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J Biol Chem, Vol. 275, Issue 10, 7071-7079, March 10, 2000

Ionomycin, a Carboxylic Acid Ionophore, Transports Pb²⁺ with High Selectivity^*

Warren L. Erdahl, Clifford J. Chapman, Richard W. Taylor^§, and Douglas R. Pfeiffer^¶

From the  Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210 and the ^§ Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb²⁺ > Cd²⁺ > Zn²⁺ > Mn²⁺ > Ca²⁺ > Cu²⁺ > Co²⁺ > Ni²⁺ > Sr²⁺. The selectivity of this ionophore for Pb²⁺ is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb²⁺ at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb²⁺ versus Ca²⁺ was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of ~450 was obtained. This rose to ~3200 when both cations were present and transported simultaneously. 1 µM Pb²⁺ inhibited the transport of 1 mM Ca²⁺ by ~50%, whereas the rate of Pb²⁺ transport approached a maximum at a concentration of 10 µM Pb²⁺ when 1 mM Ca²⁺ was also present. Plots of log rate versus log ionomycin or log Pb²⁺ concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb²⁺, but that complexes containing an additional Pb²⁺ may occur. The species transporting Pb²⁺ may include H·IPb·OH, wherein ionomycin is ionized once and the presence of OH maintains charge neutrality. Ionomycin retained a high efficiency for Pb²⁺ transport in A20 B lymphoma cells loaded with Indo-1. Both Pb²⁺ entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb²⁺, rather than Ca²⁺, of possible value for the investigation and treatment of Pb²⁺ intoxication.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lead remains as a pervasive toxin to which we are all exposed, albeit at levels that vary substantially with geographical location, occupation, and socioeconomic factors (1-3). To illustrate the magnitude of this problem, on the order of 1 in 10 American children currently possess blood lead levels above the toxic threshold of ~0.5 µM (reviewed in Ref. 4). The identification of mechanisms leading to lead toxicity is therefore of considerable interest and actively investigated. Work of this type conducted at the cellular level has been facilitated by the recent demonstration that entrapped Indo-1 can be used to monitor Pb²⁺ transport into cells (5, 6), even though this fluorescent compound is responsive to changes in the intracellular free Ca²⁺ concentration (7). Transport studies utilizing Indo-1-loaded cells of several types have shown that Pb²⁺ enters via plasma membrane Ca²⁺ channels that are activated upon depletion of internal Ca²⁺ stores (5, 6), as is also true for other toxic cations (8, 9).

The investigation of lead toxicity at a cellular level would benefit from the availability of ionophores that transport Pb²⁺ selectively in comparison with other cations; however, to date, no compound with this property has been identified. This situation may reflect the use of cells and subcellular preparations in early studies on the selectivity of ionophore-mediated transport (10). Biological systems are not stable to toxic cations such as Pb²⁺, making it difficult to use them for such purposes. In addition, the difficulty inherent in distinguishing ionophore-derived transport from transport occurring via endogenous channels and carriers may also have discouraged attempts to identify ionophores that are selective for Pb²⁺.

Unlike biological systems, phospholipid vesicles are stable to a wide range of cations and conditions and are free of endogenous transport activities. We have demonstrated that these structures, when prepared by freeze-thaw extrusion, are useful for investigating the transport mechanisms (11-13) and specificities (14, 15) of divalent cation ionophores such as A23187, 4-BrA23187, and ionomycin. In this report, we extend this work to include Pb²⁺ transport. The results indicate that ionomycin is substantially selective as an ionophore for Pb²⁺ and that this property is displayed in both phospholipid vesicles and cultured A20 B lymphoma cells. Accordingly, ionomycin may be useful for manipulating levels of Pb²⁺ during studies of its toxic properties in other cell types and at higher levels of biological organization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Synthetic 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC)¹ was obtained from Avanti Polar Lipids, Inc. Purity was confirmed by thin-layer chromatography before use. Ionomycin, A23187, and 4-BrA23187 were obtained from Sigma, Calbiochem, or Teflabs and were used without further purification. Stock solutions in ethanol were standardized spectrophotometrically using the following extinction coefficients: ionomycin, ₂₇₈ = 13,560; A23187, ₂₇₈ = 21,040; and 4-BrA23187, ₂₉₀ = 15,600. Quin-2 (K⁺ salt) from Sigma was purified by passage over Chelex 100 resin (100-200 mesh) in the Cs⁺ form as described previously (11). The nitrate and chloride salts of divalent cations were the ultrapure grade from Alfa Products. Stock solutions of these were standardized by titration with a primary standard EDTA solution (16) or by atomic absorption spectroscopy using certified solutions (Fisher).

Preparation of Phospholipid Vesicles-- Freeze-thaw-extruded POPC vesicles loaded with Quin-2 were prepared as described previously (17, 18). Briefly, 300 mg of POPC in chloroform was dried by rotation under a nitrogen stream to produce a film on the wall of a 25 × 150-mm culture tube. Residual solvent was removed under high vacuum (4 h), and the film was subsequently hydrated in 6 ml of a solution containing 5 mM purified Quin-2 (Cs⁺) and 10.0 mM Hepes adjusted to pH 7.00 with Chelex-treated CsOH (11). The mixture was vortexed, and the resulting multilamellar vesicles were frozen in a dry ice/acetone bath, thawed in lukewarm water, and vortexed again. The freeze-thaw and vortexing procedures were repeated two additional times, after which the vesicles were extruded three times through two stacked 100-nm polycarbonate membrane filters. This step was followed by six additional freeze-thaw cycles coupled with additional extrusions. The resulting preparations were applied to Sephadex G-50 minicolumns (19) to remove extravesicular Quin-2. These columns were eluted by low speed centrifugation and had previously been equilibrated with a solution containing 10 mM Hepes, pH 7.00. A single pass over such columns effectively removes the external Quin-2 (11, 17, 18).

The nominal concentration of POPC in the final preparations was determined by measurement of lipid phosphorus (20) and was near 80 mM. The average diameter of these vesicles is 71 nm as determined by freeze-fracture electron microscopy (17), and they contain entrapped solutes at the following concentrations; Quin-2, 10.5 ± 0.8 mM; Hepes, 34 ± 8 mM (pH 7.4); and Cs⁺, 60 ± 5 mM. Specific values for Quin-2 and Cs⁺ were determined for each preparation by the methods described previously (11, 12). Briefly, entrapped Quin-2 was determined by spectrophotometric titration with standard CaCl₂ following dispersion of the vesicles in deoxycholate. Entrapped Cs⁺ was determined by atomic absorption spectroscopy following replacement of the external medium with one not containing Cs⁺ and dispersion of the vesicles in 0.1 N HCl. When of interest, buffer entrapment was determined from the other values by calculation using the Henderson-Hasselbach equation, the buffer pK_a, and the internal pH. When buffer entrapment was to be determined, the vesicles also contained the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) so that the internal pH could be ascertained. The internal pH and solute concentrations differ from those of the vesicle formation medium because of a freeze-thaw-driven solute concentrating effect that operates during preparation of the vesicles (17, 18).

Vesicles loaded with BAPTA plus Indo-1 were prepared and characterized in an analogous way. For those preparations, the formation medium contained 5 mM BAPTA (in place of Quin-2) and 100 µM Indo-1. Accordingly, the resulting vesicles contained a relatively high level of BAPTA and a much lower level of Indo-1. The efficiency of BAPTA entrapment was similar to that of Quin-2 and was determined by titrating the intact vesicles with standard Ca²⁺ in the presence of excess ionomycin. Fluorescence of the Indo-1·Ca²⁺ complex was monitored at 400 nm (excitation at 336 nm) using an SLM-AMINCO 8100 spectrofluorometer operated in the analog mode. The end point can be taken to represent the end point for the simultaneous reaction of Ca²⁺ with BAPTA because the two compounds have essentially the same affinity for Ca²⁺ (7, 21), as described further under "Results." Knowledge of Indo-1 entrapment was not required during the present investigation, and that parameter was not determined.

Cell Culture and Loading of Cells with Indo-1-- A20 B lymphoma cells (ATCC TIB-208) were grown to a density of 1.5-2.5 × 10⁶ cells/ml at 37 °C under an atmosphere of 95% air and 5% CO₂. RPMI 1640 medium was employed and was supplemented with 25 mM Na⁺/Hepes, pH 7.4, 100 units/ml penicillin, 100 µM streptomycin, 2 mM L-glutamine, and 5% fetal bovine serum albumin (22). For loading with Indo-1, the cells were incubated for 45 min at 37 °C in a medium containing 4 µM Indo-1/AM plus 140 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM Na⁺/Hepes, pH 7.4, and 10 mM L-glucose (23). 1.0 mM probenecid was also present to inhibit the release of Indo-1 after loading (24). Following this, the loaded cells were maintained at 25 °C with gentle shaking and were used within 6 h.

Cation Buffers and Determination of Transport-- Pb²⁺ and Ca²⁺ were usually presented from a buffer system for transport into the vesicles or cells. 15 mM citrate was employed to buffer the concentration of cations, whereas 10 mM each Hepes and Mes were present to buffer H⁺. Seventeen equilibria involving citrate³, H⁺, Pb²⁺, and OH were accounted for when calculating the free Pb²⁺ concentration when this cation was present alone. Six additional equilibria involving Ca²⁺ were also considered when both Pb²⁺ and Ca²⁺ were present and the free concentration of both cations was of interest. The respective equilibrium constants were taken from literature sources (25, 26), and when necessary, the Davies equation (27) was used to correct these to an ionic strength of 100 mM. The species distribution program COMICS (28) was used to solve the applicable sets of simultaneous equations at experimental conditions of interest and to allow the generation of standard curves. As examples of the latter, Fig. 1A shows how free Pb²⁺ and PbOH⁺ vary in this system as a function of total Pb²⁺ when the medium pH is 7.0. Fig. 1B shows the pH dependence of this system.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Characteristics of the lead/citrate buffer system. A, shown are the calculated values of free Pb2+ () and PbOH+ () at pH 7.00 as a function of total Pb2+ in the system. The total citrate concentration and ionic strength were taken to be 15 and 100 mM, respectively. B, values like those shown in A were calculated for several pH conditions and were used to form the three-dimensional surfaces that are shown. The dark and light surfaces correspond to free levels of Pb2+ and PbOH+, respectively.

The transport of Pb²⁺ and other divalent cations into Quin-2-loaded vesicles was determined by monitoring formation of the Quin-2·cation complexes spectroscopically. Vesicles containing Quin-2 were present at a nominal POPC concentration of 1.0 mM in a medium that also contained 50 mM CsCl and 10 mM each Hepes and Mes. The medium pH ranged from 6.0 to 8.0 and was adjusted with CsOH that had been passed over Chelex 100 columns to remove contaminating divalent cations (11). To maintain internal pH at the external value, valinomycin (0.5 µM) and carbonyl cyanide m-chlorophenylhydrazone (5 µM) were also present (12). Specific concentrations of ionophores and divalent cation chlorides and pH values are given in the figure legends. Reactions were started by the addition of the divalent cation ionophore following an initial 2-3-min period that was allowed for the equilibration of transmembrane pH.

The formation of Quin-2·cation complexes was followed continuously by difference absorbance spectroscopy using an SLM-AMINCO DW2a spectrophotometer operated in the dual wavelength mode. An Oriel 59800 band-pass filter was used between the cuvette and the beam scambler-photomultipler assembly to prevent detection of the fluorescent light emitted by Quin-2. The sample wavelength used for all cations was 264 nm. The reference wavelengths were at an isosbestic point in the Quin-2/Quin-2·cation complex difference spectrum of interest. These wavelengths vary slightly from cation to cation, as described previously (14). Data were collected on disc using Unkel Scope software.

The vesicles loaded with BAPTA plus Indo-1 were used to monitor the simultaneous transport of Pb²⁺ and Ca²⁺, as further described under "Results." Pb²⁺ transport into Indo-1-loaded A20 B lymphoma cells was monitored as described by Kerper and Hinkle (5, 6), with modifications that are also described under "Results."

Other Methods-- To determine initial transport rates, an early portion of the progress curves was fit to Equation 1 using standard nonlinear least-squares methods.

(Eq. 1)

In this expression, A_T and A₀ are the observed and the initial absorbance values, respectively; B is the initial rate in units of absorbance/s; C is a correction factor for nonlinearity; and t is time. The values presented are in units of micromolar external cation transported into the vesicles/s. B values obtained from Equation 1 were converted to the latter unit by referring to a standard curve for the cation of interest that was generated by titrating the vesicles in the presence of excess ionophore or after they had been lysed with 0.33% (w/v) Cs⁺ deoxycholate (11, 14). This was with the exception of Pb²⁺, where apparent multiple equilibria between the cation and Quin-2 required a slightly modified approach (see "Results"). Transport selectivities are expressed as S values defined by Equation 2.

(Eq. 2)

When determining S, an equal concentration of the cation in question is substituted for Ca²⁺ with all other conditions held constant (14). All data were obtained at 25.0 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Use of Quin-2-loaded Vesicles to Investigate Ionophore-catalyzed Pb²⁺ Transport-- The vesicle-based transport system employed here is useful for determining selectivity and mechanisms of ionophore-mediated cation transport (11-15), but there are two features of importance that must be accounted for when applying the system to citrate-buffered solutions of Pb²⁺ (PbOH⁺). The first is an apparent slow permeation of Pb²⁺ in the absence of an ionophore, as illustrated in Fig. 2A. This is seen at much lower free Pb²⁺ concentrations than with Ca²⁺ permeation, with the uncatalyzed movements of both cations showing a complex dependence on concentration and pH (Fig. 2B). Under the conditions we utilized, the rate of uncatalyzed transport was usually negligible, but was determined, and data were corrected accordingly when it exceeded 2-3% of the ionophore-dependent rate.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Uncatalyzed transport of Pb2+ and Ca2+. A, POPC vesicles containing Quin-2 were incubated in a medium containing 15 mM citrate (Cs+) plus 10 mM each Hepes and Mes. The nominal concentrations of POPC and Quin-2 were 1.0 mM and 20 µM, respectively, and the external pH was adjusted to 7.00 using purified CsOH. Pb(NO3)2 was added to establish the calculated concentrations of free Pb2+ (PbOH+ not considered) that are indicated to the right of individual traces. From top to bottom, the corresponding total Pb(NO3)2 concentrations were as follows: 8.00, 4.00, 2.00, 1.00, 0.500, 0.125, and 0.031 mM. The entry of Pb2+ (PbOH+) was then monitored spectroscopically, as formation of the Quin-2·Pb2+ complex, and these data were calibrated as described under "Experimental Procedures." The y axis units refer to total lead from the external medium that had entered the vesicles and been complexed by Quin-2 at the time indicated on the x axis. B, data like those shown in A were obtained at three pH values as follows: pH 6.00 (), pH 7.00 (), and pH 8.00 (). Initial rates of Pb2+ accumulation were obtained from the progress curves as described under "Experimental Procedures" and are shown as a function of the free Pb2+ concentration in external medium. Also shown is the rate of uncatalyzed Ca2+ entry as a function of free Ca2+ at pH 6.00 (), pH 7.00 (), and pH 8.00 (). Conditions were the same as those used for Pb2+, except the total levels were adjusted to give the desired concentrations of free Ca2+.

The second feature relates to an apparent formation of multiple complexes between Pb²⁺ and entrapped Quin-2 and to how this impacts on the calibration procedure used to relate the Quin-2 difference absorbance measurements to Pb²⁺ transport. When the vesicles were suspended in an unbuffered Pb²⁺ solution and the total Pb²⁺ available for transport exceeded the total Quin-2 trapped within the vesicles, a biphasic progress curve was obtained upon the addition of a Pb²⁺ ionophore (Fig. 3A). During the first phase, entrapped Quin-2 approached an apparent saturation; however, as time proceeded, a second phase was observed that would indicate a partial release of previously accumulated Pb²⁺ if it were taken at face value (Fig. 3A). However, titrating vesicle-entrapped Quin-2 with Pb²⁺ in the presence of excess ionophore also produced a biphasic pattern, wherein the apparent degree of saturation decreased as Pb²⁺ was added beyond the 1:1 equivalence point (Fig. 3B). In addition, biphasic transport curves were not seen when Quin-2 was present in excess of total Pb²⁺ (data not shown), and titration data were less multiphasic when the vesicles had been lysed to dilute the internal concentration of Quin-2 that originally existed (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Multiple complexes between Pb2+ and Quin-2. A, Quin-2-loaded vesicles were incubated as described in the legend to Fig. 2, except that 50 mM CsCl was present instead of cesium citrate, and the pH was 6.65. An unbuffered Pb2+ concentration of 100 µM was established by adding that concentration of Pb(NO3)2. Where indicated, Pb2+ transport was initiated by the addition of ionomycin at 1.0 µM. B, shown is the fractional change in Quin-2 difference absorption, at 342 versus 264 nm, during titration of the compound with Pb2+ (). Quin-2 was contained in POPC vesicles, and an excess of ionomycin was present to equilibrate added Pb(NO3)2 with the internal indicator. , Quin-2 was released by lysing the vesicles with deoxycholate, as described under "Experimental Procedures," before titrating with Pb(NO3)2. The solid line shows that the two sets of data are coincident until the total concentration of Pb2+ exceeds ~22 µM. Total Quin-2 was 27 µM, as determined by titrating with Ca2+ (11).

We interpret these data to indicate that more than one complex is formed between Pb²⁺ and the indicator under conditions that pertain in the lumen of intact vesicles. There was no indication of multiple complexes when this system was employed to investigate ionophore-catalyzed transport of Ca²⁺ and other divalent cations, such that this factor was not considered in the previous studies that established procedures for utilizing the vesicle system to investigate ionophore-mediated transport (11-15, 17, 18). Here we have avoided possible errors in the reported rate and selectivity values by deriving these parameters from the initial portion of the progress curves, where internal Quin-2 was present in excess of internal Pb²⁺. There was no disagreement between the internal and external calibration methods within this region (Fig. 3B), indicating that the same complex is considered in both cases.

Selectivity and Mechanism of Transport-- Ionomycin transported Pb²⁺ more efficiently than other divalent cations as shown in Fig. 4A. This is in contrast to A23187 (Fig. 4B) and 4-BrA23187 (data not shown), where rates of Pb²⁺ transport were intermediate in comparison with the others. Selectivity for Pb²⁺ compared with Ca²⁺ is of particular interest because Pb²⁺ enters cells via channels that normally transport Ca²⁺ (5, 6, 29) and because competition between these cations at intracellular sites is important in mechanisms of Pb²⁺ toxicity (30, 31). Using Equation 2 and the data in Fig. 4, it can be shown that S_Pb is 98 for ionomycin and only 2.3 for A23187 under the conditions of this figure.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Transport of Pb2+ and other divalent cations by Ca2+ ionophores. POPC vesicles loaded with Quin-2 were incubated in a medium containing 50 mM CsNO3 plus 10 mM each Hepes and Mes. The nominal concentrations of POPC and Quin-2 were 1.0 mM and 20 µM, respectively. The external pH was adjusted to 7.00 using purified CsOH, and valinomycin (0.5 µM) plus carbonyl cyanide m-chlorophenylhydrdazone (5 µM) were present to maintain the internal pH at the external value. Divalent cations were present at 20 µM in all cases (nitrate in the case of Pb2+ and chlorides for all others). Instrumentation and other conditions were as described under "Experimental Procedures." Transport was initiated at 0 s by the addition of 0.10 µM ionomycin (A) or A23187 (B). A 2-min preincubation preceded the initiation of transport to allow the equilibration of internal and external pH (12).

The rate of ionomycin-catalyzed Pb²⁺ transport is a function of the ionophore (Fig. 5) and the Pb²⁺ (Fig. 6) concentrations, as expected for a mechanism where a discrete complex between the cation and the ionophore underlies activity. When the ionophore concentration was varied, a plot of log initial rate versus log of the ionophore concentration was well represented by a straight line of slope 1.0, as was also true for Ca²⁺ transport (Fig. 5) (11). Varying the free Pb²⁺ concentration produced a log versus log plot that also had a slope near 1 within the lower portion of the range examined, but a progressively smaller value as the free Pb²⁺ concentration rose (Fig. 6). This nonlinear behavior was less apparent with Ca²⁺ transport (Fig. 6), which resulted in lower values of S_Pb being obtained from Equation 2 as higher concentrations of the two cations were compared. To avoid this circumstance and to obtain a more representative value of S_Pb, we calculated a value based upon the x axis separation of the plots within regions where (near) linear behaviors were observed. Within these regions, both sets of data were separated by 2.65 log units, which translates into an S_Pb value of ~450.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of ionophore concentration on the initial rate of Pb2+ transport. A, experiments were conducted as described under "Experimental Procedures" and in the legend to Fig. 4, except that free Pb2+ was buffered at 1.0 µM using citrate (see legend to Fig. 2), and the ionomycin concentration was varied as shown to the right of the individual traces. B, shown is the log of the initial Pb2+ () or Ca2+ () transport rate as a function of log ionomycin concentration. The data for Ca2+ transport were determined from experiments like those shown in A, wherein 1.0 µM free Ca2+ replaced Pb2+.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of Pb2+ concentration on the initial rate of Pb2+ transport. A, experiments are analogous to those shown in Fig. 5, except that the ionomycin concentration was held constant at 0.10 µM, and the free Pb2+ concentration was varied as shown to the right of the individual traces. B, shown is the log of the initial Pb2+ () or Ca2+ () transport rate as a function of log concentration for that cation. The data for Ca2+ transport were obtained from experiments like those shown in A, wherein the free Ca2+ concentration was varied instead of free Pb2+.

Competitive Transport of Pb²⁺ Versus Ca²⁺ in Vesicles-- To determine if ionomycin retains high transport selectivity for Pb²⁺ over Ca²⁺ when both cations are present, we utilized vesicles loaded with BAPTA plus a small amount of Indo-1, rather than Quin-2. Quin-2-loaded vesicles are not suitable for investigating simultaneous transport because there is no spectral distinction between the primary Pb²⁺ and Ca²⁺ complexes that are formed with this indicator. The same is true for the corresponding BAPTA complexes; however, the Pb²⁺ and Ca²⁺ complexes of Indo-1 can be distinguished by their fluorescence properties as reported by Kerper and Hinkle (5, 6).

Fig. 7 illustrates, in part, how the differences they described can be exploited to allow initial rates to be obtained for the two cations individually when both are present. Titration of vesicles containing BAPTA plus Indo-1 with Pb²⁺ in the presence of high ionomycin allowed the external cation to equilibrate with the internal chelators, as shown earlier for Quin-2-loaded vesicles titrated with Ca²⁺ (11). As such a titration proceeded, Pb²⁺ association with Indo-1 quenched its fluorescence emission at 452 nm (Em₄₅₂), while having a much smaller effect on Em₄₀₀ (Fig. 7A). The former wavelength is an isosbestic point between the emission spectra of free Indo-1 and its Ca²⁺ complex (5, 6), whereas the latter is a maximum in the spectrum of the Ca²⁺ complex (7) and is little affected upon the binding of Pb²⁺ (6). Accordingly, the entry of Pb²⁺ during a continuous transport experiment should progressively decrease Em₄₅₂, without affecting Em₄₀₀, and this was observed (Fig. 7B). Likewise, titrating the vesicles with Ca²⁺ increased Em₄₀₀ and had a much smaller effect on Em₄₅₂ (Fig. 7C), whereas the same features were again apparent during entry of Ca²⁺ by continuous transport (Fig. 7D).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Indo-1 fluorescence distinguishes between Pb2+ and Ca2+ transport. A, POPC vesicles loaded with BAPTA plus Indo-1 were incubated in a medium containing 50 mM CsNO3 plus 10 mM each Hepes and Mes. The nominal concentrations of POPC and BAPTA were 1.0 mM and 20 µM, respectively, and Indo-1 was present at <1 µM. The external pH was adjusted to 7.00 using purified CsOH. Valinomycin (0.5 µM) plus carbonyl cyanide m-chlorophenylhydrazone (5 µM) were present to maintain the internal pH at the external value, and 1.0 µM ionomycin was present to rapidly equilibrate external Pb2+ with the internal chelators.  and , change in Indo-1 fluorescence at 400 and 452 nm, respectively (excitation at 336 nm), during titration of the system with Pb(NO3)2. B, same as A, except that 15 mM cesium citrate replaced CsNO3, and 125 µM Pb(NO3)2 was present to establish a free Pb2+ concentration of 81 nM. Transport was initiated by the addition of ionomycin, where indicated, and fluorescence at 400 and 452 nm was monitored by rapid scanning. C, same as A, except that the vesicles were initially titrated with Ca2+ rather than Pb2+, until the total Ca2+ added reached 41 µM. Thereafter, the titration was continued by adding Pb2+. Above 41 µM, the x axis units refer to the sum of Ca2+ and Pb2+ that had been added. D, same as B, except that 3.00 mM Ca(NO3)2 replaced Pb(NO3)2 to establish a free Ca2+ concentration of 89 µM.

Thus, the accumulation of Pb²⁺ or Ca²⁺ gives rise to distinct fluorescence signals from the entrapped Indo-1. However, as the accumulation of either cation proceeds, most of the internal quantity is associated with BAPTA, whereas Indo-1 equilibrates with the fraction that is free. To relate the wavelength-specific fluorescence changes to rates of transport, the relationships between fractional Indo-1 and BAPTA saturation must be taken into account. The stability constants of the BAPTA and Indo-1 complexes with Ca²⁺are similar at 6.03 × 10⁶ and 4.36 × 10⁶, respectively (7, 21). In the case of Pb²⁺, the corresponding values are 1.99 × 10¹¹ (21) and 2.88 × 10¹⁰ (5). Given the inherent uncertainties, the Ca²⁺ values could probably be considered equivalent; however, this is not true in the case of Pb²⁺, which is bound 6.9-fold more tightly by BAPTA than by Indo-1. Ignoring this discrepancy would cause the Pb²⁺ transport rate to be underestimated significantly.

To correct for the discrepancy, we used the maximum and minimum Em₄₅₂ values observed when no Pb²⁺ and saturating Pb²⁺ were present, respectively, to calculate the Indo-1/Indo-1·Pb²⁺ complex ratio at each point in the progress curves. These values were converted to free Pb²⁺ concentrations using the stability constant of the Indo-1·Pb²⁺ complex, and thereafter, the BAPTA/BAPTA·Pb²⁺ ratio could be calculated using the stability constant for the complex formed between that ligand and Pb²⁺. From the resulting values and the amount of BAPTA entrapped, Pb²⁺ accumulated as a function of time could be calculated (free Pb²⁺ and Pb²⁺ associated with Indo-1 can be ignored because they are small compared with the BAPTA-associated Pb²⁺). An analogous procedure was applied to the Ca²⁺ accumulation data obtained as Em₄₀₀.

Fig. 8 shows the results of these procedures when applied to determine S_Pb. When the medium free Ca²⁺/free Pb²⁺ ratio was ~900, the initial rate of Pb²⁺ transport was 3.5-fold greater than the rate of Ca²⁺ transport. Thus, under conditions of simultaneous transport, S_Pb is ~3200 in comparison with Ca²⁺. The release of Ca²⁺ that was seen as Pb²⁺ accumulation proceeded represents the displacement of Ca²⁺ from Indo-1 (and BAPTA) by the more tightly bound Pb²⁺ (Fig. 8). This was also seen when vesicles were first titrated with Ca²⁺ and then with Pb²⁺ (Fig. 7C).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Simultaneous transport of Pb2+ and Ca2+. Vesicles loaded with BAPTA plus 100 µM Indo-1 (rather than Quin-2) were employed. The external medium conditions were as described in the legend to Fig. 7, except that 125 µM Pb(NO3)2 and 3.00 mM Ca(NO3)2 were both present to establish citrate-buffered free Pb2+ and Ca2+ concentrations of 81 nM and 89 µM, respectively. 1.0 µM ionomycin was added at 0 s to intiate the transport of Pb2+ and Ca2+. Indo-1 was excited at 336 nm, with emission monitored at 400 nm (Ca2+ transport) and 452 nm (Pb2+ transport) by rapid scanning, and the original data were corrected for the affinity differences of BAPTA and Quin-2 for Pb2+ and Ca2+ as described under "Results." In the inset, the free Ca2+ concentration was increased to 1.0 mM, and the free Pb2+ concentration was varied as shown. Progress curves like those shown in the main panel were obtained, and initial rate values were determined.

Fig. 8 (inset) shows the dependence of Pb²⁺ and Ca²⁺ transport on Pb²⁺ concentration when the external free Ca²⁺ concentration is 1 mM. Approximately 1 µM free Pb²⁺ decreased the Ca²⁺ transport rate by 50%, whereas the rate for Pb²⁺ transport approached a maximum as the free Pb²⁺ concentration approached 10 µM. The methods we employed to correct the data for differing stabilities of the Indo-1 and BAPTA complexes with Pb²⁺ and Ca²⁺ do not account for all factors of possible interest²; however, it is clear that a very high selectivity for Pb²⁺ over Ca²⁺ is manifested by ionomycin when both cations are present.

Pb²⁺ Transport in Cells-- Ionomycin-catalyzed Pb²⁺ transport into A20 B lymphoma cells is demonstrated in Fig. 9. Fig. 9A was obtained using cells that were maintained in growth medium during loading with Indo-1. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), the membrane-permeant chelator of heavy metal cations (32), increased Em₄₅₂ when added to these cells, indicating the presence of contaminates (Fig. 9A). This was not seen when the cells were in the defined medium during loading (Fig. 9B and data not shown) so that condition was adopted. The addition of the impermeant chelator DTPA (Fig. 9B) or 1 µM free Pb²⁺ (Fig. 9C) was also without an immediate effect on Em₄₅₂, demonstrating the absence of Indo-1 and its Ca²⁺ complex in the external volume. Thus, these cells, when loaded with Indo-1, are an appropriate system for the investigation of Pb²⁺ transport.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Ionophore mediated Pb2+ transport in A20 lymphoma cells. Cells were grown and loaded with Indo-1 as described under "Experimental Procedures." They were incubated at 25 °C and 1 × 106 cells/ml in a medium containing 140 mM NaCl, 3 mM KCl, 1 mM each CaCl2 and MgCl2, 10 mM Na+/Hepes, pH 7.4, 10 mM glucose, and 1.0 mM probenecid. For all traces, a downward deflection indicates Pb2+ accumulation. In A and B, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and DTPA were added at 100 µM where indicated. In A, the cells were incubated in growth medium during loading with Indo-1, whereas in B and all other experiments, the incubation medium was employed. In C and D, 15 mM citrate and 2.0 mM PbCl2 were present to establish buffered concentrations of free Pb2+ and Ca2+ at 1.1 and 25 µM, respectively. In C, PbCl2 was added where indicated. In other cases, it was present from the beginning. In D, 0.5 µM ionomycin was added where indicated. The data in E were obtained from experiments similar to the one illustrated by D, except the level of ionomycin or Pb2+ was varied. , ionomycin was varied, and free Pb2+ was present at 0.38 µM; , free Pb2+ was varied, and ionomycin was present at 0.5 µM. Cell viability was checked at the end of individual experiments and had not decreased below the initial range of 90-95%.

As with other cell types, Pb²⁺ entered A20 lymphoma cells slowly when it was present at a free concentration of 1 µM (Fig. 9C). The rate was markedly accelerated by 0.5 µM ionomycin (Fig. 9D), demonstrating that the compound is active as a Pb²⁺ ionophore in this biological system. As with vesicles, the rate of transport is a function of the Pb²⁺ and ionomycin concentrations over a broad range of either parameter. This is shown by Fig. 9E, which summarizes concentration dependence data covering 2 orders of magnitude in both cases.

Fig. 10 demonstrates that ionomycin-mediated Pb²⁺ transport in cells is reversible. To obtain these data, the Pb²⁺/citrate buffer system was replaced by 20 µM Pb(NO₃)₂ to yield a free Pb²⁺ concentration in that range. As with other cells, Pb²⁺ rapidly entered A20 B lymphoma cells through endogenous activities when present at this higher concentration (Fig. 10, data obtained as Em₄₅₂). The entry was accelerated by ionomycin addition and reversed by the subsequent addition of excess EDTA. Like DTPA, EDTA is membrane-impermeant and binds Pb²⁺ with high affinity. Thus, the recovery of Em₄₅₂ following the addition of EDTA indicates that internal Pb²⁺ was released from Indo-1, transported out of the cells, and chelated in the external volume. Also shown are the parallel changes in Em₄₀₀, reflecting, in part, the accompanying changes in free Ca²⁺. Em₄₀₀ decreased as Pb²⁺ entered the cells initially, consistent with a displacement of endogenous Ca²⁺ from Indo-1 by the more tightly bound Pb²⁺. Conversely, Em₄₀₀ rose to a high level upon EDTA addition and Pb²⁺ depletion, indicating that the cytoplasmic Ca²⁺ concentration is increased progressively as Pb²⁺ is removed. This rising Ca²⁺ level presumably reflects an ionomycin-mediated entry of Ca²⁺ from the external volume and possibly a release of Ca²⁺ from internal stores into the cytoplasm.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Release of Pb2+ from A20 lymphoma cells. Indo-1-loaded cells were incubated as described in the legend to Fig. 9. Excitation was at 336 nm for both traces, whereas emission was monitored by rapid scanning between 452 nm (solid line) and 400 nm (dotted line). Pb(NO3)2 (20 µM), ionomycin (Iono; 0.10 µM), and EDTA (200 µM) were added as indicated.

	DISCUSSION

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Early studies utilizing solvent extraction systems, sarcoplasmic reticulum vesicles, and mitochondrial preparations established that ionomycin is a Ca²⁺ ionophore (33-35), and it has long been utilized in that regard. More recently, it has become clear that this compound binds and transports a wide range of cations, including alkaline earth and first transition series divalent cations and lanthanide series trivalent cations (14, 15, 36). Among all the cations considered to date, ionomycin is the most active by far as an ionophore for Pb²⁺. Accordingly, it seems appropriate to describe this compound as a Pb²⁺ ionophore that has a limited activity for the transport of other polyvalent cations such as Ca²⁺. Pb²⁺ transport by ionomycin is a first-order function of ionophore concentration, as indicated by the linear character and slope value of 1.0 that are seen in plots of log initial rate versus log ionomycin concentration (Fig. 5). The same is true with respect to Pb²⁺ concentration within the lower portion of the range examined (Fig. 6). Where these relationships hold, the rate of Pb²⁺ transport can be described by Equation 3, in which k_trans is a second-order rate constant that is valid at the pH and temperature employed.

(Eq. 3)

The versatility of the vesicle transport system with respect to cations that can be considered and the range of conditions that are accessible makes it possible to express the efficiency and selectivity of ionophore-catalyzed transport in terms of rate constants. These values are usually not accessible when traditional methods are employed to characterize ionophores. From the present data, k_trans values for Pb²⁺ and Ca²⁺ transport are 1.2 × 10⁶ and 2.7 × 10³ M¹ s¹, respectively.

Within the concentration regions where Equation 3 is applicable, the stoichiometry of the transporting species must be 1:1, ionophore to cation, based upon the first-order kinetic characteristics (slope values of 1.0 in the log versus log plots). Ionomycin is a monocarboxylic acid that contains an enolized -diketone moiety (37) and can therefore ionize twice to form 1:1 charge neutral complexes with divalent cations (36). Thus, it is possible that Pb²⁺ is transported as the neutral species IPb, formed according to Equation 4, wherein H₂I is the fully protonated form.

(Eq. 4)

However, monoprotonated complexes between ionomycin and several divalent cations have been identified (36), and charge neutral complexes of 1:1 stoichiometry are formed between ionomycin and trivalent lanthanide cations, apparently by the inclusion of an OH (15). Since Pb²⁺ and Pb²⁺ complexes are prone to hydrolyze (38-40), it is then possible that the weakly acidic enolized -diketone moiety remains protonated in the transporting species and that this species contains OH. Thus, a mixed complex of stoichiometry H·IPb·OH might also be responsible for transporting Pb²⁺, wholly or in part. An unambiguous identification of the transporting species must await determination of the mode of transport (electroneutral, electrogenic, or mixed) and investigation of the complexation equilibria between this ionophore and Pb²⁺.

Explanations for the progressive curvature in plots of log rate versus log Pb²⁺ concentration that is evident at higher values of free Pb²⁺ (Fig. 6) and the increase in S_Pb compared with Ca²⁺ that is seen when both cations are present (Fig. 8) also await investigation of the complexation equilibria between Pb²⁺ and ionomycin. Regarding the former, we suspect that curvature reflects an approach to saturation of the ionophore with Pb²⁺ as the free concentration of the cation rises. However, it is also possible that it reflects the reaction of a 1:1 transporting species with a second Pb²⁺ ion to form species such as IPb₂ (Equation 5, charge and hydroxylation status not specified) that are unable to cross membranes.

(Eq. 5)

If the higher order species were less stable than the transporting species, the result would be less ionophore contributing to transport as the free Pb²⁺ concentration increased, producing the type of curvature that is observed (Fig. 6).

Regarding the high value of S_Pb that is seen when Pb²⁺ and Ca²⁺ are present, it seems possible that both complexation equilibria and differing transmembrane diffusion constants contribute to the explanation. For example, if Pb²⁺ were bound more tightly than Ca²⁺, but formed a transporting complex that crossed the membrane relatively slowly compared with the complex transporting Ca²⁺, the result could be an increase in the apparent selectivity when both cations were present, which is what we observed (Fig. 8).

Legare et al. (29) reported that ionomycin transports Pb²⁺ into primary cultures of rat astroglia cells, and our results with A20 B lymphoma cells extend their findings. We cannot compare the activity values in vesicles and cells quantitatively because the relationship between free and total Pb²⁺ in the cells is unknown. In addition, the total aqueous/lipid phase volume ratios are not similar at the vesicle and cell concentrations we employed. Accordingly, and for other reasons, the fraction of ionophore that is partitioned to the lipid phase and engaged in transport is probably different in the two systems (much larger in the case of vesicles). Nevertheless, it is clear that ionomycin retains a high activity as a Pb²⁺ ionophore in cells. This is seen qualitatively in Fig. 9D, where 0.5 µM ionomycin efficiently loaded the cells with Pb²⁺ when the external free Pb²⁺ concentration was 1 µM. The former value is typical of ionomycin concentrations used to load cells with Ca²⁺, whereas the cation concentration employed in that case is normally much higher (millimolar). The Pb²⁺ transport activity of ionomycin will, presumably, be a useful addition to the approaches used for investigating the toxic activities of Pb²⁺ at a cellular level.

A final point relates to the reversibility of ionomycin-mediated Pb²⁺ transport that is demonstrated in Fig. 10. It is interesting to note that the ionophore-mediated release of Pb²⁺ appears to be more rapid than the initial accumulation. This same difference was seen earlier for Ca²⁺ transport using the vesicle system, in which Ca²⁺ release occurs 20 times faster than uptake (11). The differing rates are thought to reflect the large difference in volume of the aqueous phase compartments accessible from alternate sides of the membrane and the effect of this on ionophore levels at the two interfaces where transporting species are formed (11). It seems possible that efficient release of Pb²⁺ from cells when ionomycin is present will extend to higher levels of biological organization and reach a practical application as follows. Lead intoxication is treated by the administration of chelating agents that complex Pb²⁺ in blood and are subsequently secreted by the kidney (41, 42). The agents must be given repeatedly over a lengthy time course. There are reports that multiple hydrophilic chelators administered simultaneously remove accumulated lead more effectively than single compounds (43) and that a degree of hydrophobicity improves single compound effectiveness (44-46). Low levels of a lead ionophore administered together with a hydrophilic chelator might capture the multiple chelator advantage and the hydrophobicity advantage simultaneously. The result could be an improved treatment for lead intoxication, both in terms of the completeness of removal and a shorting of the treatment period required.

	ACKNOWLEDGEMENTS

We thank Dr. K. Mark Coggeshall and co-workers, who initially provided A20 B lymphoma cells and helped to establish the culture of these cells in our laboratory.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant HL49181 from NHLBI, National Institutes of Health, and by the Wallace Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Medical Biochemistry, Ohio State University, 1645 Neil Ave., 310A Hamilton Hall, Columbus, OH 43210-1218. Tel.: 614-292-8774; Fax: 614-292-4118.

² The amount of Ca²⁺ transported is progressively underestimated in Fig. 8 because we have not corrected the progress curve for the reduction in Indo-1 available to associate with Ca²⁺ as Pb²⁺ accumulation proceeds. The available Indo-1 is progressively decreased because Pb²⁺ is bound much more tightly than Ca²⁺, which essentially removes the Pb²⁺ associated indicator from the equilibrium involving Ca²⁺. This factor was not considered because our interpretations are based upon initial rates, which are not affected by this feature of the system.

	ABBREVIATIONS

The abbreviations used are: POPC, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; DTPA, diethylenetriaminepentaacetic acid.

	REFERENCES

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