Digestive Health Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intestinal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyrin (ZnMP), was validated as a heme homolog in uptake studies of intestinal (Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone were performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in intestinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparing. Uptake was dose- and temperature dependent, inhibited by heme competition, and saturated over time. Preincubation with succinylacetone augmented uptake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong evidence that this uptake is a regulated, carrier-mediated process.
porphyrin; iron; zinc; micronutrients; trace elements; succinylacetone
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INTRODUCTION |
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HEME (ferrous protoporphyrin IX) is an important cofactor of cellular proteins such as cytochromes, hemoglobin, and myoglobin that are involved in dioxygen transport, electron transfer reactions, and other oxidation-reduction reactions (13). Dietary heme is also the most bioavailable form of dietary iron, the preferred form for treating iron deficiency (5). For convenience, the word "heme" will be used in this article without regard for the oxidation state of the metal.
The existence of a high-affinity eukaryotic transporter has been controversial, although an increasing number of prokaryotic heme transport systems are being described (1, 22). Blood-feeding arthropods that lack heme biosynthetic pathways and thus are reliant on exogenous heme for cellular processes have been described (2), although the mechanism responsible for their cellular uptake of heme is uncharacterized. Previous studies (3, 4, 6, 7, 16, 17) suggested the existence of a heme binding protein on the surface of mammalian enterocytes, hepatocytes, and hematopoietic cell lines, cells that either internalize and/or extensively utilize heme. The role of this heme binding activity in transmembrane heme transport, however, has not been determined. Other work suggested that heme traverses cell membranes by diffusion, although these studies used a heme molecule covalently modified with carbon monoxide to inhibit aggregation (12), a change that may have significantly altered the properties of the molecule by eliminating some of the metal character of the compound (9). Thus the precise pathway of transcellular heme uptake remains obscure.
Assessment of cellular heme uptake has been confounded by reliance on compounds that contain radioisotopes in either the iron or the porphyrin ring. These compounds are expensive to prepare and require special disposal methods. In the case of uptake across the cell membrane, the heme is rapidly cleaved by intracellular heme oxygenases (HOs), with the resultant free iron and heme degradation products shunted into different pathways, complicating analysis of these studies (21). Biophysical studies such as fluorescence spectroscopy have used zinc-substituted heme analogs in heme-containing proteins such as hemoglobin to answer structural questions about heme binding (20). These zinc-containing porphyrin compounds are in vitro and in vivo inhibitors of HOs, which is of potential benefit in fluorescence uptake studies because this would prevent catabolism of the compound and would therefore amplify a weak signal by increasing cellular fluorescence (8). We developed a system that employs a zinc-substituted porphyrin as a fluorescent heme analog in living cells to determine whether cellular uptake has the characteristics of active, regulated transport.
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MATERIALS AND METHODS |
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Cell culture. Human enterocyte-like Caco-2, human fetal intestinal I-407, and human hepatoma HepG2 cell lines were obtained from the American Type Culture Collection. Caco-2 cells were propagated in DMEM high-glucose medium with 1% MEM-sodium pyruvate, 0.21% (wt/vol) sodium bicarbonate, 10% fetal bovine serum, and 1% MEM-nonessential amino acids. HepG2 cells were propagated in MEM (Eagle) with nonessential amino acids with Earle's balanced salt solution, 1% sodium pyruvate, and 10% fetal bovine serum. I-407 cells were propagated in MEM (Eagle) with nonessential amino acids with Earle's balanced salt solution and 10% fetal bovine serum. All media components were obtained from Life Technologies (Rockville, MD). Tissue culture was performed at 37°C in 5% CO2. Before the uptake experiments, the cells were trypsinized from standard tissue culture flasks and plated on Lab-Tek chamber slides (no. 177402, Nalge Nunc), where they became adherent while maintained overnight in their standard medium. The chamber was removed, and a coverslip was placed on the microscope slide for viewing the attached cells.
Chemicals. Zinc mesoporphyrin (ZnMP) and hemin were obtained from Porphyrin Products (Logan, UT). All other chemicals were obtained from Sigma (St. Louis, MO) in the purest form available. ZnMP was formulated with a published method (21) and made up as a 0.3 mM stock solution with 1% ethanolamine and 10 mg/ml of BSA. This solution was buffered to pH 7.4 with 1.0 N HCl and kept in the dark at 4°C.
Uptake studies. ZnMP was freshly diluted in uptake buffer immediately before use. The uptake buffer consisted of (in mM) 50 HEPES, pH 7.4, 130 NaCl, 10 KCl, 1 CaCl2, and 1 MgSO4. The medium was gently aspirated off the cells, and the cells were washed with uptake buffer. All subsequent steps were performed in the dark or in a tissue culture hood that was as close to total darkness as practical (8). All experiments were performed at similar cell densities. The incubation solution was placed on the cells, and the cells were incubated for the conditions of temperature, time, or substrate concentration described in each experiment. To terminate the uptake process, the incubation solution was removed, and the cells were washed with ice-cold 5% BSA in uptake buffer followed by two more washes with uptake buffer. The BSA incubation was done to remove ZnMP not internalized by the cells. The slide chamber mechanism was carefully removed, and the cells were gently covered with PBS and a coverslip before microscopy. Cell morphology was not changed by the ZnMP incubation. Trypan blue exclusion revealed no changes in cell viability as a consequence of ZnMP incubation.
Microscopy.
Studies were performed with a Zeiss Axioskop epifluorescence
microscope with images obtained on Fujichrome Provia color reversal film at 1600 ASA or obtained quantitatively on the same microscope with
a Spot-2 cooled charge-coupled display (CCD) chip camera. CCD
microscopy was performed with the set to 1 so that the
pixel numbers were linear with respect to fluorescence intensity. The linearity and dynamic range of the fluorescent images were verified with the InSpeck Orange (540/560) Microscope Image Intensity
calibration kit (6.0 µm; Molecular Probes; Leiden, OR) by following
the manufacturer's instructions for drying and mounting these beads. A
linear relationship between fluorescence intensity and pixel density
was established over a range of pixel values from 0 to 254 (data not
shown). All fluorescence studies were performed with identical camera
settings and a rhodamine-Texas red filter set in either the film or CCD camera applications. All cells received the same fluorescence exposure,
with each region of the cells being exposed to minimal fluorescence
excitation only once, to ensure that the images obtained from a given
experiment were directly comparable.
Statistics. Three or more images were used for each data point, with results expressed as means ± SE.
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RESULTS |
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Intestinal and hepatic cell lines take up ZnMP.
ZnMP was assessed as a possible fluorescent analog of heme uptake.
Human enterocyte-like Caco-2, human hepatoma HepG2, and human fetal
intestinal epithelial cell I-407 cell lines were incubated with ZnMP to
assess their ability to take up this reagent as a fluorescent
probe of cellular heme uptake. The results, shown in Fig.
1, demonstrated that all three epithelial
cell lines take up this compound and that these signals are not
confounded by background autofluorescence. The highest autofluorescence
was that of the HepG2 cells and is shown at the bottom of Fig.
1A, with its corresponding phase-contrast image.
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Heme inhibits ZnMP uptake.
To ascertain whether ZnMP uptake occurred through a dedicated heme
pathway, such as through a plasma membrane transporter, a heme
competition study was performed. A fixed concentration and incubation
time of ZnMP was used with an increasing concentration of heme in the
uptake solution. The results for HepG2 cells (Fig. 2) show that ZnMP uptake was inhibited by
an increasing concentration of heme in the uptake media.
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Temperature dependence of ZnMP uptake.
Experiments were performed for ZnMP uptake in HepG2 cells at 4°C and
at 37°C in a tissue culture incubator to determine the temperature
dependence of this process. As shown in Fig.
3, uptake is highly temperature
dependent. In this experiment, the cells at time 0 were
transiently incubated with ZnMP and then immediately washed as per our
protocol to establish the initial rate of uptake. Uptake was reduced in
cells incubated at 4°C compared with those incubated at 37°C.
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Kinetics of ZnMP uptake in Caco-2 and HepG2 cells.
Caco-2 and HepG2 cells were incubated with ZnMP in a time-course
experiment to determine the kinetics of uptake at 37°C. The cells at
time 0 were transiently incubated with ZnMP and then immediately washed to establish the initial rate of uptake. The results, shown in Fig. 4, indicate an
initial steep linear uptake followed by a plateau phase, suggesting
saturation of uptake with time. To confirm that the saturation of ZnMP
uptake over time did not represent depletion of ZnMP from the uptake
buffer, a series of additional experiments was performed. The uptake of 5 µM ZnMP by HepG2 cells was permitted to occur for 5 h
(equilibrium). Concentration in aliquots of the uptake buffer was
determined spectrophotometrically at 540 and 580 nm, the two major
peaks of ZnMP absorbance. The concentration of ZnMP in the uptake
buffer was decreased by a maximum of 7% from 0 to 4 h
(n = 8 cultures), confirming that saturation
with time is not an artifact of depletion of the substrate from the
uptake buffer.
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Inhibition of cellular heme synthesis enhances ZnMP uptake.
-Aminolevulinate (ALA) dehydratase catalyzes the fusion of
four ALA molecules into a single porphyrin ring. An inhibitor of ALA
dehydratase, succinylacetone (SA; 4,6-dioxoheptanoic acid), prevents
cellular heme synthesis by inhibiting this enzymatic step
(18). We reasoned that inhibition of endogenous heme
synthesis with SA would lead to compensatory changes in heme uptake at
the plasma membrane if cellular uptake of heme was a regulated process. This might occur through an increase in the number or activity of
specific heme transporter molecules. To test this hypothesis, we
preincubated HepG2 and Caco-2 cells with SA before ZnMP exposure. These
uptake experiments led to dose-dependent increases in ZnMP uptake as shown in Fig. 5.
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DISCUSSION |
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The use of ZnMP in a living cell system provides a novel method for further characterizing the mechanisms by which heme enters a cell and a new approach for addressing the question of whether a heme transporter exists. This fluorescent reagent allows the subcellular localization of the compound to be determined in individual living cells. This characteristic clarifies the experimental results by eliminating the nonspecific extracellular membrane binding that has confounded earlier studies that employed radiolabeled heme. We saw little uptake in fibroblast cell lines compared with that in the intestinal and hepatic cell lines, suggesting that these latter organs, which have pivotal roles in iron and heme metabolism, have specific mechanisms for the uptake of this heme analog. The temperature dependence, heme competition in coincubation experiments, and saturability of uptake in these lines is supportive of a specific protein-mediated transport process. From the comparative experiments it is clear that the HepG2 cells exhibit the highest baseline uptake, whereas our experiments with the inhibitor of endogenous heme synthesis, SA, suggest that this activity is also highly inducible in the Caco-2 enterocyte-like cell line.
Additional compelling evidence for a heme transporter is provided by the experiments with SA. The inhibition of cellular heme synthesis with SA, like iron deprivation, led to cellular changes consistent with iron deficiency, such as increased surface transferrin receptor number, in addition to having direct effects on heme synthesis (23). The ZnMP accumulation in cells at the highest SA concentrations (maximal inhibition of endogenous heme synthesis) was quite striking. This enhanced cellular accumulation of heme strongly suggested that these cells are capable of upregulation of plasma membrane heme transport. Our finding of both enhanced uptake and, in particular, the increase in the initial rate of uptake in SA-treated cells confirms the earlier concept of a dedicated mammalian heme transporter.
Although the above studies confirmed the presence of an inducible, dedicated mammalian heme transport process, the available data cannot fully characterize the precise pathway responsible for this phenomenon. The fluorescent images were not capable of distinguishing a receptor-mediated endocytic pathway from a transmembrane protein-mediated plasma membrane transporter.
Previous evidence for a protein heme transporter includes the increased intestinal brush border binding of heme and the increased rate of heme uptake in iron deficiency, suggesting functional regulation (19). High-affinity binding of heme to intact cells in culture has been found to be saturable, reversible, pH dependent, and degraded by the proteolytic enzyme trypsin, supporting the existence of a specific cellular binding protein rather than a diffusional physicochemical interaction with a phospholipid bilayer (4). Although the binding affinity for heme was high in these studies [Michaelis-Menten coefficient in the subnanomolar range (4)], a fact that supports the potential physiological importance of this receptor/transport protein, further characterization of this protein has not been achieved. On the basis of more recent biophysical studies (11, 12) with carbon monoxide-modified heme, the concept of a specific protein transporter has become controversial and the concept of passive diffusion has been entertained as the predominant mechanism for heme uptake. Although an alternative explanation may be that changes in the lipid character of the membrane might facilitate passive binding of ZnMP, we believe that the weight of the past and current evidence strongly suggests a specific protein transporter for heme.
We have used a metal-substituted porphyrin, ZnMP, in cellular uptake studies as a new approach to studying heme regulation. Although zinc protoporphyrin IX was contemplated as an alternative reagent, we found ZnMP an attractive choice because mesoporphyrin derivatives lack reactive vinyl groups and are therefore more chemically stable than protoporphyrin IX (21). Both zinc derivatives have been used safely as drugs in humans and are inhibitors of HO (21). The potency of ZnMP as an inhibitor of HO is in the micromolar range, nearly identical to that of zinc protoporphyrin (21). Interestingly, unlike tin-substituted porphyrins (also fluorescent), the zinc derivatives have been shown to be toxic to erythropoietic cells (14, 15). Our assumption was that the zinc porphyrins might be a more appropriate heme analog for fluorescence studies because the reported toxicity could be a result of global effects on heme metabolism in cellular proteins other than HO. The use of ZnMP provides a new tool for further characterizing intestinal and hepatic heme uptake.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02501 and an American Digestive Health Foundation Industry Research Scholar Award (to M. T. Worthington.).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. T. Worthington, P.O. Box 800708, UVAHSC, Charlottesville, VA 22908-0708 (E-mail: mtw3p{at}virginia.edu).
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.
Received 26 October 2000; accepted in final form 16 January 2001.
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