A2E, a Lipofuscin Fluorophore, in Human Retinal Pigmented Epithelial Cells in Culture

Abstract

purpose. To study A2E, a component of retinal pigmented epithelial (RPE) cell lipofuscin, after its internalization by cultured human RPE cells.

methods. A2E was synthesized and incubated with an adult RPE cell line devoid of native lipofuscin. To investigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Red DND-99; Molecular Probes, Eugene, OR). Plasma membrane integrity was evaluated by assaying for leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH), by fluorescence nuclear staining with a membrane-impermeant dye and by morphologic criteria. The emission spectrum of internalized A2E was also determined. The levels of A2E accumulated by the cultured cells were quantified by high-performance liquid chromatography and compared with amounts present in RPE isolated from human eyes.

results. Internalization of A2E by the RPE cells was evidenced by the acquisition of intracellular granules detectable by fluorescence confocal imaging. Internalized A2E had an emission maxima of 565 to 570 nm. The levels of A2E accumulating in cells incubated with 10 to 25μ M A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from human eyes. Colocalization of A2E and the Lysotracker probe revealed a preferential accumulation in acidic organelles. The elevated LDH levels that were measured after exposure to 50 and 100 μM A2E were attributable to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling.

conclusions. Internalized A2E has an affinity for acidic organelles. The membrane damage exhibited by A2E-accumulating RPE is dependent on the concentration of A2E and reflects the ability of this amphiphilic compound to exert detergent-like effects.

One of the hallmarks of aging and of some inherited retinal disorders is the progressive accumulation of autofluorescent membrane-bound lipofuscin in the retinal pigmented epithelium (RPE). ^{1 2 3 4 5 6} Lipofuscin is primarily responsible for the intrinsic fluorescence of the human ocular fundus, ^{7 8} and by the ninth decade of life, lipofuscin granules occupy approximately 19% of the area of a macular RPE cell. ⁵ It is now generally accepted that lipofuscin is amassed by the RPE cells, in part because of their role in phagocytosing the large number of outer segment disc membranes that are shed daily by the photoreceptor cells. The greatest accumulation of lipofuscin by RPE cells occurs in the macula, and although the reasons for this are not clear, an important factor may be the higher photoreceptor-to-RPE cell ratio. ^{5 6}  

Although RPE lipofuscin consists of a complex mixture of pigments, the major hydrophobic component is the fluorophore A2E, which arises from the reaction of two molecules of all-trans-retinal with ethanolamine, both of which are molecular components of the photoreceptor outer segment membrane. ^{9 10 11} Recent studies have confirmed the structure of A2E by total chemical synthesis ¹¹ and have led to the development of a facile biomimetic preparation of A2E. ¹² Moreover, A2E has been quantified in human donor eyes, and an additional fluorescent pigment of RPE lipofuscin, iso-A2E, has been characterized. ¹² A2E and iso-A2E readily interconvert under standard room illumination, and it is likely that the photoequilibration of these two pigments is occurring in vivo. ¹² This photoisomerization is potentially significant, given that it could involve the production of radical species. ¹³  

The issue of whether the buildup of lipofuscin by RPE cells has significant deleterious effects on the RPE cells, and thus retinal function, has been controversial. Although no evidence for a direct causal relationship exists, lipofuscin levels in RPE cells are topographically correlated with histopathologic indicators of age-related macular degeneration (AMD) ^{5 6 14} and with the loss of photoreceptor cells in aged eyes. ⁶ By taking advantage of the ability to synthesize A2E, we have demonstrated that when A2E is incubated with RPE cells in vitro, it accumulates intracellularly within acidic organelles and can exhibit detergent-like properties. 

Methods

RPE Culture and Isolation

A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA) was grown in Dulbecco’s Modified Eagle Medium (DME) (Gibco, Gaithersburg MD) with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 2 mM glutamine (Gibco), 0.1 mM minimum essential medium nonessential amino acids solution (Gibco), and gentamicin sulfate (10 μg/ml) in eight-well plastic chamber slides (Laboratory-Tek; Nunc, Naperville, IL) or 96-well tissue culture plates. 

Adult RPE cells were also harvested from human donor eyes (National Disease Research Interchange, Philadelphia, PA), as previously described, ¹⁵ and the numbers of harvested RPE cells were determined by counting in a hemocytometer. 

A2E Synthesis and Treatment

A2E (Fig. 1) was prepared from all-trans-retinal and ethanolamine (2:1 molar ratio) as previously described. ¹² A2E samples contained approximately 15% iso-A2E, determined by high-performance liquid chromatography (HPLC) analysis. A2E was stored as a stock solution in dimethyl sulfoxide (DMSO) and was kept at− 80°C in the dark. For uptake into cultured human RPE cells, A2E was delivered in 10-, 50-, 75-, and 100-μM concentrations in culture media, and confluent cultures of the cells were incubated with A2E or DMSO (control) for the indicated times. All experiments included untreated cells. 

Emission Spectra

Fluorescence spectra were obtained on a 0.22-m double spectrometer (model 1680 Fluorolog; Spex, Edison, NJ) with front-face detection. Samples were stirred during each measurement and maintained at 23°C with a bath circulator. In all cases, an excitation wavelength of 380 nm was used. The absorbance of A2E solutions was approximately 0.2 at 380 nm. To measure the emission spectra of cell-associated A2E, ARPE-19 cells were grown in 35-mm dishes and exposed to 100 μM A2E in media for 3 hours. After vigorous washing to ensure that only intracellular A2E remained in the sample, the cells were trypsinized, collected in culture media, pelleted, and resuspended in phosphate-buffered saline (PBS; pH 7.4). To obtain paraformaldehyde-fixed preparations, cells were fixed in suspension for 15 minutes in 2% paraformaldehyde, washed and resuspended in PBS. For RPE cell samples, multiple scans were measured and averaged to provide the final emission spectrum. 

HPLC

Pelleted RPE cells were homogenized with a solution of chloroform-methanol (2:1 ml), and A2E was quantified by HPLC, as previously described. ¹² The quantities of A2E in cultured RPE and in RPE isolated from human eyes were determined from integrated peak intensities and were expressed as nanograms per 10⁵ cells. 

Lactate Dehydrogenase Assay

The cytoplasmic enzyme lactate dehydrogenase (LDH) was measured in cell culture supernatants by colorimetric assay (Boehringer–Mannheim, Indianapolis, IN). The LDH assays were performed using RPE cultures grown to confluence in 96-well plates, and LDH was measured at the indicated times after A2E or DMSO treatment and at a specified interval of time (LDH release period) after the addition of fresh media (with 1% serum) to A2E-containing (or DMSO-treated) cells in culture. After collection, the culture supernatants were centrifuged at 250 g for 10 minutes to remove suspended cells. The cell-free supernatants were then assayed in either undiluted form (2–6-hour LDH release period) or were serially diluted (1:2–1:8; 24-hour LDH release period) in the appropriate culture media for a final volume of 100 μl/well. Twenty minutes after the addition of the reaction mixture, absorbance was measured at 490 nm using a microtiter plate reader (EL340; Bio-Tek Instruments, Burlington, VT). For each experiment, 3 to 6 replicates of each condition were included and background levels, determined using media not exposed to cells, were subtracted from absorbance values obtained for each condition. The data were analyzed by one-way analysis of variance, followed by the Bonferroni multiple comparisons test. 

Fluorescence Assays

For fluorescence labeling of acidic organelles, confluent cultures of ARPE-19 cells grown in eight-well chambers were incubated simultaneously with A2E (50 μM) and LysoTracker Red DND-99 (50 nM; Molecular Probes, Eugene OR) ^{16 17} in culture media for 2 hours. Subsequently, the cultures were washed, fixed with 2% paraformaldehyde in PBS and examined using a confocal laser scanning system (model 410; Carl Zeiss; Thornwood, NY) equipped with an argon-krypton laser. In control experiments, a fluorescent mitochondrial marker (MitoTracker Red CM-H₂Xros, 500 nm; Molecular Probes) was used. 

Membrane-compromised cells were visualized in confluent cultures using a fluorescence assay (Molecular Probes) in which the nuclei of nonviable cells fluoresce red because of staining by a membrane-impermeant dye (Dead Red nucleic acid stain; 1/500 dilution). The numbers of red-labeled nuclei were counted in photomicrographs of representative areas of the cultures (1.2-mm² fields) and were expressed as a percentage of the total nuclei in the field (3062% ± 54% [SEM]), the latter being determined in companion cultures permeabilized with 0.1% Triton X-100 before fluorescence staining. Data are based on counts performed in duplicate or triplicate wells in each of two experiments, with 5 to 10 fields counted per well. 

Immunofluorescence Labeling

For immunofluorescence labeling of RPE cultures, the cells were fixed in 2% paraformaldehyde in PBS for 25 minutes at room temperature. After preincubation in blocking serum containing 0.1% Triton X-100, the cells were incubated in rabbit antibody to human ZO-1 (Zymed Laboratories, South San Francisco CA) for 1 hour followed by TRITC-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with propidium iodide (Molecular Probes, Eugene OR). 

Fluorescence Microscopy

A2E accumulation in cultured RPE cells was detected using the confocal laser scanning system equipped with an argon-krypton laser. For dual analysis, the fluorescence of A2E was detected with a 515- to 540-nm band-pass filter after excitation at λ 488 nm (fluorescein isothiocyanate [FITC]–appropriate filters), and LysoTracker Red, rhodamine, and propidium idodide were excited at λ 568 nm and visualized with 670 to 810 nm (Lysotracker Red and rhodamine) and 575 to 640 nm (propidium iodide) band-pass filters. All confocal images were a single optical section (1 μm). Images were then processed for publication (Photoshop 5.0; Adobe, San Jose, CA). 

Results

By HPLC analysis, we confirmed that ARPE-19 cells (Fig. 2) do not contain endogenous A2E. After incubation of ARPE-19 cells with A2E (10, 50, and 100 μM), uptake by the cells was evidenced by the acquisition of intracellular granules, which appeared yellowish brown when viewed by transmitted illumination (Fig. 3) . A yellow hue, visible to the unaided eye, was acquired by cultures treated with 50 μM and 100 μM A2E (not shown). These yellowish brown granules were not present in cells unexposed to A2E (Fig. 3D) . The cells that had accumulated A2E also exhibited autofluorescence when viewed either by laser scanning fluorescence microscopy (Fig. 4) or epifluorescence microscopy (not shown) using FITC-appropriate filters. This autofluorescence was not present in untreated or DMSO-exposed cells (Fig. 4C) , and our ability to detect the autofluorescence in A2E-containing cells did not diminish with time in culture. Imaging by confocal microscopy in the horizontal plane at depths within the cells (Figs. 4A 4B) confirmed that the exogenously delivered A2E was internalized by the RPE cells in culture. 

The emission spectrum of internalized A2E was obtained by measuring the fluorescence of cultured cells that were exposed to A2E. Initially, emission spectra of A2E in n-butyl chloride (n-BuCl), methanol, and PBS were measured (Fig. 5 A). We found that the emission maximum of A2E was dependent on the nature of the solvent with the most hydrophobic solvent, n-BuCl, giving a blue-shifted maximum (λ_max = 585 nm) in comparison with the peaks observed in both methanol (λ_max = 600 nm) and PBS (λ_max = 610 nm). Suspended preparations of both fresh and paraformaldehyde-fixed A2E-containing RPE cells provided emissions with maxima of 565 to 570 nm (Fig. 5B) . RPE cells that had not been treated with A2E did not exhibit an emission maximum (data not shown). 

Using optimized HPLC conditions that allow us to detect as little as 5 ng of A2E, ^12. we also quantified A2E accumulation in cultured RPE cells and compared these amounts with estimates of the amounts of endogenous A2E that are present in RPE cells isolated from healthy human donor eyes at various ages (Table 1) . It was apparent that 10- to 25-μM concentrations of A2E in media were required to achieve cell-associated levels that approximated the levels of A2E measured in human RPE isolated from healthy donor eyes (34–134 ng/10⁵). These measurements are an estimate of the overall levels of A2E in the cultured cells. When observing the A2E-accumulating cultures by transmitted light and fluorescence microscopy (Figs. 3 4 6) , it was clear that the fluorophore was not distributed evenly among the cells in a single culture, with some cells having proportionately greater or lesser amounts. Similarly, RPE cells in vivo exhibit regional variability in their content of lipofuscin. ^{5 18} It was also evident from both fluorescence microscopic imaging (Figs. 4A 4B 6A) and HPLC analysis (Table 1) that the amounts of A2E internalized by the cells varied with the A2E concentration in media and with the time of incubation. We estimate that 5% to 10% of the A2E in the media was internalized by the cells. 

Given that internalized A2E becomes distributed in a punctate perinuclear pattern (Figs. 3C 4B) that is typical of lysosomes, ¹⁹ we sought to define the intracellular compartmentalization of A2E. Accordingly, cultures of ARPE-19 cells were simultaneously incubated with A2E (50 μM) and Lysotracker Red DND, a membrane-diffusible acidophilic fluorophore. On examination by confocal microscopy, many A2E-containing intracellular granules were found to colocalize with the Lysotracker probe (Figs. 6A 6B) . The colocalization is made particularly apparent when the red and green signals from A2E and Lysotracker Red, respectively, are merged to yield yellow vesicles caused by the combination of red and green (Fig. 6C) . Conversely, intracellularly accumulated A2E did not colocalize with the mitochondrion-selective dye Mitotracker Red CM-H₂XRos (Fig. 6D) . 

Because the wedge-shaped A2E and the more streamlined iso-A2E contain both hydrophobic retinoid-derived chains and the hydrophilic pyridinium head group, they can behave as amphiphilic detergents that have the potential to interact with membranes and perturb membrane integrity. Thus, we examined for evidence of membrane damage by assaying for the release of the cytoplasmic enzyme LDH into the tissue culture media. As illustrated in Figure 7 A, RPE cells exposed to 50 and 100 μM A2E exhibited elevated levels of LDH in the culture supernatants after 2 hours of treatment, followed by a 2-hour incubation in fresh media. Further increases in LDH levels were observed when the incubation in fresh media was lengthened to 4 hours. 

Using an experimental paradigm that involved a longer exposure (6 hours) to A2E, we also found LDH levels in the culture supernatants to be elevated at 1 and 3 days after incubation in 50 and 100 μM A2E (Fig. 7B) . Conversely, cultures accumulating A2E from a 10-μM concentration in media, did not exhibit elevated LDH levels. Moreover, 7 days after incubation in 50 and 100 μM A2E, the amount of LDH released from cells over a 6 hours was not significantly different from that released from DMSO-treated and untreated cells. 

The proportions of membrane-compromised cells in confluent A2E-exposed cultures were also visualized by labeling the nuclei of these cells with a red fluorescent nucleic acid stain (Fig. 8) . After a 6-hour exposure to 100 μM A2E, we determined that approximately 15% (14% ± 1%, SEM) of the total number of nuclei were labeled compared with 0.1% (0.1% ± 0.1%) of nuclei in control cultures. With a 50-μM concentration of A2E for 6 hours, considerably fewer nuclei were labeled (1.0% ± 0.2%). Similarly, when the cells were incubated for 2 hours with 100- and 50-μM concentrations of A2E in media, a small proportion of the nuclei were labeled (0.5% ± 0.1% and 0.2% ± 0.1%, respectively). 

The results of the above assays of cell viability were consistent with the microscopic appearance of the cells, 1 and 3 days after they had accumulated A2E. Among the cells that internalized readily visible amounts of A2E, some had rounded up (Figs. 3A 3B) . Nevertheless, 1 week after A2E exposure, the cultures once again appeared healthy, while continuing to show intracellular A2E (Fig. 3C) . 

Discussion

We have demonstrated by fluorescence confocal and transmitted light microscopy, by fluorescence spectroscopy, and by quantitative HPLC that cultured human RPE cells internalized A2E delivered to them in culture media. The ability of A2E to accumulate intracellularly was consistent with its amphiphilic structure, consisting of a hydrophilic pyridinium head group and two hydrophobic tails. ¹⁰ Our modeling studies indicate that iso-A2E, which is naturally occurring in aged human eye samples, constitutes approximately 15% of an A2E sample and exists in a more streamlined conformation than A2E. ¹² This light-induced isomer of A2E, may more readily penetrate phospholipid membranes. Although the structure of A2E predicts that it can insert into the plasma membrane, it is clear that it is not retained there, because we did not detect A2E autofluorescence within the plasma membrane. Instead, we observed A2E intracellularly. Similarly, it does not appear that this compound was retained within certain other phospholipid bilayers elsewhere in the cells, because, for instance, A2E was not visible in the nuclear and mitochondrial membranes (Figs. 3 4 6) . Regarding the mode of uptake of A2E, it is also conceivable that A2E was taken up in association with serum proteins, thereby undergoing “piggyback” endocytosis. ²⁰  

In vivo, native A2E and other components of RPE lipofuscin are derived from molecular components of phagocytosed photoreceptor outer segments and become concentrated in lysosomal storage bodies. ¹ Thus, the affinity of A2E for acidic organelles when delivered to the cells in culture, may replicate the compartmentalization of A2E in vivo. Holz et al. ²¹ recently reported that A2E coupled to low density lipoprotein (LDL) accumulates intracellularly in lysosomes. The targeting of LDL and A2E to lysosomes reflects a property of LDL, a ligand well known to undergo receptor-mediated endocytosis and routing to lysosomes. Conversely, our observation that uncoupled A2E also accumulates in acidic organelles may speak to a property of A2E, per se. Eldred and Laskey ⁹ and Eldred ²² speculated that A2E would exhibit lysosomotropic properties, the basis for this assumption being that, similar to alkyl amines that cross the cell membrane in a deprotonated state, A2E would become trapped in lysosomes after protonation in the acidic environment of the lysosome. ^{20 23 24} Nevertheless, the accumulation of A2E within lysosomes, as observed in the present work, is unlikely to involve the latter mechanism, because A2E is a quarternary pyridinium salt that cannot be deprotonated or reprotonated. Additionally, although there is a proton on the alcohol derived from ethanolamine, A2E cannot exist as a zwitterion with this alcohol in the deprotonated form: In neither the cytosol (pH ∼7.4) nor the lysosome (pH 5.2–5.3) would an alcohol (pK_a ∼20) lose its proton. Thus, although the mechanism by which A2E accumulates in acidic organelles is not known, it may occur by incorporation from the cytoplasm through a transmembrane mechanism. ²⁵ Alternatively, after entering the plasma membrane, A2E may enter lysosomes through endocytic vesicles that bud off the plasma membrane. Moreover, although we conclude that a significant amount of exogenously delivered A2E accumulates within acidic organelles, we do not exclude the possibility that some of the A2E is present in the extraorganelle compartment of the cytoplasm. 

Because A2E cannot undergo further protonation within the acidic environment of the lysosome, this protonation mechanism is unlikely to explain the observation that lysosomal pH is elevated after the uptake of A2E and LDL by cultured cells. ²¹ An alternative explanation for an A2E-associated change in lysosomal pH, is that it occurs as a result of a detergent-mediated perturbation of the membrane-bound adenosine triphosphatase that actively pumps protons into the lysosome. ^{26 27 28} The ability of A2E to exhibit detergent-like activity is demonstrated here. 

We have observed that the emission maximum of A2E varies with its environment, with a more hydrophobic milieu producing a blue-shifted maximum. In a previous report, in which the emission spectra of fluorophores extracted from human RPE lipofuscin were described, the fraction identified as A2E had an emission maximum of 605 nm in n-butyl chloride. ²⁹ Because it is clear that the emission profile of A2E is dependent on the local environment of the molecule, the difference between the emission of extracted and synthetic A2E in n-butyl chloride is probably attributable to the fact that the synthetic sample was the trifluoroacetate salt, whereas the extracted material was either the acetate salt (chromatographic eluant contained acetic acid) or chloride (as would be the case in vivo). That the emission maximum of cell-associated A2E (565–570 nm) corresponds most closely to that observed in n-BuCl, may indicate that intracellular A2E is localized to a hydrophobic environment. The intracellular A2E emission observed here is distinct from the spectral characteristics of fundus fluorescence, because in the latter case, a mixture of fluorophores, including A2E, are detected. ^{7 8}  

In the present work, we have used the plasma membrane, together with extracellular A2E, as a model to study the effects of A2E on a phospholipid bilayer. We have shown that A2E, when present in sufficient concentrations, can induce a loss of membrane integrity, that in its timing is not typical of cells undergoing programmed cell death. ^{30 31 32 33} The advantage of using the plasma membrane to demonstrate this property of A2E is that permeability changes in the plasma membrane can readily be assayed. Accordingly, the A2E-mediated loss in membrane integrity, which we have demonstrated by the early egress of LDH, by the labeling of nuclei with a cell-impermeant dye, and by cytotoxic indicators such as cell rounding, ^{24 34} is consistent with an A2E-mediated detergent activity. ³⁵ This surfactant property of A2E is attributable to its amphiphilic structure. ¹⁰ The contention that the concentration of A2E must reach some critical level for detergent activity to be manifest ^{9 22} is supported by several observations. For instance, although intracellular A2E accumulation from a 10-μM concentration in media, was readily demonstrable by HPLC quantitation, this concentration of A2E was not associated with elevated LDH release (Fig. 7) . Moreover, it was clear from both light and fluorescence microscopy that even at the higher concentrations of A2E in media (100 and 50 μM), the extent of intracellular accumulation of A2E was not the same for all the cells. Correspondingly, membrane damage, as evidenced by fluorescence nuclear staining (Fig. 8) and cell rounding (Fig. 3) were exhibited by a subpopulation of the cells only. In addition, 1 week after A2E incubation, both the LDH levels and the light microscopic appearance of the cultures appeared normal, despite the presence of readily detectable levels of A2E within the cells. The most parsimonious explanation for the latter finding is that the surviving cells had accumulated less A2E, presumably tolerable levels, than neighboring cells that experienced membrane disruption. This apparent concentration dependence is typical of detergents wherein the cooperative action of several molecules is required, as in the formation of detergent micelles. ²⁴ These results are also consistent with the notion that lipofuscin accumulation in RPE cells may reach a critical threshold above which disease is realized. ^{6 36}  

In our experiments, the exogenously delivered A2E likely exerted its detergent effects on the plasma membrane during transit into the cells. The importance of this observation notwithstanding, we recognize that if damage from A2E were to occur in vivo, it would emanate from inside the RPE cell, with the detergent-like activity we have demonstrated manifesting if the concentration of A2E within the lysosome reached a level sufficient to disrupt the organelle’s membrane. We are currently performing experiments that are designed to examine this concept. 

The issue of whether lipofuscin inclusions are deleterious to the RPE cell has been a matter of debate for some time, and mechanisms by which some detrimental effects may occur have been proposed. ^{1 9} Our ability to load RPE cells with A2E at levels that are comparable to that present in the eye will enable us to investigate questions related to the impact of A2E on the RPE cell. 

 Supported by Fight for Sight, unrestricted funds from Research to Prevent Blindness, National Institutes of Health Grant 34509, and a National Institutes of Health Research Service Award postdoctoral fellowship (CAP).

 Submitted for publication February 23, 1999; revised July 2 1999; accepted July 23, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

Structure of A2E and iso-A2E. X⁻, A2E counterion, which in vivo is most likely chloride.

Figure 2.

 

HPLC (UV) analysis of ARPE-19 cell extracts. Methanol-chloroform extracts of ARPE-19 cells were analyzed for endogenous A2E. Reversed-phase HPLC (Cosmosil 5C18; Nacalai Tesque, Kyoto, Japan; 150 × 4.6 mm) was used for A2E analysis (linear gradient of methanol in water; 85%–96% methanol + 0.1% trifluoroacetic acid, 1 ml/min). Under these conditions, a standard sample of A2E eluted after approximately 12 minutes. No A2E was detected in ARPE-19 cells.

Figure 3.

 

Internalization of A2E by RPE cells in culture. Phase-contrast micrographs of A2E-accumulating ARPE-19 cells, 1 day (A), 3 days (B), and 7 (C) days after incubation with 100 μM A2E for 6 hours. A2E appears as yellowish brown intracellular granules. Note that at 1 and 3 days, some of the A2E-containing cells have rounded up (arrows). (D) Control cells not exposed to A2E. n, unfilled nuclear region. Scale bar, 50 μm.

Figure 4.

 

Detection of internalized A2E by fluorescence laser scanning confocal microscopy. ARPE-19 cells were incubated with 100 μM A2E for 6 (A) or 2 (B) hours or were untreated (C). After an additional 2 days in culture, the cells were fixed, and cell borders were outlined by immunolabeling with antibody to ZO-1 (red), and nuclei were stained with propidium iodide (red). Internalized A2E appears as green autofluorescent granules because of the use of FITC filters. With moderate A2E loading, as shown in (B), the perinuclear distribution of A2E is evident. Each image is a single optical section (1 μm). Scale bars, (A) 45 μm; (B, C), 20 μm.

Figure 5.

 

Fluorescence emission spectra of A2E. (A) Emission of A2E in n-butyl chloride (n-BuCl), methanol (MeOH), and PBS. Spectra were obtained in a 1-cm² quartz cuvette, stirring at 23°C, with an excitation wavelength of 380 nm. (B) Emission of A2E in RPE cells. RPE cell cultures were treated with A2E as described in the Methods section and the emission spectrum was determined for fresh (RPE cells) and paraformaldehyde-fixed (fixed RPE cells) cells suspended in PBS. Front-face detection was used to minimize the effects of scattered light. For each RPE cell sample, five scans were accumulated and averaged to provide the final spectrum.

Table 1.

 

Quantitation of A2E in RPE by HPLC (UV) Analysis

Donor age (y)	A2E/105 cells* (ng)
RPE Isolated from Human Donor Eyes
58	57
58	96
64	134
65	80
65	99
70	128
70	102
72	55
72	75
79	34

A2E in Media, † (μM)	A2E/105 cells* , § (ng)
ARPE-19 Cells Accumulating A2E in Culture
100	1085 ± 141 (3)
75	306 ± 153 (3)
50	241 ± 70 (2)
25	104 ± 4 (3)
10	37 (1)

*  Cell preparation and A2E quantitation as described in the Methods section.

†  6-Hour incubation.

§  Mean ± SD of (n) determinations.

Figure 6.

 

Colocalization of intracellular A2E with Lysotracker Red (Molecular Probes). The fluorescence in (A) and (B) is superimposed in (C); thus, particles that colocalize in (A) and (B) appear yellow in (C), because of the combination of green and red. A2E autofluorescence was imaged using FITC-appropriate filters (excitation, 488 nm; emission 515–540 band-pass). (D) A2E autofluorescence (green signal) does not overlap with the signal (red) for the mitochondrial marker Mitotracker Red CM-H₂Xros (Molecular Probes, Eugene, OR). Representative of three experiments. Scale bar, 20μ m.

Figure 7.

 

LDH release from ARPE-19 cells accumulating A2E in culture. (A) Short term. LDH levels in culture supernatants were determined either immediately after a 2-hour period of A2E (or DMSO) accumulation or after an additional 2- or 4-hour incubation in fresh media. To control for the variability in total incubation times, the LDH values obtained for untreated cultures were subtracted from the A2E- and DMSO-treated cultures. Values are means ± SEM of two to four experiments. ∗, P < 0.05; ∗∗, P < 0.001. (B) Long term. Cultures were incubated in A2E- or DMSO-containing media for 6 hours. After the indicated number of days, LDH was measured in culture supernatants collected after a 24-hour incubation in fresh media. Values are the mean ± SEM of two to four experiments.

Figure 8.

 

Fluorescence labeling of the nuclei of membrane-compromised cells. Confluent cultures of ARPE-19 cells were exposed to 100 μM (A) and 50 μM (B) A2E for 6 hours or were untreated (C). Cultures were incubated with a cell-impermeant red fluorescent nucleic acid stain to label cells with compromised membranes. Scale bar, 200 μM.