Ion-selective microelectrode analysis of salicylate transport by the Malpighian tubules and gut of Drosophila melanogaster
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
* Author for correspondence (e-mail: odonnell{at}mcmaster.ca)
Accepted 7 October 2004
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Summary |
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Key words: salicylate, ion-selective microelectrode, organic anion transport, Malpighian tubule
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Introduction |
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The present study addresses three questions concerning organic anion
transport by insect epithelia. (1) Do Malpighian tubules of the fruit fly
Drosophila melanogaster transport the organic anion salicylate?
Salicylate is of particular interest for studies of organic anion transport by
insect tissues because it is a plant-produced signal that activates plant
defence genes after herbivory or pathogen attack. In insects, salicylate is
also known to activate cytochrome P450 genes that are associated with
detoxification, thereby protecting the insect against toxins produced by host
plants (Li et al., 2002). (2)
Does the gut of the fruit fly transport salicylate? We have recently shown
that organic cations, such as tetraethylammonium, are transported from the
haemolymph to the lumen not only by the Malpighian tubules but also across the
posterior midgut (Rheault and O'Donnell,
2004
). It is therefore of interest to determine if regions of the
gut contribute to elimination of salicylate from the haemolymph. (3) Can
transport of salicylate be measured using ion-selective microelectrodes?
Radiolabelled or fluorescent compounds have been widely used in studies of
organic anion transport by tissues, such as the vertebrate kidney, crustacean
antennal gland, and insect Malpighian tubule
(Bresler et al., 1990
;
Pritchard and Miller, 1991
;
Linton and O'Donnell, 2000
).
Our paper describes measurement of organic anion transport using rapid and
low-cost microelectrode methods that exploit the high selectivity of the anion
exchanger tridodecylmethylammonium (TDMA) for the organic anion salicylate.
Macroscopic (
1 cm diameter) electrodes based on lipophilic quaternary
ammonium salts such as TDMA chloride have previously been used to measure the
concentrations of salicylate (Rover et
al., 1998
; Creager et al.,
1995
), penicillin (Yao et al.,
1989
) and heparin, a negatively charged polysaccharide
(Ma et al., 1993
). These
electrodes are typically >2000 times more selective towards salicylate than
to Cl- (e.g. Rothmaier et al.,
1996
), the predominant inorganic anion in intracellular and
extracellular fluids.
Our first method combines the high selectivity of TDMA-based electrodes for
salicylate and the self-referencing ion-selective microelectrode technique for
non-invasive spatial and temporal analysis of ion flux
(Smith et al., 1994;
Piñeros et al., 1998
).
A salicylate-selective self-referencing microelectrode is moved between two
positions within the unstirred layer adjacent to the surface of a tissue.
Salicylate transport by the tissue perturbs the salicylate concentration in
the unstirred layer, and the difference in salicylate concentrations measured
between the two microelectrode positions can be converted into a corresponding
salicylate flux using the Fick equation. We have used this method to assess
spatial and temporal variations in salicylate flux in different regions of the
MTs and gut.
Our second method uses a salicylate-selective microelectrode to measure the concentration of salicylate in fluid droplets secreted by isolated insect Malpighian tubules set up in a Ramsay secretion assay. Flux across the entire tubule can be calculated from the product of secretion rate and secreted droplet salicylate concentration. We have also used salicylate-selective microelectrodes to measure changes in salicylate concentration in haemolymph collected after animals have been fed salicylate-enriched diets or after salicylate has been injected into the haemocoel.
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Materials and methods |
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Dissection and Ramsay assay
Malpighian tubule dissection and application of the Ramsay assay for
measurement of fluid secretion rates have been described previously
(Dow et al., 1994). The four
Malpighian tubules are arranged as an anterior and posterior pair. Each tubule
in the anterior pair consists of a distal segment, a secretory main segment
and a reabsorptive proximal segment. The lumen of the distal segment is filled
with Ca2+-rich concretions
(Dube et al., 2000
) but does
not secrete fluid or K+ (Dow et
al., 1994
; Rheault and
O'Donnell, 2001
). When bathed in saline containing 20 mol
l-1 K+ and 132 mmol l-1 Na+ the
main segment secretes a near-isoosmotic fluid containing
120 mmol
l-1 K+ and
30 mmol l-1 Na+
(O'Donnell and Maddrell,
1995
). The proximal (lower) segment secretes Ca2+ into
the lumen, acidifies the lumenal fluids and reabsorbs K+,
Cl- and water (O'Donnell and
Maddrell, 1995
). The posterior tubules are identical except that
they lack a distal segment. The anterior and posterior pairs of MTs are each
connected to the hindgut through a short ureter. Each pair of Malpighian
tubules joined by a common ureter was dissected out under Drosophila
saline consisting of (in mmol l-1): 117.5 NaCl, 20 KCl, 2
CaCl2, 8.5 MgCl2, 20 glucose, 10 L-glutamine,
10.2 NaHCO3, 4.3 NaH2PO4 and 8.6 Hepes. The
saline was titrated with NaOH to pH 7. The addition of glutamine has been
found to maintain higher and stable rates of fluid secretion for prolonged
periods (>2 h; Hazel et al.,
2003
). Pairs of isolated tubules were transferred on fine glass
probes from the dissecting saline to 20 µl droplets of saline under
paraffin oil. One tubule of each pair was pulled out of the bathing droplet
and wrapped around a fine steel pin. 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 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 min-1) was calculated by dividing the droplet
volume by the time (min) over which the droplet formed.
To collect fluid from tubules in which the lower tubule was also positioned
inside the bathing droplet, an alternative preparation was used
(O'Donnell and Maddrell,
1995). By dissecting out all four tubules plus a very short
connecting section of gut, it was possible to collect fluid from two whole
tubules, including both the main segments and the lower Malpighian tubule. One
pair of tubules was positioned inside a 40 µl bathing saline droplet. One
tubule of the other pair was removed and discarded, and the remaining tubule
was pulled out into the paraffin oil and used to anchor the preparation. Fluid
was thus collected after it had passed through the entire length of two
tubules upstream of their common ureter.
Salicylate-selective self-referencing (Sal-SeR) microelectrodes
Technical and theoretical aspects of self-referencing microelectrodes have
been described previously (Smith et al.,
1994; Piñeros et al.,
1998
). Applications of the technique to the study of K+
and tetraethylammonium transport by isolated insect Malpighian tubules have
been described previously in Rheault and O'Donnell
(2001
) and Rheault and
O'Donnell (2004
),
respectively.
Procedures for microelectrode construction were similar to those described
previously for measurement of K+ flux using valinomycin-based
K+-selective microelectrodes
(Rheault and O'Donnell, 2001).
Micropipettes were pulled to tip diameters of
5 µm on a programmable
puller (P-97 Flaming-Brown, Sutter Instrument Co., Novato, CA), silanized by
treatment with N,N-dimethyltrimethylsilylamine (200°C, 30 min), cooled and
then stored in an air-tight chamber over desiccant until use. Immediately
prior to use, microelectrodes were backfilled with 150 mmol l-1
KCl. The KCl solution was forced to the tip by positive pressure and the
microelectrode tip was then front-filled with a short column length (
100
µm) of the ion exchanger cocktail, which consisted of 9% (w/v) TDMA Cl
(Fluka, Buchs, Switzerland) in 2-nitrophenyl octyl ether. The reference
electrode consisted of a 10 cm long, 1.5 mm diameter glass capillary tube
(TW150-4) filled with a mixture of 3 mol l-1 KCl and 1% agar
inserted into a microelectrode holder Ag-AgCl half-cell filled with 3 mol
l-1 KCl.
Selectivity coefficients for TDMA-based microelectrodes were calculated by
the separate solutions method (Ammann, 1986) for salicylate relative to the
anions found in Drosophila saline (Cl-,
HCO3-, H2PO4-).
Selectivity coefficients were also determined for two substrates of organic
anion transport systems (PAH and cyclic adenosine monophosphate). All
solutions were prepared at 0.1 mol l-1 and neutral pH. All
salicylate calibration solutions were made up in Drosophila saline
containing 158.5 mmol l-1 Cl-. This method is referred
to as the `unorthodox' (Thomas,
1978) or `empirical' method
(Vaughan-Jones and Aickin,
1987
) of correction for the effects of interfering ions on
electrode response.
The development of the Sal-SeR microelectrode system has been referred to
in a recent review of insect epithelial anion transport
(O'Donnell et al., 2003) but
the details of the system's use for analysis of salicylate flux have not been
described previously. The arrangements of the electrode, positioning system,
signal amplifier and data acquisition are shown in schematic form in
Fig. 1. The Sal-SeR
microelectrode was connected through a chlorided silver wire to the amplifier
head stage, which was mounted on a set of translator stages (Newport Corp.,
Fountain Valley, CA, USA). An orthogonal array of computer-controlled stepper
motors connected to the translator stages (CMC-4, Applicable Electronics Inc.,
Forrestdale, MA, USA) moved the microelectrode in three dimensions with
submicron accuracy and repeatability. At each measurement site, the electrode
was moved perpendicular to the tissue surface between two positions separated
by 100 µm. Voltage measurements taken at the limits of the excursion were
amplified 1000-fold (IPA-2 ion/polarographic amplifier, Applicable Electronics
Inc.) and used to calculate a voltage difference over the excursion distance
of the microelectrode. This differential signal was then converted into a
salicylate concentration difference using a standard microelectrode
calibration curve that related voltage output to salicylate concentration in
saline. The highly sensitive self-referencing system resolves voltage
differences as small as 10 µV, corresponding to differences in salicylate
concentration (in bathing saline containing 500 µmol l-1
salicylate) as small as 0.04%. The Sal-SeR microelectrode was viewed using an
inverted microscope equipped with a video camera and the `move, wait and
sample' protocol at each measurement site was controlled through a PC running
Automated Scanning Electrode Technique (ASET) software (Sciencewares, East
Falmouth, MA, USA). The Sal-SeR microelectrode tip was first `moved' to a site
10 µm from the tissue surface. The microelectrode then remained stationary
during the 9 s `wait' period to allow ion gradients near the tubule to
re-establish after the localized stirring during the movement period. No data
were collected during the wait period. The microelectrode voltage was recorded
and averaged for 1 s during the `sample' period. The probe was then moved to
the other extreme of the 100 µm excursion, followed by another wait and
sample period. Each move, wait and sample cycle at each extreme of probe
excursion was complete in 10 s. Each flux determination required measurement
of the concentration difference between the two extremes of probe excursion,
for a total of 20 s. Fluxes are reported as an average of 3-5 repetitive
measurements at each site.
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For salicylate flux measurements with Sal-SeR microelectrodes, tissues were transferred after dissection to 35 mm diameter Petri dishes filled with 4 ml of saline. Dishes were pre-coated with 100 µl droplets of 125 µg ml-1 poly-L-lysine and air dried before filling with saline to facilitate adherence of the tubules to the bottom of the dish. Fluxes were measured typically at 3-8 sites in the field of view (550 µm at 10x magnification). The preparation was then moved so that sites in an adjacent region of the gut or MT could be scanned.
Voltage differences (V, µV) obtained over the amplitude of the
Sal-SeR microelectrode excursion were converted to a salicylate concentration
difference (
C, µmol cm-3) using the equation:
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where V is the differential signal measured over the amplitude of
electrode excursion, CB is the background concentration of
salicylate in the medium at a distance of >1000 um from the preparation
(µmol cm-3) and S is the slope (µV) of the electrode measured
in response to a 10-fold change in salicylate concentration. Derivations of
this equation are given by Kuhtreiber and Jaffe
(1990
) and Smith et al.
(1994
). Except where noted,
CB was 0.5 µmol cm-3 in all experiments.
Values of
C were converted into corresponding fluxes by
substitution into the Fick equation:
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where JSalicylate is the flux of salicylate (mol
cm-2 s-1), D is the diffusion coefficient for salicylate
at 25°C (0.959x10-5 cm2 s-1;
Lide, 2002) and
r is the amplitude of electrode excursion (cm).
Measurement of salicylate flux in Ramsay secretion assays
Secreted fluid droplets were collected under paraffin oil using the Ramsay
assay and the concentration of salicylate was determined using either
salicylate-selective microelectrodes or liquid scintillation counting of
14C-labelled salicylate.
The arrangements of the electrodes and recording system are shown in
schematic form in Fig. 2.
Micropipettes were silanized, backfilled with 150 mmol l-1 KCl and
front filled with TDMA Cl as described above for Sal-SeR microelectrodes.
Displacement of the ion exchanger cocktail by paraffin oil was prevented by
coating the tip of each microelectrode with a thin (1 µm) layer of
poly vinyl chloride (PVC), as described previously for use of
tetraethylammonium-selective microelectrodes under paraffin oil
(Rheault and O'Donnell, 2004
).
The reference microelectrode electrode had a tip diameter of
1 µm and
was filled with 150 mmol l-1 KCl. Both the salicylate-selective
microelectrode and reference microelectrode were connected through chlorided
silver wires to a high impedance (>1015
) electrometer,
which in turn was connected to a computerized data acquisition and analysis
system (Axotape, Burlingame, CA, USA).
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For experiments with [14C]-labelled salicylate (31 mCi mmol-1), isolated tubules were set-up in 40 µl droplets of Drosophila saline containing salicylate at a final concentration of 33 µmol l-1. Secreted fluid droplets were collected after 60 min, secretion rates determined as above and the concentration of salicylate was measured by liquid scintillation counting in a LKB Wallac 1217 Rackbeta Liquid Scintillation Counter.
Salicylate flux was calculated as the product of fluid secretion rate (nl min-1) and secreted fluid salicylate concentration (mmol l-1) determined using either technique described above.
Injection of salicylate into the haemocoel
Third instar larvae were secured to the bottom of a Petri dish using
double-sided tape. A 1 ml plastic tuberculin syringe pulled out to a fine tip
over a low flame was used to transfer 0.1 µl of 100 mmol l-1
salicylate from a Gilson micropipette into the tip of a glass micropipette
whose tip was broken back to a diameter of 3 µm. The micropipette was
mounted on a micromanipulator and the tip was introduced into the haemocoel of
the larva observed under a dissecting microscope. Air pressure supplied by a
60 ml syringe connected to the back of the micropipette through plastic tubing
was used to expel the salicylate solution into the haemocoel.
Measurement of chloride and salicylate concentrations in haemolymph samples
Chloride concentration in haemolymph samples was measured using
Cl--selective microelectrodes based on chloride ionophore I,
cocktail A (Fluka) and backfilled with 500 mmol l-1 KCl. The
microelectrodes were front-filled with the cocktail and coated with PVC as
described above. The reference microelectrode was backfilled with 1 mol
l-1 Na+ acetate near the tip and 1 mol l-1
KCl in the upper part of the barrel.
For Cl- measurements, third instar larvae were removed from the culture tubes, washed for 5-10 s in 5 ml distilled water and dried on tissue paper. The larvae were transferred to a Petri dish filled with Paraffin oil and the cuticle was torn with forceps to permit the escape of haemolymph. Care was taken to avoid damaging the gut. Haemolymph samples were transferred by micropipette to another oil-filled dish containing calibration droplets (10 mmol l-1 and 100 mmol l-1 KCl).
For salicylate measurements, haemolymph samples were collected from larvae that had been injected with salicylate or fed salicylate-rich or control diets. Haemolymph samples were collected from larvae that had been washed and dried as above, then secured to a 35 mm Petri dish with double-sided tape. This permitted collection of multiple haemolymph samples from the same larvae by inserting a glass micropipette into the haemocoel under the control of a micromanipulator and applying suction using a syringe connected to the back of the micropipette through plastic tubing. Salicylate concentration was measured using the same procedures as for secreted fluid samples except that the calibration solutions of 5, 0.5 and 0.05 mmol l-1 salicylate were made up in 30 mmol l-1 KCl.
Calculations and statistics
Values are expressed as mean
±S.E.M. Two-sample F-tests were used
to compare the variances of the data for the control and experimental groups.
Depending on the outcome of each F-test, differences between experimental and
control groups were compared using unpaired Student's t-tests
appropriate for data with either equal or unequal variances. Fluid secretion
rates of isolated tubules in salines containing different concentrations of
salicylate were analysed by one-way ANOVA (analysis of variance) with
Dunnett's post hoc test (GraphPad InStat version 3.05, GraphPad
Software, San Diego CA). Differences were considered significant if
P<0.05. The responses of the same group of tubules or guts before
and after an experimental treatment were compared using a paired
t-test. Dose-response curves relating salicylate flux or secreted
fluid salicylate concentration to bathing saline salicylate concentration were
fitted by non-linear regression (SigmaPlot 2000, SPSS Inc., Chicago).
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Results |
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The high selectivity of TDMA-based electrodes for salicylate relative to other anions was also indicated by the electrode slopes for a 10-fold change in salicylate concentration for solutions made up in Drosophila saline, which contains 158.5 mmol l-1 Cl-, 10.2 mmol l-1 HCO3- and 4.2 mmol l-1 H2PO4-. Slopes were 59.3+0.4 mV (N=16) between 10 and 1 mmol l-1 salicylate, 59.2±0.4 (N=15) between 5 and 0.5 mmol l-1 salicylate and 54.7±0.2 between 2.5 and 0.25 mmol l-1 salicylate (N=7). The slope was 31.9±1.3 mV between 0.5 and 0.05 mmol l-1 salicylate (N=15) in Drosophila saline containing 158.5 mmol l-1 Cl-. However, the slope between 0.5 and 0.05 mmol l-1 salicylate was 56.5±1.7 (N=5) in 30 mmol l-1 KCl. The latter solution was chosen to represent the upper limit of haemolymph Cl- levels in the larvae (see below).
The relatively large tip diameter (5 µm) of the microelectrodes
used in this study and the low resistivity of ion exchanger-based
microelectrodes (relative to neutral carrier-based ion-selective
microelectrodes) contributed to rapid response times. The 90% response times
for Sal-SeR microelectrodes used in this study were typically in the range of
0.3-0.7 s. Electrode noise based on the scatter of measurements with
self-referencing electrodes in saline containing 0.5 mmol l-1
salicylate was typically of the order of ±30 µV in for individual
measurements, but was reduced to ±20 µV for averages of three
measurements and to ±10 µV for averages of five measurements
(Fig. 3). At the lowest noise
level, changes in salicylate concentration of less then 0.04% could be
resolved. Salicylate flux was calculated after subtracting the noise at a
reference position 1 mm or more from the preparation from the differential
signal measured at the site of interest near the preparation. This subtraction
corrects the signals measured near the preparation for any minute regional
variations in salicylate concentration in the bathing dish. The typical signal
to noise ratio was in the range of 10-50.
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Measurement of salicylate flux using the Sal-SeR microelectrode
A representative recording of salicylate flux across the main segment of an
isolated Malpighian tubule is shown in Fig.
4A. In these experiments, a positive differential signal denotes a
decrease in the salicylate concentration of the unstirred layer near the
basolateral surface of the tubule. This decrease reflects secretion of
salicylate from the bath into the tubule (i.e. salicylate influx). In general,
there was a slight increase in the magnitude of the differential signal at all
sites over the 2 h recording period, but the data indicate substantial spatial
and temporal heterogeneity. Although most of the sites in
Fig. 4A showed relatively
constant differential signals for periods of 40-60 min, signals at some sites
varied substantially over time. Increases in differential signal were more
dramatic in the three left-most sites in
Fig. 4A. In
Fig. 4B,C, the electrode signal
has been plotted as a function of time for eight sites on two different
tubules. Fig. 4B,C shows that
substantial variations in differential signal occurred over distances which
were only a few times larger than the diameter of the principal cells in the
tubules (30 um). The largest differential signal (site 1 in
Fig. 4B) exceeded the smallest
signal (Site 4) more than 7-fold. The two sites (1 and 2) with the highest
initial signals in Fig. 4B also
showed oscillations in the differential signal. There were peaks in the
signals near 0, 30 and 80 min and troughs near 20 and 60 min. In
Fig. 4C, there was a small but
steady increase in differential signal at site 1, whereas there was a
pronounced peak in signal between 30 and 70 min for sites 3 and 4. The
significance and possible mechanisms of temporal and spatial variations in
differential signal are discussed below.
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Measurements of differential signal and calculated salicylate flux in different segments of the Malpighian tubule and gut are summarized in Fig. 5. There was a small but significant influx of salicylate in the distal segment of the anterior tubules, and the largest fluxes were observed in the secretory main segment of the tubule. There was evidence of an efflux of salicylate (from tubule lumen to bathing saline) in the lower Malpighian tubule. Preliminary measurements showed no differences in the fluxes of salicylate across the anterior versus the posterior midgut, and the data have been combined. Salicylate influxes in the midgut, ileum and rectum were 21%, 63% and 45%, respectively, of those in the main segment of the Malpighian tubule.
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The dependence of salicylate flux on active transport was assessed by comparing flux before and after inhibition of metabolism with the metabolic inhibitor sodium cyanide. Based on measurements at 13 sites in four tubules, salicylate flux changed from 8.3±0.6 pmol cm-2 s-1 in control saline to -13.2±4.2 pmol cm-2 s-1 and 1.4±0.3 pmol cm-2 s-1 by 15 and 30 min, respectively, after addition of 2 mmol l-1 NaCN to saline containing 0.5 mmol l-1 salicylate. The negative values at 15 min indicate that the flux transiently shifted from an influx to an efflux in response to cyanide. The basis for this effect is discussed below on the basis of the high lumen concentrations of salicylate maintained in metabolically active Malpighian tubules.
The finding of salicylate flux from bath to midgut lumen raised the question of whether salicylate is normally absorbed from the gut into the haemolymph if the insects feed on diets containing this compound. We therefore measured salicylate fluxes across midguts dissected from 3rd instar larvae that had been fed for 24 h on Drosophila diet containing 100 mmol l-1 salicylate. In saline containing 0.5 mmol l-1 salicylate there was a flux of salicylate from lumen to bath of 7.6±2.4 pmol cm-2 s-1 (N=20 sites in N=5 guts).
Measurements of salicylate flux using the Ramsay assay
A sample recording of the voltage from a PVC-coated TDMA-based
microelectrode is shown in Fig.
6. The 90% response time of the PVC-coated microelectrodes used to
measure salicylate concentrations in droplets under paraffin oil was 5 s.
The measurements of secreted fluid droplets before and 30 min after addition
of 0.05 mmol l-1 salicylate are bracketed by measurements of
calibration droplets containing 0.05, 0.5 and 5 mmol l-1 salicylate
in Drosophila saline. The voltage recorded in a droplet of fluid
secreted by an isolated tubule in the absence of salicylate in the bathing
saline (d1) was equivalent to that produced by 0.06 mmol l-1
salicylate in Drosophila saline. This background represents the
interference of other anions in the secreted fluid on the salicylate
microelectrode. This background must be subtracted from the signals measured
after addition of salicylate to the bathing saline. The voltage recorded in a
droplet of secreted fluid (d2) after the addition of 0.05 mmol l-1
salicylate to the bathing saline was equivalent to that produced by 4.90 mmol
l-1 salicylate. The corrected concentration of 4.84 mmol
l-1 salicylate in the secreted fluid was then calculated by
subtracting the background concentration in d1 from d2.
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Dose-response curves relating fluid secretion rate for tubules, secreted
fluid salicylate concentration and salicylate flux to bathing saline
salicylate concentration are shown in Fig.
7. Although salicylate at concentrations below 0.5 mmol
l-1 had no significant effect on fluid secretion rate for tubules
from adult flies (ANOVA, P>0.05), concentrations of 1.25 and 2.5
mmol l-1 were inhibitory (Fig.
7A). The maximum concentration of salicylate in the secreted fluid
was 7 mmol l-1 (Fig.
7B). At lower bathing saline concentrations (0.0025-0.125 mmol
l-1) the concentration of salicylate in the secreted fluid was
elevated 50-fold to 100-fold (Fig.
7B, inset). Values of Jmax (2.72 pmol
min-1) and Kt (0.046 mmol l-1) were
obtained by fitting the salicylate flux data to the Michaelis-Menten equation
(Fig. 7C). It should be noted
that these kinetic values represent the steady-state consequence of at least
two transport steps operating in series in the tubule epithelium.
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Salicylate was also secreted by isolated Malpighian tubules dissected from 3rd instar Drosophila larvae. Fluid secretion rates, secreted fluid salicylate concentration and salicylate flux for larval tubules did not differ significantly from the corresponding values for tubules from adult flies (Student's t-tests, P>0.05). Fluid secretion rates for tubules from larvae and adult flies bathed in saline containing 0.05 mmol l-1 salicylate were 0.53±0.06 nl min-1 (N=8) and 0.51±0.06 nl min-1 (N=9), respectively. Fluid secreted by tubules of larval and adult flies contained 2.58±0.35 mmol l-1 salicylate and 3.13± 0.24 mmol l-1 salicylate, respectively. Salicylate fluxes across tubules of larval and adult flies were 1.35±0.22 pmol min-1 and 1.57±0.16 pmol min-1, respectively.
The Sal-SeR microelectrode experiments indicated that salicylate was transported from bath to lumen across the main segment of the Malpighian tubule, but that there was a loss of salicylate from the lumen to the bath across the lower Malpighian tubule of adult flies (Fig. 5). Transport of salicylate from lumen to bath would be expected to reduce the lumenal concentration of salicylate. This possibility was tested by comparing the concentration of salicylate in fluid secreted by whole tubules with that of the main segments of the same tubules. Whole pairs of tubules set up in a Ramsay assay and bathed in saline containing 0.5 mmol l-1 salicylate secreted fluid containing 5.82±0.49 mmol l-1 salicylate (N=8). When the tubules were repositioned so that only the main segment of one of the tubules remained in the bathing saline, the secreted fluid contained 7.78±0.97 mmol l-1 salicylate. The significant difference (P<0.02; paired t-test) in the concentration of salicylate is consistent with the loss of salicylate from lumen to bath as the fluid passes through the lower Malpighian tubule.
Flux of [14C]-salicylate
The mean salicylate flux for isolated tubules bathed in saline containing
0.033 mmol l-1 14C-labelled salicylate was 0.95±0.10 pmol
min-1 (N=6). This value is between the values of
0.78±0.04 pmol min-1 and 1.63±0.16 pmol
min-1 based on measurements with salicylate-selective
microelectrodes for tubules bathed in saline containing 0.025 and 0.05 mmol
l-1 salicylate, respectively
(Fig. 7). Based on the
regression equation fit to the data in Fig.
7C, the flux predicted in a bathing saline concentration of 0.033
mmol l-1 salicylate was 1.14 pmol min-1.
Salicylate levels in haemolymph of Drosophila larvae fed salicylate-rich diets or injected with salicylate
We also measured salicylate in haemolymph samples collected from 3rd instar
larvae fed either the control diet or diet containing 100 mmol l-1
salicylate. There was no evidence for increased mortality or effects on
pupation or adult emergence in the experimental group relative to the
controls. The levels of salicylate in the haemolymph after feeding for 3-6 or
24 h on the salicylate diet were 0.50±0.25 mmol l-1
(N=8) and 0.40±0.09 mmol l-1 (N=15),
respectively. The level of salicylate in the haemolymph increased to
0.93±0.13 mmol l-1 (N=7) when larvae were fed on
the salicylate diet for 3-6 h and then chilled to 4°C for 30-70 min to
reduce metabolic rate. The electrode signal in haemolymph collected from
animals on the control diet without salicylate was equivalent to a salicylate
concentration of 0.006±0.002 mmol l-1 (N=7). This
level of interference is less than that of 0.06 mmol l-1
reported above for droplets of fluid secreted by Malpighian tubules
(Fig. 6), presumably due to the
lower concentration of chloride in the haemolymph (22.3±3.1 mmol
l-1; N=9) relative to that in secreted fluid. The rate of
change of salicylate concentration in the haemolymph was measured after larvae
were injected with
0.1 µl of 100 mmol l-1 salicylate and
two or more samples of haemolymph of
10 nl each were collected at
intervals of 60-100 min (Fig.
8). The mean concentration of salicylate in the first sample of
haemolymph was 3.4±0.9 mmol l-1 (N=9). The mean
rate of decline in haemolymph salicylate concentration, determined from the
concentration difference between successive samples, was 0.0136±0.0036
mmol l-1 min-1 (N=9 larvae).
Fig. 8 also shows that
haemolymph salicylate concentration declined linearly
(r2=0.998) from 3.4 to 1.2 mmol l-1 in four
samples collected from a single larva.
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Discussion |
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Salicylate-selective microelectrodes
Microelectrodes based on the anion exchanger tridodecylmethylammonium
chloride provide rapid, sensitive and low-cost methods for measurement of
salicylate concentration in biological fluids. Salicylate flux can be
calculated as the product of fluid secretion rate and secreted fluid
salicylate concentration in the Ramsay assay, or it can be measured directly
by the Sal-SeR microelectrode technique. Salicylate-selective microelectrodes
can also be used to measure salicylate concentrations in the haemolymph after
feeding animals salicylate-enriched diets or injecting salicylate into the
circulatory system. Such measurements permit estimates of the rates of
clearance of salicylate from the haemolymph.
For Malpighian tubules set up in the Ramsay assay the results show good
agreement between salicylate flux determined using salicylate-selective
microelectrodes and that determined using liquid scintillation counting of
radiolabelled salicylate. Dose-response curves relating salicylate flux across
isolated Malpighian tubules to bathing saline salicylate concentration can be
determined using the Ramsay assay and either radiolabelled salicylate or
salicylate-selective microelectrodes. The latter technique is preferable for
reasons of cost, time, safety and accuracy. The tip diameter of the PVC coated
microelectrodes used in this study (5 µm) would permit the use of
droplets as small as 2-3 times this diameter, corresponding to volume of much
less than one nanoliter. In practice, we have readily measured salicylate
concentration in 0.1 nl droplets. The maximum specific activity of
commercially available radiolabelled salicylate does not permit ready
measurement of its concentration in such small volumes. For example, the
highest commercially available specific activity of 14C-labelled
salicylate is 56 mCi mmol-1, equivalent to
25 cpm for a 1 nl
droplet containing 0.2 mmol l-1 salicylate. This level of
radioactivity is approximately equal to the typical background level (
30
cpm) of radioactivity encountered in liquid scintillation counting of
14C-labelled compounds. By contrast, the salicylate-selective
microelectrode maintains a near-Nernstian response down to concentrations of
0.2 mmol l-1 salicylate in fluids containing
150 mmol
l-1 Cl-, and can be used in droplets that are ten times
smaller (0.1 nl).
Salicylate flux measured using the Sal-SeR microelectrode technique can
also be compared with the flux calculated from the Ramsay assay. The two
techniques are complementary, as both measure steady-state rates of salicylate
transport. Comparison of main segment salicylate flux for the Ramsay technique
with the corresponding flux measured with the Sal-SeR microelectrode requires
division by the tubule surface areax60 to produce the units measured by
the self-referencing microelectrode (pmol cm-2 s-1). For
tubules bathed in saline containing 0.5 mmol l-1 salicylate, the
flux in the Ramsay assay was 2.27 pmol min-1 tubule-1
(Fig. 7C) and the flux measured
by the Sal-SeR microelectrode was 9.03 pmol cm-2 s-1
(Fig. 5). Assuming that the
tubule can be represented as a cylinder, a nominal surface area of the
basolateral aspect of the tubule can be estimated from DL, where
D is diameter and L is length. The outside diameter of
tubules measured with an eyepiece micrometer is
46 µm and the length
of the tubule in the Ramsay assay is
2.18 mm, giving a surface area of
0.0032 cm2 (Rheault and
O'Donnell, 2004
). The salicylate flux measured using the Ramsay
assay is therefore equivalent to 12.0 pmol cm-2 s-1,
which is 1.3 times greater than the flux measured directly by the Sal-SeR
microelectrode technique. This ratio is undoubtedly smaller, and the agreement
between the two techniques closer, because our estimation of tubule surface
area is based on the assumption that the tubule is a cylinder. The functional
surface area of a tubule will be larger than our estimation due to the
extensive infoldings of the basolateral membrane
(O'Donnell et al., 1985
). The
agreement between the flux of salicylate measured in the Ramsay assay with
that measured using the Sal-SER microelectrode technique indicates that the
flux measured by the latter corresponds primarily to transepithelial
salicylate transport, as opposed to transport across the basolateral membrane
followed by sequestration in intracellular organelles. However, it is
important to note that if tubule surface area is greatly underestimated, then
the flux measured by the Sal-SeR microelectrode may be larger than that
measured in the Ramsay assay. The difference could be accounted for by
sequestration of salicylate within the cells.
The self-referencing microelectrode technique is of particular advantage
for tissues, such as the gut in small insects like as Drosophila,
which may be difficult to perfuse or set up as flat sheets or everted sac
preparations, as has been done successfully for analysis of epithelial
transport in larger species, such as locusts and lepidopteran larvae (e.g.
Phillips, 1964,
Moffett, 1980
).
Self-referencing microelectrodes have previously been used in studies of
H+ and Cl- transport by the gut of the mosquito
Aedes aegypti (Boudko et al.,
2001
). The technique is also of use for comparison of associated
gut regions and the Malpighian tubules in a single preparation. The tubules
can be removed or left intact to assess the effects of high lumen
concentrations on transport across regions of the gut such as the rectum and
ileum that are downstream of the point at which the tubules join the gut.
The Sal-SeR microelectrode technique may be of use in future studies of
salicylate transport in other systems. Salicylate is known to activate heat
shock proteins in other animals (Ishihara
et al., 2003), induce apoptosis
(Lee et al., 2003
) and alter
sensory cell functioning (Cazals,
2000
), as well as act as a substrate or competitive inhibitor of
organic anion transporters (Russel et al.,
2002
).
The major limitation of the salicylate-selective microelectrode techniques described in this paper is that other organic anions may interfere with the response of the electrode to salicylate. The results indicate that the organic anions PAH and cAMP are poorly detected by the electrode and it is therefore feasible to use appropriate concentrations of these compounds and assess their impact on salicylate transport in vitro or in vivo. However, detailed studies of competitive and non-competitive inhibitors of salicylate transport that do interfere with the electrodes will require the use of alternative approaches, such as measurement of radiolabelled salicylate fluxes.
Salicylate transport by Drosophila
Our results show that the organic anion salicylate is transported at high
rates by the isolated Malpighian tubules and gut of Drosophila. This
study also documents the extraordinary capacity of insects to clear the
haemolymph of organic anions. Salicylate-selective microelectrodes can be used
to monitor the concentrations of salicylate in the haemolymph in response to
dietary loading or direct injection into the haemocoel. Measurements in
animals fed salicylate-enriched diet revealed that the haemolymph salicylate
concentration is far below that in the food. Slowing metabolism by chilling
the animals resulted in an increase in haemolymph salicylate concentration,
suggesting that metabolism maintains salicylate levels in the haemolymph below
those in passive electrochemical equilibrium with the gut contents.
The contribution of the Malpighian tubules to clearance of salicylate from
the haemolymph can be assessed by comparison of the rates of decline of
haemolymph salicylate concentration in vivo with the rates of
transport of salicylate by isolated Malpighian tubules in vitro.
Repeated measurements of salicylate concentration in the haemolymph of 3rd
instar larvae that had previously been injected with salicylate permit
indicated that salicylate concentration declines at the rate of 0.0136 mmol
l-1 min-1 (Fig.
8). For the initial mean concentration of 3.4 mmol l-1
in the haemolymph after injection of salicylate, the concentration of
salicylate in the haemolymph would decline by 50% in 125 min. This
half-time for clearance can be compared with estimates based on the salicylate
transport rates of isolated Malpighian tubules. The maximum flux across a
single isolated tubule is 2.72 pmol min-1 tubule-1.
Given that the haemolymph volume of a 3rd instar larvae is
1 µl
(Carton et al., 2002
), then
four tubules transporting at the maximal rate will reduce the salicylate
concentration from 3.4 mmol l-1 to 1.7 mmol l-1 in
156 min. This value will increase if there is reabsorption of salicylate
across the lower tubule, but it is nonetheless of the same order of magnitude
as the half-time estimated from the rate of decline in haemolymph salicylate
concentration. These calculations suggest that the Malpighian tubules may play
an important role in the elimination of salicylate.
Salicylate excretion will be further aided by transport across the gut,
which will constrain movement of salicylate from gut lumen to haemolymph when
the fly's diet contains salicylate. When very high concentrations of
salicylate are present, the capacity for haemolymph to lumen secretion of
salicylate may be exceeded, so that a net flux from lumen to haemolymph will
occur. In support of this view is our finding that fluxes measured with
Sal-SeR microelectrodes indicated transport of salicylate from lumen to bath
for guts isolated from animals fed salicylate-rich diet. Estimates of the
gut's contribution to salicylate excretion are complicated by difficulties in
estimation of gut surface area. Such estimates are required to convert the
Sal-SeR microelectrode flux data (pmol cm-2 s-1) into
flux across the entire gut. It is also worth noting that our data on the
decline of salicylate concentrations in the haemolymph do not reveal whether
salicylate has been excreted, sequestered within tissues or metabolized to
neutral compounds such as salicin and catechol
(Ruuhola et al., 2001) that
are not detected by the electrode.
As noted above, the concentration of salicylate in the lumen is as much as
50 to 100-fold higher than that of the bathing saline. This finding indicates
active transport of salicylate, since the lumen-positive transepithelial
potential of 45 mV(O'Donnell et al.,
1996) could account for an elevation of only 6-fold above that in
the bath under conditions of passive electrochemical equilibrium.
High lumenal concentrations of salicylate provide an explanation for the
transient reversal of salicylate flux in response to metabolic inhibition of
isolated Malpighian tubules. The Ramsay assays indicate that the concentration
of salicylate in the tubule lumen is 7 mmol l-1 when the
bathing saline contains 0.5 mmol l-1 salicylate. During the first
15 min after addition of NaCN the flux of salicylate reversed direction, so
that net transport from lumen to bath was observed. We suggest that this
reversal reflects passive leakage of salicylate down its concentration
gradient, from the
14-fold higher concentration in the lumen to the lower
concentration in the bath. As this concentration gradient dissipates, the
efflux progressively declines.
Our results also revealed pronounced spatial and temporal heterogeneity in
the transport of salicylate across the main segment of the Malpighian tubule.
Previous studies have shown that transport of tetraethylammonium is nearly
constant over the length of the main segment and shows little variation over
time (Rheault and O'Donnell,
2004). However, studies with self-referencing K+
microelectrodes have indicated that not all morphologically similar cells
participate equally in K+ transport, nor do they respond equally to
stimulation of K+ transport with cAMP
(Rheault and O'Donnell, 2001
).
Similarly, `hot-spots' in extracellular Cl--dependent current
density are associated with stellate rather than principal cells and current
density can vary dramatically between stellate cells in a single tubule
(O'Donnell et al., 1998
).
Salicylate influx across the MT main segment also shows pronounced spatial
variation (Fig. 4). One
possible explanation for this finding is that local variations in
transepithelial or apical membrane potential may affect either active
salicylate transport or the extent of passive backflux from lumen to bath,
thereby altering the net flux. It is worth noting in this context that the
space constant of the MTs of another dipteran, the mosquito Aedes
aegypti, is
300 µm (Veenstra
et al., 1997
), so variations in transepithelial potential along
the length of the MT are feasible.
Temporal variations in transepithelial ion flux have also been observed in
earlier studies. The rate of K+ reabsorption across the lower
Malpighian tubule oscillates with a period of 0.8 min
(Rheault and O'Donnell, 2001).
In addition, the transepithelial potential across the main segment of
Drosophila tubules oscillates with a period of 0.6 min, and it
appears that these oscillations reflect Ca2+-dependent changes in
transepithelial Cl- permeability
(Blumenthal, 2001
).
Oscillations in salicylate transport across the main segment were much slower
than either of the above phenomena, with a period of
40 min. One possible
explanation is that high intracellular or lumenal concentrations of salicylate
affect other cellular processes, as evidenced by the decline in fluid
secretion rate when the salicylate concentration in the bathing saline exceeds
0.5 mmol l-1.
Further studies are required to describe the mechanisms of transepithelial
salicylate transport by the Malpighian tubules and hindgut. It is worth noting
that salicylate has been shown in other studies to be a substrate or a
competitive inhibitor of several membrane transporters. These include the
organic anion transporter OAT1
(Apiwattanakul et al., 1999;
Russel et al., 2002
), the
voltage-dependent organic anion transporter OATV1
(Jutabha et al., 2003
), a
proton-linked monocarboxylate transporter
(Emoto et al., 2002
) and the
sodium phosphate transporter NPT1 (Uchino
et al., 2000
).
In summary, we have developed ion-selective microelectrodes suitable for measurement of haemolymph levels of salicylate and for analysis of salicylate transport across isolated tissues. We have used these techniques to demonstrate transport of salicylate by the Malpighian tubules and gut of the fruit fly. Further studies will address modulation of salicylate transport by peptides and intracellular second messengers, or in insects fed on different diets.
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