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CELLULAR AND MOLECULAR

Involvement of Endocytic Organelles in the Subcellular Trafficking and Localization of Riboflavin

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (P.W.S.); and Division of Pharmaceutics (S.-N.H., P.W.S.) and Biophysics Program (M.A.P., P.W.S.), The Ohio State University, Columbus, Ohio

Received for publication March 13, 2003
Accepted April 17, 2003.

	Abstract

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Previous studies by our laboratory have suggested the potential role of receptor-mediated endocytosis components in the cellular translocation of riboflavin (vitamin B₂). To delineate the intracellular compartments and events involved in the internalization of riboflavin, we synthesized a rhodamine-labeled riboflavin conjugate to monitor its movement via fluorescent microscopy. Cellular uptake studies in BeWo cells show that rhodamine-riboflavin conjugate exhibits similar ligand affinity toward the putative riboflavin transport system as [³H]riboflavin, whereas rhodamine does not significantly interfere with its internalization mechanism. Microscope analysis reveals rapid internalization of the rhodamine-riboflavin conjugate via a riboflavin-specific process into acidic vesicular compartments throughout the cells. The intracellular punctate distribution is comparable with that of fluorescein isothiocyanate (FITC)-transferrin, a well characterized receptor-mediated endocytosis substrate. Double-labeling fluorescence microscopy studies further confirm that with 10 min of internalization, rhodamine-riboflavin conjugate substantially concentrates within vesicular structures associated with clathrin, rab5, FITC-transferrin, and the acidotropic marker LysoTracker Blue. In summary, our studies provide, for the first time, direct morphological evidence of the involvement of endocytosis machinery in the intracellular trafficking of riboflavin. The subcellular localization of rhodamine-riboflavin conjugate suggests that, under the experimental conditions in this study, the internalization of riboflavin follows a classical receptor-mediated endocytosis pathway.

Recently, we demonstrated that riboflavin transport in the human small intestinal Caco-2 cell line is sensitive to pharmacological agents known to alter endocytic pathways (Huang and Swaan, 2000). The results indicated that microtubule-based movements and vesicular-sorting components are essential for cellular transfer of riboflavin. Previously, Low and colleagues showed that internalized bovine serum albumin-riboflavin conjugate has a distribution pattern reminiscent of endosomal compartments in carcinoma cell lines from various human tissues (Holladay et al., 1999) and in rat lung tissue both in vivo and in vitro (Wangensteen et al., 1996), although in these studies, riboflavin failed to inhibit surface binding of bovine serum albumin (BSA)-riboflavin. The finding that BSA-riboflavin only partially blocked riboflavin uptake further suggested that a nonspecific internalization mechanism might exist to accommodate this macromolecular riboflavin adduct (Holladay et al., 1999).

Direct evidence of an endocytosis mechanism usually entails microscopic analysis of subcellular distribution of ligands and receptors (Pastan and Willingham, 1985). Since the riboflavin transporters have yet to be identified, to elucidate the involvement of receptor-mediated endocytosis, we aim to monitor the cellular trafficking of riboflavin itself. Although riboflavin is intrinsically fluorescent, its emission spectrum is difficult to distinguish from cellular autofluorescence (Andersson et al., 1998). Moreover, endogenous riboflavin and riboflavin-related coenzymes further complicate signal interpretation. To avoid these complications, in the present study, we developed a strategy to conjugate riboflavin with rhodamine, a widely used red fluorescent probe. The intracellular itinerary of riboflavin is investigated by following the movements of rhodamine-riboflavin conjugate via fluorescence microscopy. We choose a human choriocarcinoma-derived cell line, BeWo, as our system. Recently, we reported the existence of high-affinity riboflavin transporter(s) on the microvillous membrane of this polarized trophoblast model (Huang and Swaan, 2001). Compared with Caco-2 cells, BeWo cells have a better-defined nucleus and cytoplasmic boundary, together with a more homogeneous cell population. More importantly, our studies showed that at physiological riboflavin plasma concentration (5 nM), riboflavin uptake activity in BeWo cells is three-fold higher than that in Caco-2 cells (S.-N. Huang and P. W. Swaan, unpublished data). The present study shows, for the first time, direct visualization of riboflavin in subcellular compartments in a punctate fashion, providing further evidence for a specific endocytosis uptake mechanism for this important vitamin.

	Materials and Methods

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Materials. Tetramethyl-rhodamine-carbonyl-azide, LysoTracker Blue-White DPX, 4,6-diamidino-2-phenylindole (DAPI), and SlowFade light antifading kit were purchased from Molecular Probes (Eugene, OR). Cell culture materials and buffer solutions were obtained from Invitrogen (Carlsbad, CA). Monoclonal antibodies to human clathrin heavy chain and rab5 were obtained from BD Biosciences Transduction Laboratories (San Diego, CA). FITC- and tetramethylrhodamine B isothiocyanate-conjugated Fc-specific goat antimouse IgG antibodies, FITC conjugation kit, human transferrin, [³H]riboflavin (20 Ci/mmol), and [¹⁴C]mannitol (60 mCi/mmol) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were from Fisher Scientific Co. (Pittsburgh, PA).

Cell Cultures. BeWo cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C, under 5% CO₂, in complete medium consisting of F-12K medium with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The culture medium was replaced every other day. The cells were harvested at 80% confluence (day 4–5) by exposure to a trypsin-EDTA solution.

Synthesis of Rhodamine-Riboflavin Conjugates. Six milligrams riboflavin and 0.8 mg tetramethyl-rhodamine-carbonyl-azide (10:1 M ratio) was dissolved in dimethyl sulfoxide and heated at 80°C for 1 h with gentle stirring. After 1 h, 40 µl of absolute ethanol was added in the reaction mixture and reacted for an additional 20 min to terminate residual isocyanates. Rhodamine-riboflavin conjugates were further purified by a Beckman System Gold high-performance liquid chromatography system (Beckman Coulter, Inc., Fullerton, CA) using a Beckman RP C-18 preparative column (octadecylsilane, 5 µm, 10 mm x 25 cm). Crude reaction mixtures were eluted using a linear gradient of 0 to 100% acetonitrile over 30 min (3 ml/min), and the eluents were collected by a fraction collector (1.5 ml/min). A Beckman 166P detector operating at 267 nm and an Applied Biosystems 980 programmable fluorescence detector (_ex, 545 nm and _em, 580 nm) (Applied Biosystems, Foster City, CA) were used to detect riboflavin and rhodamine, respectively. Comparison of the aligned UV and fluorescence chromatograms revealed that rhodamine-riboflavin conjugate and riboflavin have retention times of around 6 and 11 min, respectively. Other rhodamine byproducts are eluted after 14 min (data not shown). Rhodamine-riboflavin conjugate was subjected to mass spectrometry and fluorescent absorption scanning spectrum analysis using a PerkinElmer Luminescence spectrometer LS 50 (Boston, MA). Mass spectrometry indicated a M_r of 804.3. Compared with spectra of standard compounds, rhodamine-riboflavin shows combined riboflavin and rhodamine absorbance patterns with a slight right-shift in _abs for the rhodamine fluorophore (15 nm).

Uptake Studies. BeWo cell monolayers were grown on rat tail collagen-coated 12-well plates at a density of 5 x 10⁴ cells/cm². Confluent monolayers were formed between 3 to 5 days after seeding and were used for experiments at that time. Before studies were initiated, BeWo cell monolayers were washed twice with warm (37°C) phosphate-buffered saline (PBS) (pH 7.4). Riboflavin uptake studies were performed at 37°C in bathing medium (Hanks' balanced salt solution containing 25 mM glucose and 10 mM HEPES, adjusted to pH 7.4), with a final concentration of 5 nM [³H]riboflavin in the absence (control) or presence of rhodamine-riboflavin conjugate. [¹⁴C]Mannitol (0.37 µM) was incorporated in the incubation medium to determine the specificity of the washing steps. After 20 min, bathing medium was aspirated, and cells were washed twice with ice-cold PBS (pH 3.0) to remove free and surface-bound riboflavin. Finally, cells were lysed with 1% Triton X-100 solution, and the amount of dual-labeled radioactivity in cell lysates was quantitated using a Beckman liquid scintillation counter (model LS 6000IC).

Subcellular Localization of Rhodamine-Riboflavin Conjugate. To study the cellular internalization of riboflavin, BeWo cells were grown on BD Falcon CultureSlides (BD Biosciences, San Jose, CA) at a density of 5000 cells/cm². Nearly confluent BeWo cells (3-day-old) were briefly rinsed with ice-cold PBS and incubated with fluorescent ligands (500 nM of rhodamine-riboflavin, riboflavin, or rhodamine and 25 µg/ml iron-saturated FITC-transferrin in bathing medium) at 4°C for 2 h to allow equilibrium binding. After preequilibration, cells were washed three times with ice-cold PBS to remove unbound substrate and chased with prewarmed ligand-free bathing medium at 37°C for 10 min to allow internalization. Cells were then fixed with 4% paraformaldehyde (in PBS with 4% sucrose) at room temperature for 20 min, incubated with 300 nM DAPI for 10 min, and mounted in SlowFade antifading reagent. Samples were sealed with nail polish and examined with a Nikon Eclipse 800 fluorescent microscope (Melville, NY) equipped with FITC (_ex, 460–500 nm; _em, 505–560 nm; dichroic splitter, 505 nm), rhodamine (_ex, 530–560 nm; _em, 590–650 nm; dichroic splitter, 570 nm), and DAPI (_ex 340–380 nm; _em, 435–485 nm; dichroic splitter, 400 nm) filters. Images were captured with a Micromax cooled charge-coupled device camera (Roper Scientific, Inc., Trenton, NJ) and IPLab software (Scanalytics, Inc., Fairfax, VA) using a constant exposure time at each filter combination. Composite images were colored and assembled in Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA), with no alterations in the relative gray scale levels.

Colocalization Studies. For colocalization with LysoTracker, BeWo cells were pulsed with 500 nM rhodamine-riboflavin at 4°C for 2 h. After a three-time ice-cold PBS wash, cells were incubated with prewarmed bathing medium containing 300 nM LysoTracker Blue-White DPX at 37°C for 10 min. Cells were then immediately fixed with 4% paraformaldehyde and mounted in SlowFade.

Before immunofluorescent studies, the specificity of all primary antibodies was confirmed by Western blot analyses. Compared with the positive control cell lysates (supplied with the monoclonal antibodies by BD Biosciences), BeWo cells express high levels of clathrin heavy chain and rab5 proteins, and antibodies proved highly specific (data not shown). The absence of cross-reactivity of the secondary antibodies was also verified by omitting the primary antibodies during immunofluorescent studies.

For colocalization with clathrin or rab5 protein, BeWo cells were incubated with 500 nM rhodamine-riboflavin or 25 µg/ml iron-saturated FITC-transferrin at 4°C for 2 h and chased with prewarmed ligand-free bathing medium at 37°C for 10 or 30 min. After paraformaldehyde fixation, cells were washed twice with 25 mM glycine and permeablized with PBS containing 0.2% Triton X-100 and 1% BSA for 20 min. Clathrin or rab5 was visualized using a monoclonal mouse antihuman clathrin heavy chain or antihuman rab5 antibody (1:250; 1 h) followed by Fc-specific, FITC- or tetramethylrhodamine B isothiocyanate-labeled goat antimouse IgG (1:200; 1 h). Cells were then counterstained with 300 nM DAPI and mounted in SlowFade.

For colocalization with FITC-transferrin, cells were incubated with 500 nM rhodamine-riboflavin and 25 µg/ml iron-saturated FITC-transferrin simultaneously at 4°C for 2 h. After an ice-cold PBS wash, cells were incubated with ligand-free medium at 37°C for 15 min. Cells were immediately fixed with 300 nM DAPI for 10 min and mounted in SlowFade.

	Results

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Synthesis of Rhodamine-Riboflavin Conjugate. Compared with bioconjugation of macromolecules, direct fluorochrome labeling of small chemical entities, such as riboflavin (M_r, 376.4), is more challenging. The limited molecular size not only reduces flexibility for adduct linkage but also increases the risks of modifying the biological affinity of ligands due to impending steric hindrance. Potential conjugatable moieties in riboflavin include the hydroxyl groups of the D-ribose chain and the isoalloxazine nitrogen groups (Fig. 1). Based on our previous studies with riboflavin-structural analogs, the D-ribose side chain plays an insignificant role in the interaction between riboflavin and its placental transporter when compared with the isoalloxazine ring (Huang and Swaan, 2001). Thus, conjugation to the ribose side chain would have a minimal effect on ligand-receptor interactions.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Synthesis of rhodamine-labeled riboflavin conjugate.

Rhodamine was linked to the D-ribose chain of riboflavin using tetramethyl-rhodamine-5-carbonyl-azide. Upon heating of tetramethyl-rhodamine-5-carbonyl-azide (Fig. 1), its acyl azide group undergoes a Curtius rearrangement to yield an isocyanate group, which reacts with primary hydroxyl groups to form a carbamate linkage (Adams, 1942; Takadate et al., 1985). Since the remaining secondary nitrogen and hydroxyl groups on riboflavin are less reactive, the only primary OH on C₅ of D-ribose is expected to be the preferred target for nucleophilic attack of isocyanate. Indeed, after a 1-h reaction, only one predominant peak with significant absorbance under both riboflavin and rhodamine wavelengths was detected in our high-performance liquid chromatography analysis (data not shown). The chemical identity of the particular eluent was further verified as rhodamine-riboflavin conjugate, utilizing fluorescent scanning spectrum and mass spectrometry analyses as described under Materials and Methods.

Substrate Specificity of Rhodamine-Riboflavin Conjugate. To examine the biological specificity of rhodamine-riboflavin conjugate, we used BeWo cells, a human choriocarcinoma cell line. Previously, we reported that high-affinity riboflavin transport system is functionally expressed on the microvillous membrane of these cells (Huang and Swaan, 2001). Uptake experiments in BeWo cell monolayers revealed that 75.6% of [³H]riboflavin uptake was blocked in the presence of 1000-fold rhodamine-riboflavin, whereas rhodamine did not show significant effect (Fig. 2A). Rhodamine-riboflavin conjugate was equally effective in inhibiting [³H]riboflavin uptake compared with unlabeled riboflavin, with IC₅₀ values of 0.80 ± 0.12 and 0.97 ± 0.17 µM, respectively (Fig. 2, A and B), indicating that the conjugates have ligand affinity toward riboflavin transporter(s) comparable with that of riboflavin.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effect of rhodamine-riboflavin conjugate and rhodamine on riboflavin uptake. Uptake of [3H]riboflavin (5 nM) and [14C]mannitol (0.37 µM) were measured in the presence of 5 µM of riboflavin (Rf), rhodamine-riboflavin conjugate (Rho-Rf), or rhodamine (Rho) (A). Cells were washed twice with ice-cold acidic PBS, lysed with 1% Triton X-100, and measured for radioactivity after a 20-min uptake study. B, concentration-dependent inhibition of rhodamine-riboflavin conjugate on [3H]riboflavin uptake in BeWo cell monolayers. Uptake of [3H]riboflavin (5 nM) was measured in the presence of various concentrations of rhodamine-riboflavin conjugate (IC50, 0.80 ± 0.12 µM) or riboflavin (IC50, 0.97 ± 0.17 µM). Each value represents the mean ± S.D. for four experiments. , p < 0.001 versus controls.

Internalization and Subcellular Localization of Rhodamine-Riboflavin Conjugate. To analyze the internalization of rhodamine-riboflavin, cells were preincubated with the conjugate at 4°C for 2 h, then chased with ligand-free medium at 37°C. The subcellular distribution of rhodamine-riboflavin was visualized by fluorescence microscopy. Compared with nuclear DAPI stain, after 10 min, the rhodamine signals resided intracellularly in the perinuclear punctate regions, indicating efficient internalization and possible cytosolic accumulation of the conjugate (Fig. 3, G–I). In a parallel study, internalization of rhodamine alone was also examined to assess the basal level of nonspecific endocytosis processes in BeWo cells under the experimental condition. Contrary to the distinct localization patterns of rhodamine-riboflavin conjugate or FITC-transferrin, the majority of rhodamine signal was randomly distributed in the cells (Fig. 3, D–F). A generally weak and hazy staining was observed throughout the cells, indicating nonspecific background adsorption. Moreover, when BeWo cells were incubated with the same concentration of riboflavin, a hazy background was also observed, but distinct intracellular punctate staining patterns (Fig. 3, A–C) resembling that of rhodamine-riboflavin conjugate could also be detected.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Internalization of riboflavin, rhodamine, and rhodamine-riboflavin conjugate. BeWo cells were incubated with riboflavin, rhodamine, or rhodamine-riboflavin conjugate at 4°C for 2 h. Unbound ligands were washed off, and cells were warmed to 37°C for 10 min. Cells were then fixed immediately, incubated with nuclear DAPI stain, and processed for fluorescence microscopic analysis. Panels show distribution patterns of riboflavin (A), rhodamine (D), and rhodamine-riboflavin conjugate (G) and nuclear stain (B, E, and H) in the identical optical field. Column 3 displays the superimposed images from columns 1 and 2. Bar, 10 µm.

The specificity of rhodamine-riboflavin conjugate binding to the surface riboflavin transporter was further investigated under a microscope. In these experiments, BeWo cells were preincubated with rhodamine-riboflavin conjugate in the presence of 100 µM riboflavin at 4°C for 2 h. Compared with the cells labeled with rhodamine-riboflavin conjugate alone, significantly lower fluorescent intensities were detected in cells preincubated with both riboflavin and the conjugate (data not shown). Since excess riboflavin competed for limited riboflavin binding sites on the BeWo cell membrane (Huang and Swaan, 2001), these results provided additional support for the presence of a transporter system that specifically mediates the internalization of rhodamine-riboflavin conjugate into BeWo cells.

Colocalization Studies with FITC-Transferrin, Clathrin Heavy Chain, Rab5, and Acidotropic Marker (LysoTracker). FITC-transferrin, a well characterized receptor-mediated endocytosis substrate, was used to identify the characteristic morphology of endosomal compartments in BeWo cells. Unlike other polarized epithelia, BeWo cells express high numbers of transferrin receptor on their apical cell surface, and a specific endocytic pathway has been identified for internalization of transferrin (Cerneus and van der Ende, 1991). After 15 min, FITC-transferrin (Fig. 4C) exhibited a comparable punctate distribution as observed with rhodamine-riboflavin conjugate (Figs. 3G and 4A).

View larger version (49K): [in this window] [in a new window]   Fig. 4. Colocalization of rhodamine-riboflavin conjugate with FITC-transferrin, clathrin, rab5, and LysoTracker Blue. BeWo cells were incubated with rhodamine-riboflavin conjugate with and without FITC-transferrin at 4°C for 2 h. For cells incubated with FITC-transferrin, unbound ligands were washed off, and cells were warmed to 37°C for 15 min. Cells were then fixed immediately, incubated with nuclear DAPI stain, and processed for fluorescent microscopic analysis. For cells without FITC-transferrin, unbound ligands were washed off, and cells were warmed to 37°C for 10 min with and without LysoTracker Blue. Cells labeled with LysoTracker Blue were then fixed immediately and processed for fluorescence microscopic analysis. Remaining cells were fixed, permeabilized, and stained with anti-clathrin or anti-rab5 heavy chain monoclonal antibodies followed by FITC-labeled secondary antibody and DAPI nuclear stain. Panels show distribution patterns of rhodamine-riboflavin conjugate (A, E, I, and M), FITC-Tf (C), clathrin (G), or rab5 (K) with DAPI (B, F, and J) or LysoTracker Blue (N). Overlapping images are shown in panels D, H, L, and O. Rhodamine-riboflavin conjugate colocalization with respective markers is shown in yellow (D, H, and L) or white (O). Bar, 10 µm.

In most mammalian cells, uptake of receptor-bound ligands results mainly from clathrin-dependent endocytosis (Mellman, 1996). To investigate the potential role of clathrin in rhodamine-riboflavin internalization, we performed dual-labeling studies with monoclonal antibodies against human clathrin heavy chain. The signals of clathrin were visualized with fluorochrome-conjugated secondary antibodies. As expected, clathrin was present from the cell periphery and throughout the cell interior (Fig. 4G) (Tolbert and Lameh, 1996). After 10 min, colocalization of rhodamine-riboflavin conjugate with clathrin in the cytoplasma was observed (Fig. 4H). Consistent with colocalization of FITC-transferrin, these results suggest that the conjugate is internalized via a clathrin-mediated pathway.

In addition to clathrin and FITC-transferrin, we also used anti-rab5 antibody to further delineate the involvement of clathrin-mediated endocytosis in internalization of rhodamine-riboflavin conjugate. Rab5, a small GTP-binding protein, resides only in clathrin-coated vesicles and early endosomes (Rhodaman and Wandinger-Ness, 2000). Throughout its entire endocytic cycle, transferrin remains bound to transferrin receptor, and the majority of the complex is known to confine in early endocytic compartments (Welch, 1992). Consistent with studies by Vandenbulcke et al. (2000), rab5 signals were concentrated in vesicular compartments (Fig. 4K). After 10 min of internalization, almost all rab5-labeled regions exhibited strong rhodamine-riboflavin conjugate staining (Fig. 4, I–L).

To examine the cellular identity of the vesicular structures containing rhodamine-riboflavin, cells were treated simultaneously with rhodamine-riboflavin conjugate and LysoTracker Blue-White DPX, a membrane-diffusible probe accumulating in acidic organelles. This acidotropic marker has been used successfully to label compartments involved in endocytosis processes (Bucci et al., 2000). After 10 min, significant overlap of rhodamine-riboflavin and LysoTracker signals (as indicated by white color) revealed that the vesicular structures associated with rhodamine-riboflavin were acidic (Fig. 4, M–O).

	Discussion

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The present study demonstrates that the endocytic machinery is involved in the internalization of rhodamine-riboflavin conjugate via a highly specific riboflavin internalization mechanism. This is evidenced by 1) riboflavin-specific binding of rhodamine-riboflavin conjugate, 2) a punctate endosome-like subcellular distribution of the conjugate, 3) selective sequestration of rhodamine-riboflavin conjugate into acidic vesicular organelles, and 4) colocalization of conjugate within early endocytic compartments containing FITC-transferrin and immunostained positively for clathrin and rab5.

In contrast to the nonspecific internalization of BSA-riboflavin reported by Low and colleagues (Holladay et al., 1999), our results show that rhodamine-riboflavin conjugate significantly blocks [³H]riboflavin uptake (Fig. 2). Specific membrane binding of rhodamine-riboflavin conjugate is also substantially reduced in the presence of riboflavin. The mutual inhibition between riboflavin and rhodamine-riboflavin conjugate strongly indicates that the internalization of conjugate is mediated by membrane surface riboflavin receptors. Importantly, the preserved specificity and high affinity of rhodamine-riboflavin conjugate toward the riboflavin transporters further confirms the insignificant role of D-ribose chain in the ligand-transporter interactions (Huang and Swaan, 2001).

Receptor-mediated endocytosis plays an essential role in the selective uptake of nutrients, growth factors, and hormones into most cells (Mukherjee et al., 1997). In most cases, the endocytosis process is initiated from clathrin-coated pits, and subsequently, the ligands/receptors are internalized in clathrin-coated vesicles (Mellman, 1996). Our results show that rhodamine-riboflavin conjugate is detected in clathrin-positive clusters after short-term incubation (Fig. 4, E–H), suggesting the formation of clathrin-coated vesicles in the internalization of riboflavin. Colocalization of rhodamine-riboflavin conjugate with FITC-transferrin, a well characterized substrate of clathrin-mediated endocytosis, also supports this finding (Fig. 4, A–D). Clathrin-mediated endocytosis usually involves concentration of receptors in the clathrin-coated pits, with most receptors containing a conserved internalization motif in their cytoplasmic domain (Hirst and Robinson, 1998). Currently, it is unknown whether the present results reflect that the putative riboflavin receptor shares such a structural feature and is localized in coated pits. Further investigations are necessary to elucidate the role of clathrin in the endocytosis of riboflavin. Interestingly, we have previously shown that [³H]riboflavin uptake was not affected by phorbol-12-myristate acetate, a protein kinase C activator known to specifically block caveolae-dependent endocytosis (Smart et al., 1994; Huang and Swaan, 2001). Whether riboflavin is internalized exclusively from the clathrin-coated membrane awaits further verification.

Following the clathrin-mediated endocytosis pathway, internalized ligand-receptor complexes are delivered rapidly into early endosomal compartments (Mellman, 1996). Our results show that within 10 min of incubation, signals of rhodamine-riboflavin conjugate are noticeably accumulated within punctate perinuclear vesicular organelles, reminiscent of endosomal compartments (Figs. 3G and 4, E, I, and M). The acidic nature of many of these structures also supports our morphologic observations (Fig. 4, M–O). More importantly, colocalization of conjugate with FITC-transferrin and rab5 further confirms the molecular identities of these compartments as early endosomes (Fig. 4, A–E and I–L).

After ligand-receptor complexes are internalized into endocytic vesicles, they might undergo several sorting scenarios. The fates of ligands and receptors can vary significantly according to the specific type of receptors (Mukherjee et al., 1997). Most G-protein-coupled receptors will release their ligands in the early endosomes and then recycle back into the cell membrane, whereas others, like neurotensin receptor, will be delivered to lysosomes together with their ligands (Vandenbulcke et al., 2000). In general, clathrin-mediated endocytosis ligands are released from their receptors in the early endosome. The dissociation of ligands and receptors results from pH-dependent conformational change of receptors induced by the acidic climate of early endosomes. Previously, we have shown that dissociation of surface-bound riboflavin in Caco-2 cells is pH-dependent, with significantly higher riboflavin release at acidic pH (pH 3–5) (Huang and Swaan, 2000). Taken together, since rhodamine-riboflavin conjugate is not designed to bind to the putative riboflavin transporters irreversibly, it is likely that upon entry of early endosomes, the rhodamine-riboflavin conjugate will dissociate from the transporter. Accordingly, riboflavin transporter might not travel in parallel with the conjugate into the late endosomes.

The current observation that rhodamine-riboflavin conjugate is sorted from early endosomes to late endocytic compartments is also in line with our recent findings with endocytosis perturbants (Huang and Swaan, 2000). Previously, we showed that nocodazole, a microtubule-depolymerizing agent, significantly inhibited the apical uptake of [³H]riboflavin. Since microtubules are required for the maturation of early endosomes to late endosomes, disruption of microtubular network is expected to deter the endosomal movement (Mukherjee et al., 1997). Consequently, intracellular accumulation of riboflavin in nocodazole-treated cells would be substantially reduced. For growth factors, such as gastrin-releasing peptide receptors, late endosomal sorting and subsequently, lysosomal degradation of ligands is essential for termination of the triggered cellular response (Grady et al., 1995). At physiological concentrations (low nanomolar range), riboflavin itself does not exhibit any intrinsic activity; however, studies have reported that massive riboflavin supplement can protect against cerebral ischemic damage (Hultquist et al., 1993; Betz et al., 1994). Using cell culture and rat tissues, Daly et al. (1997) further demonstrated that riboflavin, at low micromolar concentrations, significantly inhibits adenylate cyclase and guanyl nucleotide turnover of G-proteins and is an antagonist of A₁-adenosine receptors. Currently, it is unknown whether the late endosomal sorting of riboflavin represents a regulatory mechanism against its potential pharmacological effects.

It should be noted that our present studies with riboflavin-rhodamine conjugate may not reflect the intracellular fate of native riboflavin. Like other B vitamins, riboflavin exerts its biological functions via its coenzyme forms FMN and FAD (Rivlin, 1975). As documented in hepatocytes, upon entry of the cells, riboflavin will be converted into FMN and FAD by cytoplasmic flavokinase and FMN pyrophosphatase (McCormick and Zhang, 1993). Because these biotransformation processes require a free 5'-hydroxyl group on the D-ribose chain of riboflavin, the rhodamine-riboflavin conjugate is likely to bypass the endogenous metabolic pathway.

In summary, our studies present a novel method to synthesize a rhodamine-tagged riboflavin conjugate. Since the strategy preserves the essential isoalloxazine moiety of riboflavin, the rhodamine-riboflavin conjugate successfully retains its specificity and affinity toward the putative riboflavin transport system. Using the conjugate as a probe, we report, for the first time, morphological evidence of the involvement of a classical endocytosis mechanism in the internalization of riboflavin in BeWo cell culture. Our current findings clearly identify that endocytic compartments are involved in the uptake and intracellular trafficking of riboflavin; however, they do not rule out the possible existence of a carrier-mediated transport mechanism for riboflavin in placenta or other tissues and cell lines. The results reported in this study will help guide future research intending to answer such questions and will aid in our understanding of the cellular riboflavin disposition and potential future drug targeting approaches via the riboflavin transport system.

	Footnotes

 
This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (NIDDK R01-DK56631).

DOI: 10.1124/jpet.103.051581.

ABBREVIATIONS: BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.

Address correspondence to: Peter W. Swaan, Department of Pharmaceutical Sciences, University of Maryland, 80 Penn Street, Baltimore, MD 21202. E-mail: pswaan{at}rx.umaryland.edu

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