The dg2 (for) gene confers a renal phenotype in Drosophila by modulation of cGMP-specific phosphodiesterase
1 Institute of Biomedical and Life Sciences, Division of Molecular Genetics,
University of Glasgow, Glasgow G11 6NU, UK
2 Institute of Biomedical and Life Sciences, Division of Biochemistry and
Molecular Biology, University of Glasgow, Glasgow G11 6NU, UK
Author for correspondence (e-mail:
s.a.davies{at}bio.gla.ac.uk)
Accepted 12 May 2004
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Summary |
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Thus, polymorphisms at the dg2 locus do indeed confer a cGMP-dependent transport phenotype, but this can best be ascribed to an indirect modulation of cG-PDE activity, and thence cGMP homeostasis, rather than a direct effect on cGK levels.
Key words: Malpighian tubule, cyclic nucleotide, capa-1, epithelial transport, Drosophila melanogaster, dg2 locus
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Introduction |
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An autocrine role for NO/cGMP has been proposed for tubule principal cells
(Broderick et al., 2003), with
NO/GMP signalling being compartmentalised to principal cells in the main,
fluid-secreting segment of tubules. These cells contain the electrogenic
vacuolar H+-ATPase (V-ATPase) pump
(Dow, 1999
), which energises
fluid transport. Furthermore, electrophysiological studies show that cGMP
signalling modulates V-ATPase activity
(Davies et al., 1995
),
suggesting that cGMP signalling may regulate ion transport in tubules. Major
effectors of cGMP signalling, including cGMP-dependent protein kinases (cGK)
(Vaandrager and de Jonge,
1996
) have previously been described in tubules. Furthermore,
pharmacological and transgenic modulation of cGMP-specific phosphodiesterase
(cG-PDE) activity (Broderick et al.,
2004
,
2003
;
Dow et al., 1994b
) both result
in an epithelial phenotype.
In Drosophila, cGK is encoded by two genes, dg1
(Foster et al., 1996) and
dg2. Both genes are expressed by Malpighian tubules
(Dow et al., 1994b
).
dg2 was isolated and characterised during a search for cAMP-dependent
kinase genes (Kalderon and Rubin,
1989
) and the putative cGK shown to be transcribed into three
major RNA species of different size and several minor RNA species. These main
transcripts (T1, T2 and T3) collectively code for at least three
(de Belle et al., 1993
), and
possibly more, different polypeptides. The DG2 protein shares 64% overall
homology with the prototypical bovine lung cGK, with 64% and 75% sequence
identity to the cGMP-binding and kinase domains, respectively.
Studies in Drosophila have revealed in vivo roles for
dg2 and cGK. The naturally occurring rover/sitter foraging
polymorphism in Drosophila, which defines larval food search
strategies, has been mapped to the dg2 gene (de Belle et al.,
1989,
1993
). Rovers
(forR) have significantly longer path lengths than sitters
(fors) in a nutritive environment, although both travel
similar distances when food is absent. Similarly, adult
fors animals travel shorter distances around nutrients
(Pereira and Sokolowski,
1993
). Phosphorylation studies performed on samples from adult
heads showed that fors contained slightly (10%) reduced
cGK enzyme activity compared to forR. Also, northern and
western analysis showed a small reduction in RNA and protein levels in
fors compared to forR
(Osborne et al., 1997
). It has
therefore been suggested that a reduction in amounts of cGK transcript and
protein, together with reduced cGK activity, may account for the
fors phenotype in larvae. Thus, the foraging
polymorphism points to the possibility that subtle alterations in cGK levels
can have profound effects on the whole animal.
Fluid transport assays performed on tubules from adult for lines
has demonstrated that tubules from fors flies exhibit
hypersensitivity to exogenously applied cGMP in comparison to
forR or wild-type flies
(Dow and Davies, 2001).
However, stimulation of fluid transport by leucokinin, which stimulates fluid
secretion via a calcium signal in the stellate cells, is unaltered in
the for alleles (Dow and Davies,
2001
), suggesting that effects of alterations in cGK are confined
to principal cells.
We show here that the fors allele results in hypersensitivity of tubule fluid transport (compared with forR) in response to the neurohormone, capa-1. Intriguingly, the fors mutation does not appear to affect cGK activity in tubules; rather it impacts on cGMP content, and on cG-PDE activity. Capa-1 inhibits cG-PDE, which results in increased cGMP content, and the transport phenotype observed; this also demonstrates modulation of cG-PDE activity by a neurohormone in insects, for the first time.
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Materials and methods |
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Materials
Schneider's medium (Gibco) was obtained from Invitrogen (Renfrew, UK). The
nitridergic neuropeptide capa-1 was used in this study (GANMGLYAFPRVamide)
because of its identical mode of action but slightly greater potency than
capa-2 (Kean et al., 2002) and
was synthesised by Research Genetics, Inc., now Invitrogen. Radiochemicals
were obtained from Amersham Biosciences (Chalfont St Giles, UK). All other
chemicals were obtained from Sigma-Aldrich (Gillingham, UK) unless stated
otherwise.
Locomotion activity monitoring
Fly lines were assessed by monitoring activity of adult flies using the
Drosophila locomotor activity monitor IV, (TriKinetics Inc., Waltham,
MA, USA) in order to verify previously published adult behavioural phenotypes
ascribed to for alleles.
Flies were maintained at 22°C on standard Drosophila diet over a 12 h:12 h photoperiod, with lights on at 11.30 am. Tubes were made from 7.5 cm lengths of Tygon (Charny, France) clear flexible plastic tubing (R3603, i.d. 5/32", o.d. 7/32", wall 1/32"), plugged at one end with normal fly food and sealed with clear tape. 7-day-old male flies were anaesthetized and placed singly into each tube. Ends of tubes were then plugged with cotton wool. Tubes were placed in the monitor and flies allowed to recover overnight prior to monitoring. Readings were taken every 30 min over a period of 7 days.
Transport assays
Flies were used 1 week post-emergence, cooled on ice, then decapitated
before dissection to isolate whole Malpighian tubules. Tubules were isolated
into 10 ml drops of Schneider's medium under liquid paraffin and fluid
secretion rates measured in tubules as detailed elsewhere
(Dow et al., 1994a) under
various conditions, as described in the text. Basal rates of fluid transport
were measured for 30 min, and capa-1 (10-7 mol l-1;
Kean et al., 2002
) added as
indicated, after which transport rates were measured for a further 30 min.
Assay for tubule cGK activity
The protocol, based on quantification of 32P-labelled
phosphopeptide under conditions where cGK is active, has been adapted from the
SignaTECTTM cyclic AMP-dependent protein kinase assay system (Promega,
Southampton, UK) and from Osborne et al.
(1997).
Approximately 80 tubules from either forR or fors 1-week-old adults were dissected and placed in 20 µl buffer (25 mmol l-1 Tris, pH 7.4, 150 mmol l-1 sucrose, 2 mmol l-1 EDTA, 100 mmol l-1 NaCl, 50 mmol l-1 ß-mercaptoethanol, 2 mg ml-1 leupeptin, 5 mg ml-1 aprotinin, 1 mg ml-1 phenylmethylsulphonyl fluoride). Tubules were either treated with hormone for 10 min, or left untreated, before being homogenised and centrifuged for 5 min at 13 000 g. Protein concentration of tubule homogenates was determined by the Lowry assay and homogenates adjusted to equivalent protein concentrations for use in cGK assays. cGK activity was assayed with and without cGMP for each tubule preparation.
Two reaction mixes were prepared with and without the addition of 1 µmol
l-1 cGMP, containing 25 mmol l-1 Tris, pH 7.4, 7 mmol
l-1 magnesium acetate, 1 mmol l-1 EDTA, 2 mmol
l-1 EGTA, 0.2 mg ml-1 GLASStide [RKRSRAE, a heptapeptide
cGK-specific substrate; Calbiochem, Beeston, UK
(Hall et al., 1999)], 20
µmol l-1 ATP, 0.5-2 ml [
-32P]dATP (370 MBq
µl-1, to an approximate specific activity of 4000 c.p.m.
pmol-1 ATP), 1 nmol l-1 PKI (PKA inhibitor,
TYADFIASGRTGRRNAI-NH2) and 1 mmol l-1 dithiothreitol
(DTT).
For each reaction, 40 µl reaction buffer was added to 5 µl (approximately 30 µg protein) tubule sample. This was done with both cGMP-containing (+cGMP) and cGMP-absent (-cGMP) buffer. Each tubule preparation was assayed as 2-4 separate reactions within the experiment. However, all cGK experiments were carried out on several separate biological replicates in order to obtain statistically sound data. Sample blanks were generated using 40 µl reaction buffer and 5 µl of homogenisation buffer. Reactions were incubated for 30 min at 30°C, after which 35 µl of each sample was spotted onto individual squares of P81 paper (Whatman, Maidstone, Kent). These squares of paper are referred to as reaction samples. In order to determine the specific activity of the radiolabelled ATP at the end of the reaction, several reactions were chosen randomly and 5 µl samples (representative of 1/9 of total counts) of each spotted onto individual squares of P81 paper (`total count'), allowed to dry and set aside.
The reaction samples were washed for 3x 5 min in 75 mmol l-1 phosphoric acid, then washed once for 15-20 s in ethanol and allowed to dry. All squares of paper, including the total count samples, were then transferred to scintillation vials, with the addition of 3 ml scintillation fluid and counted in a scintillation counter (Beckman, High Wycombe, UK) for 60 s.
Specific activity of [-32P]ATP was calculated (9x
mean c.p.m. of total count squares/[ATP] in reaction) and used to calculate
protein kinase activity (pmol ATP min-1 µg-1
protein), as follows: (sample c.p.m. - sample blanks / sample volume on filter
x reaction time x protein amount x specific activity).
Values for `sample c.p.m.' were based on those obtained by subtracting mean
-cGMP values from mean +cGMP values for each set of replicate reactions.
cG-PDE activity assays
Assays for cG-PDE activity in tubules were performed essentially as
previously described (Broderick et al.,
2003) using 50 tubules (20-30 µg protein) for each sample,
assayed in 0.185 kBq ml-1 3H-cGMP in 20 µmol l-1
cGMP, 10 mmol l-1 Tris, 5 mmol l-1 MgCl2, pH
7.4. For cG-PDE assays in heads, six heads from each line were dissected into
100 µl KHEM buffer (50 mmol l-1 KCl, 10 mmol l-1
EGTA, 1.92 mmol l-1 MgCl2, 1 mmol l-1 DTT, 50
mmol l-1 Hepes, pH 7.21, 1 µl Sigma P8340 protease inhibitor
cocktail), disrupted with a pestle, sonicated for 10 s and centrifuged at 15
000 g for 5 min at 4°C. Supernatants were assayed for
protein concentration, and 50 µl samples (containing 10 µg protein)
assayed for cG-PDE activity as for tubule samples. A final substrate
concentration of 10 µmol l-1 cGMP was used in reactions, as
endogenous Drosophila cG-PDEs are enzymes with high
Km (Broderick et al.,
2004
; Day et al.,
2003
). Final activity was expressed per mg protein. Protein
concentrations were assayed according to standard protocols (Lowry Assay).
Tubule cyclic GMP assays
Cyclic GMP levels were measured in pooled samples of 20 tubules by
radioimmunoassay (Amersham Biotrak Amerlex M), as previously described
(Dow et al., 1994b). Tubules
were pre-incubated with 10-8 mol l-1 of the cG-PDE
inhibitor Zaprinast (Calbiochem, Beeston, UK) for 10 min. Where required,
capa-1 (10-7 mol l-1) was added to tubules for a further
10 min. Incubations were terminated with ice-cold ethanol and homogenised.
Samples were dried down and dissolved in 0.05 mol l-1 sodium
acetate buffer (Amersham) and processed for cGMP content according to the
manufacturer's protocol.
Statistics
Data are presented as mean ± S.E.M. Where appropriate,
the significance of differences between data points was analysed using
Student's t-test for unpaired samples, taking P<0.05 as
the critical level.
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Results |
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Basal and capa-1 stimulated fluid transport rates are modulated in a dg2 allele
A significant increase in neuropeptide-induced transport is observed at 50
and 60 min in tubules from fors animals, compared to those
from forR (Fig.
2). This is especially pronounced at 60 min, when maximum fluid
transport rates are approximately 2 nl min-1 in
fors tubules, and approximately 1 nl min-1 in
forR tubules. However, no differences are observed in
basal transport rates between the two lines.
|
cGK activity is not significantly perturbed in fors tissue
It has been previously shown (Osborne
et al., 1997) that cGK activity in the heads of
fors mutants is slightly downregulated. In order to test
the effect of the fors mutation in tubules, we assayed
tubule cGK activity, as well as that from head and body. Measurements of
tubule cGK activity using a cGK-specific phosphorylation substrate showed this
to be unchanged in fors tubules compared to
forR (Fig.
3, P=0.109, unpaired t-test). Similar analyses
of bodies also failed to show any difference in cGK activity
(Fig. 3, P=0.577,
unpaired t-test); however, whilst analyses of head extract appear to
show a slight decrease in cGK activity (consistent with published results),
this proved not to be statistically significant
(Fig. 3, P=0.148,
unpaired t-test).
|
Given the similarities in cGK activity between forR and fors tubules, the epithelial phenotype characterised in the studies shown in Fig. 2 would appear to be due to modulation of other regulatory components.
The fors allele impacts on cG-PDE activity
We have previously shown that the degradation of cGMP by cG-PDE activity is
critical in the regulation of fluid transport by the tubule (Broderick et al.,
2003,
2004
). Given this, we set out
to assess any impact of the fors allele on cG-PDE activity
in both tubules and heads.
We identified a small, but nevertheless significant, increase in the cG-PDE activity of tubules from fors compared to forR flies (Fig. 4). In contrast to this, there is no significant difference in cG-PDE activity between heads from the forR and fors lines (P=0.73, unpaired t-test).
|
fors tubules contain reduced cGMP
cGMP is a direct modulator of fluid transport by Malpighian tubules
(Dow et al., 1994b), where the
use of selective inhibitors has identified cG-PDE activity as playing a key
regulatory role by manipulating cGMP levels in tubules. Here we identify
differences in cG-PDE activity in fors flies
(Fig. 4). To determine if this
could influence resting cGMP levels, we assessed the cGMP content in tubules
from both forR and fors lines
(Fig. 5). Intriguingly, basal
cGMP levels are significantly reduced in fors (28±6
fmol 20-1 tubules) compared to forR tubules
(40±4 fmol 20-1 tubules). By contrast, cGMP levels are
elevated to the same levels in both lines upon stimulation by the nitridergic
peptide, capa-1 (forR: 76±11;
fors: 69±12 pmol 20-1 tubules,
Fig. 5). This suggests that in
fors tubules there is a greater increase in cGMP content
compared to that in forR tubules, in response to capa-1
(forR: approx 187% stimulation; fors:
approx. 250% stimulation). Also, the fors allele does not
compromise the ability of these tubules to synthesise cGMP upon hormonal
stimulation.
|
Capa modulation of fluid transport via cGMP: downregulating cG-PDE
The novel transport phenotype identified here in fors
tubules is observed upon stimulation with exogenously added cGMP
(Dow and Davies, 2001) and
also with capa-1 (Fig. 2). We
thus assayed capa-1 stimulated cGK and cG-PDE activity to determine if a
change in their activities may play a role in the phenotype.
Fig. 6 shows a small reduction
in cGK activity in capa-1-stimulated forR tubules
(Fig. 6A). Interestingly,
however, no change in capa-1-stimulated cGK activity is observed in
fors flies (Fig.
6A). Thus, capa-1 modulation of cGK activity in
fors tubules is not measurable.
|
In contrast to this, capa-1 treatment results in a significant reduction in
cG-PDE activity in both forR (approx. 37% reduction,
Fig. 6B) and
fors (approx. 70% reduction,
Fig. 6B). cG-PDE activity was
also reduced in wild-type Oregon R tubules to a similar extent as
forR (data not shown). The extent of inhibition of cG-PDE
activity was greater in fors tubules compared to
forR (Fig.
6B; cG-PDE activities, expressed as pmol GMP min-1
mg-1 protein: fors control, 798±31;
fors capa-1 treated, 238±21;
forR control, 642±72; forR
capa-1 treated, 407±64) Under such conditions, reduced cG-PDE activity
can be expected to result in maintenance of high intracellular cGMP levels
(Broderick et al., 2003),
ultimately resulting in elevated fluid transport rates upon capa-1 stimulation
in fors.
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Discussion |
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While we did observe a small inhibition of cGK in the heads of
fors adults (and despite phenotypic confirmation of the
`sitter' phenotype), the reduction was not, however, statistically significant
(Fig. 2). Nevertheless, this
result does not necessarily exclude that changes in dg2 underlie the
foraging polymorphism. It is possible that subtle environmental
effects could lead to small differences in cGK assessments in different
laboratories, or that the difference in cGK activity associated with the
polymorphism is relatively modest. Furthermore, compartmentalisation of cAMP
and cGMP signalling pathways (Edwards and
Scott, 2000; Schlossmann et
al., 2000
), results in changes in phosphorylation status of
proteins associated with such `pools'. Therefore, measurements of bulk, as
opposed to localised, cGK activity, may not be sufficient to monitor subtle
changes in cGK. Perhaps most obviously, there are two cGK genes in
Drosophila, and so even substantial changes in dg2 levels
might be undetectable against a high background of dg1 protein.
Notwithstanding this, our data indicated that cGK activity was unchanged in
tubule and so clearly did not seem to form the basis of the dg2
phenotype observed in tubules. As cGMP signalling has been clearly shown to
play a pivotal role in tubule functioning, we reasoned that other components
of the cGMP pathway might be involved. In this regard, cG-PDE provides the
sole route for the degradation of the second messenger, cGMP, and as such, is
poised to play a key regulatory role in controlling cGMP signalling in cells.
Indeed, we have shown this to be the case in tubules (Broderick et al.,
2004,
2003
;
Dow et al., 1994b
), prompting
us to probe for any role of cG-PDE activity in this tubule phenotype.
Analysis of cG-PDE activity in forR and
fors adults showed that cG-PDE activity is affected in
several ways in the dg2 mutation. Firstly, in
fors animals, basal cG-PDE activity is increased in
tubules, although not in head, which results in decreased cGMP levels. This is
consistent with the role of cG-PDE in maintaining cGMP homeostasis. Secondly,
capa-1 stimulated cGMP levels are increased to similar amounts in both lines,
suggesting that the increase in cGMP is greater in fors
compared to forR, which may implicate differential
regulation of cG-PDE activity by capa-1 in fors tubules.
This is confirmed by the data on capa-1 modulation of cGK and cG-PDE
(Fig. 6). cG-PDE activity is
depressed by capa-1, resulting in increased cGMP content. This occurs to a
greater extent in fors, however, resulting in the greater
foldstimulation in cGMP content. This would appear to provide a rationale for
the transport phenotype (hypersensitivity to capa-1) observed in
fors tubules. The effects of capa-1 on cG-PDE activity are
consistent with its role in stimulated fluid transport, and show that the mode
of action of this peptide is not merely to stimulate cGMP production
via soluble guanylate cyclase
(Kean et al., 2002), but also
to act via the potent inhibition of cGMP breakdown. Manduca
sexta CAP2b (Davies et al.,
1995
), another member of the capa family
(Kean et al., 2002
), also acts
to inhibit cG-PDE in Oregon R, forR and
fors tubules (data not shown). Thus, the inhibition of
cG-PDE may be a general mechanism of action by the capa family of nitridergic
peptides. Previous work shows that regulation of cGMP breakdown via
cG-PDE, as opposed to cGMP synthesis, is a powerful modulator of fluid
transport in tubules (Broderick et al.,
2003
), suggesting that cG-PDE(s) have a central role in epithelial
transport and are thus candidate targets for nitridergic peptide action. An
analogous situation exists for cAMP signalling in tubules. A cAMP-mobilising
hormone, Corticotrophin-like Releasing Factor (CRF), has been shown to
modulate cAMP-specific PDE activity in tubules
(Cabrero et al., 2002
). Thus in
insects, as in vertebrates, modulation of PDEs by specific hormones is an
effective signalling mechanism (Dousa,
1999
).
No measurable change in cGK activity was observed in capa-stimulated fors tubules, which suggests that the neuropeptide-stimulated epithelial phenotype in fors tubules is entirely due to cG-PDE. Modulation of cGK activity is not implicated in this process. However, in forR, a small but significant decrease in tubule cGK activity is observed upon capa-1 stimulation, for which there is currently no explanation.
How can polymorphism at the for locus act on a functionally
related, but physically remote, gene? The mapping of for to the
region containing dg2 is authoritative
(Osborne et al., 1997); and
although some alleles (e.g. gamma irradiation-induced) might be expected to
impact on neighbouring genes as well as dg2 (there are several genes
within 10 kb of for), there is no cyclic nucleotide phosphodiesterase
within megabases of the for locus. Additionally, differences in cGK
levels between the non-lethal alleles of for are either modest
(Osborne et al., 1997
), or
undetectable (this work), yet there is still an impact on cGMP signalling. We
propose that a solution is offered by the concept of feedback; in order to
maintain signal integrity, relatively modest changes in cGK activity elicit
relatively large changes in cG-PDE, so compensating for differences in kinase
levels. PDEs undergo post-translational modification by phosphorylation,
interactions with other proteins and by proteolytic cleavage
(Francis et al., 2001
). It is
possible that small changes in cGK can profoundly affect the activity of
cG-PDE in tubule. Thus the polymorphism at the for locus may indeed
act to modulate cGMP signalling, but through an unexpected route. This concept
is consistent with a previous observation that tubules in which nitric oxide
synthase was overexpressed by around twofold showed only a modest increase in
stimulated secretion, because cG-PDE was upregulated by nearly tenfold
(Broderick et al., 2003
).
Accordingly, there is evidence that, in at least this tissue, cG-PDE activity
can vary over quite a wide range in order to compensate for relatively modest
perturbations elsewhere in the pathway.
We have thus uncovered a central role for cG-PDE in tubules of a dg2 allele. Furthermore, we show that the capa peptides modulate cG-PDE activity as an effective mechanism of increasing cGMP content in vivo.
There are now obvious and exciting avenues for further study: it may be that modulation of cG-PDE may provide an interesting and general explanation for the effects of the foraging polymorphisms in other contexts, such as susceptibility to parasitism and neuronal activity.
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Acknowledgments |
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Footnotes |
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