Organic cation transport in rat choroid plexus cells studied by fluorescence microscopy

David S. Miller, Alice R. Villalobos, and John B. Pritchard

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quinacrine uptake and distribution were studied in a primary culture of rat choroid plexus epithelial cells using conventional and confocal fluorescence microscopy and image analysis. Quinacrine rapidly accumulated in cells, with steady-state levels being achieved after 10-20 min. Uptake was reduced by other organic cations, e.g., tetraethylammonium (TEA), and by KCN. Quinacrine fluorescence was distributed in two cytoplasmic compartments, one diffuse and the other punctate. TEA efflux experiments indicated that more than one-half of intracellular organic cation was in a slowly emptying compartment. The protonophore monensin both emptied that TEA compartment and abolished punctate quinacrine fluorescence, suggesting that a large fraction of total intracellular organic cation was sequestered in acidic vesicles, e.g., endosomes. Finally, quinacrine-loaded vesicles were seen to move within the cytoplasm and to abruptly release their contents at the blood side of the cell; the rate of release was greatly reduced by the microtubule disrupter nocodazole.

compartmentation; confocal microscopy; endosomes; microtubules; monensin; vesicle fusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CHOROID PLEXUS ACTIVELY transports xenobiotics and metabolic wastes from cerebrospinal fluid (CSF) to blood for subsequent elimination via urine or bile (reviewed in Refs. 5 and 8). Like kidney and liver, choroid plexus possesses a specific transport system for organic cations and weak organic bases. However, compared with kidney and liver, little is known about the cellular mechanisms that drive organic cations across choroid plexus. One reason for this is that small tissue size, complex morphology, and inaccessibility have been significant impediments to the study of transport mechanisms in choroid plexus. To circumvent these difficulties, Villalobos et al. (10) recently developed a procedure to isolate and culture choroid plexus epithelial cells from neonatal rats. By morphological, biochemical, and functional criteria, these cells were shown to grow with the apical (CSF) side facing the medium and the blood side attached to the support. Villalobos et al. (10) also demonstrated that these choroid plexus cell monolayers grown on a solid support, like choroid plexus slices from neonatal and adult rats, exhibit specific and concentrative uptake of the model organic cation tetraethylammonium (TEA). Because of the orientation of the cells in the monolayer, the uptake of TEA corresponds to the first step in transport of organic cations from CSF to blood.

In the present study, conventional and confocal fluorescence microscopy and digital image analysis were used to investigate the second step in organic cation transport across choroid plexus cells, movement through the cytoplasm. To do this, we followed the uptake and intracellular distribution of a fluorescent organic base, quinacrine. The data show that quinacrine uptake was mediated by the same process responsible for TEA uptake. Once in the cells, quinacrine partitioned between cytoplasm and an acidic vesicular compartment, which accounted for a substantial fraction of total organic cation accumulation. Quinacrine-loaded intracellular vesicles were mobile and appeared to release their contents at the blood side of the cell.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. [14C]TEA bromide (53 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Quinacrine, TEA, and nocodazole were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were obtained from commercial sources and were of the highest quality available.

Cell culture. Three- to five-day-old Fischer rats reared in the animal facility at the National Institute of Environmental Health Sciences (Research Triangle Park, NC) were used in these studies. Animals were anesthetized in the cold with CO2 before decapitation and removal of the brain. For each preparation, lateral and fourth plexuses from a total of 30-36 neonatal rats were removed and placed in ice-cold DMEM-Ham's F-12 medium (DMEM/F-12) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). Cell isolation procedures are given in detail by Villalobos et al. (10). Isolated epithelial cells were suspended in MEM with D-valine substituted for L-valine and with 10% Nu-Serum IV and growth promoters (triiodothyronine, PGE1, forskolin, and epidermal growth factor). Cells were plated at a density of 4.5 × 105 cells/cm2 on individual wells of 24-well tissue culture plates ([14C]TEA uptake), glass dual-chamber microscope slides (immunostaining), or 4 × 4-cm glass coverslips in culture dishes (quinacrine accumulation). Cells were maintained at 37°C in humidified 95% air-5% CO2. On day 3, unattached cells were removed as the initial plating medium was replaced with medium containing 5%, rather than 10%, Nu-Serum IV. From day 5 on, cells were maintained with DMEM/F-12 medium containing 5% Nu-Serum IV and growth promoters (10). Medium was changed every 2-3 days. Cells were used for experiments on days 9-11.

Conventional fluorescence microscopy. Glass coverslips (4 × 4 cm) with attached cells were mounted in covered Bionique chambers under an atmosphere of 95% air-5% CO2. The medium in the chamber was an artificial CSF (aCSF; in mM: 118 NaCl, 3 KCl, 0.7 Na2PO4, 18 NaHCO3, 2 urea, 0.8 MgCl2, 1.4 CaCl2, and 12 glucose, pH 7.4), which also contained 5 µM quinacrine and, when indicated, transport inhibitors. Experiments were carried out at room temperature. The chamber containing cells was placed on the stage of a Nikon Diaphot inverted microscope fitted with epifluorescence optics, fluorescence objectives [Nikon ×40, numerical aperture (NA) 1.4; Olympus ×60 oil, NA 1.3], a 100-W mercury lamp, and a fluorescein filter set (Nikon B-1A; 460- to 485-nm band-pass excitation filter, 510-nm dichroic filter, and 515-nm long-pass emission filter). To minimize photobleaching, a neutral density filter that passed only 1 or 10% of the excitation light was kept in the light path, and fluorescence measurements were made over periods of ~1-2 s.

Epifluorescence images were acquired through the microscope side port by use of a Hamamatsu 2400 or a Paultek charge-coupled device (CCD) video camera connected to an 8-bit video image capture card (Scion Video Image LG-3 with 4 megabytes of onboard memory) in an Apple Macintosh Centris 650 computer. The video card could capture and average up to eight full frames (640 × 480 pixels) at the video rate (30 frames/s). Incoming images were displayed on a high-resolution computer monitor (Apple) using image capture and analysis software [National Institutes of Health (NIH) Image 1.58], and 8-frame averages were computed and stored on an Olympus optical disk recorder for later analysis.

To make a measurement, dye-loaded cells in the chamber were viewed under reduced, transmitted light illumination. A field was selected, and an epifluorescence image was acquired by averaging eight frames. We have found using confocal and conventional video microscopy systems and glass capillary tubes filled with solutions of known concentrations of fluorescent solutes that the relationship between image fluorescence and dye concentration is approximately linear (Ref. 3 and Miller, unpublished data). Calibration of the present systems with quinacrine showed a similar linear relationship over a 100-fold range of concentrations, from 0.25 to 25 µM. However, with concentrations >25 µM, the relationship became nonlinear, suggesting self-quenching. For example, when the quinacrine concentration was raised from 25 to 50 µM, measured fluorescence increased by 70% rather than by 100%. Quinacrine fluorescence was also pH dependent, e..g., reducing buffer pH from 7.4 to 6.5 decreased fluorescence by 60%. Because of the many uncertainties in relating cellular fluorescence to actual compound concentration in cells with complex geometry, data are reported here as average measured pixel intensity rather than as estimated fluor concentration.

Confocal fluorescence microscopy. Cells in a Bionique chamber were mounted on the stage of a Zeiss model 410 inverted laser scanning confocal microscope and viewed through a Zeiss ×100 oil immersion objective (NA 1.4). To collect fluorescent images, quinacrine-loaded cells were illuminated by an Ar-Kr laser at 488 nm. A 510-nm dichroic filter was positioned in the light path, and a 515-nm long-pass emission filter was placed in front of the detector. In some experiments, cells were loaded with quinacrine and the mitochondrial probe rhodamine 123. These cells were illuminated by both 488- and 568-nm laser lines. A double dichroic filter was used; quinacrine fluorescence was collected through a 515- to 565-nm band-pass filter and rhodamine fluorescence through a 590-nm long-pass filter.

Each collected image was a single 8-s scan. To minimize photobleaching, images were collected at 20% laser power with neutral density filters passing only 3-30% of the light. Preliminary experiments showed that, under these conditions, tissue autofluorescence was undetectable and photobleaching was minimal, i.e., fluorescence intensities in cells were reduced by <5% in consecutive 8-s scans. Confocal images (512 × 512 × 8 bits) were viewed on a high-resolution monitor, saved to optical disk, and transferred to the Power Macintosh computer for analysis.

Image analysis. Average cellular quinacrine fluorescence was determined from stored images. Each cell was outlined, and the average fluorescence intensity was measured using NIH Image software as described previously (2). One must keep an important caveat in mind when interpreting these measurements of cellular fluorescence intensity. The fluorescence signal from a probe is sensitive to probe environment, e.g., pH and solvent polarity. As a result, the relationship between probe fluorescence and concentration could and does (see Quinacrine accumulation) vary with the region of the cell.

Two procedures were used to analyze vesicle dynamics. First, the movement of fluorescent vesicles within cells was followed over time using confocal microscopy. Cells were incubated for 30 min in medium with 5 µM quinacrine. The microscope was focused at roughly the center of the cells (z-axis), and confocal images were acquired at 10-s intervals over a total of 5 min. The motion of individual fluorescent vesicles could be followed over time by displaying the sequence as a movie. To display this three-dimensional data set in two dimensions, the stack of images was projected onto a single plane using a Silicon Graphics Octane workstation and Vox Blast software (Vaytek). This software also allowed us to treat the stack as a volume (with x, y, and time as the three dimensions) and to tilt the volume around the x-axis and visualize the displacement (tracks) of individual fluorescent vesicles through time.

Second, rates of vesicle "flashing" were measured using conventional fluorescence video microscopy. Cells were loaded in medium with 5 µM quinacrine. The plane of focus was at the cell-coverslip interface, and images of a field of one or two cells were acquired at one-half the video rate (15 frames/s) over a total of 5-10 s. To determine flashing rate, each image in a sequence was subtracted pixel by pixel from the next image (NIH Image). The resulting difference image was scaled to an average pixel intensity of 128. Flashes were evident in difference images as areas of black on a field of gray. The resulting time series of difference images could then be viewed as a movie, and the number of black areas that appeared could be counted.

TEA uptake and efflux. On day 11, uptake of [14C]TEA by cells plated on 24-well tissue culture plates was measured as described previously (10). Briefly, cells were rinsed and preincubated in aCSF for 1 h at 37°C. Transport was initiated by replacement of preincubation buffer with 1 ml aCSF containing the labeled substrate; cells were incubated for 0-90 min. All incubations were conducted at 37°C under 95% air-5% CO2. To terminate uptake, transport buffer was removed, and the cells were rinsed with 3 ml isotope-free aCSF. Within the transport well, cells were solubilized in 1 N NaOH for 1 h and neutralized with 1 N HCl. An aliquot of the solubilized cell suspension (800 µl) was retained for determination of protein by a Bio-Rad microassay using BSA as standard. The remainder of the cell suspension was transferred to a scintillation vial for counting. Cellular TEA was calculated in picomoles per milligram protein.

For efflux experiments, cells were loaded with 50 µM [14C]TEA as described above, rinsed twice with TEA-free aCSF, and incubated in TEA-free aCSF. At each sampling time, an aliquot of the efflux medium was removed, and the cells were rinsed and processed for counting as described above.

Immunostaining. Cells on glass tissue culture chamber slides (Nunc, Naperville, IL) were fixed for 10 min in 2% formaldehyde and 0.1% glutaraldehyde in PHEM buffer (in mM: 60 PIPES, 24 HEPES, 5 EGTA, and 1 MgSO4, pH 6.9), permeabilized with 1% Triton X-100 in PHEM buffer for 10 min, and exposed to a primary polyclonal, anti-tubulin antibody (rabbit; Sigma) and fluorescein-labeled secondary antibody (Kirkegaard and Perry) as described previously (10). Slides were sealed with Agua-PolyMount (Polysciences, Warrington, PA) and allowed to dry overnight in the dark. Immunostained cells were viewed using the Zeiss model 410 confocal scanning laser microscope.

Statistics. Data are given as means ± SE. Means were considered to be statistically different when P < 0.05 as determined by the appropriate paired or unpaired t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quinacrine uptake and distribution. A representative conventional epifluorescence micrograph of choroid plexus cells that had been incubated for 10 min in medium containing 5 µM quinacrine is shown in Fig. 1A. Notice that the distribution of fluorescence within the cells is not uniform. There are three components to cellular fluorescence: one is nuclear, a second is cytoplasmic and diffuse, and a third is cytoplasmic and punctate. Although punctate fluorescence is seen throughout the cytoplasm, it is most concentrated in the perinuclear region, which is the thickest part of the cell. Figure 1B shows that adding the organic cation TEA to the medium substantially reduced overall cellular fluorescence. From these micrographs, it appears that TEA had the greatest effect on the fluorescence intensity of the punctate compartment.


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Fig. 1.   Conventional epifluorescence micrographs of choroid plexus cells after 10 min of incubation in medium with 5 µM quinacrine (A) or 5 µM quinacrine + 1 mM tetraethylammonium (TEA; B).

Confocal microscopy confirmed these general features of quinacrine distribution in control cells. Comparison of differential contrast interference (DIC) and confocal images of quinacrine-loaded cells showed that each punctate site of fluorescence could be localized to a vesicular structure in the DIC image (Fig. 2). However, not all vesicular structures seen in the DIC image were fluorescent. To further characterize the vesicular compartment that accumulated quinacrine, cells were incubated to steady state in medium with 5 µM quinacrine and then exposed to 0.5 µM rhodamine 123, a fluorescent dye that accumulates in mitochondria. Figure 3 shows a representative image of double-labeled cells. The quinacrine and rhodamine 123 labeling patterns differed in two important respects: 1) punctate sites of localization appeared to be compact for quinacrine (red image), whereas they were elongated for rhodamine 123 (green image), and 2) little colocalization of the labels [which would appear as yellow pixels in the red-green-blue (RGB) image] could be seen. Clearly, mitochondria did not accumulate quinacrine.


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Fig. 2.   Differential interference contrast (left) and confocal (right) images of quinacrine-loaded choroid plexus cells.


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Fig. 3.   Double-labeled confocal image of choroid plexus cells. Cells were incubated to steady state in medium with 5 µM quinacrine and then briefly exposed to 0.5 µM rhodamine 123. This image shows quinacrine labeling in red and rhodamine 123 labeling in green. Areas of colocalization appear as yellow. Rhodamine 123 labeled elongated structures, whereas quinacrine labeled roughly circular structures. Little colocalization is evident, i.e., the 2 dyes appear to label different structures.

We acquired conventional epifluorescence images from cells incubated in medium containing 5 µM quinacrine, and from these we measured average cellular fluorescence intensity. Figure 4 shows the time course of quinacrine accumulation. Cellular fluorescence increased initially and reached a steady state within 10-20 min. At steady state, the model substrate for the renal organic cation transport system, TEA, reduced average cellular fluorescence by >50% and the metabolic inhibitor, KCN, reduced fluorescence by ~75%. Other experiments showed that average, steady-state cellular fluorescence was reduced in a concentration-dependent manner by TEA (Fig. 5A). Significant reductions in steady-state cellular fluorescence were also seen with verapamil, darstine, paraquat, and tetrapentylammonium (TPA), all organic cations (Fig. 5). The organic anion p-aminohippurate, at 1 mM, was without effect (not shown). From these data, we can construct a rough order of inhibitory potency: verapamil = paraquat = TPA > TEA > darstine. We verified certain of these results using confocal microscopy. Cells were incubated in medium with 5 µM quinacrine without or with 100 µM TEA or 100 µM TPA, and confocal images were acquired after 30 min. Average, steady-state cellular fluorescence intensity was reduced 44 ± 3% by TEA and 55 ± 2% by TPA; these values are similar to those seen in experiments with conventional optics. In those cultures exposed to TEA or TPA, confocal microscopy showed that fluorescence intensity in both the diffuse and punctate compartments was reduced relative to controls. For example, in control cells, fluorescence intensities in the cytoplasm and in the punctate compartment averaged 57 ± 6 and 176 ± 19 fluorescence units, respectively; in cells incubated with 100 µM TEA, corresponding values were 35 ± 4 and 95 ± 10 fluorescence units (both significantly lower than controls, P < 0.01).


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Fig. 4.   Time course of quinacrine accumulation by choroid plexus cells. Cells were incubated in medium with 5 µM quinacrine, without (control) or with TEA or KCN. At times indicated, conventional fluorescence images were acquired. Results (means ± SE of fluorescence intensity for 30-47 cells) are from 1 experiment representative of experiments carried out with 2 cultures. TEA and KCN significantly reduced fluorescence intensity at all times tested (P < 0.01).


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Fig. 5.   Inhibition of quinacrine uptake by organic cations. Cells were incubated for 30 min in medium with 5 µM quinacrine, without (control) or with indicated additions (concentrations in µM). Each set of bars shows results (means ± SE of fluorescence intensity for 35-50 cells) from 1 experiment representative of experiments carried out with at least 2 cultures. All organic cations tested significantly reduced fluorescence intensity (P < 0.01). TPA, tetrapentylammonium; VERAP, verapamil; DARST, darstine; PARAQ, paraquat.

It is important to note that, for these choroid plexus cells in culture, the same general order of inhibitor effectiveness was found in the present experiments in which quinacrine accumulation was followed with fluorescence microscopy and in experiments in which [14C]TEA uptake was measured (10). This agreement and the finding that quinacrine accumulation was reduced by TEA suggest that uptake of quinacrine and TEA may be mediated by the same carrier protein. To investigate this point further, we determined the effects of quinacrine on the 30-min uptake of 10 µM [14C]TEA. Quinacrine at 50 and 100 µM reduced TEA uptake by 18 and 40%, respectively. Taken together, the inhibition data for quinacrine accumulation (present study) and for TEA uptake (Ref. 8 and present study) indicate that choroid plexus cells grown on solid supports accumulate both substrates through a common transporter specific for organic cations.

Organic cation efflux. Both conventional and confocal fluorescence images of quinacrine distribution in choroid plexus cells showed that a substantial fraction of cellular fluorescence was associated with a punctate compartment. Previous studies from this laboratory suggest an identity for that compartment. Using endosomes isolated from rat renal cortex, Pritchard et al. (6) demonstrated organic cation (TEA) uptake that was specific and ATP dependent and was abolished by protonophores. The mechanism of organic cation accumulation involved acidification of the intraendosomal space by a proton ATPase and TEA/proton exchange. To determine whether accumulation by an acidic vesicular compartment might contribute to organic cation accumulation in choroid plexus cells, we loaded cells to steady state in medium with 10 µM [14C]TEA, removed the cells to TEA-free medium, and followed cell TEA content for 30 min in the absence (control) and presence of the protonophore monensin. Figure 6 shows that, in controls, TEA efflux was initially rapid but then slowed. From 15 to 30 min, little additional TEA was lost from the cells, and <50% of total cellular TEA had been lost to the bath after 30 min. In contrast, when monensin was added 10 min into the experiment, TEA efflux subsequently increased, and at 30 min the total amount lost was twice that in cells not exposed to monensin (Fig. 6). Even without detailed kinetic analysis, the control data indicate that about two-thirds of cellular TEA was in a cellular compartment from which efflux was very slow. Monensin rapidly released TEA from that compartment.


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Fig. 6.   Time course of efflux of TEA from choroid plexus cells. Cells were first loaded with 50 µM [14C]TEA. After 30 min, they were washed twice in TEA-free medium and then incubated in TEA-free medium; in 1 set of cultures, 5 µM monensin was added at 10 min (arrow). TEA content of medium samples and cell extracts was determined by liquid scintillation counting, and data (means ± SE of 3 experiments) are expressed as amount of TEA remaining in cells. Monensin significantly reduced TEA content at both times tested (P < 0.01).

Fluorescence microscopy of quinacrine-loaded cells incubated in quinacrine-free medium showed that monensin had two effects on intracellular fluorescence distribution patterns (Fig. 7). First, monensin rapidly increased overall cellular fluorescence. Preliminary measurements of average cellular fluorescence showed that it had increased two to three times within 2 min of monensin exposure. This increase in cellular fluorescence occurred even though cells were in quinacrine-free medium. Second, monensin greatly reduced vesicular fluorescence (Fig. 7). Because quinacrine fluorescence is quenched by low pH and at high concentrations (see Conventional fluorescence microscopy), the increase in overall cellular fluorescence caused by monensin most likely reflects alkalinization of the vesicular compartment by the protonophore and movement of quinacrine into the cytoplasm, where fluorescence was no longer quenched.


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Fig. 7.   Effects of monensin on quinacrine fluorescence in choroid plexus cells. Cells were loaded for 30 min in medium containing 5 µM quinacrine and then transferred to quinacrine-free medium with no additions (control) or with 20 µM monensin. A: conventional epifluorescence image of control cells 5 min after transfer. Substantial diffuse and punctate fluorescence is evident. B: conventional epifluorescence image of monensin-treated cells 5 min after transfer. Note loss of punctate fluorescence and increase in overall cellular fluorescence intensity.

Vesicle dynamics. To document the movements of quinacrine-containing intracellular vesicles, choroid plexus cells were incubated to steady state in medium with 5 µM quinacrine and then observed using fluorescence microscopy. Two time frames were employed: in the slow time frame, events occurring over tens of seconds to minutes were followed; in the rapid time frame, events occurring over tens of milliseconds were followed.

Confocal microscopy was used to record the slow movement of fluorescent vesicles. In these experiments, a single optical section was taken every 3-10 s at a level of the cell that was 2-4 µm above the coverslip, roughly at the middle of the cell. These time sequences were displayed sequentially on the computer monitor as a movie showing vesicle dynamics within a narrow volume of focus (depth <1 µm). About 10-30% of the vesicles moved little in recordings lasting 2-5 min. The remaining vesicles showed several types of movement. About 50% of the vesicles exhibited saltatory movements, which resulted in no obvious net change in position within an optical slice. About 20-40% of the vesicles clearly changed position. Some were lost from view, suggesting that they had moved out of the optical section. Some appeared suddenly, suggesting that they had moved into the section from a volume of cytoplasm above or below. Others moved within the plane. For some of these, the path of movement through the cytoplasm appeared to be nearly linear, suggesting vesicle movement along a cytoskeletal element.

Figure 8 shows some elements of vesicle motion from one time sequence of confocal images. The first image gives the position of vesicles at the start of the sequence (Fig. 8A). The second image is a projection of all 30 images onto a single plane; it shows the integrated movement of these vesicles over 90 s as lines or tracks (Fig. 8B). Also shown are plots of the movement of four of the vesicles (Fig. 8C) and of their linear velocities for each sampling period (Fig. 8D). Vesicles 1 and 2 show directed movement with a total displacement in the 10- to 20-µm range. Vesicles 3 and 4 show little net displacement but rather appear to move within a restricted area. In spite of the differences in net displacement, linear velocities calculated for each sampling period are comparable for the four vesicles. Clearly, this representative sequence of confocal images shows that, on the time scale of tens of seconds, a substantial fraction of quinacrine-loaded vesicles move within the cytoplasm.


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Fig. 8.   Vesicle motion within quinacrine-loaded choroid plexus cells. Cells were incubated for 30 min in medium with 5 µM quinacrine. Confocal images of a single field were acquired at 3-s intervals for a total of 1.5 min (plane of focus at nucleus; 1.4-s scans of a 174-line region of interest). A: first image of time sequence. Numerous fluorescent vesicles are evident in cytoplasm. B: a projection of all 30 images onto a single plane. Vesicle motion within optical section is seen as a series of white tracks, some of which extend for several µm. C: analysis of movement of individual vesicles. Four vesicles were selected (vesicles 1-4), and their positions were followed and plotted. D: linear velocities of vesicles 1-4 for each consecutive sampling period.

In initial experiments concerned with more rapid elements of vesicle dynamics, we used conventional epifluorescence optics to follow cells for periods of 5-10 s under continuous illumination from a Hg lamp (images collected at 15 frames/s). With appropriate neutral density filters to attenuate the light and a sensitive CCD camera to monitor cell fluorescence, little photobleaching of quinacrine was observed. In the course of these experiments, we were surprised to see repeated, local, transient spreading of fluorescence that appeared to arise from some of the vesicles. These were most evident when a high-numerical-aperture objective was focused at the level of the cell-glass support interface. In these polarized cells, the plane of focus was at the plasma membrane that would face the blood side of the choroid plexus epithelium. Because of the transient nature of these changes in fluorescence pattern and because of an initial increase in local fluorescence, we called the phenomenon flashing. Figure 9 shows a representative sequence of video frames acquired at 15 frames/s. The arrows indicate areas in which flashes have occurred. The flashes appeared to arise from single vesicles or clusters of vesicles and remained visible for two or three frames (100-200 ms). Close examination of electronically magnified image sequences showed that the phenomenon involved two phases, an initial increase in fluorescence followed by a rapid decline (Fig. 10A). Other vesicles in close proximity do not appear to be altered (Fig. 10, B-F).


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Fig. 9.   Sequence of video images demonstrating phenomenon of "flashing." Cells were incubated for 30 min in medium with 5 µM quinacrine and viewed continuously with conventional epifluorescence optics. Plane of focus was at cell-coverslip interface. Images were acquired at rate of 15 frames/s for 7 s and numbered sequentially. Arrows point to regions within cell where fluorescence intensity has transiently increased.


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Fig. 10.   Fate of single quinacrine-loaded vesicles. Top: sequence of images shows a magnified and enhanced region from a time series collected as in Fig. 7. Arrow points to a vesicle that exhibits an increase in fluorescence intensity followed by an abrupt decrease. Notice that fluorescence intensities of neighboring vesicles appear unchanged. Bottom: vesicle fluorescence (average measured pixel intensity) vs. time for vesicle indicated by arrow at top (A) and other vesicles nearby (B-F). Increase in fluorescence intensity in A is limited by signal saturation at a value of 255. Broken line in A indicates background level of diffuse cytoplasmic fluorescence.

Confocal microscopy was used to characterize the flashing phenomenon further. Although confocal optics increase spatial resolution in three dimensions and allow optical sectioning of cells, for most confocal systems the price one pays for enhanced spatial resolution is limited temporal resolution. For our microscope, useful full-frame images (512 scan lines) of quinacrine-loaded choroid plexus cells could only be obtained at a rate of one every 2-4 s, too slow to be able to investigate the flashing seen with conventional optics. However, fluorescence from a single laser scan line could be imaged on a millisecond time scale. Figure 11 shows the results of scanning, at high magnification, a single line across a quinacrine-loaded choroid plexus cell every 5 ms for a total of 2.5 s. In this experiment, the microscope was focused at the interface between the cell and the coverslip, and the confocal pinhole was closed down so that the axial resolution of the system was maximal (0.5-1 µM). The position of the scan line was chosen to include several fluorescent vesicles, and each vertical line shows the history of one or more vesicles over 2.5 s (512 scans). Many of the lines appear uninterrupted, with little change in fluorescence intensity or position with time. However, several show an abrupt broadening followed by a sharp decline in fluorescence. The broadening represents the spread of fluorescence into the cytoplasm surrounding the vesicle. In some cases, punctate fluorescence is no longer detectable after this broadening. Fluorescence intensity scans of single-vesicle tracks are shown in Fig. 12. Each track shows a roughly constant level of fluorescence interrupted by a sharp decline. A small, short-lived increase in local fluorescence is evident just before the decline. Note that no flashing was evident when the plane of focus was raised several micrometers into the cells (not shown). These confocal images show the same vesicle-derived flashing seen with conventional optics; they again demonstrate that the phenomenon was localized to the region near or on the membrane at the blood side of the cells.


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Fig. 11.   Confocal analysis of flashing. Repeated scan of a single line across a quinacrine-loaded cell. Plane of focus was at cell-coverslip interface, and confocal pinhole was narrowed to give a thin optical section (<1 µm in depth with 1.4-numerical-aperture, ×100 objective used). Scans are 2.5 ms apart. Vesicle histories appear as vertical lines. Each flash is evident as a lateral spreading of fluorescence followed by an abrupt decrease in fluorescence intensity.


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Fig. 12.   Scans of intensity vs. time for selected single vesicles from Fig. 11.

Using conventional fluorescence optics, the video system, and digital image analysis, we collected time series of fluorescent images at 15 frames/s and, from these, measured the rate of flashing in fields of choroid plexus cells. In all of these experiments, the microscope was focused at the interface between the cells and the coverslip support. To determine the rate of flashing within a time series, each image was subtracted pixel by pixel from the next image in the series. Flashes showed up in the difference images as areas of black on a field of gray. The resulting time series of difference images could then be viewed as a movie. The number of black spots arising during this movie was taken as the number of flashes. Figure 13 shows flashing rates from a typical experiment in which choroid plexus cells were incubated in medium with 5 µM quinacrine and 2- to 5-s time series were acquired at various times. The rate of flashing increased over the first 15 min of exposure to quinacrine and then reached a plateau. On the plateau, the rate of flashing was ~6 s-1. In 53 experiments using 32 coverslips from 8 cultures, the average control rate after 15-45 min of exposure to quinacrine ranged from 3.5 to 8.2 s-1, with a grand mean of 5.3 s-1.


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Fig. 13.   Analysis of flashing rate using conventional fluorescence microscopy. Cells were incubated for time indicated in medium with 5 µM quinacrine and viewed with conventional epifluorescence optics. Plane of focus was at cell-coverslip interface. At each time, images were acquired at rate of 15 frames/s for 5-7 s. To determine flashing rate, each image in a sequence was subtracted pixel by pixel from next image. Resulting difference image was scaled to an average pixel intensity of 128. Flashes were evident in difference images as areas of black on a field of gray. Resulting time series of difference images could then be viewed as a movie, and black areas were counted. A: representative pair of sequential images (left and middle) and resulting difference image (right). Arrow, flash region. B: rate of flashing as a function of time in medium with quinacrine. Data are from 1 experiment that is representative of experiments carried out with 3 cultures. Each point gives flashing rate for a single 5- to 7-s sequence of 75-105 images.

We previously speculated that endosome recycling, guided by cytoskeletal elements, may contribute to organic cation secretion in renal proximal tubule (6). To determine whether flashing in organic cation-loaded vesicles involved microtubules, we loaded choroid plexus cells to steady state in medium with 5 µM quinacrine, exposed the cells to nocodazole, a microtubule disrupter, and determined rates of flashing. Figure 14A shows that 20 µM nocodazole caused a time-dependent decrease in flashing rate. A significant reduction in the rate was seen after 20 min, and by 30 min the rate was reduced by 70%. Although 20 µM nocodazole reduced flashing by >50%, it had only a small effect on average cellular fluorescence (10-20% reduction; data not shown). Figure 14B shows that nocodazole effects on flashing were concentration dependent, with a significant reduction occurring at 5 µM and a further reduction at 20 µM.


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Fig. 14.   Effects of nocodazole on flashing rate. A: cells were preincubated in medium containing 5 µM quinacrine. After 20 min, a short time sequence of images was acquired as described in Fig. 11, and then nocodazole was added to a concentration of 20 µM. Additional sequences were acquired at times indicated. B: cells were preincubated in medium containing 5 µM quinacrine for 20 min. Then 0-20 µM nocodazole was added, and image sequences were acquired after 30 min. All image sequences were analyzed as described in Fig. 11. Each point represents mean ± SE of flashing rate for 4-6 cultures.

To establish that the concentrations of nocodazole used here actually disrupted microtubules in choroid plexus cells, we incubated cells for 30 min in medium without (control) and with 5-10 µM nocodazole, fixed and permeabilized the cells, and immunostained them using an anti-tubulin primary antibody and a fluorescein-labeled secondary antibody. Antibody distribution was visualized using confocal microscopy. Control cells exhibited a well-developed filamentous network of microtubules (Fig. 15A). Nocodazole at 5 and 10 µM caused nearly total disruption of this network (Fig. 15, B and C). Other experiments with cells labeled with anti-actin antibodies showed that nocodazole did not disrupt microfilaments (not shown). Thus the action of this drug appeared to be specific for microtubules.


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Fig. 15.   Immunostaining of tubulin in control and nocodazole-treated choroid plexus cells. Cells were exposed to 0 (A), 5 (B), or 10 (C) µM nocodazole for 20 min, fixed, and then immunostained for tubulin (see MATERIALS AND METHODS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quinacrine accumulation. It is well established that choroid plexus epithelial cells possess one or more specific transporters for organic cations on the apical (CSF-facing) plasma membrane and that these contribute to the transport of positively charged xenobiotics and metabolites from CSF to blood (5, 8). However, little is known about subsequent steps in transport. In the present study, we used a recently developed primary culture of rat neonatal choroid plexus epithelial cells, a fluorescent organic cation, fluorescence microscopy, and image analysis to investigate the mechanisms by which organic cations move across the cellular compartment. Conventional and confocal microscopy showed that the distribution of quinacrine fluorescence within choroid plexus cells was not uniform. Quinacrine was detected in both the nucleus and cytoplasm. Within the cytoplasm, fluorescence was distributed between diffusive and punctate compartments. The latter appeared to be vesicular, and the fluorescence intensity in the vesicular compartment was substantially higher than in bulk cytoplasm, suggesting accumulation of quinacrine by these vesicles. A similar pattern of vesicular quinacrine accumulation was also found in intact choroid plexus from adult and neonatal rats (Miller, unpublished observations). It is not surprising to find a complex intracellular distribution pattern for quinacrine, since this weak organic base has been shown to bind to a variety of polyanions, including RNA, DNA, and ATP, and to accumulate within acidic intracellular compartments (see, e.g., Refs. 4, 11, 13).

Apical uptake of quinacrine (average cellular fluorescence) by choroid plexus cells was specific and energy dependent. Uptake was reduced by several organic cations, including TEA, but not by the organic anion p-aminohippurate. The order of inhibitory effectiveness of the tested organic cations on total quinacrine uptake paralleled that found for uptake of [14C]TEA by Villalobos et al. (10). In addition, we found that TEA reduced quinacrine uptake and that quinacrine reduced [14C]TEA uptake (present study). Finally, analysis of confocal micrographs showed that TEA and TPA reduced quinacrine fluorescence in both the cytoplasmic and vesicular compartments. Taken together, these results are consistent with TEA and quinacrine sharing a common, organic cation-specific entry step at the apical (CSF) side of the choroid plexus cells.

In addition to providing direct evidence for quinacrine accumulation in a vesicular compartment, the present results also suggest intracellular compartmentation for the model organic cation TEA. [14C]TEA efflux experiments indicated that substantially more than one-half of organic cation within choroid plexus cells was in a slowly exchanging compartment that could be the same vesicular compartment that accumulated quinacrine. By themselves, these findings have important implications with regard to the energetics of organic cation transport in choroid plexus. They argue that the cell-to-medium concentration ratio calculated from tracer uptake experiments greatly overestimates the actual cytoplasm-to-medium concentration ratio. As a result, cells would be directing less metabolic energy to organic cation uptake at the apical plasma membrane and more to uptake by vesicles. In addition, the actual organic cation concentration gradient available to drive transport from cytoplasm to blood should be less than that predicted based on average cellular concentration, possibly requiring a higher than expected direct or indirect input of metabolic energy to overcome the electrical potential energy barrier to efflux at the blood side plasma membrane.

What is the nature of the vesicular compartment for organic cations? Previous experiments with isolated endosomes from rat renal cortex and liver showed ATP-dependent accumulation of organic cations (6, 9). Data for endosomes from both tissues were consistent with organic cation accumulation being due to proton/organic cation exchange that was energetically coupled to a proton ATPase that acidified the vesicle interior. Membrane-permeable protonophores, e.g., monensin, both collapsed the pH gradient across the endosomal membrane and greatly reduced organic cation uptake (6, 9). In the present experiments with choroid plexus cells, we used monensin as a tool to implicate an acidic intracellular compartment in TEA and quinacrine sequestration. TEA efflux from preloaded cells was resolved into a fast component and a very slow component, with the latter being the larger of the two. More than one-half of the TEA in this slow cellular compartment was rapidly released by monensin, suggesting that TEA was sequestered in an acidic intracellular compartment. It is important to note that, unlike quinacrine, which is a weak base, TEA is an organic cation. Accumulation of TEA in an acidic compartment cannot be driven by nonionic diffusion followed by pH trapping of the conjugate acid. Rather, the present data for choroid plexus cells suggest that, as in kidney (6) and liver (10), TEA accumulation in the acidic compartment is specific, possibly mediated by a proton/organic cation exchanger.

As seen in similarly designed quinacrine efflux experiments, monensin effects on quinacrine fluorescence appeared to be more complicated. Monensin caused a rapid increase in cellular fluorescence followed by a decline. In monensin-treated cells, punctate sites of intense fluorescence were greatly reduced. During the efflux phase of the experiment, the cells were in quinacrine-free medium, so the increase in cellular fluorescence could not be due to increased uptake. The data are consistent with the following explanation. The immediate effect of monensin was to collapse the pH gradient across the vesicle membrane. This had two consequences. First, because quinacrine fluorescence is quenched by low pH (see Conventional fluorescence microscopy), the intrinsic fluorescence of the dye increased and total cellular fluorescence rose. Second, with the increase in pH of the vesicle interior, the driving force for quinacrine accumulation was dissipated, and quinacrine diffused into the cytoplasm and then rapidly out of the cells; an additional increase in intrinsic fluorescence could have occurred as dye was released (relief of self-quenching). Thus, in TEA and quinacrine efflux experiments, monensin effects can be interpreted within the context of the ionophore releasing sequestered organic cation from an acidic, vesicular compartment, e.g., endosomes.

Note that the results of the quinacrine efflux experiments indicate that for this fluorescent organic cation measurements of average vesicular fluorescence clearly underestimate total cell quinacrine content. As a result, imaging should not be used to quantitate the actual total uptake of quinacrine by choroid plexus cells or the distribution of quinacrine among the various cellular compartments. For quinacrine, imaging should only be considered to be a semiquantitative tool with which to characterize uptake and intracellular distribution.

Vesicle dynamics. Fluorescence microscopy and image analysis have also allowed us to investigate vesicle dynamics within choroid plexus cells. In these experiments, only those vesicles that concentrated the fluorescent organic cation, quinacrine, were visible. At present, we do not know whether other vesicle populations within these cells behave similarly. On a slow time scale (tens to hundreds of seconds), many quinacrine-loaded vesicles were seen to move in three dimensions. Two types of vesicle motion were observed: saltatory motion within a limited volume and directed movements over several micrometers. We have observed similar movement of vesicles in experiments with choroid plexus slices from adult rats. Preliminary experiments with intact tissue and cells in culture showed that vesicle motion was reduced following exposure to the microtubule disrupter nocodazole (Miller, unpublished observations). Further experiments are needed to determine the mechanistic significance of this result. However, it could signify that some of the vesicles that accumulate organic cations are associated with microtubules and that the microtubules and associated molecular motors provide the respective tracks and engines that direct vesicle motion (1, 7, 12).

An unexpected finding of the present study was the phenomenon we have called flashing. This was revealed in time series of images acquired using both wide field and confocal fluorescence microscopy. Flashing appeared as an abrupt but transient increase in fluorescence around a quinacrine-loaded vesicle, followed by a spread of fluorescence through the immediate area. Each event (local increase in fluorescence followed by the spread of fluorescence) occurred on a time scale of 100-200 ms. After the flash, the fluorescence intensity of the original vesicle was abolished or greatly diminished, although fluorescence intensities in adjacent vesicles remained unchanged. Confocal microscopy confirmed that flashing occurred in the immediate vicinity of the basal (blood side) plasma membrane but not several micrometers into the cell interior. Thus it is unlikely that these flashes represent release of vesicle contents due to photodynamic damage. Preliminary experiments with choroid plexus cells loaded with another fluorescent organic cation, daunomycin, also revealed occasional vesicle-derived, abrupt spreads of fluorescence; these were, however, more difficult to detect, since daunomycin fluorescence is relatively insensitive to pH and there was no initial increase in fluorescence (Miller, unpublished findings). No such phenomenon was seen in cells that were not exposed to fluorescent organic cations. Finally, nocodazole both disrupted the microtubular network and reduced the rate of flashing. We interpret these observations to mean that quinacrine was rapidly lost from vesicles at the basal pole of choroid plexus cells and that this loss was microtubule dependent.

We recently proposed a sequence of events whereby endosomal sequestration of organic cations could contribute to transepithelial transport in renal proximal tubule (6): 1) endosomes, which are known to cycle between the luminal membrane and intracellular organelles, concentrate organic cations from the cytoplasm by an ATP-dependent process, and 2) endosomal contents are delivered to the urinary space when the endosomes fuse with the luminal membrane. On the basis of the present results, we suggest that such a vesicle-based transport mechanism could contribute to the flux of organic cations from CSF to blood in choroid plexus. In choroid plexus cells, a substantial fraction of organic cation (TEA and quinacrine) was sequestered within an acidic vesicular compartment. The organic cation-containing vesicles were seen to move within the cells. Vesicles near the plasma membrane at the blood side of the cells appeared to release their contents; release was not seen in vesicles removed from the plasma membrane. Finally, the effects of nocodazole on microtubule integrity, vesicle motion, and quinacrine release suggest that microtubules are involved in both phenomena, perhaps serving as tracks along which vesicles coupled to molecular motors might move. Although these results are indeed consistent with membrane fusion and exocytosis, additional experiments are required to 1) definitively establish vesicle-membrane fusion as the source of flashing and 2) directly demonstrate that the vesicular phenomena reported here contribute to the flux of organic cations from CSF to blood.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. S. Miller, LPC, NIH/NIEHS, PO Box 12233, Research Triangle Park, NC 27709 (E-mail: miller{at}niehs.nih.gov).

Received 15 April 1998; accepted in final form 13 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Cole, N. B., and J. Lippincott-Schwartz. Organization of organelles and membrane traffic by microtubules. Curr. Opin. Cell Biol. 7: 55-64, 1995[Medline].

2.   Miller, D. S. Daunomycin secretion by killifish renal proximal tubules. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R370-R379, 1995[Abstract/Free Full Text].

3.   Miller, D. S., and J. B. Pritchard. Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1470-R1477, 1991[Abstract/Free Full Text].

4.   Mitchell, C. H., D. A. Carre, A. M. McGlinn, R. A. Stone, and M. M. Civan. A release mechanism for stored ATP in ocular ciliary epithelial cells. Proc. Natl. Acad. Sci. USA 95: 7174-7178, 1998[Abstract/Free Full Text].

5.   Pritchard, J. B., and D. S. Miller. Mechanisms mediating renal secretion of organic anions and cations. Physiol. Rev. 73: 765-796, 1993[Free Full Text].

6.   Pritchard, J. B., D. B. Sykes, R. Walden, and D. S. Miller. ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F966-F976, 1994[Abstract/Free Full Text].

7.   Sheetz, M. P. Microtubule motor complexes moving membranous organelles. Cell Struct. Funct. 21: 369-373, 1996[Medline].

8.   Suzuki, H., T. Terasaki, and Y. Suguyama. Role of efflux across the blood-brain barrier and blood-cerebrospinal fluid barrier on the disposition of xenobiotics in the central nervous system. Adv. Drug Delivery Res. 25: 257-286, 1997.

9.   Van Dyke, R. W., E. D. Faber, and D. K. Meijer. Sequestration of organic cations by acidified hepatic endocytic vesicles and implications for biliary excretion. J. Pharmacol. Exp. Ther. 261: 1-11, 1992[Abstract].

10.   Villalobos, A. R., J. T. Parmelee, and J. B. Pritchard. Functional characterization of choroid plexus epithelial cells in primary culture. J. Pharmacol. Exp. Ther. 282: 1109-1116, 1997[Abstract/Free Full Text].

11.   Wadsworth, S. J., A. R. Spitzer, and A. Chander. Ionic regulation of protein chemical (pH) and electrical gradients in lung lamellar bodies. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L427-L436, 1997[Abstract/Free Full Text].

12.   Walker, R. A., and M. P. Sheetz. Cytoplasmic microtubule-associated motors. Annu. Rev. Biochem. 62: 429-451, 1993[Medline].

13.   White, P. N., P. R. Thorne, G. D. Housley, B. Mockett, T. E. Billett, and G. Burnstock. Quinacrine staining of marginal cells in the vascularis of the guinea pig cochlea: a possible source of extracellular ATP? Hear. Res. 90: 97-105, 1995[Medline].


Am J Physiol Cell Physiol 276(4):C955-C968