Organic cation transport by Malpighian tubules of Drosophila melanogaster: application of two novel electrophysiological methods
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Canada L8S 4K1
* Author for correspondence (e-mail: rheaulmr{at}mcmaster.ca)
Accepted 29 March 2004
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Summary |
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Key words: organic cation transport, tetraethylammonium, ion-selective microelectrode, Malpighian tubule, Drosophila melanogaster
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Introduction |
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There are no reports of TEA and/or choline transport by the Malpighian
(renal) tubules of insects, although mechanisms of inorganic ion transport and
the control of these processes by hormones and second messengers have been
studied extensively (O'Donnell and Spring,
2000; Dow and Davies,
2001
). Drosophila has four Malpighian tubules arranged in
anterior and posterior pairs. 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
).
The main segment secretes a near-isosmotic fluid containing
120 mmol
l1 K+,
30 mmol l1
Na+ and
150 mmol l1 Cl
(O'Donnell and Maddrell,
1995
). The proximal (lower) segment of the Malpighian tubule (LMT)
secretes Ca2+ into the lumen, acidifies the luminal fluids and
reabsorbs K+, Cl and water
(O'Donnell and Maddrell,
1995
). The posterior tubules are identical except that they lack a
distal segment.
Insect Malpighian tubules also exhibit mutually competitive transport of
nitrogenous bases such as morphine, nicotine and atropine, suggesting the
presence of a multi-alkaloid transporter
(Maddrell and Gardiner, 1976).
Studies of isolated Malpighian tubules of Manduca larvae by Gaertner
et al. (1998
) suggest that the
insect alkaloid transporter involves a p-glycoprotein-like mechanism.
Manduca tubules also excrete basic (cationic) dyes such as methyl
green and methylene blue (Nijhout,
1975
). There are also reports of organic anion transporters for
sulphonates and carboxylates in tubules of Drosophila and many other
insect species (Maddrell et al.,
1974
; Bresler et al.,
1990
; Quinlan and O'Donnell,
1998
; Linton and O'Donnell,
2000
).
This paper describes the application of new electrophysiological techniques
for assessing the fluxes of tetraalkylammonium compounds, especially TEA, and
has applied these techniques to analysis of organic cation transport by
isolated tissues of Drosophila melanogaster. The techniques exploit
the high selectivity of the cation exchanger potassium
tetra-p-chlorophenylborate (Corning 477317) for quaternary ammonium
compounds such as TEA and tetramethylammonium (TMA). Although originally used
for measurement of K+ activity, the selectivity of microelectrodes
based on this exchanger for TEA and TMA exceeds that for K+ by
factors of 107 and 102.7, respectively
(Oehme and Simon, 1976;
Ammann, 1986
). Ion-selective
microelectrodes based on cation exchangers have previously been used to reveal
changes in extracellular space volume and tortuosity in the mammalian central
nervous system through measurement of the extracellular concentration of TMA
(Nicholson and Phillips,
1981
). Microelectrode measurement of TMA concentration has also
been used in studies of cell volume regulation in epithelial cells of the
gallbladder of Necturus maculosus
(Reuss, 1985
). In this
procedure, cells are loaded with TMA by transient exposure to a solution of
high TMA concentration containing the pore-forming antibiotic nystatin. Upon
removal of nystatin, in the continued presence of TMA, spontaneous restoration
of the native ionic permeability of the cell membrane has been observed. After
resealing of the cell membrane and removal of bathing saline TMA,
intracellular TMA concentration can be measured using microelectrodes based on
the cation exchanger potassium tetra-p-chlorophenylborate (Corning
477317). Changes in intracellular TMA concentration, and hence in cell volume,
elicited by alterations of the bathing medium osmolality, can thus be recorded
(Reuss, 1985
).
Our first method combines the high selectivity of the Corning 477317 ion
exchanger for TEA and the self-referencing ion-selective microelectrode
technique for spatial and temporal analysis of ion flux
(Smith et al., 1994;
Piñeros et al., 1998
).
A TEA-selective self-referencing (TEA-SeR) microelectrode is moved between two
positions within the unstirred layer next to the surface of a cell, and the
measured difference in TEA concentration between the two positions is
converted into a corresponding TEA flux. We have used this method to assess
spatial and temporal variations in TEA flux in different regions of the
Malpighian tubules and gut.
Our second approach uses TEA-selective microelectrodes to measure the concentration of TEA 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 TEA concentration.
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Materials and methods |
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Dissection and Ramsay assay
Procedures for dissection of Malpighian tubules and fluid secretion assays
based on a modification of the original Ramsay
(1954) technique have been
described previously (Dow et al.,
1994
). The anterior and posterior pairs of Malpighian tubules 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 l1): 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 10 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 until the lower tubule and the common
ureter of the tubule pair was positioned in the oil just outside the bathing
droplet. The lower tubule and ureter were thus positioned outside of the
bathing saline, and so 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 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
min1) was calculated by dividing 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 20 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.
Self-referencing ion-selective (SeRIS) microelectrode systems
Technical and theoretical aspects of SeRIS microelectrodes have been
described previously (Smith et al.,
1994; Piñeros et al.,
1998
). Application of the technique to the study of K+
transport by isolated insect Malpighian tubules was described in Rheault and
O'Donnell (2001
).
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 on a programmable puller (P-97 Flaming-Brown; Sutter
Instrument Co., Novato, CA, USA), 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 back-filled with 100 mmol l1 TEA
Cl. Inclusion of TEA in the backfilling solution prevents non-Nernstian
electrode responses to tetraalkylammonium ions
(Nicholson and Phillips,
1981
). The TEA Cl 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 Corning ion exchanger 477317 (IE190; WPI, Sarasota,
FL, USA). Electrical contact between the microelectrode and the head stage of
the self-referencing probe was made through a chlorided silver wire (EHBI;
WPI). 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 l1
KCl and 1% agar inserted into a microelectrode holder half-cell (MEH3S; WPI)
filled with 3 mol l1 KCl. In some experiments, the transport
of tetramethylammonium (TMA), tetrapropylammonium (TPA) or tetrabutylammonium
(TBA) was measured. Corresponding self-referencing microelectrodes for TMA,
TPA or TBA were also based on Corning ion exchanger 477317, but the
backfilling solutions were 100 mmol l1 TMA Cl, TPA Cl or TBA
Cl, respectively. Preliminary experiments showed that electrodes based on the
same exchanger responded poorly to changes in the concentration of
tetrapentylammonium Cl, and thus the transport of this compound was not
examined.
Briefly, the SeRIS used in this study utilized an orthogonal array of
computer-controlled stepper motors (CMC-4; Applicable Electronics Inc.,
Forrestdale, MA, USA) fitted to a set of translator stages (Newport Corp.,
Fountain Valley, CA, USA). The stepper motors 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. The electrode was moved only at
right angles to the long axis of the electrode because movement along the long
axis affects stability of the ion exchanger column, as discussed by Smith et
al. (1994). Voltage
measurements taken at the limits of the excursion were amplified 1000-fold
using an IPA-2 ion/polarographic amplifier (Applicable Electronics Inc.).
These measurements were used to calculate a voltage difference over the
excursion distance of the microelectrode. This differential signal was then
converted into a TEA concentration difference using a standard microelectrode
calibration curve that related voltage output to TEA concentration in saline.
The highly sensitive self-referencing system resolves voltage differences as
small as 10 µV in a bathing medium containing 4 mmol l1
TEA, corresponding to a difference in concentration as small as 0.04%. The
TEA-SeR microelectrode was viewed using an inverted microscope equipped with a
video camera. A Pentium PC running automated scanning electrode technique
(ASET) software (Sciencewares, East Falmouth, MA, USA) controlled the `move,
wait and sample' protocol at each measurement site. The TEA-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.
Lastly, 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 were
reported as an average of 35 repetitive measurements at each site.
TEA-SeR microelectrode measurement of TEA flux: electrode slope, selectivity and efficiency
In saline containing 20 mmol l1 K+, the
electrode slope for a change from 0.1 to 1 mmol l1 TEA was
58.2±0.1 mV (n=50 microelectrodes). Under the conditions of
our experiments, application of the NicolskyEisenman equation
(Ammann, 1986) indicates that
the interference of K+ on the differential signal recorded by the
TEA-SeR microelectrodes is negligible (<0.01 µV). Slopes of
microelectrodes based on Corning ion exchanger 477317 for a 10-fold change in
the concentration of TMA, TPA and TBA from 0.4 mmol l1 to 4
mmol l1 in saline were 57.7 mV (N=2), 58.5 mV
(N=2) and 59.6 mV (N=1), respectively.
Previous studies have shown that because of the time constant associated
with measurements made with high-impedance electrodes, self-referencing
microelectrodes for other ions may measure less than the actual gradient if
vibrated between the two excursion limits at frequencies of >0.3 Hz. Under
these circumstances, the electrode may measure only 65% of the true signal for
Ca2+ (Smith et al.,
1994), 85% for K+ or 63% for H+
(Faszewski and Kunkel, 2001
)
and must be corrected to provide estimates of true flux
(Smith et al., 1994
). Recent
papers suggest that measurements of Cl can be achieved with
near 100% efficiency (Land and Collett,
2001
). We have used a move, wait and sample protocol that involves
sampling once every 10 s. These low sampling rates are appropriate because of
the relatively large size of the signals recorded by TEA-SeR microelectrodes
(typically 1001000 µV) relative to the rate of drift of the
electrode potential
0.4 µV S1). Efficiency of
TEA-SeR microelectrodes was determined using a gradient established by leakage
of TEA from a source microelectrode filled with 1 mmol l1
TEA, which was placed in a bath containing 100 µmol l1
TEA. The efficiency of TEA-SeR microelectrodes was not significantly different
from 100%, based on 31 self-referencing measurements of gradients between 700
and 1200 µV and comparing these with measurements made at intervals of 60 s
using static TEA-selective microelectrodes.
For TEA-flux measurements with SeRIS microelectrodes, tissues were transferred after dissection to 35 mm-diameter Petri dishes filled with 4 ml saline. Petri dishes were pre-coated with 100 µl droplets of 125 µg ml1 poly-L-lysine and air dried before filling with saline to facilitate adherence of the tubules to the bottom of the dish. Fluxes were typically measured at 38 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 Malpighian tubule could be scanned. When there were significant spatial variations in TEA flux, these have been plotted as a function of distance from some readily identifiable morphological feature such as the junction of the ureter and hindgut or the junction of the ureter and the two lower Malpighian tubules. When there was no evidence of spatial variation, the data were pooled for each region (midgut, hindgut).
Calculation of TEA flux
TEA-specific signal differences (V; measured in µV)
obtained over the amplitude of the TEA-SeR microelectrode excursion were
corrected for the background concentration of TEA and converted to a TEA
concentration difference (
C; µmol cm3)
using the equation:
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Effects of variations in bathing saline K+ or drugs on TEA electrode signal
For experiments involving application of drugs or changes in bathing saline
K+, the response of the electrode to a 10-fold change in [TEA] was
measured in the presence or absence of the drug or change in [K+].
This was necessary because electrodes based on
tetra-p-chlorophenylborate respond to organic cations other than TEA.
All drugs were dissolved in saline. A reduction in slope of >2 mV was
considered the upper limit of acceptability for the drug at the concentration
used. There was no effect of cimetidine (0.1 mmol l1),
verapamil (5 µmol l1) or vinblastine (2 µmol
l1) on electrode response to TEA (0.11 mmol
l1). For 10 µmol l1 quinidine, the
slope of the electrode response to a 10-fold change in TEA concentration was
reduced by 2.5 mV. For 2 µmol l1 quinidine, there was no
effect on electrode slope. There was no effect on TEA-SeR microelectrode slope
when [K+] in the bathing saline was increased from 2 to 20 mmol
l1 or decreased from 100 to 10 mmol
l1.
Measurement of TEA in secreted fluid
In the second technique, TEA flux was calculated as the product of fluid
secretion rate (nl min1) and secreted fluid TEA
concentration. Secreted fluid droplets were collected under paraffin oil using
the Ramsay assay, and the concentration of TEA was determined using
TEA-selective microelectrodes. One problem with the use of ion-selective
microelectrodes for these measurements is that the paraffin oil tends to enter
the tip of the silanized micropipette tip by capillarity, displacing the
ionophore cocktail. In the past, we have used careful adjustment of the
silanization time so that the micropipette is sufficiently hydrophobic to
retain the ionophore cocktail when the micropipette tip is in aqueous
solutions but not so hydrophobic as to cause movement of oil into the tip.
Although this approach works well with ionophore cocktails based on neutral
carriers in nitrophenyl octyl ether, we have found the success rate to be low
for electrodes filled with Corning 477317. Instead, we have found that the
displacement of the ionophore cocktail in silanized micropipettes placed in
paraffin oil can be prevented by coating the tip of the micropipette with a
thin layer of high-molecular-mass poly vinyl chloride (PVC; 81392,
Sigma-Fluka, Oakville, ON, Canada). It is worth noting that macroscopic
ion-selective electrodes are often fabricated using PVC membranes and
appropriate ionophores (Ammann,
1986). Micropipettes were silanized, backfilled with 100 mmol
l1 TEA Cl and front-filled with Corning 477317 as described
above for TEA-SeR microelectrodes. The tip of the micropipette was then dipped
in a 5% (w/v) solution of PVC in tetrahydrofuran for
0.5 s. The dipping
was repeated 34 times at intervals of a few seconds. Evaporation of the
tetrahydrofuran resulted in a thin
1 µm) coating of PVC around the
distal 200500 µm of the micropipette. Electrode slope and
selectivity were unaltered by coating with PVC.
Calculations and statistics
Values are expressed as means ±
S.E.M. for the indicated number (n)
of sampling sites on the indicated number of tubules (N). 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 assuming either equal or
unequal variances. Differences were considered significant if
P<0.05. The responses of the same group of tubules before and
after an experimental treatment were compared using a paired t-test.
Concentrationresponse curves relating TEA flux or secreted fluid TEA
concentration to bathing saline TEA concentration were fitted using a
commercial graphics and analysis package (Igor; Wavemetrics Inc., Lake Oswego,
OR, USA) and an associated set of analysis procedures written by Dr F. Mendez
(Patcher's Power Tools;
http://www.wavemetrics.com/Users/ppt.html).
The iterative procedure allowed estimation of the kinetic parameters
Jmax and Kt (see below) for TEA
transport.
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Results |
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Previous studies using the Ramsay technique for fluid collection from isolated Malpighian tubules under paraffin oil have not examined the transport properties of the ureter because this region of the tubule is necessarily positioned outside the bathing saline droplet to permit collection of secreted fluid droplets. The SeRIS permits direct measurement of ion flux across the ureter. TEA influx across the lower Malpighian tubule and the adjacent regions of the ureter were of similar magnitude (Fig. 3). Influx was reduced in the proximal ureter closest to the junction with the gut (Fig. 3B).
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Scans of the gut with TEA-SeR microelectrodes revealed that TEA is secreted by the posterior half of the midgut. The mean flux of 0.99±0.09 pmol cm2 s1 (n=45 sites in eight preparations) was of similar magnitude to that across the main segment of the Malpighian tubules. Scans of the anterior half of the midgut, including the proventriculus, showed that the magnitude of TEA flux (0.03±0.04 pmol cm2 s1; n=45 sites in four preparations) in this region was not significant. By contrast, there was a small but significant efflux of TEA of 0.12±0.03 pmol cm2 s1 (n=43 sites in four preparations) across the hindgut and rectum.
Kinetics and selectivity of tetraalkylammonium transport
Measurements of TEA flux in bathing saline TEA concentrations between 0.05
and 4 mmol l1 were used to construct
concentrationresponse curves for TEA transport by the lower tubule, the
main segment and the posterior midgut (Fig.
4). The values of the MichaelisMenten parameters
Jmax and Kt for the main segment and
the posterior midgut were similar, whereas TEA flux across the lower tubule
was associated with a lower Kt and a
Jmax value approximately 4-fold higher than in the other
two tissues (Fig. 4). The
transport efficiency for each tissue was calculated as
Jmax/Kt. Such calculations showed that
the transport efficiency of the lower tubule was 5.8-fold greater than that of
the main segment and 7.9-fold greater than that of the posterior midgut. It
should be noted that the presence of an unstirred layer around the tubule
necessitated that our reported Kt, which reflected the
bulk medium TEA concentration, was an overestimate of the true
Kt at the membrane surface
(Winne, 1973). This was
corrected by subtracting the
C in the unstirred layer from the
concentration in the bulk medium to yield the concentration of TEA at the
membrane surface. Kinetic values were then recalculated using the
concentrations of TEA at the membrane surface and compared with those
calculated using bulk medium TEA concentrations. Using the data from our
experiments, we have approximated this overestimate to be
10 µmol
l1 for our reported Kt of
100200 µmol l1. This error is relatively
small compared with our reported values and has been ignored so that our
kinetic values measured using the TEA-SeR technique can be compared with those
in subsequent secreted fluid experiments where no estimate of unstirred layer
dimensions is possible. It should also be noted that all reported kinetic
values represent the steady-state consequence of at least two transport steps
operating in series in the tubule epithelium.
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Analysis of signal-to-noise ratios shows that the TEA-SeR microelectrodes
were sufficiently sensitive to resolve TEA fluxes over the entire
concentration range used in the experiments described in
Fig. 4. The voltage difference
(V) recorded in the unstirred layer of the lower segment in
the lowest TEA concentration (50 µmol l1) was
2485±364 µV, and the corresponding background signal in the bulk
medium where there was no gradient was 35±12 µV. The signal-to-noise
ratio was therefore 2485/35=71. At the highest concentration of TEA (4 mmol
l1), the recorded values for
V in the
unstirred layer and bath were 239±30 µV and 12±4 µV,
respectively, corresponding to a signal-to-noise ratio of 20. The
signal-to-noise ratios for the main segment at the lowest (50 µmol
l1) and highest (4 mmol l1) TEA
concentrations were 9 and 7, respectively.
In addition to TEA, self-referencing ion-selective microelectrodes based on the Corning ion exchanger were used to study transport in the lower tubule of three other quaternary ammonium compounds, TMA, TPA and TBA. Sequential comparisons of TEA flux versus TMA, TPA or TBA on the same tissue using appropriately backfilled self-referencing ion-selective microelectrodes and substitution of the bath tetraalkylammonium compounds were performed (Table 1). Fluxes of TEA were significantly larger than those of TMA, TPA or TBA at the same concentration (100 µmol l1).
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Effects of bathing saline [K+] on TEA fluxes
Previous studies have shown that carrier-mediated uptake of TEA varies when
membrane potential is altered by changing saline K+ concentration
(Smith et al., 1988).
Basolateral membrane potential in the main segment of Drosophila
tubules changes by
40 mV when bathing saline potassium concentration is
changed from 10 to 100 mmol l1 K+
(O'Donnell et al., 1996
).
Fig. 5 shows the effects of
changes in bathing saline K+ concentration on fluxes measured with
TEA-SeR microelectrodes. TEA fluxes in the lower tubule decreased by 64%,
within 6 min, when bathing saline K+ concentration was increased
from 10 to 100 mmol l1
(Fig. 5A). The decrease was
partially reversed within 6 min of restoring bath K+ concentration
to 10 mmol l1. TEA flux in the main segment increased by
176%, within 6 min, when bath K+ concentration was reduced from 20
to 2 mmol l1 (Fig.
5B).
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Pharmacology
TEA influx in the lower Malpighian tubule was reduced 53% within 30 min of
the addition of 5 µmol l1 verapamil
(Fig. 6). There was no effect
of verapamil on TEA flux in the main segment or the distal tubule. TEA influx
in the lower tubule was also reduced by 70% within 10 min of the addition of 2
µmol l1 quinidine, from 3.97±1.24 pmol
cm2 s1 (N=6 tubules) to
1.01±0.43 pmol cm2 s1. TEA influx
in the lower tubule was also reduced by 84% within 1020 min ofthe
addition of 100 µmol l1 cimetidine, from 4.34±
0.69 pmol cm2 s1 (N=5 tubules) to
0.69±0.24 pmol cm2 s1. Exposure to
2 µmol l1 vinblastine resulted in a 33% decrease in TEA
influx within 30 min, from 5.01±0.45 pmol cm2
s1 to 3.32±0.53 pmol cm2
s1. TEA influx recovered to 4.64±0.58 pmol
cm2 s1 within 10 min of washing off
vinblastine. There was no effect of these pharmacological treatments on the
basolateral membrane potential of Malpighian tubules (N=47
measurements for each drug concentration).
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Ramsay assay of transepithelial secretion of TEA
TEA-selective microelectrodes were first positioned in droplets collected
from isolated whole Malpighian tubules set up in a Ramsay assay for 20 min
prior to addition of TEA. These background measurements tested for the
presence of endogenous compounds within the droplets that might interfere with
the electrode signal. The electrode voltage in these droplets was equivalent
to that produced by 0.03±0.0 mmol l1 TEA. After 0.1
mmol l1 TEA was added to the bathing saline, the whole
tubules secreted droplets containing 1.90±0.17 mmol
l1 TEA (N=12) within 2040 min. When the
lower segment of the Malpighian tubule was pulled out of the bathing saline
into the paraffin oil, the main segments of the tubules secreted fluid
containing 0.72±0.09 mmol l1 TEA.
Fig. 7 shows a typical voltage
recording in which TEA concentration was measured in fluid secreted by a pair
of whole tubules before and after the addition of 0.1 mmol
l1 TEA to the bathing droplet and by the main segment of one
tubule from the same pair.
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Concentrationresponse curves for secretion of TEA by isolated tubules set up in Ramsay assays are shown in Fig. 8. The maximum concentration of TEA in fluid secreted by the main segment was 2.5 mmol l1 (Fig. 8A). The corresponding Jmax and Kt for TEA transport by the main segment were 1.39 pmol min1 tubule1 and 0.22 mmol l1, respectively (Fig. 8C). The maximum concentration of TEA in fluid secreted by the whole tubule was 3.28 mmol l1 (Fig. 8B). The corresponding Jmax and Kt values for TEA transport by the whole tubules were 1.52 pmol min1 tubule1 and 0.18 mmol l1, respectively (Fig. 8D). The transport efficiency (Jmax/Kt) of whole tubules was 1.3-fold greater than that of the main segments alone. At the lowest concentration of TEA in the bathing saline (5 µmol l1), the concentration of TEA was increased 12-fold for the droplets collected from the main segments and 52-fold for droplets collected from whole tubules.
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The effects of cimetidine on TEA secretion by isolated whole tubules bathed in saline containing 0.1 mmol l1 TEA are shown in Fig. 9. Addition of 0.1 and 1.0 mmol l1 cimetidine reduced secreted fluid TEA concentration by 58% and 86%, respectively. Addition of 0.1 mmol l1 cimetidine did not significantly alter the electrode signal relative to that in droplets secreted before addition of the drug. It is worth noting that there was no significant difference in the final concentration of TEA in droplets collected from tubules pre-incubated in cimetidine before addition of TEA and in droplets from tubules exposed first to TEA and then to cimetidine.
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Discussion |
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Use of tetraalkylammonium-selective microelectrodes for studies of organic cation transport
The two electrophysiological methods described in this paper facilitate
non-invasive spatial and temporal analysis of the transport of TEA and other
quaternary ammonium compounds. In comparison with radioisotopic methods, the
TEA-SeR microelectrode technique permits low-cost, direct measurements of TEA
flux with a spatial resolution of 50 µm. Measurement of flux is
provided in near real time, whereas fluxes determined from measurements using
radiolabelled probes require calculation from the rate of change in cellular
concentration of the probe. An additional problem with the use of
radiolabelled compounds is that the highest commercially available specific
activity of radiolabelled TEA is still too low to permit accurate measurement
of TEA fluxes across isolated Drosophila tubules unless droplets are
either collected over long periods of time, typically >30 min, or are
pooled from several tubules. The TEA-SeR microelectrode technique also has an
advantage over the use of radiolabelled TEA for the determination of
concentrationresponse experiments. The concentrationresponse
relationship between saline TEA concentration and TEA flux is determined
directly, whereas the use of radioisotopes requires dilution of
14C-labelled TEA with `cold' TEA and attendant changes in specific
activity. The TEA-SeR technique is not limited to secretory epithelia or those
that can be perfused and is also of use for multilayered or semi-opaque
tissues such as the gut, where the overlying musculature may confound
measurements of fluorescence intensity of probes (e.g. NBD-TMA) transported by
underlying epithelial cells.
Our second technique, the use of TEA-selective microelectrodes for analysis of secreted fluid TEA or other quaternary ammonium compounds is suitable for secretory cells or those that can be perfused. In contrast to localized measurements of TEA flux by the TEA-SeR microelectrodes, flux is calculated as the product of fluid secretion rate and secreted fluid TEA concentration, thus providing an integrated measurement of flux across the entire length of the tubule in contact with the bathing saline. Measurements of transepithelial TEA secretion by analysis of secreted fluid droplets demonstrate that the flux recorded by the TEA-SeR microelectrodes is due to transepithelial secretion of TEA, as opposed to fluxes reflecting transport of TEA across the basolateral membrane only and subsequent intracellular sequestration.
The two techniques are complementary, as both measure steady-state rates of
TEA transport. Comparison of main segment maximal TEA flux
(Jmax) for the modified Ramsay technique (1.39 pmol
min1 tubule1;
Fig. 8C) with the corresponding
maximal flux measured with the TEA-SeR microelectrode (4.7 pmol
cm2 s1;
Fig. 4) requires division by
the tubule surface area x 60 to produce the units measured by the
self-referencing microelectrode (pmol cm2
s1). 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. Outside diameter of tubules measured with an eyepiece micrometer
was
46 µm and the active length of the tubule in the modified Ramsay
assay was
2.18 mm, giving a surface area of 0.0032 cm2. The
Ramsay assay TEA flux is therefore equivalent to 7.2 pmol
cm2 s1, which is 1.5 times greater than
the flux measured directly by the TEA-SeR 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 actual surface area of a tubule will be larger than our
estimation due to the extensive infoldings of the basolateral membrane
(O'Donnell and Maddrell,
1983
).
The major limitation of both of our techniques is that ion exchanger electrodes based on potassium tetra-p-chlorophenylborate are sensitive to compounds other than TEA. Although interference from K+ or other inorganic cations is negligible, the electrodes will respond to other organic cations, including quaternary ammonium compounds. Determining the relative transport of TEA and TBA, for example, requires sequential measurements of the transport of each, rather than simultaneous competition of the two species for cellular transporters. This problem extends to the use of pharmacological reagents. Verapamil, for example, interferes significantly with the electrode at high concentrations (>10 µmol l1). Analysis of TEA transport by Drosophila Malpighian tubules using TEA-selective microelectrodes is feasible nonetheless because the concentrations of verapamil that block TEA transport are well below those that cause significant interference. For application of TEA-selective microelectrodes to studies of organic cation transport by other tissues, appropriate control experiments must be conducted to determine whether the concentrations of each drug used interfere significantly with the electrode voltage.
Organic cation transport by Drosophila Malpighian tubules and gut
Our results demonstrate TEA transport by the Malpighian tubules and
posterior midgut. Given that the transepithelial potential is of the order of
3080 mV lumen-positive (O'Donnell
et al., 1996) and that the lumen concentration of TEA is
12-fold above that in the bath for main segments bathed in 5 µmol
l1 TEA, this indicates that TEA is actively transported
across the main segment against an opposing electrochemical gradient.
Similarly, the transepithelial potential in the region of the lower Malpighian
tubule closest to the main segment is
5 mV lumen-positive
(O'Donnell and Maddrell,
1995
), again consistent with active transport of TEA. The
transepithelial potential in the proximal portion of the lower tubule is
15 mV lumen-negative (O'Donnell and
Maddrell, 1995
), which could account for a near doubling of the
concentration of TEA if the cation was in passive electrochemical equilibrium
with the transepithelial potential. However, the finding that the
concentration of TEA in the lumen is
52-fold above that in the bath for
whole tubules bathed in 5 µmol l1 TEA indicates that
active transport is a feature of this region of the tubule as well. Moreover,
TEA flux was maintained, albeit reduced, when the transepithelial potential
was made more lumen-positive by bathing in saline containing 100 mmol
l1 K+. The possibility that this 52-fold increase
in lumen-to-bath TEA concentration in the lower tubule is a consequence of TEA
secretion by the main segment followed by fluid reabsorption in the lower
tubule can be ruled out. The lower segment of the Malpighian tubule has been
shown to reabsorb
12% of the fluid secreted by the main segment
(O'Donnell and Maddrell,
1995
). This fluid reabsorption would increase the lumen-to-bath
ratio of TEA from 12-fold in the main segment to
13.5-fold in the lower
tubule. The latter value is much smaller than the observed value of 52-fold in
the lower segment. We therefore conclude that the luminal concentration of TEA
in the lower segment is due primarily to active transport of TEA in that
segment and not to secretion of TEA by the main segment and subsequent
downstream reabsorption of fluid by the lower tubule.
The novel finding that the lower Malpighian tubule actively secretes the
organic cation TEA is important when one considers this in the context of the
physiology of the lower tubule. Previous studies have shown that the lower
Malpighian tubule is involved in acidification of the urine and in active
secretion of Ca2+ and reabsorption of K+ and
Cl (O'Donnell and
Maddrell, 1995). Our findings suggest that TEA, a known
K+ channel blocker, is not using K+ channels in the
lower tubule as a pathway for secretion, because TEA is secreted by lower
tubules whereas K+ is reabsorbed in the lower tubule. In addition,
it has been proposed in vertebrate models of organic cation transport that the
apical transport of organic cations may involve an exchange of the organic
cations for protons (Pritchard and Miller,
1991
). The finding that the lower tubule is a favoured site for
TEA secretion, together with previous evidence for acidification of the
luminal fluid by the lower tubule, leads us to suggest that a mechanism for
organic cation/proton exchange may be present in the apical membrane of the
Malpighian tubule of Drosophila and deserves further
investigation.
Although we have not fully characterized the mechanism of TEA transport by
the Malpighian tubules of Drosophila in this paper, we have found
considerable evidence suggesting some similarity to mechanisms previously
observed in vertebrate renal systems. The value of Kt for
the lower segment of the Malpighian tubule is similar to that previously
reported for members of the organic cation transporter (OCT) family. In
particular, our reported Kt of 132 µmol
l1 for TEA secretion by the lower Malpighian tubule is of
similar magnitude to values reported previously for vertebrate renal
transporters. In rat kidneys, both high-affinity
(Kt=3050 µmol l1) and
low-affinity (Kt=200300 µmol
l1) basolateral sites of TEA uptake have been identified
(Goralski and Sitar, 1999).
The Kt for steady-state TEA secretion from the bath to the
lumen of snake renal tubules is
20 µmol l1
(Hawk and Dantzler, 1984
),
whereas the Kt for TEA uptake across the basolateral
membrane of flounder renal tubules is 80 µmol l1
(Miller and Holohan, 1987
).
The values of Kt for cloned organic cation transporters
from rabbits expressed in COS-7 cells are 188 and 125 µmol
l1 for rbOCT1 and rbOCT2, respectively
(Zhang et al., 2002
).
In vertebrate studies, transport of TEA by OCTs is typically blocked by
other organic cations such as quinidine, cimetidine and verapamil. Similarly,
the results of our study show that TEA secretion by the Malpighian tubules of
Drosophila was blocked by competing organic cations. In addition, our
study has shown that TEA influx is potential dependent. TEA does not block
potassium channels in the Malpighian tubules of Drosophila (M.R.R.
and M.J.O'D., unpublished), so the effects of saline K+
concentration on membrane potential are not altered by the presence of TEA. In
the main segment, bathing saline manipulations that depolarized the
basolateral membrane potential resulted in decreased TEA influx while in the
main segment bathing saline manipulations that hyperpolarized the basolateral
membrane potential increased TEA influx. These results parallel those observed
in the transport of TEA by flounder renal tubules
(Smith et al., 1988).
It is worth noting that an orthologue of the basolateral OCTs of vertebrate
kidney, designated Orct, has been demonstrated in a larval Drosophila
cDNA library (Taylor et al.,
1997). Tissue-specific expression patterns and substrate
affinities of Orct remain to be elucidated in both adult and larval
Drosophila. TEA transport across the lower tubule was sensitive to
verapamil, whereas there was no effect of the drug on TEA transport across the
main segment. This may suggest the involvement of more than one transporter
for TEA secretion by the LMT. Specifically, in addition to our suggestion
above of an organic cation/proton exchanger, inhibition by verapamil suggests
the additional involvement of a p-glycoprotein-like transport mechanism for
TEA in the lower tubule that is not involved in the secretion of TEA by the
main segment.
We also demonstrated transport of TEA by the posterior midgut. The fluxes across the anterior midgut near the proventriculus were negligible, whereas a small efflux of TEA was recorded across the hindgut and rectum. The latter observation may reflect passive leakage of TEA from the lumen of the hindgut/rectum after accumulation of TEA upstream in the lumen of the midgut and/or Malpighian tubules and ureter. The role of TEA transport by the posterior midgut may be to minimize absorption of potentially toxic organic cations from the gut lumen into the haemolymph.
In summary, we have used TEA-selective microelectrodes to show for the first time that the insect Malpighian tubule and gut secrete the prototypical organic cation TEA with characteristics reminiscent of those observed in studies of vertebrate renal epithelia. Further understanding of organic cation secretion in insects will require a detailed characterization of the mechanisms of basolateral uptake and apical transport of TEA in the Malpighian tubules.
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