Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines

Mark T. Worthington, Steven M. Cohn, Suzanne K. Miller, Roger Qi Luo, and Carl L. Berg

Digestive Health Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma  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.

Digital image analysis was performed with the program NIH Image 1.62 in grayscale mode, with black = 255 and white = 0. For the quantitative studies, the background portion of the image containing the cells was identified with the use of the program's density slice function, and the mean pixel intensity was then calculated for both cells and background. The density slice function was less operator dependent and more reproducible than outlining groups of cells by hand. Cells without ZnMP could be identified from the background on the CCD camera images because of their faint autofluorescence. This autofluorescence was barely perceptible with the unaided eye as opposed to the readily identified ZnMP signal.

Statistics. Three or more images were used for each data point, with results expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Uptake of zinc mesoporphyrin (ZnMP) by Caco-2 and I-407 intestinal and HepG2 hepatoma cell lines. ZnMP concentrations of 0-20 µM and incubations lasting 0-3 h were used. Images were acquired with the rhodamine-Texas red filter set of a Zeiss Axioskop epifluorescence microscope, and the images were captured on 1600 ASA film. HepG2, Caco-2, and I-407 cells all internalized ZnMP. HepG2 cells uniformly took up 10 µM ZnMP in a 1-h incubation (A, top). There was minimal background autofluorescence in cells not exposed to ZnMP (A, bottom left). Corresponding phase-contrast images of these untreated cells are also shown (A, bottom right). HepG2 cells demonstrated the highest autofluorescence of the 3 cell lines studied, a level barely perceptible to the unaided eye. B: Caco-2 cells demonstrated the dose dependence of this uptake process. Three-hour incubations with 1, 10, and 20 µM ZnMP were performed. The phase-contrast view of the 20 µM image is shown at bottom right. The uptake was similarly dose dependent for the HepG2 and I-407 cells (data not shown). C: human fetal intestinal I-407 cells, a noncancer cell line, were assessed for their ability to take up this heme analog. Top, fluorescence photomicrograph of a 20 µM, 3-h incubation and the corresponding phase-contrast image documenting the uniformity of uptake by all cells. Bottom, cells without ZnMP incubation in fluorescence (left) and phase-contrast (right) views.

The experiment to determine uptake by the human fetal intestinal I-407 cell line was performed to address the possibility that ZnMP uptake was only a property of malignant cells. All three cell lines showed diffuse cytoplasmic accumulation of this fluorochrome with nuclear sparing. This diffuse cytoplasmic fluorescence was compatible with ZnMP binding to recognized cytoplasmic heme binding proteins such as heme binding protein-23, liver fatty acid binding protein, and glutathione-S-transferase (10). Although the results shown in Fig. 1B are those for Caco-2 cells, all three cell lines showed dose-dependent uptake of this reagent. HepG2, Caco-2, and I-407 cells were incubated with ZnMP in uptake buffer at various concentrations on the same multiwell tissue culture-coated microscope slide and were photographed with the same camera settings so that the results within a given experiment could be directly compared. Uptake was not a universal phenomenon; cell lines such as mouse fibroblasts took up little ZnMP (data not shown).

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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Fig. 2.   Heme inhibits ZnMP uptake. ZnMP uptake was examined in the presence of 0 (A), 0.1 (B), 1 (C), and 10 (D) µM heme during the ZnMP uptake reaction on the same multiwell microscope slide. Uptake was carried out in HepG2 cells with 1 µM ZnMP for 30 min. Images were obtained on 1600 ASA reversal film.

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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Fig. 3.   Temperature dependence of ZnMP uptake. HepG2 cells were incubated in 5 µM ZnMP for 6 h in a humidified 5% CO2 atmosphere at 4°C or 37°C. Time 0 cells were briefly incubated with ZnMP and immediately washed to establish the initial rate of uptake. Photography was performed with the microscope set at the same parameters and filter settings in both conditions. All digital images for quantitation used a gamma  set to 1.0, and background was subtracted from the cells with a density slice method. Results are means ± SE.

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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Fig. 4.   Kinetics of ZnMP uptake in Caco-2 and HepG2 cells. A: ZnMP incubation (5 µM) from 0 to 6 h was used to determine the kinetics of uptake in Caco-2 cells. Error bars, SE. Where error bars are not shown, they are smaller than the symbol. B: corresponding uptake curve for HepG2 cells incubated with 5 µM ZnMP. These experiments were performed simultaneously with the one depicted in Fig. 3, and the pixel results are directly comparable. Data were obtained with a charge-coupled display camera.

Inhibition of cellular heme synthesis enhances ZnMP uptake. delta -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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Fig. 5.   Effect of the cellular heme synthesis inhibitor succinylacetone (SA) on ZnMP uptake. HepG2 (A) and Caco-2 (B) cells were incubated overnight in tissue culture medium containing 0, 250, and 1,000 µM SA, an inhibitor of the delta -aminolevulinate (ALA) dehydratase enzyme required for endogenous heme biosynthesis. ZnMP incubation and photography were performed as described with 5 µM ZnMP for 1 h. Cell viability was determined before the uptake experiment with the use of trypan blue exclusion in parallel wells and was >95%. Images were obtained on 1600 ASA reversal film. SA pretreatment from 0 to 1,000 µM (left to right) results in increasing cellular ZnMP fluorescence.

To determine whether SA inhibition of ZnMP uptake led to an increase in the kinetics of uptake, we performed a time-course experiment of ZnMP uptake by Caco-2 cells with and without overnight SA pretreatment. A change in the initial rate of uptake would strongly suggest a change in the uptake properties of the membrane, such as increased number or activity of a plasma membrane transporter. SA (250 µM) in complete medium or complete medium alone was placed on the cells, a 5 µM ZnMP uptake experiment was performed the next day, and the amount of uptake was compared on the digital microscopy images. The results, shown in Fig. 6, reveal that heme synthesis inhibition by SA enhanced the rate of ZnMP uptake. At the zero time point, performed without ZnMP incubation, SA treatment did not significantly increase background autofluorescence.


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Fig. 6.   Uptake of Caco-2 cells with and without 250 µM SA. Caco-2 cells were incubated with () and without (black-lozenge ) the heme synthesis inhibitor SA, and a time-course experiment was performed with 5 µM ZnMP to determine if the rate of uptake was changed by this treatment. Incubation with ZnMP was not performed at time 0 to eliminate the possibility that SA led to an accumulation of endogenous porphyrins. Error bars, SE. Where error bars are not shown, they are smaller than the symbol. Data were obtained from images using a charge-coupled display camera.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 280(6):G1172-G1177
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