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Proceedings of the Society for Experimental Biology and Medicine 224:178-186 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Albumin Facilitates Zinc Acquisition by Endothelial Cells

David J. Rowe and Dennis J. Bobilya1,


Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, New Hampshire 03824


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Albumin has long been observed to have a marked influence on the delivery of zinc to cells, but the mechanism of the interaction remains elusive. We examined whether albumin facilitates the acquisition of zinc by endothelial cells. Cultures of endothelial cells were used to analyze binding and acquisition of zinc and albumin to test this interaction. Our results indicate that albumin plays a role in facilitating the physiological delivery of zinc to endothelial cells. Albumin receptors that preferentially recognize albumin molecules carrying a zinc atom were demonstrated on the endothelial cell surface. Endocytosis is instrumental in albumin uptake, which was also consistently true of zinc uptake. Zinc and albumin were acquired by the cells in a 1:1 molar stoichiometry during the first 20 min of incubation in a medium with equimolar concentrations of zinc and albumin. The amount of albumin associated with the cells stabilized after 30 min, whereas the amount of zinc continued to increase. One possible explanation for this result is that a physiological route for zinc delivery into endothelial cells is by co-transport with albumin via receptor-mediated endocytosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Zinc, a trace element essential for all living cells, is a component of over 300 enzymes involved in protein, nucleic acid, carbohydrate, and lipid metabolism; it also influences gene transcription (1, 2). Zinc uptake into cells has been investigated extensively without obtaining an adequate explanation of the process. A number of researchers have observed that albumin is influential in zinc transport (3-7). Generally, albumin has been shown to decrease zinc uptake into cells (3-5, 8). Since greater than 99% of the labile zinc in plasma that is available to cells is bound to albumin (7, 9), the influence of this protein on zinc uptake could be considered as modulatory or regulatory rather than antagonistic.

Albumin was previously viewed as a passive vehicle of nutrients in the plasma, with no significant role in nutrient acquisition by cells. The transport of albumin was previously thought to be a nonspecific, bulk fluid-phase process in which albumin would be endocytosed or transcytosed through the nonspecific invagination of the cell plasmalemma. More recent evidence implicates a more complex process (10). Albumin receptors that are saturable and specific for either ligand-complexed albumin or free albumin have now been identified on the surface of endothelial cells (11, 12). Moreover, receptor-mediated uptake of albumin is enhanced in endothelial cells when glucose (13) or fatty acids (14) are bound to albumin. Transcytosis of albumin by endothelial cells also is enhanced when zinc is bound to the albumin (15). This report describes evidence of a supportive role for albumin in the delivery of zinc to endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture.
Bovine pulmonary artery endothelial cells (BPAEC) were obtained from American Type Culture Collection (#CCL-209; Rockville, MD) at passage 16. Cells were grown in minimum essential medium Eagles (MEM) with 20% fetal bovine serum (FBS, Summit Biotechnology, Ft. Collins, CO) without antibiotics. The growth medium contained 12 µM zinc and 75 µM albumin by analysis. Cultures were at 37°C, 95% relative humidity, and 5% CO2. Experiments were conducted using cells at passages 18–23. Cells for experiments were subcultured into 25 cm2 plastic culture flasks (Corning Costar Lab. Sci. Co., Park Ridge, IL) at a density of 15,000 cells/cm2. The cells reached confluency in 4 days, with each flask containing an endothelial monolayer of approximately 3 million cells. Experiments were performed at 3 days postconfluency, so that the cells would exhibit a relatively stable rate of metabolism in contrast to when they were dividing rapidly. The usual physiological status of endothelial cells in vivo is nonmitotic.

During this project, we observed that the relative proportion of specific acquisition of albumin (relative to nonspecific albumin acquisition) is higher in porcine venous endothelial cells (PVEC) than in BPAEC, so we employed PVEC in some studies to further characterize the zinc-albumin interaction. PVEC were isolated from the inferior vena cava of weanling Yucatan miniature pigs (Sus scrofa). The pigs were euthanized in compliance with the institutional animal care and use committee. The inferior vena cava was removed and transferred to a Petri dish and cut lengthwise to expose the vessel lumen. Collagenase (Type IA, 380 IU/ml) was applied and incubated for 10 min at 37°C. Endothelial cells were removed with a sterile cotton swab and seeded into fibronectin-coated (2 µg/cm2) plastic tissue culture dishes in 10% FBS in MEM plus 100 mg heparin/l and 50 mg hypothalamic extract/l. The hypothalamic extract contained an endothelial cell growth supplement and was prepared by the method of Maciag et al. (16). The medium was changed every 2 days, and cells were subcultured at confluence in a 1:4 split ratio. Experiments were performed with cells in passage 4–6 at 2 days postconfluency.

Endocytosis of rhodamine-labeled acetylated-low-density lipoprotein (Ac-LDL) (17) and the presence of Factor VIII-related antigen (18, 19) were used as biochemical markers for endothelial cell characteristics. Endothelial cell cultures were also examined daily for morphology using phase-contrast microscopy to avoid use of senile cell cultures. Senility is characterized by endothelial cells with enlarged or multiple nuclei and an irregularly shaped plasma membrane. If senile cells began to develop, the cultures were promptly discarded.

Zinc Acquisition.
After removal of the growth medium, the flasks were rinsed twice with 37°C HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). Then, 2.0 mL of labeling medium containing 9.25 x 106 Bq 65Zn/l (carrier-free 65ZnCl2, Amersham Life Science, Arlington Heights, IL) was applied. The final composition of the labeling medium varied depending upon the experimental question, but all were MEM-based and always contained some albumin. Incubation times also varied depending upon the question being considered (e.g., 10 min when only zinc acquisition was measured, 30 min when albumin and LDL acquisition were measured, and 60 min when endocytotic inhibitors were included in the protocol). We previously demonstrated that zinc acquisition by endothelial cells is linear over the first 60 min (20; Fig. 5Go). Following the timed incubation at 37°C on a rotary shaker (30 r.p.m.), the labeling medium was removed, and the cell monolayer was washed briefly (5–6 sec) with cold (4°C) HEPES buffer plus 10 mM EDTA. After two subsequent rinses with cold HEPES buffer, the cells were solubilized in 2.0 ml of 0.01% sodium dodecylsulfate (SDS) in 0.2 M NaOH for analysis. Solubilized cells were analyzed for radioactivity using a {gamma} emissions detector (#1282 CompuGamma, Universal Gamma Counter; LKB Nuclear Inc., Gaithersburg, MD). Rate of zinc acquisition was calculated as pmoles zinc/mg cellular protein based upon the specific activity of the transport medium (typically 2 x 1012 Bq/mole zinc).



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  		Figure 5.   Time-dependent association of zinc (65Zn) and albumin (FITC-BSA) with PVEC. Flasks of cells were incubated at 37°C with medium containing 3 µM zinc and 3 µM FITC-BSA in MEM. After incubation for the indicated lengths of time, the flasks were washed three times with HEPES buffer (no EDTA). Data are presented as means ± SE (n = 6 flasks of cells), though some SE bars may be obscured by the symbols.

 
Albumin Binding and Acquisition.
Albumin binding and acquisition were measured similarly to the procedures of Schnitzer et al. (21) and others (22). Procedures were the same as those for measuring zinc acquisition, with the following exceptions. There was a 10 min preincubation in MEM without albumin to reduce prior albumin binding to the surface of the cells. The labeling medium contained radioactively labeled bovine serum albumin (9.25 x 106 Bq 125I-BSA/l) or fluorescently labeled bovine serum albumin (50 mg FITC-BSA/l). 125I-BSA was prepared using iodogen (Pierce Chemical Co, Rockford, IL) to react 2 x 107 Bq Na125I with 100 µg BSA (ICN Biomedical Co., Costa Mesa, CA) (21). Specific activity was 0.2–2.0 x 105 Bq/µg BSA. 125I interfered with our ability to measure 65Zn so a fluorescent label was more suitable in some studies. FITC-BSA was obtained from Sigma Chemical Co. (St. Louis, MO) and hydrated to a stock concentration of 5 g/l in phosphate-buffered saline (PBS). The 125I-BSA and FITC-BSA were passed through a desalting column (10DG, Bio-Rad Laboratories, Richmond, CA) to remove unbound label from the labeled albumin before its addition to the labeling medium, immediately prior to the experiments. Albumin binding was measured during a 5-min incubation at 4°C, which followed a 10-min preincubation in MEM at 37°C and a subsequent 10-min preincubation in MEM at 4°C. Albumin acquisition was measured during a 30-min incubation at 37°C, following a 10-min preincubation in MEM at 37°C. After incubation, the cells were washed three times with cold PBS and solubilized in 2.0 ml of 0.01% SDS in 0.2 N NaOH for analysis. The amount of albumin bound by or taken into the cells was calculated based upon the specific activity of the labeling medium. Nonspecific binding and acquisition were determined by performing the assay in excess unlabeled albumin (100 g/l). Specific binding and acquisition were derived by subtracting nonspecific values from total values.

Endocytic Inhibition.
Four commonly accepted strategies for chemically disrupting endocytosis were employed. Microtubule and microfilament structure and function were altered with 5 mg cytochalasin B/l or 10 mg colchicine/l, respectively (23, 24). Recycling of receptors involved in coated pit receptor-mediated endocytosis was inhibited by treatment with 18 mg chlorpromazine/l (25). Formation of clathrin-coated pits was inhibited using K+-depletion medium (50 mM HEPES and 100 mM NaCl) (26, 27). Appropriate controls were included in all treatments (e.g., cytochalasin B was solubilized in absolute ethanol at 200X final concentration so a control with 0.5% ethanol was also included). A preincubation of 10–30 min was employed to initiate disruption of endocytosis by the inhibitor. This was followed by a 60-min incubation with the inhibitors in the labeled medium to determine zinc and albumin acquisition. Visual observation with phase-contrast microscopy at 100x, 200x, and 400x magnification revealed greater than 95% reduction in the number of vesicles in the treated cells without significant loss in viability as determined by trypan blue exclusion.

Endocytosis was also temperature dependent. Experimental medium consisted of 1% BSA in MEM with 15 µM zinc and either 9.25 x 106 Bq 65Zn/l, 9.25 x 106 Bq 125I-BSA/l, or 2.5 mg DiI-Ac-LDL/l (acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Flasks were incubated for 30 min at 4°C, 25°C, or 37°C. Flasks for measuring LDL acquisition were preincubated for 24 hr with 1% BSA in MEM to enhance the sensitivity to Ac-LDL (17). Following the incubations, the cells were washed and solubilized in 2.0 ml of 0.01% SDS in 0.2 N NaOH for analysis. DiI-Ac-LDL was measured fluorometrically in the cell digest using an excitation of 540 nm and an emission of 585 nm.

Additional Analyses.
The amounts of 65Zn and 125I-BSA in the cell digest were determined with a {gamma} scintillation detector. The amount of FITC-BSA in the cell digest was determined fluorometrically with filters of 490 nm for excitation and 515 nm for emission. The zinc concentrations of the medium were measured directly using flame atomic absorption spectrophotometry (Smith Hieftje 12, Thermo Jarrel Ash Co., Franklin, MA). Reference standards were prepared in 157 mmoles NHO3/liter of deionized water from Fisher Scientific certified standards (Pittsburgh, PA) and the linear detection range was 1–20 µM Zn. Albumin was measured by adding bromcresol green and measuring absorbance at 628 nm (28). Protein was measured by using the bicinchoninic acid method of Smith et al. (29). Based upon the specific activity of the labeling medium, the amounts of zinc (or albumin) acquired by the cells during the incubation were calculated. For example, the zinc concentration and radioactivity of the labeling medium were measured, providing the dpm of 65Zn per mole of zinc (dpm of 125I-BSA or FITC-BSA fluorescence per mole of albumin). Results are reported as pmoles zinc (or albumin) per mg of cellular protein. Unless otherwise stated, all reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Statistical Analyses.
Flasks were the experimental units, and they were randomly assigned to treatments. Analysis of variance was performed with the Crunch Statistical Package (Version 4, Crunch Software Corp., Oakland, CA). Fishers Protected LSD (Least Significant Difference) test was used for pair-wise comparisons of multiple groups. Dunnett's test was used for comparison of multiple groups with a control group. Differences were considered significant if P < 0.05. The kinetic parameters KD (concentration at which binding is half-maximal) and BL max (maximum rate of specific binding, receptor number) were estimated by nonlinear fitting of the experimental data to the equation BL = (BL max * [L])/(KD + [L]), where L = ligand concentration, using Systat computer software (Version 7.0.1, Chicago, IL), rather than by using Scatchard plot analysis that tends to exaggerate errors through linear transformations of the data (30). Comparisons of the regression curves were performed as described by Zarr (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The research model was validated by testing whether the current sources of labeled zinc and albumin were metabolized by the endothelial cells as we had previously observed (6). The effects of zinc concentration, albumin concentration, and fluorescent labeling of albumin on zinc (65Zn) acquisition by BPAEC are presented in Figure 1Go. Linkage of FITC to BSA did not affect zinc acquisition under any of the test conditions, indicating that FITC-albumin was recognized by zinc and the cells as native albumin. Increasing the zinc concentration from 3 µM to 15 µM increased (P < 0.01) the rate of zinc acquisition by approximately three-fold, whether the albumin concentration was either 15 µM or 75 µM. Increasing the albumin concentration from 15 µM to 75 µM decreased (P < 0.01) the rate of zinc acquisition at both zinc concentrations. These results validated the model system, since similar zinc and albumin results were obtained previously when using these concentrations (6, 15, 32).



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  		Figure 1.   Rates of zinc uptake into BPAEC, evaluating the influence of FITC-BSA as compared with native BSA at different zinc and albumin concentrations in MEM. Zinc concentrations were established with ZnCl2. Albumin concentrations were established with either FITC-labeled BSA or unlabeled (native) BSA. 65Zn was used to determine zinc uptake during a 10-min incubation, followed by one 5-sec wash with 4°C HEPES buffer containing 10 mM EDTA and two subsequent washes with HEPES buffer. Results are presented as means ± SE (n = 6 flasks of cells).

 
Albumin acquisition is recognized as occurring via endocytosis (10, 33, 34). If albumin has a role in delivering zinc into cells, disrupting endocytosis would influence zinc uptake. Endocytosis is temperature-dependent. LDL accumulation, a sensitive marker for endocytosis, decreased (P < 0.0001) to 19% of controls at 25°C and to 1.4% of controls at 4°C (Fig. 2)Go. Acquisition of zinc and albumin by BPAEC were also significantly (P < 0.0001) influenced by temperature (Fig. 2)Go. Zinc acquisition was reduced to 64% of control (37°C) values at 25°C and further reduced to 37% of controls at 4°C. Albumin accumulation was decreased to 57% of control values at 25°C, and further reduced to 48% of control values at 4°C. Further evidence of a role for endocytosis in zinc uptake was obtained with the use of four strategies to inhibit endocytosis: cytochalasin B, colchicine, chlorpromazine, and K+ depletion, all of which reduced (P < 0.0001) zinc and albumin acquisition (Fig. 3)Go. Some zinc and albumin were still acquired by the cells under the influence of the drugs, but this probably reflected a combination of i) our inability to measure these processes with complete accuracy and ii) their acquisition by the cells through alternative pathways not blocked by that particular inhibitor. Acquisition of zinc and albumin were both consistently inhibited by all of our experimental strategies for disrupting endocytosis. Since each of these strategies for inhibiting endocytosis have secondary effects on the cells, it was important that these results were observed consistently.



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  		Figure 2.   Uptake of zinc, albumin, and LDL into BPAEC at different temperatures during a 30-min incubation. The experimental medium consisted of 75 µM BSA and 15 µM zinc in MEM, labeled with 65Zn, 125I-BSA, or DiI-Ac-LDL as described in the Materials and Methods section. Data are presented as means ± SE (n = 5 flasks of cells). Bars with different letters within a group are statistically different with at least P < 0.05.

 


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  		Figure 3.   Effect of endocytic inhibitors on 65Zn and 125I-BSA accumulation into BPAEC during a 60-min incubation. The incubation medium consisted of the appropriate inhibitor with 15 µM zinc and 75 µM BSA in MEM as described in the Materials and Methods section. Data are presented as means ± SE (n = 6 flasks of cells). All the treatment means were different from the control group at P < 0.0001.

 
Confident that endocytosis was instrumental in zinc uptake, we next tested whether it is coincidental that zinc and albumin both rely upon endocytosis. One indication of a functional role for albumin in zinc delivery would be that the cells could differentiate between albumin molecules, depending on whether or not albumin was carrying a zinc atom. So, we examined the influence of zinc on the binding kinetics of albumin to endothelial cells. Albumin binding was determined in the presence or absence of an equimolar concentration of zinc. It was assumed that a high percentage of the zinc atoms would be in association with an albumin molecule at equimolar concentrations, bound in a 1:1 stoichiometry (35). Figure 4Go is a plot of albumin bound by the cells at different concentrations of albumin, with and without zinc. Albumin binding was greater (P < 0.05) with a zinc atom than without zinc at 25, 75, and 150 µM albumin. The binding of albumin carrying a zinc atom displayed different (P < 0.05) kinetics than did albumin binding without zinc (Table I)Go. The estimated KD for albumin binding with zinc was 85.2 ± 19.4 µM and the KD for albumin binding without zinc was 123.8 ± 52 µM. The estimated BL max for the binding of albumin was similar whether albumin carried zinc (3.47 ± 0.29 nmoles/mg cell protein) or did not carry zinc (3.24 ± 0.58 nmoles/mg cell protein). We observed a similar difference in binding kinetics in three other studies that employed different albumin concentration ranges and therefore could not be combined with this statistical analysis. We consistently observed a greater binding of albumin to the endothelial cells when the albumin carried a zinc atom.



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  		Figure 4.   Effect of zinc on FITC-BSA binding to PVEC. Flasks were incubated at 4°C with 3 µM FITC-BSA, plus 0–300 µM unlabeled BSA in MEM, followed by three 15-sec washes with HEPES buffer. The "with zinc" medium had equimolar concentrations of albumin and zinc at each level. The zinc concentrations in the "without zinc" medium were all {approx} 1 µM. Data are presented as means ± SE (n = 5 flasks of cells). Nonspecific BSA binding (25% of total BSA binding) was determined by including excess BSA (1500 µM) in incubation medium, and was subtracted from total binding to obtain specific binding. The saturation-binding data were evaluated by a computerized nonlinear regression analysis to estimate the kinetic parameters presented in Table IGo, rather than undertake a linear transformation of the data for a Scatchard plot. The two curves are significantly different (P < 0.05). Data points are from a representative experiment that was repeated three times with similar results.

 

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  		Table I.   Influence of Zinc on the Kinetic Parameters of Albumin Binding to Porcine Venous Endothelial Cells (PVEC)a
 
Further evidence of a co-transporter role for albumin in zinc uptake into cells would be that they both enter in a similar manner over time. Figure 5Go is a plot of the simultaneous association of zinc and albumin with PVEC over time. (This is total acquisition of zinc and albumin, which would include both specific and nonspecific mechanisms.) The incubation medium contained an equimolar concentration of albumin and zinc (3 µM). During the first 20 min, association of both zinc and albumin with the cells occurred linearly and in a 1:1 molar stoichiometry. Thereafter, albumin accumulation increased only very slowly over time, whereas zinc accumulation continued to increase substantially over time. This pattern of albumin accumulation in endothelial cells was also observed by measuring 125I-BSA acquisition by BPAEC over a longer period (Fig. 6)Go, though their absolute numbers differed because of the difference in cell lines and different procedures for measuring albumin acquisition. Albumin binding to the cells occurred within 1 min, but significant uptake required 10–30 min, which would agree with the observations of others (10, 34, 36). The 125I-BSA content of the cells stabilized after 30 min and remained fairly constant over the next 24 hr. The lack of further accumulation of the labeled albumin with time indicated that acquired protein was either leaving or being degraded in the cells at a constant rate, without being retained in significant amounts.



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  		Figure 6.   Time-dependent uptake and retention of 125I-BSA by BPAEC. Flasks were incubated at 37°C with medium containing 20% FBS in MEM. Data are presented as means ± SE (n = 5–11 flasks of cells per time point). These data from one experiment are presented as three frames, representing different ranges of time, to permit relative changes to be discerned that would not be possible if the data were plotted together on one graph. The first frame shows saturation of albumin binding, whereas the second and third frames show saturation of albumin uptake.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the current investigation was to examine whether albumin is a vehicle for the physiological delivery of zinc to endothelial cells. Zinc can enter cells in the absence of albumin (6, 37), and zinc uptake decreases with increasing concentrations of albumin during in vitro experiments (6). Furthermore, genetic mutations that result in analbuminemia apparently do not impair zinc status (38, 39). Albumin is surely not required for zinc to enter cells. However, {approx} 70% of zinc in plasma is bound to albumin in healthy individuals (1). The remaining 30% of plasma zinc is tightly incorporated into {alpha}2-macroglobulin. The Zn-{alpha}2-macroglobulin complex does not appear to be an available source of zinc for most cells (1). Albumin-bound zinc represents 99% of the total exchangeable zinc in plasma, with the residual 1% of exchangeable zinc associated primarily with histidine and cysteine (40, 41). The association between zinc and albumin is considered to be of high affinity, yet fairly labile (42). Albumin might release zinc, thus permitting the free Zn+2 ion to enter the cell. However, zinc transport across an endothelium was more dependent upon the Zn-albumin concentration than the free Zn2+ concentration in vitro (15). Free zinc (Zn+2) accounts for only 0.001% of total plasma zinc and 0.5% of the zinc that is not bound by high-molecular-weight ligands (43). Results about zinc transport from medium without albumin are difficult to interpret, as they typically are performed with free Zn+2 ion concentrations that are not present in vivo in mammals.

Does this interaction between zinc and albumin in the plasma influence their metabolism by cells? The results in Figure 1Go confirm the observations of many others (3-7) that albumin significantly affects zinc acquisition by cells. The results in Figure 4Go demonstrate that zinc can also influence albumin acquisition by cells. Albumin carries a variety of substances in the plasma and certainly binds to cells without the participation of zinc. However, the significantly positive influence that zinc exerted on the binding kinetics of albumin demonstrates that receptors exist on the endothelial cell surface that preferentially recognize albumin molecules that are carrying a zinc atom. A similar enhancement has been demonstrated for the uptake of albumin when it is carrying glucose (13) or fatty acids (14). Multiple receptors for albumin are currently under investigation (12, 34). Zinc has previously been shown to influence the receptor-ligand interaction of adenosine (44), orosomucoid (45), human growth hormone (46), GABA (47-49), and NMDA (50). Under physiological circumstances, only about 1 in 40 albumin molecules carries a zinc atom. This enhancement could serve as a physiological mechanism to insure that there is preference for albumin molecules carrying zinc, thereby enhancing the delivery of this essential nutrient to the endothelial cell surface.

One potentially confusing observation in Figure 1Go is that uptake of zinc decreased as the molar ratio of albumin:zinc increased. It might seem contradictory that increasing the amount of a putative carrier molecule would decrease uptake of the bound substance. However, excesses of zinc-free albumin would compete with zinc-albumin for a limited number of albumin receptors, thereby blocking zinc-albumin access to the cell and reducing zinc uptake. The enhanced affinity provides a competitive advantage to the zinc-albumin complex for binding to the receptor, but the difference is not great enough to preclude competition by excessive zinc-free albumin.

The ability of the endothelial cell to discern whether or not albumin is carrying a zinc ion might reflect different subspecies of albumin receptors. Ideally, this could be demonstrated by a competition experiment; but, it is not possible to combine zinc-albumin and zinc-free–albumin without provoking a redistribution of zinc among the entire population of albumin molecules in the experimental medium (i.e., it would not be possible to distinguish between the two original albumin species). Our current evidence indicates that the quantity of binding sites (BL max) was similar for both species of albumin. This similarity suggests (but does not demonstrate) that the receptor is the same, and only affinity is affected.

Zinc uptake into mammalian cells frequently has been observed to possess a saturable, as well as a nonsaturable, component (37, 51, 52). The findings in this report might explain these observations. Albumin transport also possesses both saturable and nonsaturable processes (21). Albumin uptake is mediated via endocytic vesicles, by both receptor-mediated and bulk fluid-phase endocytosis (53). The results in Figure 2Go demonstrate that zinc uptake into endothelial cells decreased at lower temperatures. We previously thought that this observation indicated a reliance on cellular energy for zinc transport. However, when inhibitors of metabolic energy were evaluated, N-ethylmaleimide (NEM) was the only chemical that diminished zinc uptake (32, 52). NEM is also a potent inhibitor of endocytosis and albumin uptake (33). To confirm the importance of endocytosis, we employed a battery of five additional strategies to disrupt endocytosis, and all consistently inhibited both zinc and albumin acquisition. These results indicate that endocytosis represents one, but not the only, mechanism of zinc uptake by endothelial cells.

Endocytosis encompasses a variety of endosomes and transcytotic vesicles with and without clathrin-coats, caveolae, receptor-mediated vesicles, and bulk fluid-phase vesicles (54). Each of the test inhibitors targeted a specific component of these endocytotic processes, and they all had an impact on zinc and albumin acquisition. Cytochalasin B and colchicine were used to alter microtubule and microfilament structure and function (23, 24). Chlorpromazine interferes with recycling of receptors involved in coated-pit receptor-mediated endocytosis (25), whereas potassium depletion inhibits the formation of clathrin-coated vesicles (receptor-mediated endocytosis) (26, 27). Since they all had an impact, zinc must be acquired through more than one vesicle species. This agrees with the observation that albumin is present in coated vesicles and noncoated vesicles (caveolae) (55). Antohe et al. (53) reported that 75% of albumin is transported by receptor-mediated endocytosis, and 25% is transported by nonreceptor-mediated endocytosis. In contrast, Ghitescu et al. (36) reported that albumin transport by endothelial cells was restricted to uncoated pits and plasmalemmal vesicles (caveolae). Disruption of caveolae formation with nystatin almost completely blocked zinc uptake by cultured fibroblasts (56). In general, the evidence indicates that disrupting caveolae formation has the greatest impact on albumin and zinc uptake. Identification of the specific nature of the vesicles transporting albumin and zinc merits further investigation.

The endothelial cells continued to accumulate 65Zn with time, whereas the albumin content stabilized. During endocytosis albumin would have been internalized in membrane-limited intracellular vesicles. When the labeling medium was replaced with unlabeled medium, the amount of labeled albumin in the cells declined dramatically within 20 min. This suggests that the vast majority of albumin that enters an endothelial cell subsequently exits the cell, which agrees with the observations of others (10, 12, 34, 36, 57). Because 65Zn continued to accumulate within the cells, a portion of zinc that entered the cell with albumin remained within the cell after albumin exited.

It is possible that the zinc disassociates from albumin within the vesicle when the intravesicular concentration of hydrogen ions increases. Acidification of the interior of endosomes typically occurs within 5–10 min (54). The affinity of zinc for albumin rapidly declines with decreasing pH (58). Release of zinc from albumin would dramatically increase the free Zn+2 ion concentration inside the vesicle. Evidence of zinc transporters (ZnT-2 and ZnT-4) in the membranes of intracellular vesicles has been presented (59, 60) although they appear to transport zinc across the membrane from the cytosol into the vesicles.

How does this new evidence influence our understanding of cellular zinc homeostasis? Cellular zinc homeostasis might be affected by whether acquired zinc is transferred from the endosome to the cytosol, or remains in the endosome until it is exocytosed. Detachment of zinc from albumin inside the endocytic vesicle could be under regulation. As long as albumin retains a high affinity for zinc, the Zn+2 atom will likely exit along with albumin when it is exocytosed (recognized as transcytosis). Transport across the vesicle membrane into the cytosol is presumably by a specific transport protein that is potentially regulatable. Alternatively, zinc homeostasis might also be rigorously maintained at an excretion pathway. A zinc export protein (ZnT-1) has been demonstrated in the plasma membrane of mammalian cells (61, 62).

This report provides evidence that the zinc-albumin complex is an important, but surely not the only, physiological source of zinc for delivery to endothelial cells. Further evidence of albumin's importance in zinc acquisition by endothelial cells was reported by Bax and Bloxam (7). Endothelial cells are in direct juxtaposition to plasma (and high albumin concentrations); it is unclear whether this source of zinc is important to cells in other locations of the body. Taylor and Simons (8) concluded that albumin was not a significant factor in zinc uptake by hepatocytes. Ackland and McArdle (37) concluded that no specific ligand was instrumental in zinc uptake into fibroblasts, based largely on their observation that free Zn2+ entered the cells in a concentration-dependent manner that was saturable. However, their saturation occurred at free Zn2+ concentrations that are 1000-fold higher than are likely to be present in the extracellular fluids. Since zinc uptake was measured during incubations that lasted 60 min, this evidence might reflect uptake from a variety of routes, potentially including an endocytotic step. Grider and Vazquez (56) recently observed that endoctyosis via caveolae is very important for zinc acquisition by fibroblasts, suggesting that our observations are not unique to endothelial cells.

In summary, endothelial cells preferentially bind albumin molecules if they possess a zinc atom. Endocytosis is a significant mechanism for zinc (and albumin) uptake by endothelial cells. Most of the albumin, and some of the zinc, that is in the endosome is subsequently released (transcytosis) whereas some of the zinc is retained by the cell. Therefore, some zinc is acquired by endothelial cells through endocytosis in association with albumin. This pathway is unlikely to be the only route of zinc entry into endothelial cells, and may not be as significant for nonendothelial cells with less contact by plasma albumin. This pathway permits zinc to enter the cell in a vesicle, but insights about the transfer of zinc from the endosome to the cytosol are needed.


   Acknowledgments
 
The authors thank Barb Brothwell, Jen Fruci, Livia Gorini, Holly Lehmann, Jennifer Mateleska, James McClung, and Laurie Volak for their excellent technical assistance. The authors also thank Chris Neefus, UNH Office of Biometrics, for his expert statistical support.


   Footnotes
 
This is scientific contribution number 2014 from the New Hampshire Agricultural Experiment Station. Partial support was also provided by National Institutes of Health grant R15 NS35285–01.

1 To whom requests for reprints should be addressed at the Department of Animal & Nutritional Sciences, Kendall Hall, University of New Hampshire, Durham, NH 03824–3590. E-mail: DBobilya{at}cisunix.unh.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 12, 1999. Accepted for publication February 24, 2000.




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H. M. Lehmann, B. B. Brothwell, L. P. Volak, and D. J. Bobilya
Zinc Status Influences Zinc Transport by Porcine Brain Capillary Endothelial Cells
J. Nutr., September 1, 2002; 132(9): 2763 - 2768.
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