Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function
Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK
* Author for correspondence (e-mail: j.a.t.dow{at}bio.gla.ac.uk)
Accepted 10 August 2005
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Summary |
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Radiolabelled glibenclamide is both potently transported and metabolised by tubule. Furthermore, glibenclamide is capable of blocking transport of the organic dye amaranth (azorubin S), at concentrations of glibenclamide much lower than required to impact on fluid secretion. Glibenclamide thus interacts with tubule in three separate ways; as a potent inhibitor of fluid secretion, as an inhibitor (possibly competitive) of an organic solute transporter and as a substrate for excretion and metabolism.
Key words: ir, irk2, irk3, glibenclamide, inward rectifier channel, Malpighian tubule, functional genomics, Drosophila melanogaster
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Introduction |
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Drosophila melanogaster, despite its small size, offers unique
advantages for the study of epithelial physiology, and accordingly, the
molecular basis of D. melanogaster renal function is now understood
in considerable detail (Dow and Davies,
2001,
2003
). Among the tools
available to the Drosophila community is a comprehensive Affymetrix
microarray, which allows gene expression to be studied quantitatively over
space and time. A recent such study, comparing adult Drosophila
tubule with whole flies, identified genes that are either abundantly expressed
or highly enriched in tubule (Wang et al.,
2004
). Thus, this study both identifies candidate genes for known
transport and signalling functions and suggests new, previously unsuspected
functions. Among the significant findings of this study was the observation
that, of the very large K+ channel gene family of
Drosophila, all three of the inward rectifier Drosophila
genes (ir, irk2 and irk3) were both highly abundant and
strongly enriched (to much greater extents than any other K+
channels), clearly implicating inward rectifier potassium channels (IRKs) in
epithelial function.
Mammalian IRKs form both homo- and hetero-tetramers and are sensitive to
barium and to the antidiabetic sulphonylureas such as glibenclamide
(Bryan and Aguilar-Bryan,
1997). Possible orthologues expressed in mammalian kidney include
Kir6.x and Kir1.x. These channels are often associated with ABC transporters,
such as cystic fibrosis transmembrane conductance regulator (CFTR) and
sulphonylurea receptor (SUR), and complexes with these receptors render the
channels more sensitive to glibenclamide
(Ruknudin et al., 1998
).
The highly upregulated expression pattern of the IRKs prompted our investigation into the effect of sulphonylureas on the Malpighian tubules. Not only do sulphonylureas inhibit fluid secretion, but glibenclamide blocks organic dye excretion by the tubule, and glibenclamide itself is both excreted and modified by the tubule.
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Materials and methods |
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Secretion assay
Fluid secretion assays were performed as described
(Dow et al., 1994), taking
readings every 10 min. Glibenclamide was added after the tubule basal
secretion rate had been measured for 30 min. The secretion rate continued to
be recorded for a further hour. Inhibition was calculated as the difference
between the basal rate (measured over the first 30 min) and the secretion rate
at 50 min. To study the effects of glibenclamide on neuropeptide-stimulated
secretion, glibenclamide was added at 30 min, and the diuretic peptides
Drosophila leucokinin (dLK;
Radford et al., 2002
;
Terhzaz et al., 1999
) and
Drosophila corticotropin releasing factor-like peptide (dCRF, DH;
Cabrero et al., 2002
) were
added at 60 min, then the secretion rate recorded for a further hour. In this
way, the impact of sulphonylureas could be assessed both on resting and
maximally stimulated fluid secretion.
Real-time quantitative PCR (qPCR)
qPCR was performed as described previously
(McGettigan et al., 2005).
Briefly, mRNA was prepared from 7-day-old Oregon R flies or their tubules
using Qiagen RNAeasy column, following the manufacturer's methods (Qiagen,
Crawley, West Sussex, UK). Reverse transcription was carried out using
Superscript II (Invitrogen) using oligo-dT primer. For each sample, 500 ng of
cDNA was added to 25 µl of SYBR Green reaction mix (Finnzymes, GRI,
Braintree, Essex) and appropriate primers
(Table 1). An Opticon 2
thermocycler (MJ Research, now Bio-Rad, Hemel Hempstead, Herts, UK) was set as
follows; the recommended 15 min HotStart Taq activation time, then 40 cycles
of (denaturing at 94°C for 30 s, annealing for 30 s at primer-dependent
temperature, 30 s of extension at 72°C, and 10 s absorption reading at
80°C), followed by a 5 min final extension at 72°C and a melting curve
from 70°C to 90°C. The ribosomal rp49 gene was used as a
standard in all experiments. Data were then expressed as fold difference of
tubule cDNA compared with whole fly cDNA (± S.E.M.).
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In situ hybridization
Tubules were dissected, fixed and subjected to in situ
hybridization according to published protocols
(Siviter et al., 2000) and the
Berkeley Drosophila Genome Project (BDGP) 96-well in situ
protocol
(http://www.fruitfly.org/about/methods/RNAinsitu.html).
In situ probes were directed towards the 3' UTR of each gene to
minimise cross-hybridisation between related genes. PCR products derived from
the 3' UTR of each gene were cloned into pBluescript or pCRII vectors
(Invitrogen), and DIG-labelled RNA probes were generated by in vitro
transcription. Adult tissues comprising gut, testes, ovaries and Malpighian
tubules were dissected in Schneider's medium (Invitrogen) and placed into
wells of a Millipore 96-well plate (MAGVN22 or MAGVS22) with 100 µl
Schneider's medium. Schneider's medium was removed using a vacuum pump, and
postfix solution [10 mmol l-1 potassium phosphate buffer (pH 7.0)
containing 140 mmol l-1 NaCl, 0.1% Tween 20 and 5% v/v
formaldehyde] was added for 20 min, followed by three washes with PBT [10 mmol
l-1 potassium phosphate buffer (pH 7.0) containing 140 mmol
l-1 NaCl and 0.1% v/v Tween 20]. The tissues were incubated with
proteinase K in PBT (4 µg ml-1) for 3 min at room temperature.
The reaction was stopped with two washes of PBT containing 2 mg
ml-1 glycine. The samples were washed twice with PBT before
incubating with postfix for a further 20 min at room temperature. The tissues
were washed with five changes of PBT, followed by one wash with 50%
hybridisation buffer [5x SSC containing 50% v/v formamide, 10 mmol
l-1 kPO4, 140 mmol l-1 NaCl, 1 mg
ml-1 glycogen, 0.2 mg ml-1 sheared salmon sperm DNA and
0.1% v/v Tween 20 (pH 7.0)] plus 50% PBT. The samples were washed once with
hybridisation buffer, prior to a 1 h preincubation with hybridization buffer
at 55°C, and subsequently incubated for 43 h at 55°C with 100 µl of
hybridisation buffer containing 10-500 ng of either the sense or antisense
riboprobe, taking care to seal the wells with ParafilmTM to prevent
evaporation. Following hybridisation, the samples were washed four times with
hybridization buffer at 55°C, followed by a final wash overnight with
hybridization buffer at 55°C. Samples were washed once with 50% v/v
hybridisation buffer and 50% v/v PBT, followed by four washes with PBT and
then incubated overnight at room temperature with 100 µl of pre-absorbed
alkaline phosphatase-conjugated anti-digoxigenin Fab fragment (Roche Molecular
Biochemicals, Lewes, East Sussex, UK) diluted 1:2000 with PBT. The unbound
antibody was removed with extensive washing in PBT (at least 10 times for
5-10 min). The samples were incubated with DIG detection buffer [100 mmol
l-1 Tris-HCl (pH 9.5), 100 mmol l-1 NaCl, 50 mmol
l-1 MgCl2] for 5 min then repeated again. The colour
reaction was initiated by the addition of DIG detection buffer +
5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT)
and left for 10 min-2 h at room temperature. Development was stopped with
extensive washing with PBT containing 50 mmol l-1 EDTA, and the
tissues were removed from the wells, mounted on slides with 70% glycerol and
viewed on an Axioscope microscope equipped with an Axiocam imaging system
(Zeiss, Welwyn Garden City, UK).
Confocal imaging
Tubules were dissected and mounted on poly-lysine-coated slides in
Schneider's medium. Glibenclamide labelled with Texas Red (Molecular Probes,
now Invitrogen) was added to a final concentration of 0.1 mmol l-1.
A time series was generated under fluorescence with a Zeiss 510 Meta Confocal
system. All images were taken at the same gain and exposure and processed
using Axiovision 3.0.6 software.
Glibenclamide transport assays
Glibenclamide, at 10-6-10-3 mol l-1, was
labelled with 0.05 µCi 125I (Amersham Pharmacia, Amersham, UK,
specific activity, 2000 Ci mmol l-1) and added to tubule secretion
assays, performed as described above. Secreted fluid was collected for 1 h,
the volume measured and counted in Optiflow SAFE scintillant (Fisher
Scientific UK, Loughborough, Leicestershire, UK). Transport ratios were
calculated as the ratio of specific activities of secreted drop: reservoir
bubble (values >1 thus imply concentration of the label by the tubule).
Thin layer chromatography (TLC)
Samples of 1 µl of both authentic [125I]glibenclamide (0.05
µCi) and secreted fluid, obtained by the same method as the glibenclamide
transport assay, were dried onto a 10x20 cm Polygram Sil G/UV254 plate
(Machery-Nagel GmbH, Düren, Germany) and run out using an eluent of 85%
v/v ethanol, 15% v/v PBS (pH 7) and 0.1% w/v SDS. Plates were visualized with
a Fuji PhosphorImager.
Statistics
Where errors are shown, these represent the standard error of the mean
(S.E.M.). Where appropriate, the significance of differences was
tested with Student's t-test (two-tailed), taking a critical level of
P<0.05.
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Results |
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One of these genes, irk2, was known to have two splice forms (Fig. 1), although the Affymetrix probes (against the 3' end of the mRNA) would not have distinguished them. qPCR measurement allowed the identification of irk2-RA as the major transcript (Table 1). These results also suggested the presence of an additional splice form, verified by cloning and sequencing the PCR product. This new splice form, which will be denoted RC, has an extra 21 bases prior to the translated region of the RA splice form (Fig. 1). It appears to be a low-abundance RNA product in the tubules, comprising only 6.2±0.6% of the expression of Irk2-RA. Irk2-RC did not reliably produce a qPCR product from the whole-fly cDNA because of its very low copy number. No other D. melanogaster IRK-encoding genes are known to have alternative splice forms.
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The other two genes gave fainter signals, but both were still clearly
expressed in tubule principal cells. However, ir was most abundantly
expressed in the ureter (Fig.
2), whereas irk2 was most abundant in spermatheca
(Fig. 3A). It is thus clear
that, although all genes contribute to main segment function, ir and
irk3 are also found in lower tubule, a region associated with fluid
reabsorption (O'Donnell and Maddrell,
1995). IRKs may thus play a role both in secretion and
reabsorption in distinct spatial regions of the tubule. As the genes are
extensively co-expressed in the tubule, and as IRKs can form hetero-tetramers,
there is a possibility that the properties of the channels in vivo
may not resemble those measured by heterologous expression in vitro
(Doring et al., 2002
).
As the electrochemical gradient at the apical membrane does not favour net
K+ secretion (Beyenbach et al.,
2000), it is also likely that IRKs will be located on the
basolateral membrane. Could they then constitute a major route for
K+ entry?
Sulphonylureas block fluid secretion
The cardinal inhibitors of IRKs are the antidiabetic sulphonylureas such as
glibenclamide (Inagaki et al.,
1996). Consistent with a major role for IRKs in tubule, fluid
secretion was found to be inhibited by sulphonylureas. Glibenclamide inhibited
fluid secretion with an IC50 of 0.78±0.03 mmol
l-1 (Fig. 5).
Maximal inhibition was observed 20-40 min after the drug was added, depending
on concentration. The IC50 value is comparable with those found in
mammalian Kir1.1 channels in the absence of SUR2B
(Konstas et al., 2002
) and
Tenebrio molitor tubules (Wiehart
et al., 2003
).
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Further secretion assays demonstrated that Phenol Red and amaranth did not impact on the inhibition of fluid secretion by glibenclamide. In secretion assays where no dye was added to the reservoir bubble, the reduction of secretion caused by glibenclamide was not significantly different (Fig. 9).
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The nature of the glibenclamide transport was investigated further using I125-labelled glibenclamide. Measurements of the radioactivity in the secreted drops and reservoir bubbles revealed that the level of radioactivity was approximately 8-fold higher than the bathing solution in the secreted drops across all glibenclamide concentrations investigated (Fig. 11B). The rate of secretion rose with the glibenclamide concentration (Fig. 11A). The rate and ratio measurements show that glibenclamide transport certainly occurs and that the mechanism was saturated at the highest concentration we were able to employ. Thin layer chromatography (TLC) on the secreted and original radiolabelled glibenclamide showed that the secreted glibenclamide was modified (Fig. 11C). This modification precludes the conclusion that glibenclamide is actively transported, as we cannot confirm the existence of transport across an adverse electrochemical gradient. However, for practical purposes, glibenclamide is rapidly and avidly removed from the basolateral surface of the cell.
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Glibenclamide inhibition mimics the effect of K+-free saline
Glibenclamide, although a classical inhibitor of IRKs, has also been shown
to act on a wide range of targets. For example, it blocks ABC transporters
other than SUR, such as CFTR (Zhang et
al., 2004). It has been argued to affect L-type calcium channels
and the Na+/K+-ATPase
(Lee and Lee, 2005
) and to
induce a chloride conductance in mitochondria that collapses the inner
membrane potential (Fernandes et al.,
2004
). So, it is premature to ascribe the potent inhibition of
tubule secretion by glibenclamide to blockade of IRKs. However, our confidence
is increased by the fact that a range of sulphonylureas, and not just
glibenclamide, are effective (Fig.
6). Is it possible to go further?
The effects of glibenclamide were compared with exposure of the tubule to
K+-free saline. Drosophila tubule secretion has been
reported to persist for some hours in K+-free saline
(Linton and O'Donnell, 1999),
so if the action of glibenclamide was at a site distinct from IRKs, then
secretion in K+-free saline should still be sensitive to
glibenclamide. However, in our experiments, K+-free saline led to a
total inhibition of fluid secretion, with a time course very similar to that
of glibenclamide addition, and so glibenclamide has no further effect on
tubules in K+-free saline (Fig.
12). We thus consider that the action of glibenclamide is
consistent with a run-down of cellular K+, similar to that produced
by removing external K+, although, given the multiple potential
targets of glibenclamide, we cannot rule out more complex possibilities.
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Discussion |
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As would be expected, fluid secretion was shown to be sensitive to a wide
range of the antidiabetic sulphonylureas that are selective inhibitors of IRKs
in vertebrates. The sensitivity to these drugs, however, was not sufficiently
high to suggest that these IRKs are associated with Drosophila SUR or
a related ABC transporter; in support of this, a known inhibitor of
Drosophila SUR had no effect on fluid secretion. However, it was
possible to completely inhibit fluid secretion with sulphonylureas
(Fig. 6), whereas inhibitors of
Na+/K+-ATPase and
Na+/K+/2Cl- co-transport achieve only partial
inhibition of fluid secretion (Ianowski
and O'Donnell, 2004; Torrie et
al., 2004
), and so, with this broad panel of selective inhibitors,
we can confidently assert a major role for IRKs in tubule.
Do IRKs participate in basolateral entry of K+?
The extraordinary abundance of mRNA for ir, irk2 and irk3
in tubule (Wang et al., 2004;
Table 2) suggests an important
role for these channels. The sulphonylurea pharmacology, although less
conclusive, shows a similar effect to K+-free saline, suggesting a
possible role for IRKs in the entry step. This is consistent with many models
for tubule function, in which basolateral K+ channels have featured
(Beyenbach et al., 2000
;
Dow and Davies, 2003
;
Wiehart et al., 2003
).
However, important basolateral roles have also been shown both for
Na+/K+/2Cl- co-transport and the
Na+/K+-ATPase
(Ianowski and O'Donnell, 2004
;
Torrie et al., 2004
), and a
small (9 mV) outward electrochemical gradient for K+ has been shown
for the basolateral membrane of resting Drosophila tubule
(Ianowski and O'Donnell,
2004
). Can this conflicting data be reconciled? We suggest that
IRKs play only a minor role in basal secretion: their rectification properties
imply that they are unlikely to let much K+ out, down its
electrochemical gradient. However, when the tubule is maximally stimulated,
and the apical electrogenic V-ATPase is driving K+ exit across the
apical membrane, we suggest that basolateral IRKs allow K+ entry to
support the active apical membrane. IRKs could thus be seen as adaptations to
facilitate the remarkably high per-cell secretion rates that seem to be
characteristic of insect tubules (Dow and
Davies, 2003
; Maddrell,
1991
). Consistent with this, all the sulphonylureas showed a more
marked effect on stimulated, rather than basal, fluid secretion
(Fig. 6).
Transport and metabolism of glibenclamide
We also observed two novel effects of glibenclamide: (1) the tubule itself
both excretes and modifies the compound and (2) glibenclamide inhibits the
excretion of a range of organic dyes. Although the sulphonylureas are valuable
human therapeutic agents, the molecular mechanisms of their clearance are not
understood in detail; for example, it is not clear whether SUR is itself a
sulphonylurea transporter. However, our results may impinge on clearance
mechanisms. Although we did not rigorously show competition between these
solutes, it would be a very plausible explanation, and this paper does
identify a range of interesting dyes that may help in such studies.
Demonstration of glibenclamide transport further raises the possibility
that the apparent IC50s for sulphonylureas may be distorted by
rapid removal from the basolateral membrane infoldings of the principal cell,
as has been shown for ouabain (Torrie et
al., 2004). This would have the effect of protecting basolateral
IRKs by only exposing them to a concentration of sulphonylureas that was
significantly lower than the macroscopic bath concentration, thus producing
anomalously high IC50s. We have not yet identified an inhibitor of
glibenclamide transport, so we cannot unmask a higher affinity effect of
glibenclamide, as was possible for ouabain
(Torrie et al., 2004
). We must
thus be cautious about asserting an accurate IC50 for
glibenclamide; this will need to be established by heterologous expression in
culture, where such confounding elements do not appear.
Phylogenetic scope of the model
Comparison of the Drosophila and Anopheles genome
projects reveal that IRKs are very similar between these dipteran species;
indeed, there appears to have been a gene expansion in Anopheles
(McCormack, 2003).
Glibenclamide sensitivity (at relatively high concentrations: 5 mmol
l-1) has also been documented in Tenebrio molitor, a
beetle (Wiehart et al., 2003
).
In Tenebrio, the effect of glibenclamide was similar to that of
barium, another characteristic of IRKs. Barium sensitivity has also been
widely documented in tubules of Drosophila
(Ianowski and O'Donnell, 2004
;
Wessing et al., 1993
), the
locust Schistocerca gregaria
(Hyde et al., 2001
) and the
yellow fever mosquito, Aedes aegypti
(Beyenbach and Masia, 2002
)
Consistent with this, Drosophila IRKs reveal weak barium sensitivity
when expressed heterologously in S2 cells
(Doring et al., 2002
). Our
results, showing sensitivity of fluid secretion to a spectrum of sulphonylurea
antidiabetics, and localising all three IRKs to the same cell type as the
apical V-ATPase, may thus have broad scope.
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Acknowledgments |
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