Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets
Department of Physiology, University of Otago, New Zealand
* Author for correspondence at present address: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 (e-mail: odonnell{at}mcmaster.ca)
Accepted 3 October 2005
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Summary |
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Key words: p-glycoprotein, multidrug resistance associated protein, Malpighian tubule, confocal microscopy
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Introduction |
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P-glycoproteins in insects merit study because such transporters may
contribute to insecticide resistance. Organochlorine and organophosphorous
pesticides such as chlorpyrifos bind to P-gp, and exposure to such compounds
increases MDR gene expression
(Bain and LeBlanc, 1996;
Lanning et al., 1996
).
Exposure to the P-gp inhibitor verapamil increases the toxicity of ivermectin
in chironomids (Podsiadlowski et al.,
1998
) and the toxicity of three insecticide classes (cypermethrin,
ivermectin and endosulphan) in mosquitoes
(Buss et al., 2002
).
A few studies have examined the possible role of P-gps and MRPs in the
Malpighian tubules (MTs) of insects. Although there are, to date, no
functional studies of P-gps in the MTs of the fruit fly Drosophila
melanogaster, three MDR genes have been identified
(Wu et al., 1991;
Gerrard et al., 1993
), and
deduced amino acid sequences are
40% identical to those of mammalian
homologs. P-glycoprotein-like transporters in the MTs of the tobacco hornworm,
Manduca sexta, transport P-gp substrates such as nicotine and
vinblastine into the lumen, and transport is reduced by the P-gp inhibitor
verapamil (Gaertner et al.,
1998
). Immunofluorescence studies have also indicated the presence
of P-gp-like proteins in the MTs (Murray
et al., 1994
). However, although fluorescent P-gp substrates such
as daunorubicin (daunomycin) and rhodamine 123 are taken up into the tubule
cells of Manduca, these fluorochromes do not appear in the lumen of
isolated tubules examined using confocal laser scanning microscopy (CLSM;
Gaertner and Morris, 1999
).
These authors suggested that reflection, refraction or absorption of exciting
or emitted wavelengths by structures such as uric acid crystals in the lumen
may have prevented detection of the fluorescent compounds in the lumen.
MRPs have been identified in the MTs of the cockroach Periplaneta
americana and the cricket Acheta domesticus (Karnaky et al.,
2000,
2001
,
2003
). MTs of both species
transport the fluorescent MRP2 substrate Texas Red (sulphorhodamine 101)
through the cells and into the lumen. Confocal microscopic studies of isolated
MTs indicate that the concentration of the fluorochrome in the lumen is
dependent upon metabolism and is inhibited by the non-fluorescent MRP2
substrate chlorodinitrobenzene. An ortholog of human MRP genes has
been identified in Drosophila, although transport of MRP substrates
by the MTs has not been tested. The Drosophila MRP (dMRP)
gene contains 19 exons that are alternatively spliced, resulting in multiple
isoforms (Tarnay et al.,
2004
).
The present study describes secretion of fluorescent P-gp and MRP substrates by the MTs of two insect species. Isolated tubules were set up in Ramsay secretion assays and nanolitre samples of secreted fluid were collected in optically flat glass capillaries. The concentration of fluorescent P-gp and MRP substrates was determined using CLSM. The analysis of secreted droplets circumvents many of the problems associated with analysis of isolated tubules, in which the presence of opaque concretions in the cells and/or lumen interferes with CLSM. In addition, measurements of fluid secretion rate during flux measurements permit non-specific toxicity of inhibitors of P-gps and MRPs, or of high concentrations of the fluorescent compounds themselves, to be distinguished from specific inhibition of multidrug-resistant transporters. We report here the first measurements of the transepithelial flux for P-gp and MRP2 substrates across isolated MTs. Flux is calculated as the product of fluid secretion rate, determined in the Ramsay assay, and the concentration of the compound of interest in the secreted fluid. We have determined kinetic parameters (Jmax and Kt) for transport of P-gp and MRP2 substrates. We also show that the method is suitable for fluorescent substrates of other transporters, such as those involved in transepithelial secretion of organic anions such as fluorescein and organic cations such as quinacrine.
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Materials and methods |
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Crickets (Teleogryllus commodus Walker) were obtained from Biosuppliers (Auckland, New Zealand) and were maintained on ground Purina rat chow and water ad libitum, supplemented with lettuce leaves. Tubules were dissected from last instar larvae and adult males under saline containing (in mmol l1) NaCl (100), KCl (8.6), CaCl2 (1.5), MgCl2 (8.5), NaHCO3 (4), NaH2PO4 (4.0), Hepes (25), L-glutamate (10), sucrose (56) and glucose (24). Saline was titrated to pH 7 with NaOH.
Ramsay assays and collection of fluid secreted by isolated Malpighian tubules
Isolated tubules of each species were transferred to 20 µl droplets of
saline under paraffin oil for Ramsay assays. One tubule of each pair of
isolated Drosophila tubules was pulled out of the bathing droplet and
wrapped around a steel minuten pin (0.15 mm diameter) stuck into the
SylgardTM bottom of the assay dish. The lower tubule and ureter were
positioned outside of the bathing saline, so that the composition of the
secreted fluid was determined by transport activity of the main segment only.
The lower tubule was readily identified by the absence of stellate cells.
Secreted droplets formed at the end of the ureter and were collected after
4560 min with a fine glass probe. Droplet diameters (d) were
measured using an ocular micrometer, and droplet volume (nl) was calculated as
d3/6. Secretion rate (nl min1) was
calculated by dividing the droplet volume by the time (min) over which the
droplet formed.
The blind end of each cricket tubule was placed in the saline droplet and the open end, formed where the tubule was broken from its junction with the ampulla, was wrapped around a steel pin. The tubule was then squeezed between forceps at a point halfway between the pin and the droplet, thereby rupturing the tubule wall and permitting the escape of secreted fluid.
For analysis by CLSM, secreted droplets were collected in optically flat hollow rectangle glass capillaries (VitroTubes; VitroCom, Mountain Lakes, NJ, USA). Most of the experiments described below used borosilicate capillaries with a path length of 50 µm, a wall thickness of 50 µm and a width of 500 µm. Capillaries were supplied in 50 mm lengths and were scored with the edge of a carborundum stone and broken into lengths of 810 mm. Some experiments used capillaries with a path length of 20 µm, wall thickness of 20 µm and width of 200 µm. Capillaries were held with forceps and inserted through the paraffin oil of the Ramsay assay dish and into a droplet of secreted fluid or calibration solution. The aqueous sample was taken up by capillarity and was thus enclosed by glass on four sides and by paraffin oil on two sides. Luminal concretions were sometimes expelled into the droplets secreted during the Ramsay assay. The concretions settled to the bottom of the droplet and were not collected when the fluid was taken up into the hollow rectangle glass capillary.
For Drosophila, transepithelial dye flux (fmol min1 tubule1) was calculated by multiplying fluid secretion rate (nl min1 tubule1) by the dye concentration (µmol l1) measured by CLSM. Cricket tubules varied in length by >25% within an individual and in different insects. Moreover, vigorous contractions of the muscles in each cricket tubule prevented precise measurement of tubule length, so we could not normalize fluid secretion rates per unit length. We therefore report only secreted fluid dye concentrations rather than dye flux for individual cricket tubules. However, approximate values for transepithelial dye flux across cricket tubules could be estimated from secreted fluid dye concentration and a mean secretion rate based on measurements of >50 tubules from several insects.
Confocal laser scanning microscopy
A chamber made from a 6-well tissue culture dish was used for CLSM of MTs
or optically flat glass capillaries. A 15 mm hole was drilled in the centre of
each well and a 22 mm square glass cover slip was secured across the opening
with melted dental wax. The cover slips were pre-coated with 100 µl
droplets of 125 µg ml1 poly-L-lysine (70 kDa)
to facilitate adherence of isolated MTs. Tubules were transferred into 2 ml
saline in the bottom of each well and Texas Red was added from stock solutions
to achieve concentrations of 0.5 µmol l1 to 50 µmol
l1. Glass capillaries containing secreted fluid or
calibration solutions were placed in empty wells on uncoated glass cover
slips.
Confocal fluorescent images were obtained with a Zeiss LSM 510 confocal
microscope (Carl Zeiss, Oberkochen, Germany). The software used to obtain and
analyse the images was Zeiss LSM5 (Carl Zeiss). A 40x water-immersion
objective was used for isolated tubules, and a 20x objective was used
for samples collected in hollow rectangle glass capillaries. The system
consisted of an inverted microscope, a mixed argon/krypton-ion laser with 458,
477, 488 and 514 nm lines and heliumneon lasers with 543 and 633 nm
lines. For measurement of rhodamine 123, the 514 nm laser line, a 458/514 nm
dichroic filter and a 530600 nm band pass emission filter were
employed. For measurement of daunorubicin, the 543 nm laser line, a 488/543 nm
dichroic filter and a 560 nm long-pass emission filter were employed. For
measurement of fluorescein, the 488 nm laser line, a 488 nm dichroic filter
and a 505550 nm band pass emission filter were employed. For
measurement of quinacrine, the 458 nm laser line, a 488 nm dichroic filter and
a 505 nm long-pass emission filter were employed. The Texas Red
(sulphorhodamine 101) absorbance spectrum extends between 500 nm and 650 nm,
with a primary peak at 600 nm and a secondary peak at
550 nm.
Previous studies of Texas Red transport using CLSM have used either the 529 nm
Ar ion laser line (Miller et al.,
1998
) or the 568 nm krypton ion laser line
(Miller et al., 2001
). These
lines were not available on our system, and we used the 543 nm HeNe
laser line with a 488/543 nm dichroic filter and 560 nm long-pass emission
filter. Because of the high concentrations of Texas Red in secreted fluid
droplets (4400 µmol l1; see Results), the 514 nm
Ar/Kr laser line with a 458/514 nm dichroic filter could also be used.
CLSM of isolated Malpighian tubules
Tubules in the chamber were viewed under reduced, transmitted light
illumination, and a field containing 2030 cells was selected. The focus
was adjusted so that the optical slice was through the centre of the lumen.
Then, in confocal fluorescence mode, two successive 8-s scans of the field
were collected. Laser power was adjusted to minimize photobleaching, as
determined from a decline in fluorescence intensity of the second image
relative to the first. The confocal image (512x512 pixels) was viewed on
a high-resolution monitor, and fluorescence intensity was determined as the
mean value for five regions of interest of 200500 pixelsselected in the
cellular and luminal compartments and adjacent bathing saline. Detector gain
and offset were adjusted so that fluorescence intensity was 2000 in the
lumen on a scale of 04095 (12 bits) and the pinhole was set to
75
µm, corresponding to an optical slice thickness of
10 µm. Laser
power was reduced to minimize photobleaching, and detector gain was adjusted
so that when the average pixel intensity in the tubule lumen was
2000, MT
autofluorescence was undetectable.
Data analysis
Data are expressed as means ±
S.E.M., unless otherwise indicated. Student's
t-test was used to evaluate statistical significance, and means were
considered significantly different when P<0.05. Kinetic parameters
describing maximal dye flux (Jmax), maximal secreted fluid
dye concentration ([Dye]sf,max), and the bathing saline dye
concentrations corresponding to half maximal flux or secreted fluid dye
concentration (Kt) were calculated from
concentrationresponse curves fitted by non-linear regression to the
MichaelisMenten equation (SigmaPlot 2000, SPSS Inc., Chicago, IL,
USA).
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Results |
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CLSM of fluid samples collected in hollow rectangle glass capillaries
Because of concerns that the concretions in the cells or lumen of the
tubules of Drosophila and many other species (e.g.
Wessing and Zierold, 1999)
would confound both quantitative and qualitative analysis of dye accumulation
in the tubule lumen, we developed a method for measuring the concentration of
Texas Red, daunorubicin and other fluorochromes in the fluid secreted by MTs
set up in Ramsay assays. Secreted fluid droplets were collected in optically
flat glass capillaries, as described in the methods.
Fig. 3A shows a sample of fluid
containing Texas Red. The plane of focus was adjusted so that the optical
slice comprised approximately the middle 50% of the light path along the
Z-axis. For capillaries with a path length of 50 µm, a slice
thickness of 28 µm was used. For capillaries with a 20 µm path length,
pinhole diameter was reduced for a slice thickness of 10 µm. The
sensitivity of FI measurement was maximized by using thick optical
slices for CLSM analysis of secreted fluid samples collected in hollow
rectangle capillaries. The choice of slice thickness represented a compromise
between two factors. Reducing the thickness of optical slices by decreasing
pinhole diameter facilitated positioning of the optical slice at the
Z-axis midpoint of the light path through the glass capillary, but
FI also declined linearly as the optical slice thickness was
reduced.
Two methods were used to position the optical slice at the Z-axis midpoint of the fluid in the capillary. In the first method, a series of XY images was collected at intervals along the X-axis using the Z-stack feature of the LSM 510 software. The first and last slices of the stack were selected by manually focusing just outside the upper and lower surfaces of the capillary. A pinhole corresponding to a slice thickness of 28 µm was selected, and a Z-stack of 10 images at 5 µm intervals was then collected. A region of interest (ROI) corresponding to 50100% of the fluorescent region within the image was then selected, and the FI of each slice of the Z-stack was determined. Fluorescence intensity was maximal for 23 slices near the middle of the stack, and the maximal values were used to calculate the fluorochrome concentration from the corresponding calibration curve (described below). The first method typically required 60100 s to scan a complete stack.
To facilitate more rapid analysis of 2550 capillaries typically used in each experiment, a second method was developed. The microscope was initially focused approximately in the middle of the fluid within the capillary. An XZ scan was then done, based on 20 slices at 4 µm or 5 µm intervals. The images were thus collected from some distance below, through and above the column of fluid in the capillary. Using the LSM software, the plane of focus was then adjusted to the Z-axis midpoint of the column of fluid by moving the horizontal line corresponding to the plane of focus until the band of fluorescence in the XZ image was divided into two mirror images. Fig. 3B shows the images of XZ scans made at the apparent midline of the image, defined as a Z-position of 0 µm. Fig. 3B also shows the XZ scans made when the plane of focus was adjusted 10 µm above or below the midline. A series of XY images were then collected over a range of Z-positions, and a plot of FI as a function of Z-position is shown in Fig. 3C. The plot shows that FI in the XY images varied by less than 5% when the plane of focus was set 5 µm above or below the apparent midline. With practice, the plane of focus could be adjusted to within 12 µm of the position corresponding to the maximum FI. Fluorescence intensity declined at distances greater than 10 µm from the Z-axis middle of the column of fluid within the capillary (Fig. 3C), consistent with inclusion of part of the upper or lower glass wall of the capillary in the optical section.
After the optical slice was adjusted to the Z-axis midpoint of the
fluid column within the glass capillary, a time series of two
XY images was collected. Using the 20x
objective, the samples typically filled 2575% of the
XY image, corresponding to 60 000200 000
pixels. The mean FI for the sample was subsequently calculated using
the ROI feature of the LSM software. Laser strength was adjusted to avoid any
photobleaching, evident as a decline in mean FI of the second image
relative to the first.
Concentrations of dyes in secreted fluid samples collected in hollow
rectangle glass capillaries were calculated from a calibration curve
constructed for each experiment. The curve was based on FI
measurements of samples of known dye concentrations that bracketed the range
of interest. For most experiments, four calibration droplets whose
concentrations extended over an 8-fold range were used. Detector gain and
offset were adjusted so that the FI (04095, 12 bit) in the
highest concentration was 3900. For concentrationresponse curves,
several sets of calibration samples were required to bracket the wide range of
dye concentrations in the secreted droplets.
Fig. 3D shows a representative
calibration curve for Texas Red. Each curve relating FI to dye
concentration was fitted by regression analysis. Although the relationship of
FI to dye intensity was approximately linear
(r2>0.8), fits with r2 values near
unity (r2>0.999) were obtained for 2nd or 3rd order
polynomials. The non-linearity was attributable to self-quenching of the dye
at higher concentrations. Preliminary experiments revealed that fluid
secretion rates and secreted fluid dye concentrations could vary as much as
twofold on different days due to variations in room temperature
(1723°C). Concentrationresponse curves in the Results below
were therefore done in the same assay dish with equal numbers of tubules at
each concentration on each day to avoid any bias as a result of differences in
temperature.
Ramsay assays: secretion of Texas Red
Concentrationresponse curves for secreted fluid Texas Red
concentration as a function of bathing saline Texas Red concentration for
cricket tubules are shown in Fig.
4. The inset in Fig.
4 shows the ratio of Texas Red concentration in the secreted fluid
to that in the bathing medium (S/M) as a function of bathing saline Texas Red
concentration. The value of S/M for bath Texas Red concentrations of
0.11 µmol l1 was in the range of 3040,
similar to the value of 40 for the lumen/bath FI ratio in
isolated MTs (Fig. 2). Secreted
fluid Texas Red concentration declined at a bath concentration of 50 µmol
l1, and experiments with Drosophila tubules
described below suggest that high concentrations of the dye are toxic. The
data point at 50 µmol l1 was therefore excluded from the
curve-fitting procedure used to estimate the kinetic parameters. The length of
cricket tubules varies within and between animals, so fluid secretion rates
were not calculated in most experiments. However, an approximate flux can be
calculated from an estimate of fluid secretion rates of 53 tubules from three
insects (0.40±0.04 nl min1). Using the latter value,
a maximum flux of 163 fmol min1 tubule1 is
estimated at the maximum secreted fluid concentration of 405 µmol
l1.
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Concentrationresponse curves for Texas Red flux across Drosophila MTs and corresponding S/M ratios are shown as functions of bathing saline Texas Red concentration in Fig. 5. Both fluid secretion rate and secreted fluid dye concentration declined in saline containing 50 µmol l1 Texas Red relative to the values at lower concentrations, so the data point at 50 µmol l1 Texas Red was excluded from the curve-fitting analysis used to estimate the kinetic parameters Jmax and Kt. The S/M ratios for Drosophila tubules were somewhat lower than those observed for crickets, in the range of 2030 for bathing saline Texas Red concentrations between 0.1 µmol l1 and 5 µmol l1.
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Secretion of the organic anion fluorescein
Fluorescein is a substrate of organic anion transporters in tubules of
Drosophila and other species
(Neufeld et al., 2005;
Bresler et al., 1990
). We
wished to compare transport of smaller, more hydrophilic organic anions such
as fluorescein with the transport of larger, amphiphilic MRP2 substrates such
as Texas Red. S/M ratios and concentrationresponse curves for
fluorescein flux are shown in Fig.
9. It is worth noting that Jmax for
fluorescein transport is
2.5-fold higher than that for Texas Red, but the
value of Kt is
4.5-fold higher than that of Texas
Red. As a consequence, the transport efficiency
(Jmax/Kt) for Texas Red is 1.8-fold
higher than that for fluorescein.
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Transport of Texas Red was also inhibited by probenecid (Fig. 10B,C). The concentration of 114.7±5.4 µmol l1 (N=10) Texas Red in fluid secreted by Drosophila tubules bathed in saline containing 5 µmol l1 Texas Red was reduced by 63%, to 42.0±7.2 µmol l1, in the presence of 1000 µmol l1 probenecid (Fig. 10B). However, fluid secretion rate was also reduced significantly, from 0.39±0.06 nl min1 in the controls to 0.26±0.05 nl min1 in the presence of probenecid. The 78% reduction in Texas Red flux was thus partly due to the effects of probenecid on fluid secretion rate, which suggested non-specific effects of the drug at this concentration. At a lower concentration of 200 µmol l1 probenecid, the flux of 0.5 µmol l1 Texas Red was reduced by 43% (Fig. 10C) and there was no significant effect on fluid secretion rate.
Although probenecid is a known inhibitor of both MRP2 and also
Na+-dependent organic anion transport systems
(Horikawa et al., 2002),
transport of Texas Red was not reduced in Na+-free saline. In
salines containing 5 µmol l1 Texas Red, the concentration
of the dye in the secreted fluid was 92±16 µmol l1
(N=10) for tubules in control saline and 105±5 µmol
l1 for tubules in Na+-free saline. Texas Red flux
was 62.9±14.5 fmol min1 tubule1 in
control saline and 52.3±5.0 fmol min1
tubule1 in Na+-free saline. The differences are
not significant (P>0.05).
Secretion of the organic cation quinacrine
We examined transport of quinacrine by Drosophila tubules because
it is both a P-gp modulator and it is also a substrate or inhibitor of organic
cation transporters such as rOCT2 (Sweet
and Pritchard, 1999; Dohgu et
al., 2004
). Kinetic analysis showed that the
Jmax for transport was 27.7 fmol min1
tubule1 and the associated Kt was 21.4
µmol l1 (Fig.
11).
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Although quinacrine may itself be transported by the organic cation transport system or by P-gps in other cells, neither the P-gp inhibitor verapamil nor the OCT substrate tetraethylammonium (TEA) reduced the flux of quinacrine by Drosophila tubules. Transepithelial quinacrine flux across tubules bathed in saline containing 5 µmol l1 quinacrine and 500 µmol l1 verapamil (5.99±0.72 fmol min1 tubule1; N=10) did not differ from those of the corresponding controls in 5 µmol l1 quinacrine alone (6.31±0.77 fmol min1 tubule1; N=9). Similarly, transepithelial quinacrine flux across tubules bathed in saline containing 5 µmol l1 quinacrine and 500 µmol l1 TEA (4.28±0.78 fmol min1 tubule1; N=10) did not differ from those of the corresponding controls (3.72±0.66 fmol min1 tubule1; N=10). There was no effect of verapamil or TEA on fluid secretion rate or the concentration of quinacrine in the secreted fluid (data not shown).
Effects of p-glycoprotein inhibitors on transport of daunorubicin and rhodamine 123
The concentration of daunorubicin in the fluid secreted by cricket tubules
bathed in saline containing the dye (5 µmol l1) was
reduced 71% by 100 µmol l1 verapamil
(Fig. 12A) but was unaffected
by 100 µmol l1 TEA
(Fig. 12B). The concentration
of rhodamine 123 in the fluid secreted by cricket tubules bathed in saline
containing the dye (0.5 µmol l1) was reduced 60% by 100
µmol l1 verapamil
(Fig. 12C) but was unaffected
by 100 µmol l1 TEA
(Fig. 12D).
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Discussion |
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Transport of MRP2 substrates
Previous studies by Karnaky et al.
(2000,
2001
,
2003
) demonstrated active
accumulation of the MRP2 substrates Texas Red and 5-chloromethylfluorescein
(CMF) in the cells and lumen of the MTs of crickets (Acheta
domesticus) and cockroach (Periplaneta americana). Texas Red
transport is dependent on metabolism and is reduced by the MRP2 inhibitor
chlorodinitrobenzene, which does not inhibit transport of chlorophenol red, a
substrate for the classic organic anion transporter system. Texas Red
transport is unaffected by a 50-fold excess of para-aminohippurate (PAH), a
substrate of the organic anion transporter, by TEA, a substrate of the organic
cation transporter, or by verapamil, an inhibitor of p-glycoprotein. An apical
location of an MRP2-like transporter in cricket MTs has been demonstrated by
immunocytochemical staining with an antibody to a sequence of rat MRP2
(Karnaky et al., 2001
).
However, an apical location of an MRP2-like transporter cannot account for
accumulation of Texas Red in the cells of cricket tubules. Cellular
accumulation requires an additional mechanism for transport of the dye across
the basolateral membrane. The characteristics of such basolateral transport
could be assessed in cricket tubules with few or no intracellular concretions
(as in Fig. 1A) so that the
cytoplasm is relatively translucent and fluorescence intensity could be
measured.
Our analyses of both isolated MTs and of secreted fluid samples collected
in glass capillaries demonstrated that Texas Red concentrations are elevated
2040-fold above those in the bathing medium when the latter contains
Texas Red at concentrations near or below the Kt values of
5.5 µmol l1 for cricket tubules and 7.1 µmol
l1 for Drosophila tubules. The typical
transepithelial potential across Drosophila tubules is 3045 mV
lumen-positive (O'Donnell et al.,
1996,
1998
), which could only
account for a 36-fold elevation of the concentration of the anion Texas
Red by passive means. The higher S/M ratios measured in the present paper are
therefore indicative of active transepithelial transport of Texas Red.
Possibly toxic effects of high concentrations of Texas Red are evident in the reduction of luminal Texas Red concentration in both species and the reduction in fluid secretion rate of Drosophila tubules when the concentration of Texas Red in the bathing medium is increased above 20 µmol l1. By contrast, fluid secretion rate was unaffected by fluorescein at concentrations as high as 100 µmol l1. In broad terms, the results of our kinetic analyses suggest that Texas Red transport mechanisms are of higher affinity but lower capacity than those responsible for elimination of organic anions such as fluorescein. The finding that high concentrations of Texas Red inhibit fluid secretion provides an important caveat for studies based only on measurement of dye concentrations in the lumen. Measurement of fluid secretion rate not only permits calculation of transepithelial dye flux but also provides an independent measurement of the non-specific toxicity of dye substrates or inhibitors (see below).
Transport of Texas Red was inhibited by the MRP2 inhibitors MK-571 and
probenecid. Although probenecid inhibits both MRP2 and organic anion
transporters, the latter pathway is Na+ dependent. Secretion of
organic anions such as PAH is inhibited 80% by removal of Na+ from
the bathing medium (Linton and O'Donnell,
2000). By contrast, transport of Texas Red was unaffected by
Na+-free conditions. This finding clearly distinguishes transport
of Texas Red from that of compounds such as fluorescein and indicates that
inhibition by probenecid must therefore reflect an effect upon a transporter
distinct from the classic organic anion transport pathway. Reductions of fluid
secretion rate by high (1000 µmol l1) concentrations of
probenecid were associated with a 33% reduction in fluid secretion rate. The
use of lower concentrations of probenecid permitted inhibition of Texas Red
transport to be detected in the absence of any effect on fluid secretion rate.
The relative concentrations of substrates and inhibitors that we used are
similar to those of previous studies of MRP2. Transport of 0.05 µmol
l1 estradiol glucuronide by rabbit MRP2 expressed in Sf9
cells is reduced to 33% of the control value by 5 µmol l1
MK571 (van Aubel et al.,
1998
). Transport of 3 µmol l1 saquinavir is
reduced to 17% of the control value by 75 µmol l1 MK571
(Williams et al., 2002
).
Transport of 4 µmol l1 N-ethylmaleimide
glutathione by human MRP2 expressed in Spodoptera frugiperda ovarian
cells is reduced 50% by 1000 µmol l1 probenecid
(Bakos et al., 2000
) and
transport of 1 µmol l1 methotrexate by a human carcinoma
cell line is reduced 50% by 500 µmol l1 probenecid
(Hoijberg et al., 1999).
Transport of p-glycoprotein substrates
Previous studies have reported uptake of daunorubicin and rhodamine 123
into the cells but not the lumen of the MTs of the cricket Acheta
domesticus (Karnaky et al.,
2001) and the tobacco hornworm, Manduca sexta
(Gaertner and Morris, 1999
).
The latter authors suggested that accumulation of the dyes in the cells is a
form of xenobiotic scavenging unrelated to P-gp. Our results, based on
collection of secreted fluid, clearly indicate that transepithelial secretions
of both rhodamine 123 and daunorubicin are saturable forms of transport that
are inhibitable by verapamil and quinacrine. It is possible that species
differences underlie the differences between our results and those of Gaertner
and Morris (1999
) or that the
toxic effects of rhodamine 123 noted in both Drosophila melanogaster
and Teleogryllus commodus also apply to the tubules of Manduca
sexta. Gaertner and Morris
(1999
) suggested several
possible explanations to account for transepithelial transport of
505000 µmol l1 nicotine observed in their earlier
study (Gaertner et al., 1998
),
whereas 510 µmol l1 daunorubicin and rhodamine 123
were not accumulated in the lumen. In particular, they note the possibility
that `exciting and/or emitted light was quenched, reflected or absorbed by
the tissue and opaque uric acid crystals present within the lumen.' We
also noted that it was not feasible to clearly visualize dyes in the lumen of
many cricket MTs (T. commodus) and all Drosophila tubules
because of opaque concretions in the cells and/or lumen. A reexamination of
the possible secretion of fluorescent P-gp substrates by lepidopteran tubules
may be worthwhile, using the method developed here for analysis of secreted
fluid samples collected in hollow rectangle glass capillaries. Tubules of the
cabbage looper, Trichoplusia ni, might be suitable for such study
because they secrete fluid at high rates and for prolonged periods (M. R.
Rheault, J. Plaumann and M. J. O'Donnell, unpublished).
Transport of daunorubicin by tubules of both species was reduced by the
P-gp inhibitor verapamil, consistent with involvement of a P-gp-like
transporter. The relative concentrations of substrate and inhibitor that we
have used are broadly similar to those used in previous studies. For example,
transport of 1 µmol l1 rhodamine 123 or 3 µmol
l1 doxorubicin is reduced 50% by 20 µmol
l1 verapamil in several cell lines
(van der Sandt et al., 2000
).
Transport of 1 µmol l1 daunorubicin is reduced 50% by
10.3 µmol l1 verapamil in lymphocytes
(Green et al., 2001
).
Daunorubicin transport by Drosophila tubules is also inhibited by
quinacrine, an organic cation that is also a P-gp modulator, and by high
concentrations of TEA. A role for both P-gp and organic cation/H+
exchange has been proposed to account for the effects of daunorubicin
transport in flounder renal tubules
(Miller, 1995
). We suggest
that in Drosophila tubules as well, daunorubicin may be transported
both by P-gp-like transporters and by the organic cation transporters involved
in transepithelial secretion of quaternary ammonium compounds such as TEA
(Rheault and O'Donnell,
2004
).
Analysis of transepithelial transport of rhodamine 123 is complicated by
the inhibitory effects of this P-gp substrate on fluid secretion, particularly
in the case of isolated Drosophila tubules. Transport of rhodamine
123 is saturable in cricket tubules and is inhibited by verapamil. Fluid
secretion by Drosophila tubules is inhibited by rhodamine 123 at
concentrations above 0.1 µmol l1, and transport at such
low concentrations may involve mechanisms distinct from saturable transport by
P-gp-like transporters. Moreover, the transport of rhodamine 123 by
Drosophila tubules is unaffected by very high concentrations of
verapamil, again suggesting processes unrelated to P-gp. A further
complication is that the principal cells of dipteran tubules retain rhodamine
123 for a short period and the stellate cells reabsorb the dye from the tubule
lumen (Meulemans and De Loof,
1992). The dye accumulates in the stellate cell mitochondria and
eventually in intensely fluorescing vesicles, probably lysosomes. Endocytotic
uptake has been ruled out. In addition, rhodamine 123 precipitates on the
luminal concretions in the distal segment of the anterior tubules (Meulemeans
and De Loof, 1992). Given the inhibitory effects of rhodamine 123 on fluid
secretion rate and the complexities in the routes of transport, we suggest
that it is an inappropriate substrate for further analysis of P-gp-like
transporters in dipteran MTs.
Transport of the organic cation quinacrine is also saturable, with
Drosophila tubules accumulating the compound in the lumen at
concentrations as high as 17-fold above those in the bath. However, in
contrast to quinacrine transport by the rat choroid plexus, transport by
Drosophila tubules is unaffected by either verapamil or TEA. We had
examined the effects of quinacrine on daunorubicin transport because it is a
known P-gp modulator (Tiberghien and Loor,
1996). Although we have not examined quinacrine transport in
detail, these results suggest that there may be differences in the mechanisms
of quinacrine transport in Drosophila MTs and vertebrate tissues.
Moreover, it appears that the tubules possess a mechanism of organic cation
transport distinct from that utilized for the P-gp substrate daunorubicin and
the organic cation TEA (Rheault and
O'Donnell, 2004
).
CLSM analysis of secreted droplets collected in optically flat glass capillaries
Dye concentration in nanolitre samples of secreted fluid droplets collected
in optically flat glass capillaries can readily be measured using CLSM. In
conjunction with Ramsay assays, these measurements permit calculation of
transepithelial dye flux. The Ramsay assays also provide a means for
determining the effects of the dyes or transport inhibitors on processes other
than dye transport. Rhodamine 123, for example, clearly inhibits fluid
secretion by tubules of Drosophila and crickets. This finding raises
the possibility that in instances where fluid secretion is reduced, any
associated reduction in dye transport may reflect impairment of cellular
metabolism or other transporters involved in cellular homeostasis. Rhodamine
123 is known as a mitochondrial dye, so its effects on fluid secretion
transport may reflect impairment of cell metabolism. Analysis of dye transport
solely by image analysis of cellular and luminal compartments would not
provide this additional information about the effects of the dye on cell
function.
We developed the use of optically flat glass capillaries for analysis of
transepithelial dye transport because of concerns that opaque cellular or
luminal concretions would block or interfere with laser light transmission.
Although we have previously used CLSM for visualization of fluorescein
transport by Drosophila tubules
(Linton and O'Donnell, 2000),
the presence of concretions alters the amount of laser light that reaches a
given region of the tubule, thereby making quantitative analysis unfeasible.
There are also concerns that the concretions move in the lumen during fluid
secretion, thereby altering the amount of laser light passing through the
lumen, and that cellular concretions may be transferred from cell to lumen
under certain conditions (Hazelton et al.,
2001
). Another advantage of analysis of secreted fluid droplets by
CLSM is that laser strength can be optimized to minimize photobleaching but
without the additional concerns over the effects of the laser light on
cellular components. Low concentrations of fluorochromes can be measured
quickly and accurately because fluorescence intensity measurements are based
on large numbers of pixels (typically >60 000) relative to isolated tubules
and because the thickness of the optical slice can be increased. Estimates of
dye concentration are less affected by variations in pH of the cytoplasmic
milieu, and the calibration solutions can be adjusted to resemble closely the
ionic strength and pH of the secreted fluid. Secreted fluid samples from large
numbers of tubules (typically 20 per experiment) in each Ramsay assay can be
analysed much more rapidly than is possible if multiple regions of interest
within the lumen and bath are selected for analysis in isolated tubules
examined by CLSM. It is important to point out that the methods described here
for analysis of nanolitre samples of fluid containing fluorescent transporter
substrates provide a faster and cheaper alternative to the use of
radiolabelled substrates. Lastly, it is worth noting that, although we have
analysed fluid samples collected in hollow rectangle glass capillaries by
CLSM, fluorescence intensity could also be measured by epifluorescence
microscopy of the capillaries. Transport of fluorescein by isolated tubules of
the cricket A. domesticus has recently been studied by means of
quantitative fluorescence microscopy
(Neufeld et al., 2005
).
The flux measurements reported here can be used to estimate the time
required to clear the haemolymph of a given concentration of dye. Haemolymph
volume in Drosophila is of the order of 0.1 µl in control flies
but is as high as 0.32 µl in desiccation-resistant strains
(Folk et al., 2001). For Texas
Red, each tubule secretes
80 fmol min1 when bathed in
saline containing the dye at a concentration of 20 µmol
l1 (Fig. 5).
The four tubules will thus clear the haemolymph content of 2 pmols (=0.1 µl
x 20 µmol l1) of the dye in
6 min. Similar
calculations indicate clearance times of
10 min for daunorubicin,
12
min for fluorescein and
25 min for quinacrine. These estimates suggest
that the MTs may play an important role in the elimination of potentially
toxic P-gp substrates and MRP2 substrates from the haemolymph.
In summary, our results show that transport of fluorescent substrates
analysed by CLSM of secreted fluid samples collected in hollow rectangle glass
capillaries indicates the presence of P-gp-like and MRP2-like transporters in
the MTs of two insect species. Demonstration of secretion of P-gp and MRP2
substrates by Drosophila tubules is of particular interest because of
previous molecular genetic evidence for the presence of MDR and
MRP genes in this species (Wu et
al., 1991; Tarnay et al.,
2004
). Our results set the stage for further analysis of the
effects of treatments, such as cadmium exposure and heat shock
(Tapadia and Lakhotia, 2005
),
known to alter MRP or MDR gene expression on transport of
P-gp and MRP2 substrates by isolated MTs and the possible role of such
transport in insecticide resistance. In view of recent evidence showing
modulation of organic cation transport by dietary loading, peptides, amines
and intracellular second messengers
(Bijelic and O'Donnell, 2005
;
Bijelic et al., 2005
), it will
also be of interest to examine whether transport of P-gp and MRP2 substrates
is also modulated by such treatments.
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