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J. Biol. Chem., Vol. 277, Issue 41, 38111-38120, October 11, 2002

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Monensin Mediates a Rapid and Selective Transport of Pb²⁺

POSSIBLE APPLICATION OF MONENSIN FOR THE TREATMENT OF Pb²⁺ INTOXICATION^*

Shawn A. Hamidinia, Olga I. Shimelis^§, Bo Tan^§, Warren L. Erdahl, Clifford J. Chapman, Gordon D. Renkes^¶, Richard W. Taylor^§, and Douglas R. Pfeiffer

From the  Departments of Molecular and Cellular Biochemistry and the ^¶ Department of Chemistry, Ohio State University, Columbus, Ohio 43210 and the ^§ Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

Received for publication, June 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The carboxylic acid ionophore monensin, known as an electroneutral Na⁺ ionophore, an anticoccidial agent, and a growth-promoting feed additive in agriculture, is shown to be highly efficient as an ionophore for Pb²⁺ and to be highly selective for Pb²⁺ compared with other divalent cations. Monensin transports Pb²⁺ by an electroneutral mechanism in which the complex PbMonOH is the transporting species. Electrogenic transport via the species PbMon⁺ may also be possible. Monensin catalyzed Pb²⁺ transport is little affected by Ca²⁺, Mg²⁺, or K⁺ concentrations that are encountered in living systems. Na⁺ is inhibitory, but its effectiveness at 100 mM does not exceed ~50%. The poor activity of monensin as an ionophore for divalent cations other than Pb²⁺ is consistent with the pattern of complex formation constants observed in the mixed solvent 80% methanol/water. This pattern also explains why Ca²⁺, Mg²⁺, and K⁺ are ineffective as inhibitors of Pb²⁺ transport, but it does not fully explain the actions of Na⁺, where kinetic features of the transport mechanism may also be important. When given to rats at 100 ppm in feed together with Pb²⁺ at 100 ppm in drinking water, monensin reduces Pb accumulation in several organs and tissues. It also accelerates the excretion of Pb that was accumulated previously and produces this effect without depleting the organs of zinc or copper. Monensin, used alone or in combination with other agents, may be useful for the treatment of Pb intoxication.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ionophores are lipophilic chelating agents that transport cations across phospholipid bilayer membranes, such as the plasma and subcellular membranes of cells. The naturally occurring compounds (about 100) are antibiotics produced primarily by soil bacteria of the Streptomyces genus (1). They are distinguished from pore-forming antibiotics such as gramicidin by the involvement of a discrete complex in the transport mechanism. Thus, true ionophores can be highly selective for particular cations (2, 3). The known compounds are generally divided into two groups: the so-called electrogenic and the electroneutral ionophores (4, 5). Electrogenic ionophores are typified by the well known compound valinomycin (6). They are neutral molecules that form cation complexes that carry a net positive charge, such that transbilayer charge movements accompany transport catalyzed by this class. Accordingly, the rate of transport is influenced by membrane electrical potential, which also determines the transmembrane distribution of the transported cation at equilibrium. Compounds such as monensin, nigericin, and A23187 (Fig. 1) typify electroneutral ionophores, also called carboxylic acid or polyether ionophores. The anionic forms of these compounds complex the cation, which is exchanged for H⁺, or another cation, without net charge movement. Transport catalyzed by this class is therefore influenced by transmembrane pH gradients, as is the equilibrium distribution of cations.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structure of representative carboxylic acid ionophores.

Among the carboxylic acid ionophores, it has been common to consider individual compounds as able to complex/transport a small group of cations having the same charge, with transport occurring through a single species by a mechanism that is purely electrogenic or electroneutral. Thus, monensin and nigericin were known as ionophores for Na⁺ and K⁺, respectively, with transport occurring via a 1:1 complex and electroneutral mechanism; A23187 was known as a Ca²⁺ ionophore transporting via a 1:2 complex and an electroneutral mechanism, and so forth. In work conducted over a period of time, we showed that the model depicted in Fig. 2 is a more accurate representation of the factors that establish the transport properties of a carboxylic acid ionophore (7-16). The equilibrium shown at the top of Fig. 2 represents formation of 1:1 complexes and emphasizes that carboxylic acid ionophores react with a broad range of cations (n = 1-3), not just those with a particular charge as often assumed. The 1:1 complexes then react to form higher order species by competing equilibria I-III, which involve free ionophore (A), hydroxide ion (OH), and other anions (X), respectively. Complexes of differing stoichiometry therefore arise, which include mixed species containing OH and X. Within this scheme, the mode and overall rate of transport will reflect the distribution of ionophore between these various complexes and their respective transmembrane diffusion constants. As a corollary of this interpretation, for a given ionophore and set of conditions, transport selectivity will also depend on how these factors vary with properties of the cation. The latter include size, charge, coordination and hydration number, preferred donor atom geometry, Lewis acidity, and ease of hydrolysis.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Equilibria between an ionized carboxylic acid ionophore (A1), a metal cation of charge n+ (Mn+), and selected anions (A, X, and OH). Following formation of a 1:1 complex between the ionophore and a cation (MAn  1), additional species can arise that may contribute to transport. In I, a 1:2 complex is formed by reaction with a second ionophore molecule. In II and III, mixed complexes arise by reaction with OH or another anion (X), respectively.

In considering the above model, the characteristics of the donor atoms in typical ionophores, and the metal ion complexation properties of analogous synthetic polyethers (17), we realized that some of the antibiotic compounds might be efficient ionophores for Pb²⁺ and for other cations with biological toxicity. Subsequently, we showed that ionomycin transports Pb²⁺ rapidly and with selectivity over Ca²⁺ near 10³ when both cations are present simultaneously. Ionomycin was also found to affect an efficient transport of Pb²⁺ into cultured cells as well as to facilitate the depletion of Pb²⁺ when the cells had been previously loaded. We indicated that ionomycin should be considered primarily as an ionophore for Pb²⁺, rather than Ca²⁺, and suggested that its Pb²⁺-transporting activity might be adapted to improve existing treatments for Pb²⁺ intoxication (15).

In the present report, we extend the investigation of ionophore-mediated Pb²⁺ transport by demonstrating that monensin is also effective as a Pb²⁺ ionophore and is more selective in that regard than is ionomycin. We also show that monensin promotes the excretion of Pb²⁺ from rats when the cation has been previously provided in drinking water and the ionophore is administered in feed. Aspects of these data have been presented in abstract form (18).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Solvents-- High purity nitric acid (trace metal; Fisher) and perchloric acid (double-distilled; GFS Chemicals) were obtained from commercial sources. 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. For the transport studies, monensin and ionomycin were obtained from Calbiochem and used without further purification. Ionomycin stock solutions were prepared in ethanol and standardized spectrophotometrically using an extinction coefficient of 13,560 at 278 nm. In the case of monensin, standardization was gravimetric or by titration with Me₄NOH. Quin-2 (K⁺ salt) from Sigma was purified by passage over Chelex 100 resin (100-200-mesh) in the Cs⁺ form as described previously (19), or in the Na⁺ form when Na⁺-loaded vesicles were employed. The nitrate and chloride salts of divalent cations were the ultrapure grade from Alfa Products. Stock solutions were standardized by titration with a primary standard EDTA solution (20) or by atomic absorption spectroscopy using certified solutions (Fisher).

For solution chemical studies, a mixed solvent of 80% (w/w) methanol in water was prepared gravimetrically, using distilled deionized water and reagent grade methanol (Fisher) that had been freshly distilled. The Et₄NClO₄ that was used to maintain ionic strength in this solvent was prepared by reaction of Et₄NOH (Aldrich) with 70% perchloric acid (distilled; GSH Chemicals). The salt obtained was recrystallized four times from water. Solvent containing Et₄NClO₄ and H⁺ buffering compounds was further deionized by passage over Chelex 100. For this purpose, the resin was in the Et₄N⁺ form, which was prepared as previously described (8).

Preparation of Phospholipid Vesicles-- The preparation of freeze-thaw-extruded POPC vesicles loaded with Quin-2 has also been described previously (21, 22). 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 6.6 mM purified Quin-2 and 10.0 mM Hepes buffer adjusted to pH 7.00 with Chelex-treated CsOH or NaOH (19), depending on the internal composition required. 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 (23) 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 buffer, pH 7.00. A single pass over such columns effectively removes the external Quin-2 (19, 21, 22).

The nominal concentration of POPC in the final preparations was determined by measurement of lipid phosphorus (24) and was near 80 mM. The average diameter of these vesicles is 71 nm as determined by freeze-fracture electron microscopy (21), 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⁺/Na⁺, 60 ± 5 mM. Specific values for Quin-2 and Cs⁺/Na⁺ were determined for each preparation by the methods described previously (19, 25). Briefly, entrapped Quin-2 is determined by spectrophotometric titration with standard CaCl₂ following dispersion of the vesicles in deoxycholate. The entrapped monovalent cation is determined by atomic absorption spectroscopy, following replacement of the external medium with one containing a different cation, and dispersion of the vesicles in 0.1 N HCl. When of interest, buffer entrapment is determined from the other values by calculation, using the Henderson-Hasselbach equation, the buffer pK_a, and the internal pH. When buffer entrapment is to be determined, the vesicles also contain the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, so that the internal pH can 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 (21, 22).

Pb²⁺ Buffers and the Determination of Transport-- A buffer system was used to control the concentration of Pb²⁺ available for transport into the vesicles. 15 or 5 mM citrate was employed to buffer the concentration of this cation, whereas 10 mM each of 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 Ca²⁺ or Mg²⁺ was to be buffered simultaneously, five additional equilibria involving those cations were also considered. The respective equilibrium constants were taken from literature sources (26, 27), and, when necessary, the Davies equation (28) was used to correct these to an ionic strength of 100 mM. The species distribution program COMICS (29) was used to solve the applicable sets of simultaneous equations at experimental conditions of interest and to allow the generation of standard curves. Examples of the latter were shown previously (15).

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. Unless otherwise indicated, 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 of Hepes and Mes. The medium pH ranged from 6.00 to 9.50 and was adjusted with CsOH that had been passed over Chelex 100 columns to remove contaminating divalent cations (19). In some cases, valinomycin (0.5 µM) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (5 µM) were also present to maintain internal pH at the external value and to dissipate any transmembrane electrical potential that might otherwise arise (25). Specific concentrations of ionophores, divalent cations, and pH values are given in the figure legends. Reactions were started by the addition of the ionophore, following an initial 2-min preincubation to allow equilibration of transmembrane pH.

The formation of Quin-2-cation complexes was followed continuously by difference absorbance spectroscopy, using an Aminco DW2a spectrophotometer operated in the dual wavelength mode. An Oriel 59800 band pass filter was used between the cuvette and the beam scambler-photomultiplier 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 previously described (13). Data were collected on disk using Unkel Scope software (Unkel Software, Inc., Lexington, MA).

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 in seconds. The values presented are in units of µM external cation transported into the vesicles per second. 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) of Cs⁺-deoxycholate (13, 19). Transport selectivities are expressed as S values defined by Equation 2.

(Eq. 2)

When determining S, the concentration of the Pb²⁺ or Ca²⁺ was 20 µM, and all other conditions were held constant. All transport data were obtained at 25.0 °C.

Potentiometric Titrations and the Determination of pH in Aqueous Methanol-- The protonation constants and complex formation constants of monensin were measured by potentiometric methods in the mixed solvent 80% (w/w) methanol/water. For these studies, Na⁺ monensin was purified by column chromatography on silica gel, using ethyl acetate as the eluent. The Na⁺ salt was converted to the acid form by repeated back-extraction of a CHCl₃ solution with 1.0 M HCl. The CHCl₃ solution was then washed three times with distilled water, the solvent was removed, and the product was dried under vacuum. Analysis by flame atomic emission showed that less than 0.05% Na⁺ (w/w) remained.

Pb(ClO₄)₂ and Zn(ClO₄)₂ were prepared by reaction of the metal oxide (lead, 99.9995%; zinc, 99.99%; Aesar) with 70% HClO₄ (distilled; GFS Chemicals). CaCl₂·xH₂O (99.999%) and MgCl₂·6H₂O (99.995%; Aldrich) were used as received. Stock solutions of Pb²⁺, Zn²⁺, Ca²⁺, and Mg²⁺ salts were standardized by titration using EDTA (20). NaCl (99.999%; Aldrich) and KCl (99.997%; Aesar) were dried at 110 °C, and stock solutions of these salts were prepared gravimetrically.

Test solutions for potentiometric titration typically contained 0.5-1.0 mM monensic acid (HL) and, where appropriate, the metal ion (M) at concentration ratios ([HL]/[M]) in the range of 1.0-3.0. In the case of Mg²⁺ and K⁺, [HL]/[M] ratios of 0.2-0.25 were also used. Ionic strength was maintained at 0.050 using Et₄NClO₄, except in the case of K⁺, where Et₄NCl (>99%; Fluka) was used. The titrations were carried out using a digital burette, (Metrohm, model 665) and pH meter (Fisher, model 825MP) that was interfaced to a computer (11). pH* measurements were made using double junction combination electrodes (Sensorex S1021CD, Orion Ross 8175BN, Thomas 4080-B49), where the external filling solution was replaced with 0.1 M Et₄NClO₄ or 0.1 M Me₄NCl in 20% methanol/water.

The pH meter-electrode system was calibrated in aqueous solution using standard buffers (Gram-Pac; Fisher); then the electrodes were equilibrated in 80% methanol/water for at least 2 h prior to measurement. The operational pH* scales developed by de Ligny et al. (30, 31) and Gelsema et al. (32, 33) were utilized to determine the value of pH*. The term pH* is defined as log a_H*, where a_H* is the activity of H⁺ in the mixed solvent. Accordingly, the term pH* when used in reference to a specific methanol/water mixture has the same meaning as the term pH when used in reference to an aqueous solution (see Ref. 34 and references therein).

All titrations were carried out at 25.0 °C using a thermostatted cell and 20 mM Me₄NOH as the titrant. A nitrogen or argon atmosphere was maintained to minimize contamination by CO₂. The Me₄NOH was standardized using KH₂PO₄ and checked for carbonate content (35). Typical titrations consisted of 50-100 pairs of pH* versus ml Me₄NOH readings. The titration data were analyzed using the computer programs PKAS for the protonation constants (36) and BEST for the metal ion complexation constants (37).

Treatment of Experimental Animals-- Male rats were utilized when investigating the effects of monensin on the pathophysiology of Pb²⁺. They were housed in AALAC-approved animal facilities at the College of Medicine, Ohio State University. A 12-h light-dark cycle, single housing in plastic cages, or in metabolic cages, and conditions of constant temperature and humidity were employed. One week was allowed for acclimation before an experimental protocol began. During this period, a standard laboratory chow was employed, whereas the AIN-93 M diet containing 0.5% calcium was employed thereafter. During the administration of Pb²⁺ and/or monensin, water and feed were provided ad libitum, and records of consumption were maintained, together with periodic measurements of body weight.

At the beginning of an experimental protocol, the rats weighed 245-255 g. They were divided randomly into groups of six or eight, and the administration of Pb²⁺ was begun. It was provided at 100 ppm in the drinking water and was in the form of Pb(acetate)₂. The water was rendered slightly acidic with acetic acid to prevent the precipitation of PbCO₂ (38). When utilized, monensin was provided in the feed, also at 100 ppm.

Determination of Pb in Biological Samples-- Blood samples were taken periodically from the tail artery, and blood Pb levels were determined by electrothermal atomic absorption spectroscopy against certified standards (39). For the determination of Pb in urine and feces, total outputs were collected over 2-day periods from each rat individually. The urine samples were neutralized by the careful addition of nitric acid while the samples were stirred, and the pH was monitored with an electrode. Samples were then extracted with methyl isobutyl ketone in the presence of excess ammonium pyrrolidine dithiocarbamate, which quantitatively extracts Pb²⁺ into the organic layer and concentrates it compared with the original concentration in urine (40). The Pb content was thereafter determined by flame atomic absorption spectroscopy. For the determination of Pb in feces, weighed samples comprising ~1.0 g were dispersed in 10 ml of concentrated nitric acid and allowed to stand at room temperature overnight. They were then concentrated to ~1.5 ml by heating on a hot plate and diluted to a total volume of 10.0 ml with water, and Pb was determined by flame atomic absorption spectroscopy (41).

At the completion of experimental protocols, the rats were sacrificed by the injection of excess Nembutal and perfused briefly with Hepes-buffered 0.9% NaCl, via the left ventricle, to remove blood from the organs. Organs and tissues of interest were then removed and stored at 20 °C. For Pb analysis, the samples were thawed, and weighed portions were digested with 9 volumes of nitric/perchloric acid (3:1). After reducing the volume to 1 ml by heating, the samples were diluted to a total volume of 10.0 ml with a solution containing 140 mM NH₄OH, 29 mM NH₄(H₂PO₄), and 3 mM EDTA. For samples containing relatively high levels of Pb, such as bone and kidney, an initial dilution with water preceded that step. Following all of the above steps, the Pb content was determined by electrothermal atomic absorption spectroscopy (42). The resultant values and other parameters pertaining to rats were examined by univariant analysis of variance to determine whether the differences observed were statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Monensin-mediated Pb²⁺ Transport-- Fig. 3 compares the efficiency of ionomycin and monensin as ionophores for Pb²⁺ and contrasts their selectivity for Pb²⁺ compared with other divalent cations. Under the conditions employed, the two compounds are similarly effective from the perspective of rate, whereas monensin is much more selective. Selectivity for Pb²⁺ over Ca²⁺, for example, as defined by Equation 2, is ~100 for ionomycin and ~3400 for monensin.²

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Transport of Pb2+ and other divalent cations by ionomycin (A) and monensin (B). POPC vesicles loaded with Quin-2 (Cs+) were prepared as described under "Experimental Procedures." They were incubated at 25 °C and a nominal phospholipid concentration of 1.0 mM. The external medium contained 50 mM CsNO3, 10 mM each of Mes and Hepes (both Cs+), pH 7.00, 0.5 µM valinomycin, 5.0 µM CCCP, and a 20 µM concentration of the indicated divalent cation (nitrate in the case of Pb2+, chlorides for all others). After a 2-min preincubation, 0.10 µM ionophore was added to initiate transport (designated as 0 s in the figure). The sequence of cations shown on the right corresponds to their relative rate of transport as observed directly for A or on an expanded scale for B. The y axis unit refers to cation concentration in the external medium. When this parameter reaches 20 µM, all of the cation that was originally present in the external medium has been transported into the vesicles and sequestered by Quin-2.

The stoichiometry of the complex between ionomycin and Pb²⁺ that is responsible for transport is 1:1, cation/ionophore, based upon plots of log rate versus log ionomycin or log Pb²⁺ concentration, which both display slopes of 1.0 (15). By the same criteria, monensin-mediated Pb²⁺ transport also occurs through formation of a 1:1 complex (Fig. 4), although the plot of log rate versus log Pb²⁺ concentration progressively deviates from a slope of 1 as the free Pb²⁺ concentration rises above 1 µM. This negative deviation suggests that the steady state fraction of monensin that is located at the external membrane interface approaches saturation with Pb²⁺ at concentrations above that value.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Pb2+ transport dependence on the concentrations of Pb2+ and monensin. Data were obtained as described in the legend to Fig. 3 except that the concentrations of Pb2+ and monensin were varied. In addition, the external free Pb2+ concentration was established by a 15 mM citrate-based buffer system, as described under "Experimental Procedures." A, the monensin concentration was 0.10 µM, and the concentration of free Pb2+ was varied from 10 nM (bottommost curve) to 5.63 µM (topmost curve). B, the free Pb2+ concentration was 1.0 µM, and the concentration of monensin was varied from 1.17 nM (bottommost curve) to 2.40 µM (topmost curve). C, the data from A and B are shown as log initial rate versus log Pb2+ () or log monensin () concentration. Initial rates were obtained from the individual progress curves as described under "Experimental Procedures."

Ionomycin is dibasic, because both the carboxylic acid function and the enolized  diketone moiety can be ionized (Fig. 1) (43). Accordingly, ionomycin can form uncharged complexes with divalent cations, presumably including Pb²⁺, and can exchange these for 2H⁺ in an electroneutral manner (19, 25). In contrast, monensin is monobasic (Fig. 1) (44) and would form a 1:1 complex with Pb²⁺ having a net charge of +1. Thus, the 1:1 complex between Pb²⁺ and monensin might result in electrogenic Pb²⁺ transport, or it might associate with an anion as depicted in Fig. 2, to produce transport through a charge neutral mechanism. To examine the mode of Pb²⁺ transport, we initially determined complex stability constants of potential transporting species. Potentiometric titration methods were employed using 80% methanol/water as the solvent. This mixed solvent provides an effective polarity similar to that experienced by ionophores at a POPC membrane interface (7, 12, 45, 46). Analogous complexation equilibria for other cations were also examined to provide insight into the basis of the high selectivity that is seen in Fig. 3B.

Fig. 5A shows examples of the primary data obtained, whereas Fig. 5B reports the complex stability constants. It is seen that the complex PbMon⁺ is relatively stable, displaying a log K value of 7.25. The uncharged complex PbMonOH that is formed upon reaction of PbMon⁺ with OH is nearly 4 orders of magnitude more stable than the 1:2 complex formed from PbMon⁺ and a second molecule of the ionized ionophore. When these stability constants and the protonation constant of monensin are used to generate a species distribution diagram (29) (Fig. 5C), it is seen that significant levels of both PbMon⁺ and PbMonOH are expected at the membrane interface during Pb²⁺ transport, within the pH range of 6.5-7.5. In contrast, the species Pb(Mon)₂ is negligible (<0.003% of total monensin) throughout the broad range of pH considered (not shown). Thus, the complex stability data indicate that monensin might transport Pb²⁺ by a mixed mode (electrogenic and electroneutral transport occurring simultaneously) and that the fraction transported by the neutral process would probably occur via the species PbMonOH.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Equilibrium behavior of monensin and selected cations. A, potentiometric titrations of 1.0 mM monensin in an 80% methanol/water mixed solvent were conducted as described under "Experimental Procedures." The temperature was 25 °C, and the ionic strength was 0.1 M, maintained by the presence of tetraethylammonium percholorate. I, monensin alone; II, monensin in the presence of 0.5 mM Pb2+; III, monensin in the presence of 1.0 mM Pb2+. B, equilibrium constants of interest, determined from data analogous to those shown in A. The symbol L refers to the monensin carboxylate anion. C, the species distribution diagram for a solution containing a total of 0.10 µM monensin and with the Pb2+ concentration fixed at 0.10 µM. Individual values were calculated using the program COMICS (29), the equilibrium constants of interest from B, and a pKa of 7.96 for the hydrolysis of Pb2+ in 80% methanol/water (77). In C, Mon represents monensin in the carboxylate anion form.

To further examine these possibilities, the effect of external pH on the rate of Pb²⁺ transport was examined, using Na⁺-containing vesicles with valinomycin and CCCP excluded. These conditions limit transport to the fraction occurring by neutral mechanisms, because no provision is made to collapse the membrane potential arising from electrogenic transport, which is very effective at limiting the process (e.g. Refs. 14 and 16). In addition, the use of Na⁺ rather than Cs⁺-containing vesicles allows the ionophore to exchange Pb²⁺ for Na⁺, ensuring that the rate of Pb²⁺ transport does not become limited by the protonation of monensin at the internal membrane interface. As seen in Fig. 6A, monensin remains active as an ionophore for Pb²⁺ under these conditions, demonstrating that the electroneutral mode is indeed active.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of external pH on ionophore-mediated Pb2+ transport. Vesicles were prepared as described under "Experimental Procedures" except that Na+ rather than Cs+ was employed as the counter ion to Quin-2. The external medium contained 10 mM each of Mes, Hepes, and Ches, to provide for H+ buffering over a broad range of pH, and 5 mM citrate was present to buffer free Pb2+. K+ was used as the external counter ion for citrate and the H+ buffers. The external pH was set at a value of interest, and the concentration of Pb(NO3)2 required to produce a free Pb2+ concentration of 0.10 µM was added to the cuvette. Pb2+ transport was initiated by the addition of 0.1 µM monensin. A, individual progress curves covering the pH region of 6.5-9.0. The earliest portions are omitted, because the initial equilibration of H+ and monovalent cation gradients perturbs the UV-visible spectrum of Quin-2. Accordingly, rates pertaining at 140 s were determined rather than the initial rates. B, rates obtained from A at the pH indicated. Values are the mean of duplicate determinations. They were fit to the Henderson-Hasselbalch equation to obtain the solid line.

Regarding the species PbMonOH, the data in Fig. 6B show that increasing external pH enhances the rate of transport, consistent with a model involving dissociation of a single proton from hydrated PbMon⁺ to form PbMonOH. This finding and a comparison of the transport data with the pH dependence for PbMonOH formation shown in Fig. 5C indicate that PbMonOH is indeed the major transporting species. In Fig. 6, the half-maximal rate was seen at pH 7.8 (pOH 6.2) or at pPbOH 6.9 if it is considered that that species is the one which is actually transported.

Competitive Relationships between Pb²⁺ and Other Cations-- Given the potential use of ionophores to manipulate Pb²⁺ in living systems, we considered the possibility that the relatively high concentrations of other cations found in blood and intracellular compartments might substantially prevent monensin from transporting Pb²⁺ in vivo. Specifically, the rates of Pb²⁺ transport were compared when the free Pb²⁺ concentration was buffered at 1.0 µM alone and when 1.0 mM free Ca²⁺ or Mg²⁺ was also present. Only negligible differences were seen (Fig. 7, A and B), as might be expected based upon the complex stability constants shown in Fig. 5B. Potential interference by K⁺ and Na⁺ was also examined. The actions of K⁺ were again negligible when it was present at 5.0 or 100 mM, approximately the levels of this cation in blood and cytoplasm, respectively (data not shown). The same concentrations of Na⁺ did have modest inhibitory effects (Fig. 7C); however, these were smaller than expected based on the K_ML values listed in Fig. 5B. In other words, the ratio K_Pb/K_Na is ~300, whereas at a ratio of free Na⁺/Pb²⁺ of 10⁵ (i.e. when Na⁺ was present at 100 mM and Pb²⁺ at 1.0 µM), the rate of Pb²⁺ transport is reduced by only a factor of <2.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Actions of physiological cations as inhibitors of Pb2+ transport. Vesicles were prepared as described under "Experimental Procedures" using Cs+ as the counter ion to Quin-2. The external medium contained 5 mM citrate (Cs+) plus Pb(NO3)2 sufficient to produce a free Pb2+ concentration of 1.0 µM (except for traces labeled Pb2+), and 5 mM each of Mes and Hepes (both Cs+), pH 7.0. As indicated, the medium also contained CaCl2 sufficient to produce a free Ca2+ concentration of 1.0 mM (A), MgCl2 sufficient to produce a free Mg2+ concentration of 1.0 mM (B), or the indicated concentration of NaCl (C). Transport was initiated by the addition of 0.1 µM monensin.

To further examine the effectiveness of Na⁺ as an inhibitor of monensin-mediated Pb²⁺ transport, the Na⁺ concentration was varied from 0 to 100 mM while another solute was varied in the opposite direction, so as to maintain the external ionic strength and/or the osmotic pressure at constant values. As seen in Fig. 8, Na⁺ is more effective when used to replace Cs⁺, compared with K⁺ or tetraethylammonium cation, and it is ineffective when used to replace mannitol. These data presumably reflect modest differences in the stability of complexes between monensin and monovalent cations (47-49) together with effects of ionic strength on complex stability. However, of greater importance, they further indicate that concentrations of Na⁺ found in living systems have little effect on the efficiency of monensin as a Pb²⁺ ionophore.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Inhibition of Pb2+ transport by Na+. Vesicles were prepared as described under "Experimental Procedures," using Cs+ as the counter ion to Quin-2. The external medium contained 5 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb2+ concentration of 1.0 µM, and 5 mM each of Mes and Hepes, pH 7.0. At NaCl = 0 the medium also contained 100 mM CsCl (), 100 mM KCl (), 100 mM tetraethylammonium percholorate (TEAP; ), or 200 mM mannitol (). Cs+, K+, or tetraethylammonium cation was used as the counter ion for the external buffers, so as to match the cation that was added at 100 mM. In the case of mannitol, Na+ was used. As the NaCl concentration was increased, the concentration of the external cation was decreased by an equal value or was decreased by twice that value in the case of mannitol. Monensin was used at 0.1 µM.

Monensin Promotes the Excretion of Pb²⁺ in Rats-- Given that the above data are consistent with the notion that monensin might alter the dynamics of Pb²⁺ in whole organisms, we determined the effect of monensin on the accumulation and disposition of Pb in rats. In one experiment, the ionophore and Pb²⁺ were administered simultaneously, at 100 ppm in feed and 100 ppm in drinking water, respectively, or Pb²⁺ was administered without the ionophore. Blood samples were taken at weekly intervals over a 28-day period, and thereafter the rats were sacrificed to allow the determination of Pb in organs and tissues. Throughout the entire experimental period, the average concentration of Pb in blood was lower by ~25% in the rats that had received monensin, and this difference was significant at p = 0.05 (Table I and data not shown). Monensin also reduced the accumulation of Pb in several organs, with a particularly large effect seen in heart, where it was reduced by 66% (Table I). The reduced values seen in brain, muscle, and bone were also significant.

In a second experiment, the rats were loaded with Pb²⁺ over a 21-day period and in the absence of monensin. Thereafter, they were divided into groups and given drinking water that did not contain Pb²⁺. One group was given monensin at 100 ppm in feed, whereas another was given the same feed without monensin. After an additional 21 days, the rats were sacrificed, and lead in organs and tissues was determined. This time there was no difference in blood lead, either at the time of sacrifice (Table II) or at earlier sampling times (data not shown). However, the rats given monensin had lower levels of lead in brain, kidney, liver, and bone. The greatest effect was seen in kidney (a 55% reduction), with the effect on liver being of similar magnitude. Consistent with the transport data shown in Fig. 3 and the complex stability constants shown in Fig. 5B, the reduced levels of Pb in organs were obtained without reducing the levels of zinc or copper (Table III). Thus, the actions of monensin on lead are not accompanied by perturbation of these trace elements having biological roles.

During this second experiment, the rats were housed in metabolic cages so that urine and feces could be collected to determine the fate of Pb released from the organs. Amounts excreted in urine were low and were unaffected by the presence or absence of monensin (Fig. 9). Determining excretion via the feces is more problematic, because the gastrointestinal tract in rats rejects most Pb that is ingested, resulting in high levels of Pb in feces during the loading period (Refs. 50 and 51 and data not shown). When Pb²⁺ is withdrawn, feces Pb remains high for several days while the contents of the gastrointestinal tract turn over. These large values of excreted Pb obscure smaller differences between groups, such as differences that might be produced by monensin. Nevertheless, at later times, a statistically significant effect of monensin on Pb in feces was observed. In other words, during the second and third week of monensin administration, the treated rats lost lead at a rate of ~120 nmol/day, whereas without monensin the rate was about half that value. As further considered below, these data suggest that the lead mobilized from organs by monensin is ultimately excreted via the feces.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Effect of monensin on Pb excreted in urine. Two groups of eight rats were given 100 ppm Pb2+ in drinking water, for 21 days, as further described under "Experimental Procedures." The rats were housed in metabolic cages, allowing total urine output by each rat to be collected over 2-day intervals. Lead was determined by atomic absorption spectroscopy and expressed as nmol of lead excreted per rat per day. Up to and including day 21, the values plotted are means of those obtained for all 16 rats. On day 22, Pb2+ was withdrawn, and one group was shifted to a diet containing 100 ppm monensin, while the second was maintained without monensin. , lead excreted by rats receiving monensin; , lead excreted by rats not receiving monensin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Monensin was among the first group of carboxylic acid ionophores to be discovered, a group that also includes dianemycin, nigericin, compound X-206, and lasalocid A (reviewed in Ref. 5). Soon after these compounds were reported to be potent anticoccidial agents (52) and to derive this activity through a direct interaction with monovalent cations, leading to altered cation transport (53). Among the monovalent alkali metal cations, monensin was subsequently shown to be most effective as an ionophore for Na⁺, and it has long been utilized as a research tool in that regard (reviewed in Refs. 2 and 54). It has also been used as a feed additive in agriculture, because of the anticoccidial activity and because it promotes the growth of several animal species that are used in that industry (55, 56).

Relatively recent studies have shown that monensin also forms complexes with several divalent cations and that they are generally more stable than those formed with monovalent cations when compared in nonaqueous solvents such as methanol or diethylformamide (47, 48, 57-59). More limited data have furthermore shown that monensin displays some capacity to extract divalent cations from a bulk aqueous to a bulk organic phase (57) and to convey these cations through three phase bulk solvent systems (60) and polyvinyl chloride membranes in ion-selective electrodes (61, 62). Thus, previous studies have suggested that monensin might not be strictly an ionophore for monovalent cations, but they did not reveal that it is actually a highly selective and efficient ionophore for Pb²⁺ when phospholipid membranes are employed. That activity is newly discovered and is shown in the present report (Figs. 3 and 4).

The stoichiometry of the complex that transports Pb²⁺ is clearly 1:1, ionophore/cation, based upon the relationships between log rate and log monensin or log Pb²⁺ concentration, which are both linear over a wide range and display slopes of 1.0 (Fig. 4). As illustrated in Fig. 10, the 1:1 complex PbMon⁺ may then be active as a transporting species and would function by an electrogenic mechanism. This type of mechanism may have contributed to the transport seen in Figs. 3 and 4, because valinomycin and CCCP were present during those experiments. Valinomycin and CCCP, acting together, are fully effective at collapsing an induced membrane potential in the present vesicle system (14, 16), which is required for sustained electrogenic transport.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Potential mechanisms of monensin catalyzed Pb2+ transport. The curved lines together represent a phospholipid bilayer, whereas the heavy arrows indicate transmembrane diffusion of a lead-monensin complex. The various species illustrated are assumed to be present near the membrane-bulk aqueous phase interface.

No provision was made to collapse membrane potential during the experiments reported in Figs. 6-8, so only electroneutral transport was contributing. The transporting species under those conditions is PbMonOH, as shown by the relationship between rate and external pH (Fig. 6) and by comparing that relationship to the distribution of Pb/monensin species that arises when the pH is varied (Fig. 5C). PbMonOH can form by the pathways shown by Reaction 1 or 2, as further illustrated in Fig. 10, and both of these pathways can be considered to involve the acid ionization of a hydrating water molecule associated with Pb²⁺.

REACTIONS 3 AND 4

The present data do not provide insight into which of the pathways may be the most significant, but that may not be a point of central interest, assuming that all equilibria shown in Fig. 10 remain at or near equilibrium during monensin-catalyzed Pb²⁺ transport.

Apart from the pathway forming PbMonOH, the extent of equilibration between reactants forming the transporting species is of interest when the origin of high selectivity for Pb²⁺ transport is considered, compared with the transport of other divalent cations. If near equilibrium conditions are maintained, selectivity is expected to arise from differing complex stabilities, together with variation in the transmembrane diffusion constants of the various metal cation-monensin complexes. Several alkaline earth and first transition series divalent cations are poorly shielded when complexed by monensin (57), a factor that reduces transmembrane diffusion constants. However, the role of differential shielding in establishing the selectivity of monensin as an ionophore for Pb²⁺ is uncertain, because shielding in Pb²⁺-monensin complexes has not been investigated.

Regarding complex stabilities, the CaMon⁺ and the MgMon⁺ complexes are less stable than the PbMon⁺ complex by ~4 orders of magnitude (Fig. 5B; Refs. 57 and 59), and in addition, no interaction of these complexes with OH was detected during the potentiometric titrations. The latter finding was expected, given the hydrolysis properties of these cations (63), but more importantly, these considerations further explain why monensin has little or no activity as an ionophore for Ca²⁺ (Fig. 3) and why neither Ca²⁺ nor Mg²⁺ is effective as an inhibitor of Pb²⁺ transport (Fig. 7). Likewise, Zn²⁺ is bound by monensin with low affinity compared with Pb²⁺, although the ZnMon⁺ and PbMon⁺ complexes bind OH with similar affinities (Fig. 5A). From those considerations, one might expect monensin to have some activity as an ionophore for Zn²⁺, and that was observed in the vesicle system (Fig. 3). Thus, relative complex stabilities clearly contribute to establishing the transport selectivity sequence seen in Fig. 3.

On the other hand, Na⁺ is much less effective as an inhibitor of monensin-mediated Pb²⁺ transport than would be expected on the basis of complex stability constants alone, were near equilibrium maintained between monensin, OH, and the two competing cations (Figs. 7 and 8). Thus, kinetic constants of the cation complexation/decomplexation reactions, transmembrane diffusion constants, or a differing tendency of Pb²⁺ and Na⁺ to accumulate at the membrane interface may contribute to the selectivity of monensin as an ionophore for Pb²⁺ when significant concentrations of Na⁺ are also present.

The current data obtained in model systems suggest that, like ionomycin (15), monensin might be useful in the treatment of lead intoxication; however, there are no studies that have tested that possibility in animals. Accordingly, we determined whether monensin alters the accumulation or distribution of lead when the two agents are given simultaneously and whether monensin affects the disposition of lead that was accumulated previously. The level of Pb²⁺ employed and the method of administration are typical of those used when investigating Pb toxicity in rats (64-67). The level of monensin is the same as that usually given to chickens as an agricultural practice (56) and is below the toxic threshold for monensin in rats (68, 69).³ Monensin was administered as a component of feed, because that is the practice in agriculture and because activity upon oral administration is considered to be advantageous for agents used to treat lead intoxication.

The data show that in both types of experiment monensin reduced the level of lead in several organs and tissues (Tables I and II). The design of studies that investigate the efficiency of agents used to treat lead intoxication varies widely, so it is difficult to compare the effectiveness of monensin with that of the traditional agents (e.g. EDTA and dimercaptosuccinate) in a succinct manner. Nevertheless, monensin appears to be similarly effective to these better investigated compounds (e.g. compare with Refs. 70-72), although the dose employed here, the treatment interval, etc. have not been optimized. In addition, monensin reduces Pb in bone and brain, which is not true for the traditional compounds under some circumstances (64, 73), and does not deplete the tissues of zinc and copper, which can occur as an unwanted side effect with some of the traditional compounds (74-76). Thus, monensin may indeed be useful for promoting the elimination of lead from animals.

At this early stage, it is appropriate to consider the possibility than monensin acts differently than the traditional compounds as regards the mechanism by which lead excretion occurs. The traditional compounds are hydrophilic cation chelators that circulate in blood and form Pb²⁺ complexes having stability constants of ~10¹⁵ and higher. They are not membrane-permeant and are excreted together with Pb²⁺, typically via the kidney. The lead-monensin complex is much less stable (K = 10^7.25 in 80% methanol/water) and has little solubility in an aqueous environment. Moreover, the complex easily crosses a phospholipid membrane, whereas Pb excretion occurs primarily via the colon. Thus, monensin may act primarily by transporting Pb out of cells, where it eventually returns to the lumen of the gastrointestinal tract and is excreted by a normal mechanism, possibly involving the enterohepatic circulation. Since monensin is highly active as an ionophore for Na⁺ as well as for Pb²⁺, its presence will couple the gradients of these two cations across cell membranes, providing a driving force for Pb transport out of the cell. Overall, this potential mechanism suggests that the application of monensin, together with a hydrophilic chelator, might be particularly effective at promoting the excretion of Pb from animals. That possibility is currently under investigation.

	FOOTNOTES

^* This research was supported by the Wallace Research Foundation, by American Heart Association Grant 0255017B, by National Institutes of Health Grant GM66206, and Oklahoma Center for the Advancement of Science and Technology Grant HR00-030.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; E-mail: pfeiffer.17@osu.edu.

Published, JBC Papers in Press, June 21, 2002, DOI 10.1074/jbc.M205590200

² When both cations are present simultaneously, the selectivity of ionomycin for Pb²⁺ compared with Ca²⁺ is similar to the value for monensin derived from Fig. 3B (15).

³ Consistent with the reports cited, we did not find a significant effect of monensin at 100 ppm on growth (weight gain) or on the consumption of feed and water.

	ABBREVIATIONS

The abbreviations used are: POPC, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Ches, 2-(cyclohexylamino)ethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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