norpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function
Valerie P. Pollock1,
Jonathan C. Radford1,
Susan Pyne2,
Gaiti Hasan3,
Julian A. T. Dow1 and
Shireen-A. Davies1,*
1 Institute of Biomedical and Life Sciences, Division of Molecular Genetics,
University of Glasgow, Glasgow G11 6NU, UK
2 Department of Physiology and Pharmacology, Strathclyde Institute for
Biomedical Sciences, University of Strathclyde, Glasgow G4 ONR, UK
3 National Centre for Biological Sciences, UAS-GKVK Campus, Bangalore
560065, India
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Fig. 1. An epithelial phenotype for norpA: phospholipase Cß
(PLCß) is required for neuropeptide stimulation of principal and stellate
cells. Fluid transport assays were performed on intact tubules from wild-type
(Oregon R), norpAH52 and norpAP24
flies as described in the Materials and methods. Either (A) 10-7
moll-1 CAP2b or (B) 10-7 moll-1
Drosokinin were added at 30 min (arrow), and transport rates were measured for
a further 30 min. Data are expressed as mean secretion rates ± S.E.M.
(N=8).
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Fig. 2. Resting and CAP2b-stimulated inositol (1,4,5)-trisphosphate
(IP3) levels in itpr mutants. (A) Resting IP3
levels are shown for tubules from the following lines: Oregon R (control),
itprXR12/+, itpr90B.0/+,
itpr1664/+, itpr1664/itprXR12,
itpr1664/itpr90B.0,
itpr1664/itpr1664 and
itprWC361/itprUG3. In order to aid comparison
between experiments, data are shown as the % difference between IP3
levels in itpr mutants compared with wild type (100%) ± S.E.M.
(N=4). Typical IP3 content of wild-type tubules was as
described in Table 1. (B)
CAP2b stimulates IP3 production in itpr lines.
Stimulated IP3 levels were measured in CAP2b-stimulated
tubules (10-7 moll-1, 5s). Data are expressed as the %
increase of unstimulated IP3 levels (calculated as [stimulated
IP3]/[resting IP3]x100%; [IP3] measured
in pmol µg protein-1) ± S.E.M. (N=3-4).
Significant differences between IP3 content in wild-type and
itpr lines are denoted by * (P<0.05, Student's
t-test, unpaired samples).
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Fig. 3. CAP2b- and Drosokinin-stimulated fluid transport are inhibited
in itpr mutants. Fluid transport assays were performed on intact
tubules as described in Fig. 1
for the following lines: Oregon R (control), itprXR12/+,
itpr90B.0/+, itpr1664/+,
itpr1664/itprXR12,
itpr1664/itpr90B.0,
itpr1664/itpr1664 and
itprWC361/itprUG3. Either (A) 10-7
moll-1 CAP2b or (B) 10-7 moll-1
Drosokinin were added at 30 min, and transport rates were measured for a
further 30 min. No change in basal secretion rate was observed in
itpr mutants. Furthermore, kinetics of the fluid secretion response
in all lines were similar (data not shown). To aid comparison between
stimulated transport rates, data are expressed as the % stimulation of
secretion [(maximal stimulated rates minus the mean of three basal secretion
rate readings)/(mean basal rate)x100% ± S.E.M.; N=15-20]
upon stimulation with CAP2b or Drosokinin. Stimulated fluid
transport rates that are significantly different from wild type are denoted by
* (P<0.05, Student's t-test, unpaired
samples).
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Fig. 4. itpr rescues the transport phenotype of the
itpr90B.0 allele. Fluid transport assays were performed on
the hsGAL4;itpr90B.0 line. The data show that
CAP2b-stimulated fluid transport is decreased in this line. Rescue
of hsGAL4;itpr90B.0 with UAS-itpr results in
wild-type levels of stimulated fluid transport. Fluid secretion rates were
measured for 30 min prior to addition of neuropeptide (arrow), after which
measurements were taken for a further 30 min. Data are expressed as mean fluid
secretion rates (nl min-1) ± S.E.M. (N=6-10).
UAS-itpr tubules display similar secretion rates to those of
wild-type tubules (data not shown).
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Fig. 5. CAP2b-induced cytosolic calcium signals in itpr
mutants. (A) Typical traces of changes in intracellular Ca2+
concentration ([Ca2+]i) in tubule principal cells
stimulated by 10-7 moll-1 CAP2b (arrows) in
the following lines: (i) aeq;hsGAL4;+ (control), (ii)
aeq;hsGAL4;itprXR12/+, (iii)
aeq;hsGAL4;itpr90B.0/+, (iv)
aeq;hsGAL4;itpr1664/+, (v)
aeq;hsGAL4;itpr1664/itpr1664 and (vi)
aeq;hsGAL4;itprWC361/itprUG3. Each sample
contains 20 intact tubules. While no changes in the resting
[Ca2+]i is seen in any of the mutants, changes in
amplitude of the primary and/or secondary response can be observed in all
lines (also in B). (B) Pooled results of changes in tubule
[Ca2+]i in itpr mutants in response to
10-7 moll-1 CAP2b are shown. Results are
expressed as means ± S.E.M. (N=8) for background (open bars),
CAP2b-stimulated primary peaks (filled bars) and
CAP2b-stimulated secondary peaks (hatched bars) for the lines
described in A. The measure of secondary peak is taken as the average
[Ca2+]i over 4 min post-stimulation with
CAP2b. CAP2b-stimulated primary peaks that are
significantly different from aeq;hsGAL4 tubules are denoted by *,
and statistically significant differences in secondary peaks compared to wild
type are denoted by  (P<0.05, Student's t-test,
unpaired samples).
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Fig. 6. Drosokinin-induced cytosolic calcium signals in itpr mutants. (A)
Typical traces of changes in intracellular Ca2+ concentration
([Ca2+]i) in tubule stellate cells stimulated by
10-7 mol l-1 Drosokinin (arrows) in the following lines:
(i) aeq;hsGAL4;+ (control), (ii) aeq;hsGAL4; itprXR12/+,
(iii) aeq;hsGAL4;itpr90B.0/+, (iv)
aeq;hsGAL4;itpr1664/+, (v)
aeq;hsGAL4;itpr1664/itpr1664 and (vi)
aeq;hsGAL4;itprWC361/itprUG3. Each sample
contains 20 intact tubules. While no changes in the resting
[Ca2+]i is seen in any of the mutants, changes in
amplitude of the calcium peak can be observed in
aeq;hsGAL4;itpr1664/itpr1664 and
aeq;hsGAL4;itprWC361/itprUG3 (also in B). (B)
Pooled results of changes in tubule [Ca2+]i in
itpr mutants in response to 10-7 moll-1
Drosokinin. Results are expressed as means ± S.E.M. (N=8) for
background (open bars) and Drosokinin-stimulated peaks (filled bars) for the
lines described in A. Drosokinin-stimulated primary peaks that are
significantly different from aeq;hsGAL4;+ tubules are denoted by *
(P<0.05, Student's t-test, unpaired samples).
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© The Company of Biologists Ltd 2003
