracellular calcium stores by thapsigargin and manipulation of calcium presence in the extracellular environment were used to determine the source of calcium responsible for the transient increases seen upon treatment with DMSA. In the presence of 2 mM calcium chloride in the extracellular buffer, addition of stock DMSA solution to give a final concentration of 30 mM DMSA in the test vessel, caused an immediate, transient [Ca2 + ]i spike which returned to near-basal levels (Fig. 1B). In the ‘absence’ of extracellular calcium by use of ‘calcium-deficient’ analysis buffer, treatment with DMSA provoked no calcium signal although ionomycin mobilized intracellular stores to give a transient spike (Fig. 3). Adding thapsigargin (final concentration of 3 mM) after a initial DMSA transient produced a characteristic rapid, but moderate increase in intracellular calcium levels. Cell viability was then Fig. 4. Calcium response in Fura-2 loaded LLC-MK2 cells to addition of DMSA (100 ml of 300 mM) and thapsigargin (100 ml of 33 mM) into 1.0 ml analysis buffer in the presence (A) or absence (B) of 2 mM extracellular calcium. Cellular integrity was demonstrated by the clearance of a ionomycin-induced transient (100 ml of 60 mM). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 3. Absence of calcium response to DMSA treatment in the presence of calcium-deficient analysis buffer. Arrows indicate addition of 100 ml of a 300 mM DMSA stock solution to 1.0 ml of analysis buffer. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
established by clearance of an ionomycin-induced calcium transient at the end of the experiment (Fig. 4A). In calcium-deficient analysis buffer, thapsigargin treatment elicited a steeper transient peak and ionomycin a lower transient [Ca2 + ]i peak (Fig. 4B). When endoplasmic reticulum Ca2 + stores were depleted by adding thapsigargin, a calcium response was still observed with subsequent DMSA treatment in the presence of 2 mM extracellular calcium (Fig. 5). To determine if the sulfhydryl functional groups present in DMSA may be responsible for the calcium transients elicited, DMSA was com-
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pared to other sulfhydryl-containing compounds. Cysteine, glutathione, and 2-mercaptoethanol (final concentrations of 110 mM, 2 and 500 mM respectively) each elicited transient increases in intracellular calcium levels. Cysteine and glutathione produced peak shapes remarkably similar to those seen with DMSA (Fig. 6). Another
Fig. 7. Oxidized DMSA (100 ml of 600 mM) does not elicit a calcium response. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 5. Calcium response to DMSA treatment (100 ml of 300 mM) in Fura-2 loaded LLC-MK2 cells after depletion of thapsigargin (100 ml, 33 mM) sensitive stores. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 8. Absence of calcium response upon addition of increasing concentrations of TPEN (100 ml of 0.55 mM) to Fura-2 loaded LLC-MK2 cells. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 6. Calcium responses in Fura-2 loaded LLC-MK2 cells to addition of various sulfhydryl-containing compounds (arrows indicate addition to final concentrations: 110 mM cysteine, 2 mM GSH, 500 mM 2-ME). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
observation supporting the functionality of reduced sulfhydryl groups was that oxidized solutions of DMSA failed to elicit a calcium response (Fig. 7). To determine if DMSA and the other sulfhydryl agents were chelating zinc off of membrane proteins, the zinc chelator tetrakis(2pyridylmethyl)ethylenediamine (TPEN) was added to Fura-2 loaded LLC-MK2 cells. TPEN (final concentrations of 50–150 mM) elicited no calcium response at any concentration tested (Fig. 8). Adenosine 5%-trisphophate (ATP) treatment,
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however, stimulated a transient increase in intracellular calcium levels (Fig. 9A), similar to that of DMSA, and transient spikes were absent upon ATP addition in calcium-deficient analysis buffer (Fig. 9B).
4. Discussion This work has identified a novel and unsuspected effect of the chelating drug DMSA: the influx of calcium from the extracellular environment into Rhesus monkey kidney cells. DMSA may be causing activation of calcium transients by (1) reduction of unidentified membrane protein
Fig. 9. ATP-induced (100 ml of 50 mM) calcium response in Fura-2 loaded LLC-MK2 cells in the presence (A) or absence (B) of 2 mM extracellular calcium. Cellular integrity was demonstrated by the clearance of an ionomycin-induced transient (100 ml of 60 mM). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
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sulhydryls by the sulfhydryl groups of DMSA resulting in calcium channel gate openings, (2) activating receptor-operated calcium channels, (3) stimulating membrane IP3 receptor-mediated calcium release, and/or (4) acting upon an as of yet undetermined membrane receptor which allows calcium influx to momentarily occur. While the FDA has approved DMSA as an orally administered drug for the treatment of lead poisoned subjects (FDA, 1991), little is known about its site and mechanism of action. It has been shown in rats that treatment with DMSA reduces the kidney burden of lead, but the mechanism of action for which some body stores of lead are available for chelation and not others is still unknown. 99mTc-DMSA has specifically been used for renal scintigraphy which lends credibility that DMSA may interact with and/or target certain cells of the kidney, notably proximal tubular cells (DeLange et al., 1989; Muller-Suur and Gutsche, 1995; Hecht et al., 1996), and may cause or potentiate a physiological or biochemical change within those cells. Additionally, we chose to work with concentrations of DMSA which are within the concentration ranges (10–30 mM) typically seen in the plasma of human volunteers (Aposhian et al., 1989; Dart et al., 1994; Asiedu et al., 1995). Whether DMSA enters cells or not is an active and conflicting area of research. Aposhian et al. (1992), has concluded that DMSA does not enter liver cells. On the other hand, DMSA may undergo enterohepatic circulation, suggesting some entry into hepatocytes (Asiedu et al., 1995), and DMSA is thought to enter erythrocytes (Dart et al., 1994). Our experiments did not measure DMSA uptake and thus provide no direct evidence supporting or denying movement of DMSA into cells. Our experiments do provide evidence that extracellular calcium is the source for the DMSA-induced calcium transients. We base our conclusions that extracellular calcium is the source for the following reasons: Firstly, thapsigargin, an inhibitor of microsomal Ca2 + pumps (Ca-ATPases) located within the membrane of the endoplasmic reticulum, releases sequestered Ca2 + from the endoplasmic reticulum lumen (Thastrup et al., 1990; Kijima et al., 1991). DMSA, in the presence of extracellular calcium, provoked tran-
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sient increases in intracellular calcium both before and after depletion of the intracellular calcium stores by thapsigargin. These results indicate that DMSA has the ability to mobilize calcium into the cytoplasmic domain of the cell from sources other than intracellular compartments which are depleted by thapsigargin. Secondly, DMSA failed to elicit any transient increases in the presence of a calcium-deficient extracellular environment. If DMSA acts intracellularly, regardless of the intracellular site or receptor mechanism, it would be expected to release calcium from intracellular stores, giving a response similar to that seen with thapsigargin in a calcium-deficient extracellular environment. These observations pose interesting theories to consider regarding the largely unknown pharmacodynamic properties of DMSA. DMSA is known to bind lead with high affinity. Furthermore, lead is known to mimic calcium in many biochemical and cellular processes. In the absence of lead, DMSA may mobilize calcium because of calcium’s similar physical properties to lead. During in vivo studies in humans, investigators have noted differences in DMSA metabolism in leadpoisoned subjects versus non-lead-poisoned subjects (Dart et al., 1994; Mortensen, 1994; Asiedu et al., 1995). Our experiments here utilize concentrations of DMSA similar to those found in plasma and might be considered a mechanistic or cellular approach to how specific, individual cells react to DMSA in the absence of lead. Further work by our laboratory has shown that this mobilization of calcium by DMSA as reported here is inhibited when cells have been pre-exposed to lead. (Pokorski et al., 1999). The mechanism by which DMSA causes transient increases in [Ca2 + ]i is not fully understood. Other sulfhydryl-containing compounds elicited transient increases in intracellular calcium levels, suggesting functional group (SH) involvement. Sulfhydryl-reactive reagents have recently been shown to cause increases in intracellular calcium levels by a variety of mechanisms which can act both intracellularly on ryanodine-sensitive stores and stimulate IP3-mediated calcium release at the site of the membrane (Shachar et al., 1994; Tanaka and Tashjian, 1994; Abramson et al.,
1995). Other sulfhydryl reagents such as thimerosal act primarily at the site of the membrane IP3 receptor (Berridge, 1992; Hilly et al., 1993; Laudanna et al., 1994) or at the sites of specific membrane ion channels (Karhapa¨a¨ et al., 1996). In our experiments, we demonstrated that in the presence of extracellular calcium, the sulfhydryl-containing compounds cysteine, glutathione and 2-mercaptoethanol elicited similar, but not identical calcium transients compared to those elicited by DMSA. Because of the observation that oxidized solutions of DMSA are unable to elicit a calcium response, it is likely that the calcium influx seen with treatment of DMSA may be due to DMSA’s sulfhydryl groups reducing membrane proteins which are associated with calcium channel regulation. Some may argue that given the presence of sulfhydryl groups in the DMSA molecule and that other sulfhydryl-containing compounds elicit calcium transients, it should be expected that DMSA too will cause calcium transients. Despite the assumptions, this work is the first documentation of such effects of DMSA, and furthermore, we have shown that a difference in DMSA’s ability to elicit these calcium transients is affected by the presence or absence of lead (Pokorski et al., 1999). Another possible mechanism that could explain the calcium response to DMSA is activation of receptor-operated calcium channels (ROCCs) by DMSA. Adenosine 5%-triphosphate (ATP) acts as an activator of a specific ATP-activated P2purinergic receptor which opens a non-selective cation channel, allowing extracellular calcium to enter the cell (Dave and Mogul, 1996; Yamada et al., 1996; Squires et al., 1997). ATP increases [Ca2 + ]i levels in numerous cell culture models by this mechanism (Kamada et al., 1994; Morley et al., 1994; Lee et al., 1996). Additionally, in some cells, ATP increases [Ca2 + ]i levels by mobilizing intracellular calcium stores (Yamada et al., 1996; Squires et al., 1997). In our experiments with LLC-MK2 cells, ATP evoked transient increases in [Ca2 + ]i levels only in the presence of 2 mM extracellular calcium. To investigate the possibility that DMSA is chelating zinc from membrane proteins, thereby leading to calcium influx, tetrakis-(2-pyridyl-
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methyl)ethylenediamine (TPEN) a zinc chelator was tested to see if it would duplicate the results seen with DMSA. The failure of zinc chelation by TPEN to elicit transient increases in [Ca2 + ]i ruled out this possibility. It is possible but unlikely that DMSA is acting as a calcium ionophore. Most ionophores readily cross membranes to release Ca2 + from intracellular stores. In contrast, DMSA does not appear to cross the plasma membrane (Aposhian et al., 1992) and our results with DMSA do not support the conclusion that DMSA mobilizes Ca2 + from intracellular stores. Additionally, the DMSA molecule is not structurally similar to other calcium ionophores, e.g. Ionomycin. The toxicological significance of these findings is not yet completely understood, due to the fact that little is known of the cellular mechanisms by which DMSA decreases tissue lead levels. Taking our findings that DMSA exposure causes an influx of calcium into proximal tubular cells, with understanding the processes by which cells maintain intracellular calcium levels gives rise to interesting speculation on how DMSA may reduce the cellular burden of lead. Elevations in [Ca2 + ]i are counteracted by the activation of Ca2 + -ATPases which either pump Ca2 + into the endoplasmic reticulum, or pump Ca2 + out of the cell. Although the experiments described herein did not measure [Pb2 + ]i, some physiological stimuli that elicit Ca2 + signals appear to mobilize Pb2 + as well (Pounds and Mittelstaedt, 1983). Since Pb2 + mimics Ca2 + , and actually has a higher affinity for calcium pumps than Ca2 + itself, after a Ca2 + transient, it is speculated Pb2 + may be more efficiently pumped out of the cell, thus lowering intracellular lead (tissue) levels. Because proximal tubular cells are involved in urine production, DMSA and Ca-activated Ca2 + -ATPase activity may provide a possible explanation for the increased lead found in urine after DMSA chelation therapy. In conclusion, while testing the hypothesis that DMSA treatment does not affect intracellular calcium levels in LLC-MK2 cells, an unanticipated finding occurred: elicitation of calcium transients by DMSA in untreated, non-lead-exposed LLC-MK2 cells. Subsequent follow-up ex-
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periments showed that the source of calcium responsible for the calcium transients was from the extracellular environment, and DMSA elicited calcium responses in a concentration-dependent fashion. Other selected sulfhydryl-containing compounds were also successful at eliciting a calcium response. The calcium responses to DMSA were not due to zinc chelation from membrane proteins because the zinc chelator TPEN did not mimic the effects of DMSA. This work may be significant towards understanding the cellular mechanism by which DMSA chelates lead and from which possible organ stores of lead upon which DMSA acts.
Acknowledgements Support for this research was provided by a grant (ES04040) to JGP from the National Institute of Environmental Health Sciences, National Institutes of Health, and involving the services of the Imaging and Cytometry Facility Core supported by NIEHS center grant P30 ES06639.
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cellular calcium oscillations in single isolated human granulosa-lutein cells. Endocrinology 137, 3756 – 3761. Mas-Oliva, J., 1989. Effect of lead on the erythrocyte (Ca2 + , Mg2 + )-ATPase activity: calmodulin involvement. Mol. Cell Biochem. 89, 87 – 93. McLorie, G.A., Aliabadi, H., Churchill, M.M.., Ash, J.M., Gilday, D.L., 1989. 99mTechnetium dimercapto-succinic acid renal scanning and excretory urography in diagnosis of renal scars in children. J. Urol. 142, 790 – 792. Morley, P., Vanderhyden, B.C.., Tremblay, R., Mealing, G.A.R., Durkin, J.P., Whitfield, J.F., 1994. Purinergic receptor-mediated intracellular Ca2 + oscillations in chicken granulosa cells. Endocrinology 134, 1269 – 1276. Mortensen, M.E., 1994. Succimer chelation: what is known? J. Pediatr. 125, 233 – 234. Muller-Suur, R., Gutsche, H.U., 1995. Tubular re-absorption of technetium-99m-DMSA. J. Nucl. Med. 36 (9), 1654 – 1658. Pocock, G., Simons, T.J.B., 1987. Effects of lead ions on events associated with exocytosis in isolated bovine adrenal medullary cells. J. Neurochem. 48, 376 – 382. Pokorski, P.L., McCabe, M.J. Jr., Pounds, J.G., 1999. Lead inhibits meso-2,3-dimercaptosuccinic acid induced calcium transients in cultured rhesus monkey kidney cells. Toxicology 134, 19 – 26. Pounds, J.G., Mittelstaedt, R.A., 1983. Mobilization of cellular calcium-45 and lead-210: effect of physiological stimuli. Science 220, 308 – 310. Pounds, J.G., Long, G.J., Rosen, J.F., 1991. Cellular and molecular toxicity of lead in bone. Environ. Health Perspect. 91, 17 – 32. Pounds, J.G., 1984. Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: a review. Neurotoxicology 5 (3), 295 – 332. Rosen, J.F., Chesney, R.W., Hamstra, A., DeLuca, H.F., Mahaffey, K.R., 1980. Reduction in 1,25-dihydroxyvitamin D in children with increased lead absorption. New Eng. J. Med. 302, 1128 – 1131. Schanne, F.A.X., Dowd, T.L., Gupta, R.K., Rosen, J.F., 1989a. Lead increases free Ca concentration in cultured osteoblastic bone cells: Simultaneous detection of intracellular free Pb2 + by 19F-NMR. Proc. Natl. Acad. Sci. U.S.A. 86, 5133 – 5135. Schanne, F.A.X., Moskal, J.R., Gupta, R.K., 1989b. Effect of lead on intracellular free calcium ion concentration in presynaptic neuronal model: 19F-NMR study of NG108-15 cells. Brain Res. 503, 308 – 311. Shachar, I., Rabinovich, E., Kerem, A., Bar-Nun, S., 1994. Thiol-reducing agents and calcium perturbants alter intracellular sorting of immunoglobulin M. J. Biol. Chem. 269, 27344 – 27350. Simons, T.J.B., 1988a. Active transport of lead by the calcium pump in human red cell ghosts. J. Physiol. London 405, 105 – 113. Simons, T.J.B., 1988b. Lead – calcium interactions and lead toxicity. Handbook Exp. Pharmacol. 83, 509 – 525.
P.L. Pokorski et al. / Toxicology 138 (1999) 81–91 Smith, D.R., Flegal, A.R., 1992. Stable isotopic tracers of lead mobilized by DMSA chelation in low lead-exposed rats. Toxicol. Appl. Pharmacol. 116, 85–91. Snowdowne, K.W., Freudenrich, C.C., Borle, A.B., 1985. The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells. J. Biol. Chem. 260, 11619 – 11626. Squires, P.E., Lee, P.S.N., Ho, Y.B., Leung, P.C.K., Buchan, A.M.J., 1997. Mechanisms involved in ATP-evoked Ca2 + oscillations in isolated human granulosa-luteal cells. Cell Calcium 21 (5), 365 –374. Tanaka, Y., Tashjian, A.H., 1994. Thimerosal potentiates Ca2 + release mediated by both the inositol 1,4,5-triphosphate and the ryanodine receptors in sea urchin eggs. J.
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meso-2,3-Dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells Philip L. Pokorski, Michael J. McCabe Jr., Joel G. Pounds * Department of Pharmaceutical Sciences, Institute of Chemical Toxicology, Wayne State Uni6ersity, 2727 Second A6enue Room 4000 MCHT, Detroit, MI 48201, USA Received 6 May 1999; accepted 7 July 1999
Abstract The maintenance of intracellular Ca2 + homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2 + ]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimercaptosuccinic acid (DMSA) evoked transient increases in [Ca2 + ]i. Changes in [Ca2 + ]i were monitored using the Ca2 + indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2 + , DMSA treatment caused a concentration-dependent (15 – 500 mM) transient increase in [Ca2 + ]i returning to baseline levels within 30–60 s. Pharmacologic concentrations of DMSA (30 mM) stimulated a three-fold increase in [Ca2 + ]i, which was spatiotemporally comparable to Ca2 + transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2 + ]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2 + ]i, suggesting that Ca2 + mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2 + pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5%-trisphosphate (ATP) also elicited transient increases in [Ca2 + ]i similar to those of DMSA. No transient increases in [Ca2 + ]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2 + messenger system. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DMSA; Ca2 + transients; Proximal tubular cells; Lead
1. Introduction Calcium acts as an important second messenger in many cell signaling pathways and cellular pro* Corresponding author. Tel.: +1-313-577-0100; fax: + 1313-577-0082. E-mail address: [email protected] (J.G. Pounds)
cesses. Hormones, electrical signals, antigens, neurotransmitters, toxicants, and therapeutic agents can act extracelluarly to influence free intracellular calcium levels ([Ca2 + ]i), resulting in many varied responses such as muscle contraction, cell growth, metabolism changes, alterations in gene expression, secretion and exocytosis, and even cell
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 9 2 - X
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death. Because of the potential for toxicity due to increases in [Ca2 + ]i, intracellular calcium homeostasis is tightly regulated. An important effect of lead at the cellular level is the perturbation or disruption of cellular calcium homeostasis. Lead inhibits calcium entry through voltage-gated calcium channels in neuronal cells (Simons 1988a,b) where it is thought that lead inhibits this calcium transport by entering the channel and competing with calcium at a single Ca2 + binding site, but not by transversing the channel (Busselberg et al., 1991). In bovine adrenal medullary cells, lead blocks calcium movement across the plasma membrane by blocking calcium channels (Pocock and Simons, 1987). Calcium pumps or Ca2 + -ATPases are present in both the plasma membrane and in the endoplasmic reticulum. Calcium pumps transport calcium ions from the cytoplasm to the extracellular environment or from the cytoplasm to the endoplasmic reticulum lumen. Lead has been shown to affect these pumps in two ways; lead is an effective substrate for these pumps (Simons, 1988a), and lead may inhibit Ca2 + -ATPases, depending on the ratio of calcium present compared to lead (Mas-Oliva, 1989). Lead has been known for some time to disrupt intracellular calcium homeostasis, but this phenomenon remains poorly understood (Pounds, 1984; Pounds et al., 1991). Intracellular calcium homeostasis is a tightly regulated process since any changes from the norm can cause secondary effects on intracellular signaling mechanisms, many of which are calcium dependent. In general, exposure of isolated or cultured cells to lead causes an increase in total cytoplasmic calcium levels (free Ca2 + plus bound Ca2 + ). These elevated calcium levels in turn overstimulate the calcium signaling processes. For example, treatment of neuroblastoma ×glioma cells with 5 mM Pb for 2 h produced a two-fold increase in [Ca2 + ]i (Schanne et al., 1989b). Rat osteosarcoma cells (ROS 1712.8) exposed to 5 and 25 mM Pb, sustained 50 and 120% increases in [Ca2 + ]i when measured by 19F-BAPTA NMR (Schanne et al., 1989a) In contrast, neither acute nor chronic exposure to 5–50 mM Pb caused an increase in basal [Ca2 + ]i levels in LLC-MK2 cells (Pokorski et al., 1999).
The importance of investigations to define the effects of lead on calcium metabolism in renal proximal tubular cells is two-fold. First, if lead perturbs calcium homeostasis in proximal tubular cells, the disruption may lead to inhibition of the calcium-dependent 1a-hydroxylase, which is responsible for the synthesis of l,25-dihydroxyvitamin D3. If 1a-hydroxylase is inhibited by a lead-induced increase in [Ca2 + ]i, this could be used as evidence to explain the clinical observation that serum 1,25-dihydroxyvitamin D3 levels are decreased in lead-poisoned children (Rosen et al., 1980). Second, the kidney and proximal tubular cells are a site of effective chelation of lead by meso-2,3-dimercaptosuccinic acid. In rats and mice, DMSA has been shown to reduce the lead burden of soft organs, including the kidney (Jones et al., 1984; Cory-Slechta, 1988; Smith and Flegal, 1992). In any chelation therapy regime, not only is the physical removal of the metal from the body of greatest importance, but also the restoration of affected organ and cellular processes. However, no clinical studies have been performed to monitor serum 1,25-dihydroxyvitamin D3 levels before and after DMSA therapy. Given the pathophysiological importance of renal proximal tubular cells in vitamin D3 metabolism, LLC-MK2 cells are a rational model for mechanistic studies pertaining to the mode of DMSA action. More evidence supporting proximal tubular cells as a potential site of DMSA-lead chelation comes from the fact that 99mTc-DMSA has been used in renal scintigraphy for over the past 20 years. The mechanism of uptake is unclear, but it is known that 99mTc-DMSA accumulates in renal proximal tubular cells (DeLange et al., 1989). Additionally, 99mTc-DMSA has been shown to have high binding affinity for the proximal convoluted tubules, providing good imaging of the renal parenchyma (Vanlic´-Razumenic´ and Pertovic´, 1982; McLorie et al., 1989). Since both lead and DMSA may share a common cellular target (i.e. renal proximal tubular cells), and since we have already shown that Pb does not increase [Ca2 + ]i in the LLC-MK2 cell line, attention now can be focused on the effects of DMSA, if any, on renal proximal tubular cell calcium homeostasis. It is well known that certain
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pharmaceutical agents (e.g. the antibiotic gentamicin) exhibit organ toxicity specific to kidney cells. Little is known pertaining to the potential toxic effects of DMSA on kidney cells. Specifically, the question asked in this investigation was: does DMSA treatment affect intracellular calcium levels in LLC-MK2 cells. During experimentation, we encountered a novel, previously unknown, and unexpected pharmacodynamic property of DMSA: the ability to elicit transient increases in [Ca2 + ]i. This observation warranted further study and characterization which is described in this paper.
2. Materials and methods
2.1. Cell culture Rhesus Monkey Kidney cells (LLC-MK2, macca mullata) were obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were initially cultured in M199 supplemented with 1% horse serum, penicillin (10 000 U/ml), and streptomycin (10 000 mg/ml). Cells were gradually adapted to Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (Hyclone,) with penicillin and streptomycin. The cell culture medium was replaced at 48-h intervals until cells reached confluency. Upon reaching confluency, cells were detached from T75 culture flasks using a solution of 0.1% trypsin-EDTA in calcium and magnesium free Hank’s Balanced Salt Solution (HBSS, l× ) and plated onto experimental dishes. At all times cells were incubated in a humidified 95% air:5% CO2 environment. All cell culture medium and supplements were from Gibco, BRL (Grand Island, NY).
2.2. Fura-2 loading Approximately 104 LLC-MK2 cells were plated onto 35-mm glass bottom microwells (MatTek Corp. Ashland, MA) and incubated for 3 – 4 days to near confluence. The kidney cells were then incubated with 2 mM of Fura-2-acetoxymethyl ester (Fura-2AM, Molecular Probes, Eugene, OR)
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in DMEM for l h at 37°C. The timing of Fura-2 loading was established by experimentation and observation of maximal fluorescent emission at 380 nm. After l h loading, the microwells were rinsed three times with analysis buffer (5.3 mM KCl, 44 mM KH2PO4, 137 mM NaCl, 4.2 mM NaHCO3, 0.33 mM Na2HPO4, 11 mM D-glucose, 5 mM HEPES, 2 mM CaCl2, 0.1% BSA, from Sigma Chemical Co, St. Louis), and incubated at room temperature for 15 min for dye equilibration (unesterified). The cells were then rinsed twice with analysis buffer and the microwell was examined for fluorescent emissions. For ‘calciumdeficient’ experiments, CaCl2 was omitted from the analysis buffer and excess EGTA (500 mM) was added. Measurement of free Ca2 + in this ‘calcium-deficient’ analysis buffer confirmed levels less than 1 nM.
2.3. Single cell fluorescence imaging Ca2 + measurements were made using Fura-2 ratio fluorescence from several individual cells in the microscope field using digitized images captured by an intensified video camera. Experiments were stopped and discarded if at any time during the monitoring period cells lifted off the coverslip and floated from their original position. Experiments were carried out at room temperature in a darkened environment with a Nikon TMS inverted microscope using an InCa+ +™ dual wavelength illumination system (Intracellular Imaging Inc., Cincinnati, OH). Fluorescent measurements were obtained by recording the intensities of Fura-2’s emission at a single wavelength (510 nm) due to excitation at two wavelengths (340 and 380 nm). The ratio of the two fluorescent emission intensities (340/380) was automatically calculated and converted to calcium concentrations according to Grynkiewicz et al. (1985) by comparison to a within-run calibration curve performed with the hexapotassium salt form of Fura2 and commercial calcium solution standards buffered by EGTA (Molecular Probes, Eugene, OR). Ten–15 single cells were selected for study based upon their intensity of Fura-2 emission at 380 nm. Typical experiments were between 7 and 9 min long, consisting of an initial 90 s period to
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measure basal [Ca2 + ]i followed by addition of specified treatments. The 35-mm dishes held 1 ml of analysis buffer and were open to room air. Most experiments were ended by addition (5 – 6 mM) of the calcium ionophore ionomycin (Sigma) to test the responsive integrity of the cells. Other treatment compounds used in this investigation, i.e. thapsigargin, ATP, were obtained from Sigma and stock solutions were made using analysis buffer. All experiments were replicated in triplicate using separate 35-mm dishes.
Fig. 2. DMSA concentration-response curve of the intensity of calcium transients. Data represent the mean 9SEM of triplicate dishes of the peak height of individual cell responses (n = 6 – 12 cells/dish) from a representative experiment.
2.4. DMSA treatment Pharmaccutical grade meso-2,3-dimercaptosuccinic acid (DMSA) was kindly provided by McNeil Consumer Products, Co. (Skillman, NJ). Fresh stock solutions of DMSA were prepared daily in degassed analysis buffer. Cells were treated by addition of DMSA to a pre-determined final concentration within the test microwell by pipette.
3. Results
Fig. 1. Calcium transients in Fura-2 loaded LLC-MK2 cells elicited by DMSA. Arrows indicate addition of 100 ml of an 150 mM DMSA stock solution (A) or of a 300 mM DMSA stock solution to 1.0 ml of analysis buffer (B). In all experiments described, stock solutions of treatment compounds were made in extracellular analysis buffer.
While performing control experiments to investigate the action of lead, we discovered that addition of DMSA to Fura-2 loaded LLC-MK2 cells elicited immediate intracellular calcium transient spikes which returned to near basal levels within 30 s. Further investigation demonstrated that repeated additions of DMSA elicited additional Ca2 + spikes in a concentration-dependent manner (Fig. 1). Addition of a stock solution of DMSA to give a final concentration of 5 mM DMSA caused a 2099 nM increase in peak [Ca2 + ]i, 30 mM DMSA; 97 9 26 nM; 60 mM DMSA; 1029 41 nM and, 150 mM DMSA caused an average peak calcium increase of 124916 nM calcium (Fig. 2).
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Basal intracellular calcium levels in Fura-2 loaded, untreated, control LLC-MK2 cells were 78.89 8 nM (n= 18 cells per microscope field, data not shown). In comparison to aequorin techniques used by other investigators for measuring [Ca2 + ]i levels in the LLC-MK2 cell line (Borle and Snowdowne, 1982; Snowdowne et al., 1985), Fura-2 appears to be an accurate and reliable indicator for intracellular calcium measurement. Depletion of intracellular calcium stores by thapsigargin and manipulation of calcium presence in the extracellular environment were used to determine the source of calcium responsible for the transient increases seen upon treatment with DMSA. In the presence of 2 mM calcium chloride in the extracellular buffer, addition of stock DMSA solution to give a final concentration of 30 mM DMSA in the test vessel, caused an immediate, transient [Ca2 + ]i spike which returned to near-basal levels (Fig. 1B). In the ‘absence’ of extracellular calcium by use of ‘calcium-deficient’ analysis buffer, treatment with DMSA provoked no calcium signal although ionomycin mobilized intracellular stores to give a transient spike (Fig. 3). Adding thapsigargin (final concentration of 3 mM) after a initial DMSA transient produced a characteristic rapid, but moderate increase in intracellular calcium levels. Cell viability was then Fig. 4. Calcium response in Fura-2 loaded LLC-MK2 cells to addition of DMSA (100 ml of 300 mM) and thapsigargin (100 ml of 33 mM) into 1.0 ml analysis buffer in the presence (A) or absence (B) of 2 mM extracellular calcium. Cellular integrity was demonstrated by the clearance of a ionomycin-induced transient (100 ml of 60 mM). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 3. Absence of calcium response to DMSA treatment in the presence of calcium-deficient analysis buffer. Arrows indicate addition of 100 ml of a 300 mM DMSA stock solution to 1.0 ml of analysis buffer. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
established by clearance of an ionomycin-induced calcium transient at the end of the experiment (Fig. 4A). In calcium-deficient analysis buffer, thapsigargin treatment elicited a steeper transient peak and ionomycin a lower transient [Ca2 + ]i peak (Fig. 4B). When endoplasmic reticulum Ca2 + stores were depleted by adding thapsigargin, a calcium response was still observed with subsequent DMSA treatment in the presence of 2 mM extracellular calcium (Fig. 5). To determine if the sulfhydryl functional groups present in DMSA may be responsible for the calcium transients elicited, DMSA was com-
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pared to other sulfhydryl-containing compounds. Cysteine, glutathione, and 2-mercaptoethanol (final concentrations of 110 mM, 2 and 500 mM respectively) each elicited transient increases in intracellular calcium levels. Cysteine and glutathione produced peak shapes remarkably similar to those seen with DMSA (Fig. 6). Another
Fig. 7. Oxidized DMSA (100 ml of 600 mM) does not elicit a calcium response. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 5. Calcium response to DMSA treatment (100 ml of 300 mM) in Fura-2 loaded LLC-MK2 cells after depletion of thapsigargin (100 ml, 33 mM) sensitive stores. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 8. Absence of calcium response upon addition of increasing concentrations of TPEN (100 ml of 0.55 mM) to Fura-2 loaded LLC-MK2 cells. Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
Fig. 6. Calcium responses in Fura-2 loaded LLC-MK2 cells to addition of various sulfhydryl-containing compounds (arrows indicate addition to final concentrations: 110 mM cysteine, 2 mM GSH, 500 mM 2-ME). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
observation supporting the functionality of reduced sulfhydryl groups was that oxidized solutions of DMSA failed to elicit a calcium response (Fig. 7). To determine if DMSA and the other sulfhydryl agents were chelating zinc off of membrane proteins, the zinc chelator tetrakis(2pyridylmethyl)ethylenediamine (TPEN) was added to Fura-2 loaded LLC-MK2 cells. TPEN (final concentrations of 50–150 mM) elicited no calcium response at any concentration tested (Fig. 8). Adenosine 5%-trisphophate (ATP) treatment,
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however, stimulated a transient increase in intracellular calcium levels (Fig. 9A), similar to that of DMSA, and transient spikes were absent upon ATP addition in calcium-deficient analysis buffer (Fig. 9B).
4. Discussion This work has identified a novel and unsuspected effect of the chelating drug DMSA: the influx of calcium from the extracellular environment into Rhesus monkey kidney cells. DMSA may be causing activation of calcium transients by (1) reduction of unidentified membrane protein
Fig. 9. ATP-induced (100 ml of 50 mM) calcium response in Fura-2 loaded LLC-MK2 cells in the presence (A) or absence (B) of 2 mM extracellular calcium. Cellular integrity was demonstrated by the clearance of an ionomycin-induced transient (100 ml of 60 mM). Data represent time course of [Ca2 + ]i in individual cells from a representative experiment.
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sulhydryls by the sulfhydryl groups of DMSA resulting in calcium channel gate openings, (2) activating receptor-operated calcium channels, (3) stimulating membrane IP3 receptor-mediated calcium release, and/or (4) acting upon an as of yet undetermined membrane receptor which allows calcium influx to momentarily occur. While the FDA has approved DMSA as an orally administered drug for the treatment of lead poisoned subjects (FDA, 1991), little is known about its site and mechanism of action. It has been shown in rats that treatment with DMSA reduces the kidney burden of lead, but the mechanism of action for which some body stores of lead are available for chelation and not others is still unknown. 99mTc-DMSA has specifically been used for renal scintigraphy which lends credibility that DMSA may interact with and/or target certain cells of the kidney, notably proximal tubular cells (DeLange et al., 1989; Muller-Suur and Gutsche, 1995; Hecht et al., 1996), and may cause or potentiate a physiological or biochemical change within those cells. Additionally, we chose to work with concentrations of DMSA which are within the concentration ranges (10–30 mM) typically seen in the plasma of human volunteers (Aposhian et al., 1989; Dart et al., 1994; Asiedu et al., 1995). Whether DMSA enters cells or not is an active and conflicting area of research. Aposhian et al. (1992), has concluded that DMSA does not enter liver cells. On the other hand, DMSA may undergo enterohepatic circulation, suggesting some entry into hepatocytes (Asiedu et al., 1995), and DMSA is thought to enter erythrocytes (Dart et al., 1994). Our experiments did not measure DMSA uptake and thus provide no direct evidence supporting or denying movement of DMSA into cells. Our experiments do provide evidence that extracellular calcium is the source for the DMSA-induced calcium transients. We base our conclusions that extracellular calcium is the source for the following reasons: Firstly, thapsigargin, an inhibitor of microsomal Ca2 + pumps (Ca-ATPases) located within the membrane of the endoplasmic reticulum, releases sequestered Ca2 + from the endoplasmic reticulum lumen (Thastrup et al., 1990; Kijima et al., 1991). DMSA, in the presence of extracellular calcium, provoked tran-
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sient increases in intracellular calcium both before and after depletion of the intracellular calcium stores by thapsigargin. These results indicate that DMSA has the ability to mobilize calcium into the cytoplasmic domain of the cell from sources other than intracellular compartments which are depleted by thapsigargin. Secondly, DMSA failed to elicit any transient increases in the presence of a calcium-deficient extracellular environment. If DMSA acts intracellularly, regardless of the intracellular site or receptor mechanism, it would be expected to release calcium from intracellular stores, giving a response similar to that seen with thapsigargin in a calcium-deficient extracellular environment. These observations pose interesting theories to consider regarding the largely unknown pharmacodynamic properties of DMSA. DMSA is known to bind lead with high affinity. Furthermore, lead is known to mimic calcium in many biochemical and cellular processes. In the absence of lead, DMSA may mobilize calcium because of calcium’s similar physical properties to lead. During in vivo studies in humans, investigators have noted differences in DMSA metabolism in leadpoisoned subjects versus non-lead-poisoned subjects (Dart et al., 1994; Mortensen, 1994; Asiedu et al., 1995). Our experiments here utilize concentrations of DMSA similar to those found in plasma and might be considered a mechanistic or cellular approach to how specific, individual cells react to DMSA in the absence of lead. Further work by our laboratory has shown that this mobilization of calcium by DMSA as reported here is inhibited when cells have been pre-exposed to lead. (Pokorski et al., 1999). The mechanism by which DMSA causes transient increases in [Ca2 + ]i is not fully understood. Other sulfhydryl-containing compounds elicited transient increases in intracellular calcium levels, suggesting functional group (SH) involvement. Sulfhydryl-reactive reagents have recently been shown to cause increases in intracellular calcium levels by a variety of mechanisms which can act both intracellularly on ryanodine-sensitive stores and stimulate IP3-mediated calcium release at the site of the membrane (Shachar et al., 1994; Tanaka and Tashjian, 1994; Abramson et al.,
1995). Other sulfhydryl reagents such as thimerosal act primarily at the site of the membrane IP3 receptor (Berridge, 1992; Hilly et al., 1993; Laudanna et al., 1994) or at the sites of specific membrane ion channels (Karhapa¨a¨ et al., 1996). In our experiments, we demonstrated that in the presence of extracellular calcium, the sulfhydryl-containing compounds cysteine, glutathione and 2-mercaptoethanol elicited similar, but not identical calcium transients compared to those elicited by DMSA. Because of the observation that oxidized solutions of DMSA are unable to elicit a calcium response, it is likely that the calcium influx seen with treatment of DMSA may be due to DMSA’s sulfhydryl groups reducing membrane proteins which are associated with calcium channel regulation. Some may argue that given the presence of sulfhydryl groups in the DMSA molecule and that other sulfhydryl-containing compounds elicit calcium transients, it should be expected that DMSA too will cause calcium transients. Despite the assumptions, this work is the first documentation of such effects of DMSA, and furthermore, we have shown that a difference in DMSA’s ability to elicit these calcium transients is affected by the presence or absence of lead (Pokorski et al., 1999). Another possible mechanism that could explain the calcium response to DMSA is activation of receptor-operated calcium channels (ROCCs) by DMSA. Adenosine 5%-triphosphate (ATP) acts as an activator of a specific ATP-activated P2purinergic receptor which opens a non-selective cation channel, allowing extracellular calcium to enter the cell (Dave and Mogul, 1996; Yamada et al., 1996; Squires et al., 1997). ATP increases [Ca2 + ]i levels in numerous cell culture models by this mechanism (Kamada et al., 1994; Morley et al., 1994; Lee et al., 1996). Additionally, in some cells, ATP increases [Ca2 + ]i levels by mobilizing intracellular calcium stores (Yamada et al., 1996; Squires et al., 1997). In our experiments with LLC-MK2 cells, ATP evoked transient increases in [Ca2 + ]i levels only in the presence of 2 mM extracellular calcium. To investigate the possibility that DMSA is chelating zinc from membrane proteins, thereby leading to calcium influx, tetrakis-(2-pyridyl-
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methyl)ethylenediamine (TPEN) a zinc chelator was tested to see if it would duplicate the results seen with DMSA. The failure of zinc chelation by TPEN to elicit transient increases in [Ca2 + ]i ruled out this possibility. It is possible but unlikely that DMSA is acting as a calcium ionophore. Most ionophores readily cross membranes to release Ca2 + from intracellular stores. In contrast, DMSA does not appear to cross the plasma membrane (Aposhian et al., 1992) and our results with DMSA do not support the conclusion that DMSA mobilizes Ca2 + from intracellular stores. Additionally, the DMSA molecule is not structurally similar to other calcium ionophores, e.g. Ionomycin. The toxicological significance of these findings is not yet completely understood, due to the fact that little is known of the cellular mechanisms by which DMSA decreases tissue lead levels. Taking our findings that DMSA exposure causes an influx of calcium into proximal tubular cells, with understanding the processes by which cells maintain intracellular calcium levels gives rise to interesting speculation on how DMSA may reduce the cellular burden of lead. Elevations in [Ca2 + ]i are counteracted by the activation of Ca2 + -ATPases which either pump Ca2 + into the endoplasmic reticulum, or pump Ca2 + out of the cell. Although the experiments described herein did not measure [Pb2 + ]i, some physiological stimuli that elicit Ca2 + signals appear to mobilize Pb2 + as well (Pounds and Mittelstaedt, 1983). Since Pb2 + mimics Ca2 + , and actually has a higher affinity for calcium pumps than Ca2 + itself, after a Ca2 + transient, it is speculated Pb2 + may be more efficiently pumped out of the cell, thus lowering intracellular lead (tissue) levels. Because proximal tubular cells are involved in urine production, DMSA and Ca-activated Ca2 + -ATPase activity may provide a possible explanation for the increased lead found in urine after DMSA chelation therapy. In conclusion, while testing the hypothesis that DMSA treatment does not affect intracellular calcium levels in LLC-MK2 cells, an unanticipated finding occurred: elicitation of calcium transients by DMSA in untreated, non-lead-exposed LLC-MK2 cells. Subsequent follow-up ex-
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periments showed that the source of calcium responsible for the calcium transients was from the extracellular environment, and DMSA elicited calcium responses in a concentration-dependent fashion. Other selected sulfhydryl-containing compounds were also successful at eliciting a calcium response. The calcium responses to DMSA were not due to zinc chelation from membrane proteins because the zinc chelator TPEN did not mimic the effects of DMSA. This work may be significant towards understanding the cellular mechanism by which DMSA chelates lead and from which possible organ stores of lead upon which DMSA acts.
Acknowledgements Support for this research was provided by a grant (ES04040) to JGP from the National Institute of Environmental Health Sciences, National Institutes of Health, and involving the services of the Imaging and Cytometry Facility Core supported by NIEHS center grant P30 ES06639.
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