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First published online January 16, 2009
Journal of Experimental Biology 212, 329-340 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024646
Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules
1 Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca, NY
14853, USA
2 Proteomics and Mass Spectrometry Core Facility, 143 Biotechnology Building,
Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: kwb1{at}cornell.edu)
Accepted 6 November 2008
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Key words: cAMP, PKA, Ca2+, PKC, V-type H+ ATPase, endoplasmin, actin, annexin, adducin
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). The
cockroach Leucophaea has eight leucokinins, the yellow fever mosquito
Aedes has three aedeskinins and Drosophila has only one
drosokinin (Holman et al.,
1987; Terhzaz et al.,
1999; Veenstra et al.,
1997). All three kinins in Aedes have diuretic activity
in Malpighian tubules in vitro (S. A. Schepel, A. J. Fox, F. Tiburcy,
A. W. Blum, K.L., T. Sou, R. Nachman and K.W.B., unpublished observations). In
particular, aedeskinin-III stimulates the transepithelial secretion of both
NaCl and KCl, and it hyperpolarizes the basolateral membrane voltage while
reducing the input resistance of principal cells (S. A. Schepel, A. J. Fox, F.
Tiburcy, A. W. Blum, K.L., T. Sou, R. Nachman and K.W.B., unpublished
observations). These effects duplicate those of leucokinin-VIII in
Aedes Malpighian tubules
(Beyenbach, 2003;
Pannabecker et al., 1993). As
shown in Fig. 1,
leucokinin-VIII brings about the non-selective stimulation of both NaCl and
KCl secretion by increasing the transepithelial secretion of
Cl– (Yu and Beyenbach,
2001; Yu and Beyenbach,
2002; Yu and Beyenbach,
2004). Specifically, the kinins increase the paracellular
Cl– conductance to such an extent as to produce nearly a
transepithelial short-circuit, as indicated by the sudden drop of the
transepithelial voltage from 59 mV to 5 mV
(Fig. 1C). In parallel, the
transepithelial resistance plummets from 18.4 k
cm to 3.2 kcm,
reflecting the increase in paracellular Cl– conductance, and
the apical and basolateral membrane voltages converge to values only 5 mV
apart (Fig. 1B,C).
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;
Beyenbach, 2003). Thus, for
every cation secreted via the transcellular pathway, a Cl–
ion is secreted through the paracellular pathway, bringing about equimolar
secretion rates of cations and anions (Fig.
1D).
The on/off effects of kinins on the paracellular Cl–
conductance proceed with switch-like speed
(Fig. 1C), suggesting
post-translational modifications of the paracellular pathway. Changes in
intracellular [Ca2+] appear to mediate the switch-like changes of
the paracellular Cl– conductance. How Ca2+ effects
sudden changes in paracellular Cl– conductance has piqued our
curiosity. Using the methods of proteomics, we looked for cytosolic proteins
associated with Ca2+ signaling to the paracellular pathway. We
compared the cytosolic proteome and phosphoproteome of Malpighian tubules
before and after stimulation with 10–7 mol
l–1 aedeskinin-III for only 1 min. From the many proteins
that were down- or up-regulated in the cytosol, we present here those that
might be associated with stimulating transepithelial transport. We found
evidence for aedeskinin-III signaling to the cytoskeleton, where
Ca2+ and protein kinase C appear to trigger the remodeling of
septate junctions with likely consequences for the integral membrane proteins
that define the paracellular Cl– permselectivity and
conductance. In addition, we found evidence for signaling to the transcellular
pathway, where protein kinase A (PKA) might induce the assembly and activation
of the V-type H+-ATPase at the apical membrane through a
cAMP-independent mechanism. Alternatively, protein kinase C could bring about
the assembly and activation of the V-type H+-ATPase. Thus, insect
kinins appear to activate both paracellular and transcellular transport
pathways. The additive or perhaps synergistic effects of stimulating both
transport pathways are consistent with the rapid increase in the
transepithelial secretion of NaCl and KCl that we and others have previously
reported (Coast, 1995;
Hayes et al., 1989;
O'Donnell and Spring, 2000;
Pannabecker et al., 1993).
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Materials and methods |
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).
Malpighian tubules were dissected exclusively from adult female mosquitoes
(3–7 days old). After cold-anesthetizing a mosquito on ice, the mosquito
was decapitated and submerged in Ringer solution. By holding the thorax with
one pair of forceps (Dumont #5, Fine Science Tools, Foster City, CA, USA) and
gently pulling at the rectum with another pair of forceps, the intestine and
Malpighian tubules were freed from the abdomen. The five Malpighian tubules
were removed from their attachment to the intestine and transferred to a 1.5
ml low-adhesion microcentrifuge tube (USA Scientific, Ocala, FL, USA)
containing 0.5 ml Ringer solution at room temperature. The Ringer solution
contained the following, in mmol l–1: 150 NaCl, 3.4 KCl, 25
HEPES, 1.8 NaHCO3, 1.0 MgSO4, 1.7 CaCl2 and
5.0 glucose. The pH was adjusted to 7.1 using 1 mol l–1
NaOH. To isolate enough cytosolic protein for proteomic analyses, it was necessary to collect more than 2000 Malpighian tubules each for both a control group (C) and an aedeskinin-treated (T) group. On a typical tubule collection day, several volunteers in the lab collected about 250 tubules each for C and T groups. The tubules of each group were pooled into separate microcentrifuge tubes containing 200 µl of Ringer. In the case of C tubules, the Ringer solution was withdrawn after the tubules had settled to the bottom of the vial. The tubules were subsequently frozen in liquid nitrogen. In the case of T tubules, they were resuspended in 200 µl of Ringer containing 10–7 mol l–1 aedeskinin-III. After treating the tubules for only 1 min with aedeskinin-III, the Ringer was removed and the tubules were frozen in liquid nitrogen. C and T tubules were stored at –80°C until the extraction of cytosolic proteins. The isolation sessions were repeated until we had collected 2378 tubules for the control group and 2468 tubules for aedeskinin-treated group.
Extraction of cytosolic proteins
On the day of the extraction, C and T tubules were thawed on ice. Ice-cold
extraction buffer (100 µl) was added to each. The extraction buffer
contained, in mmol l–1: 50 HEPES (pH 7.1), 10 dithiothreitol,
1 PMSF (phenylmethylsulfonyl fluoride) and 5 EDTA (ethylenediaminetetraacetic
acid). The extraction buffer was supplemented with 1% Halt Protease Inhibitor
Cocktail and 2% Phosphatase Inhibitor Cocktail (Pierce Biotechnology,
Rockford, IL, USA) and 0.10% Triton X-100. The tubules were then homogenized
using a plastic pestle. In addition, the tubules were rapidly passed back and
forth through a 200 µl pipette tip in order to mechanically dissociate and
lyse cells. As multiple microcentrifuge tubes of C and T tubules were
collected, the homogenates were pooled for each group. The pooled homogenates
were brought up to 1 ml volume with extraction buffer. As a final disruption
and lysis step, the pooled homogenates were sonicated with a Model 250
Ultrasonic Cleaner (RAI Research, Hauppauge, NY, USA) for 30 s.
To precipitate cell debris, the pooled homogenates were centrifuged at 3000
g at 4°C for 10 min. The supernatant was subsequently
removed and centrifuged further at 100,000 g and 4°C for
60 min using an OptimaMax ultracentrifuge (Beckman). The high-speed
centrifugation separated the cytosolic-protein fraction (supernatant) from the
membrane-protein fraction (pellet). The cytosolic fraction (
900 µl)
was transferred to a low-adhesion microcentrifuge tube (USA Scientific) and
kept on ice, whereas the membrane fraction was washed three times with
extraction buffer and then stored at –80°C. To estimate the
concentration of protein in the cytosolic fraction, a Bradford protein assay
(BioRad, Hercules, CA, USA) was performed on a 20 µl aliquot using bovine
serum albumin as a reference standard. The protein concentration in the
extract prepared from C tubules was 0.68 µg µl–1 and
0.84 µg µl–1 in that prepared from T tubules. Total
protein amounted to 789 µg in the case of C tubules and 966 µg for T
tubules. The cytosolic extracts were submitted for proteomic analysis to the
Cornell University Life Sciences Proteomics and Mass Spectrometry Core
Facility (Ithaca, NY, USA).
Proteomic analysis
The technical details of the proteomic analysis are described by Yang and
colleagues (Yang et al.,
2007). For the reader of The Journal of Experimental
Biology, we focus here on the major proteomic steps and on the
experimental design and data analysis.
Our study aimed to identify the cytosolic proteins that change after stimulating Malpighian tubules with aedeskinin-III using the methods of 2-D gel electrophoresis and mass spectrometry (Fig. 2). Approximately 180 µg of cytosolic protein from the control (C) and aedeskinin-III-treated (T) tubules were used for the 2-D gel analyses. The first-dimensional separation was performed by immobilized pH gradient isoelectric focusing (24 cm IPG, nonlinear pH 3–10 strips; GE Healthcare, Piscataway, NJ, USA). For electrophoresis in the second dimension, 12.5% homogenous SDS–polyacrylamide DALT gels were cast using the DALTsix gel casting system (GE Healthcare). Peppermint Stick molecular mass markers were applied to each gel at a concentration of 0.25 µg/protein.
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The image (spot) analysis software (Image Master 2D Platinum, 6.0; GE
Healthcare) produces 3-D images of spot volumes and determines the volume of
each spot in the gel (Fig. 3).
As duplicate gels yield two C spot volumes and two T spot volumes, a
significant difference between C and T spot volumes is evaluated by the C/T
ratio, using values that minimize the difference between T and C spot volumes.
The following example illustrates the calculation. Suppose that the two C spot
volumes are 33 and 35 (arbitrary units), and the two T spot volumes are 25 and
27. The minimum difference between C and T is between 33 and 27. The C/T ratio
is therefore 33/27=1.22. A negative sign is assigned to that value
(–1.22) to indicate a decrease in spot volume in the T gel. Thus, our
criterion of C/T
±1.5 selects obvious spot volume changes. A
criterion of C/T±2.0 would have been too stringent and excluded all
changes in spot volume.
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).
Gel-extracted peptides were loaded onto a C18 PepMap trap column and eluted in
a 30-min gradient of 5% to 45% acetonitrile in 0.1% formic acid. The eluted
peptides were fed into an in-line hybrid triple-quadrupole linear ion trap
mass spectrometer (4000 Q Trap from ABI/MDS Sciex, Framingham, MA, USA,
equipped with a Micro Ion Spray Head II ion source).
The mass spectrometry (MS) data were acquired using the Analyst 1.4.2 software (Applied Biosystems, Foster City, CA, USA) in the positive ion mode for information-dependent acquisition (IDA) analysis. The IDA analysis surveyed each scan between m/z 375 and m/z 1550, where m/z is the mass-to-charge ratio. A subsequent enhanced resolution scan selected the three highest intensity ions (peptides) with multiple charge states for tandem MS (MS/MS).
The MS/MS data generated from the IDA analysis were submitted to Mascot 2.2
for database search using an in-house licensed Mascot local server and the
combined Aedes aegypti and Anopheles gambiae databases
downloaded from NCBInr (November 2007), allowing for one missed cleavage site
by trypsin. The peptide tolerance was set to 1.2 Da and MS/MS tolerance was
set to 0.6 Da. The following were set as variables: carbamidomethyl
modification of cysteine, methionine oxidation and phosphorylation of
serine/threonine and tyrosine. Protein identifications were considered for
peptides only if their score defined by Mascot probability analysis was
greater than their `identity' score
(www.matrixscience.com/help/scoring_help.html#PBM).
For multiple proteins identified in single spots, an exponential modified
protein abundance index (emPAI) was used to correctly distribute the change in
expression determined by the in-gel staining and image analysis to each of the
proteins comprising a spot (Yang et al.,
2007).
Ramsay fluid secretion assays
The rate of transepithelial fluid secretion was measured in isolated
Malpighian tubules by a modified method of Ramsay
(Ramsay, 1953). After
isolating a Malpighian tubule from a female mosquito, the tubule was
transferred to a Ringer droplet of 50µl under light mineral oil. The
proximal end of the tubule was pulled into the oil with the aid of a glass
hook, leaving the distal blind end in the Ringer droplet. Half-way between the
glass hook and the oil–water interface, a stellate cell was nicked with
a fine needle so that fluid secreted by the epithelial cells could exit the
lumen into the oil. Timed volumes of secreted fluid were measured with an
ocular micrometer, taking into consideration the volume occupied by the tubule
segment within the secreted droplet. After measuring control secretion rates
for 30 min, aedeskinin-III was added to the peritubular medium to yield a
concentration of 10–6 mol l–1. After another
30 min, H89 was added to the peritubular Ringer solution, to test the effect
of this PKA inhibitor on transepithelial fluid secretion.
All aedeskinins were synthesized in the laboratory of Nachman
(Zubrzak et al., 2007). The
calcitonin-like diuretic peptide AnogaDH31 was a gift of David Schooley
(University of Nevada). The inhibitor of protein kinase A – H-89 –
was purchased from Sigma (St Louis, MO, USA). It is well known that inhibitors
are not ideally selective, and inhibitors of kinases are no exception
(Davies et al., 2000). In the
present study, we used H-89 at a concentration that is 2.5 times lower than
the concentration used to establish the role of PKA in the activation of the
V-type H+ ATPase in the blowfly salivary gland
(Voss et al., 2007).
cAMP assay
Cytosolic cAMP concentrations were measured in sets of 20 Malpighian
tubules isolated from adult female mosquitoes 3–7 days old. One set of
tubules served as the control group (C) and the other as the
aedeskinin-treated (T) group. The tubules were pooled together in 1.5 ml
low-adhesion microcentrifuge tubes (USA Scientific) containing 0.1 ml Ringer
solution supplemented with 0.5 mmol l–1
isobutylmethylxanthine (IBMX, Sigma), an inhibitor of phosphodiesterase. After
15 min incubation at room temperature, sets of tubules were treated with
Ringer solution (negative control), aedeskinin-I, –II or –III
(10–6 mol l–1) or Anoga DH31
(10–6 mol l–1) for 2 min at room
temperature. Anoga DH31 is a calcitonin-like peptide that is known
to increase the intracellular concentrations of cAMP in Aedes
Malpighian tubules (Coast et al.,
2005). Thereafter, the Ringer solution was removed and the tubules
were frozen in liquid nitrogen. The tubules were then stored at
–80°C.
For the extraction of cAMP, the tubules were thawed on ice and homogenized in their microcentrifuge tube with a plastic pestle in 100 µl of ice-cold 100% ethanol containing 0.5 mmol l–1 IBMX. After rinsing the pestle with 100 µl of ice-cold 100% ethanol containing 0.5 mmol l–1 IBMX, the tubules were further homogenized by (1) passing them back and forth through a 200 µl pipette and vortexing them for 1 min and then (2) sonicating them for 1.5 min.
The homogenates were centrifuged at 3000 g at 4°C for
10 min. The supernatant was transferred to a new microcentrifuge tube, whereas
the remaining pellet (containing precipitated protein) was used to determine
the protein concentration by using a BCA protein assay (Thermo Fisher
Scientific, Rockford, IL, USA). The microcentrifuge tube containing the
supernatant was placed in a water bath at 100°C for 11 min in order
to evaporate the ethanol and to bring the isolated cAMP to dryness. The
resulting residue was subsequently resuspended in 200 µl of cAMP Assay
Buffer (provided in the CatchPoin cAMP Assay Kit, Molecular Devices,
Sunnyvale, CA, USA) containing 0.5 mmol l–1 IBMX.
Cyclic-AMP concentrations were measured with a competitive immunoassay, the CatchPoint cyclic-AMP Fluorescent Assay Kit (Molecular Devices, Sunnyvale, CA, USA). In brief, samples and cAMP standards were placed in a 96-well plate coated with goat anti-rabbit IgG. Rabbit anti-cAMP antibody and horseradish-peroxidase-labeled cAMP conjugate were added to each well and left to incubate at room temperature for 2 h. The wells were then washed six times with 200 µl 1xWash Buffer (containing 0.02 mol l–1 Tris, 150 mmol l–1 NaCl, 0.05% Tween 20 and 0.05% Proclin 200, pH 7.4). After a 10-min incubation with a fluorogenic substrate of horseradish peroxidase (Stoplight Red), the plate was read at 540 nm using the Biotek Synergy 2 Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT, USA). A standard curve of cAMP concentrations was generated with the GraphPad Prism software (GraphPad Software, La Jolla, CA, USA), and cAMP concentrations of unknown samples were determined from this standard curve.
Electron microscopy
Malpighian tubules were prepared for electron microscopy as described
previously (O'Connor and Beyenbach,
2001). Sections were cut to a thickness of 70 nm and stained with
osmium tetroxide. Electron micrographs of the tubules were produced with a
Philips Tecnai 12 Biotwin transmission electron microscope (FEI, Eindhoven,
Netherlands).
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The 2-D gel electrophoresis detected a total of 1488 spots in SYPRO Ruby and Pro-Q Diamond stains (Fig. 2). Applying the criterion of a ±1.5-fold change in C/T ratio between C and T gels, the SYPRO Ruby stain detected 26 spots in the C gel that decreased in the T gel. Of these 26 spots, 12 did not appear in the T gel and are therefore considered `unmatched'. The SYPRO Ruby stain also found 21 spots that increased in intensity in the T gel. Of these 21 spots, five are uniquely present in the T gel and are considered `unmatched'.
The Pro-Q Diamond stain identified 81 spots in the C gel and 75 spots in the T gel (Fig. 2). Thus, approximately 5% of the spots detected by SYPRO Ruby contain phosphoproteins. The criterion of a ±1.5-fold change in C/T ratio revealed 19 spots in the C gel that decreased in intensity in the T gel. Of these 19 spots, three did not appear in T gel and are considered `unmatched'. Of the 75 spots staining positively for Pro-Q Diamond in the T gel, seven spots increased in volume over those in the C gel. The loss of phosphoproteins in 19 spots and the increase in phosphoproteins in seven spots suggest that, overall, aedeskinin-III causes more dephosphorylation than phosphorylation.
The protein identification in spots with C/T ratios >±1.5 yielded 128 cytosolic proteins that are affected by the 1 min treatment with aedeskinin-III. From these, we consider here only those proteins that might be associated with aedeskinin signaling and the consequent increase in fluid secretion (Table 1).
Subunit A of the V-type H+ ATPase
Subunit A of the V-type H+ ATPase was found in a single spot
(#565) in both C and T gels (Table
1; Fig. 3). With a
protein score of 639 and 26 unique peptides, it is the best identified protein
of them all. Unique peptides are those peptides (amino acid sequences) that
collectively are unique for the protein thus identified. Comparing C and T
gels reveals that aedeskinin-III decreases the quantity of subunit A in the
cytosol of Malpighian tubules (Fig.
3). Comparing SYPRO Ruby and Pro-Q Diamond ratios, –0.97 and
–1.54, respectively, indicates that the loss of phosphoprotein is
greater than that of protein.
Subunit B of the V-type H+ ATPase
Subunit B of the V-type H+ ATPase and calreticulin shared spot
782 in the C gel (Table 1). The
values of protein coverage indicate that subunit B is the less-abundant
protein, contributing about 21% of the protein present in this spot. The SYPRO
Ruby and Pro-Q Diamond ratios, –0.92 and –1.62, respectively,
mirror the fate of subunit A. Again, the loss of phosphoprotein from the
cytosol is greater than the loss of protein, consistent with the movement of
subunits A and B from the cytosol to the plasma membrane
(Table 1).
Of interest is the appearance of subunit B in a new location of the T gel, appearing in spot 710 as the third of four proteins present in this spot (Table 1). As spot 710 in the T gel is at a more alkaline location – that is, to the right of spot 782 in the C gel (Fig. 2) – spot 710 might contain unphosphorylated subunit B.
Calreticulin
Calreticulin is the major protein (79%) in spot 782 shared with subunit B
in the C gel (Table 1).
Calreticulin is a ubiquitous low-affinity and high-capacity
Ca2+-binding protein. The negative C/T ratios of SYPRO Ruby and
Pro-Q Diamond stains reflect the removal of calreticulin from the cytosol
after treating tubules with aedeskinin-III. Although subunit B in spot 782
might be phosphorylated, we cannot rule out that calreticulin is also
phosphorylated.
Endoplasmin
Endoplasmin drew our attention in view of a Pro-Q Diamond ratio of
–1.67 as a single protein in spot 352 of the C gel
(Table 1). The SYPRO Ruby ratio
is –1.0, which indicates that aedeskinin-III removes endoplasmin from
the cytosol. The Pro-Q Diamond ratio indicates that the loss of phosphoprotein
from the cytosol is greater than the loss of protein. Like calreticulin,
endoplasmin is a major Ca2+-binding protein. Endoplasmin is also a
molecular chaperone of the heat-shock protein 90 class located in the
endoplasmic reticulum.
Actin
Like endoplasmin, actin appeared as the single protein in a spot on the C
gel, spot 1079. The high Pro-Q Diamond ratio of –1.67 signals the loss
of phosphoprotein from the cytosol when tubules are treated with
aedeskinin-III (Table 1). The
SYPRO Ruby ratio of +1.21 indicates the increase in cytosolic actin after
treating tubules with aedeskinin. Thus, aedeskinin-III appears to increase
cytosolic actin at the expense of phosphorylated actin.
Annexin
Spot 1437 contains the single protein annexin. The SYPRO Ruby C/T ratio of
–1.57 indicates the significant reduction of annexin in the cytosol
after treating tubules with aedeskinin-III
(Table 1). Although annexin is
known to be phosphorylated (Rescher et
al., 2008), the Pro-Q Diamond stain did not detect phosphoproteins
in spot 1437.
cAMP-dependent PK type II regulatory subunit
The regulatory subunit II of the cAMP-dependent protein kinase surfaced in
spot 1062 as one of four proteins (Table
1). As the second most-abundant protein in spot 1062, the
regulatory subunit II contributes approximately 29% of the protein present in
spot 1062. The C/T ratios of –2.20 and –1.21, respectively, for
SYPRO Ruby and Pro-Q Diamond stains indicate a marked reduction of protein in
the cytosol, far greater than the reduction in phosphoprotein, consistent with
protein degradation.
rab GDP-dissociation inhibitor
Spot 971 consists of four proteins, including the rab GDP-dissociation
inhibitor (GDI), which contributes approximately 13% of the protein in this
spot. The similar C/T ratios, –1.71 and –1.86, respectively, for
SYPRO Ruby and Pro-Q Diamond stains, suggest that the removal of protein from
the cytosol is the primary mechanism for removing the phosphate signal.
Adducin
Spot 913 in the T gel is occupied by two proteins: adducin and the
β-subunit of the mitochondrial ATPase
(Table 1). Adducin accounts for
52% of the protein present in spot 913. The similar C/T ratios, +1.69 and
+1.62 for SYPRO Ruby and Pro-Q stains, respectively, indicate (1) a major
increase in cytosolic adducin and/or the β subunit of the mitochondrial
F0F1 synthase, and (2) a major increase in the
phosphorylated form of one or both proteins, after treating Malpighian tubules
with aedeskinin-III. Thus, aedeskinin-III adds phosphoproteins of adducin
and/or β subunit to the cytosol. In view of the 14 and seven unique
peptides identified in the case of adducin and the β subunit of the
F0F1 synthase, respectively, we have higher confidence
in the increase of adducin than in the increase in the β subunit of the
F0F1 synthase.
Regucalcin
Spot 1314 in the T gel reveals two proteins – regucalcin and serpin-4
(Table 1). As regucalcin
contributes 91% of the protein in the spot, the significant increase in C/T
ratios is likely to include the arrival of new regucalcin in the cytosol after
treating Malpighian tubules with aedeskinin-III. The new regucalcin arrives in
the cytosol as a phosphoprotein. Regucalcin is a Ca2+-binding
protein.
Actin-depolymerizing factor
The SYPRO Ruby stain reveals spot 2103 present in the T gel but not in the
C gel. The unmatched presence of this spot in the T gel indicates the arrival
of a new protein in the cytosol when Malpighian tubules are stimulated with
aedeskinin-III (Table 1). The
new arrival is actin-depolymerizing factor (ADF). It is the single protein of
spot 2103. The positive Pro-Q Diamond C/T ratio of 1.05 identifies ADF as a
phosphoprotein in the cytosol.
As the proteomic data indicated that treating Malpighian tubules with aedeskinin-III for 1 min results in the cytosolic loss of (1) two subunits of the V-type H+ ATPase and (2) the regulatory subunit of protein kinase A (PKA), we examined the role of PKA in mediating the diuretic effects of aedeskinin by using a PKA inhibitor in the Ramsay fluid secretion assay and measuring intracellular cAMP concentrations after stimulating tubules with aedeskinin-III.
Ramsay fluid secretion assay
Using the modified method of Ramsay, we examined whether the mechanism of
action of aedeskinin-III involves PKA. The rate of fluid secretion was
0.19±0.04 nl min–1 in 10 Malpighian tubules under
control conditions (Fig. 4).
The addition of 1 µmol l–1 aedeskinin-III to the
peritubular bath significantly (P<0.0018) increased the rate of
fluid secretion to 0.63±0.13 nl min–1 in the same 10
tubules. The subsequent addition of 20 µmol l–1 H89
– an inhibitor of PKA – to the peritubular bath significantly
reduced the rate of fluid secretion back to control rates.
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). Under non-stimulated conditions (control), the mean
cAMP content of Malpighian tubules was 0.18±0.03 pmol
µg–1 protein in 12 determinations of 20 tubules each. As
shown in Fig. 5, none of the
aedeskinins had significant effects on intracellular cAMP concentrations
relative to control tubules even though (1) aedeskinin concentrations were
10-fold greater than in the proteomic protocol and (2) tubules were exposed to
aedeskinins for 2 min rather than 1 min in the proteomic protocol. By
contrast, cytosolic cAMP levels increased more than six fold in the presence
of Anoga-DH31 compared with controls. Thus, the activation of PKA
(suggested by the proteomic data and the effects of H89) is apparently
mediated by a `non-traditional' mechanism that is independent of intracellular
cAMP concentrations.
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Electron microscopy
Electron micrographs of the paracellular pathway in Aedes
Malpighian tubules reveal that septate junctions occupy most of the
paracellular pathway from the apical to basal poles of epithelial cells
(Fig. 6A,B). Along the length
of a given septate junction, the septa can occur at regular intervals,
irregular intervals or there might be no septa at all
(Fig. 6C). The intracellular
meshwork immediately below the plasma membrane of a septate junction might
present components of the cytoskeleton
(Fig. 6C). Further into the
cytoplasm is evidence of microtubules running perpendicular to or parallel
with septate junctions (Fig.
6C). The paracellular pathway of invertebrate epithelia is thought
to include a subapical region, an adherens junction and septate junctions.
Distinct subapical regions and adherens junctions were not observed in the
present set of electron micrographs of Aedes Malpighian tubules,
which does not rule out their existence. In some images, the septate junction
extended all the way to the brush border apical membrane
(Fig. 6C).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Hypothesis 1: aedeskinin-III signals to the V-type H+ ATPase
The V-type H+ ATPase is a proton pump that consists of two
multi-subunit ring structures: a cytoplasmic V1 complex and a
membrane-embedded V0 complex
(Fig. 7A). Subunits A and B are
part of the catalytic V1 complex that mediates the hydrolysis of
ATP. Subunits c are part of the V0 complex that mediate the
`pumping' of H+ across the membrane. Mechanically, the V-type
H+ ATPase is a motor, with V1 and V0
complexes operating as a stator and a rotor, respectively
(Fig. 7B). The energy of ATP
hydrolysis can drive transmembrane H+-transport only when the
stator and rotor are physically joined
(Beltran and Nelson, 1992;
Gräf et al., 1996;
Zhang et al., 1992) and when
the V1 complex is anchored to the cytoskeleton to prevent its own
rotation.
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).
Likewise, glucose (nutrient) deprivation causes the V1 complex to
separate from the V0 complex in yeast
(Kane, 1995). Should the need
for H+ transport arise again, the V1 complex leaves the
cytoplasm to join its partner in the membrane again.
In view of the central role of the V-type H+ ATPase in
transepithelial electrolyte and fluid secretion in Malpighian tubules of
insects (Beyenbach et al.,
2000), it is expected that an increase in transepithelial
electrolyte and fluid secretion – stimulated by a diuretic agent such as
aedeskinin – increases the transport activity of the V-type
H+ ATPase (Beyenbach,
2001). One way to increase transport activity is to increase the
number of active transport pumps by joining V1 and V0
complexes, which removes the V1 complex from the cytoplasm, as
witnessed in the present study by subunits A and B diminishing in the cytosol
after tubules have been stimulated with aedeskinin-III
(Table 1).
Subunit C of the V1 complex (not to be confused with subunit c
of the V0 complex) appears to play the pivotal role in joining the
V1 complex to the V0 complex
(Fig. 7C), as gleaned from
studies of salivary glands of the blowfly. In brief, serotonin, the natural
stimulant of the salivary gland, stimulates fluid secretion by means of cAMP
and protein kinase A (Dames et al.,
2006; Rein et al.,
2008). Protein kinase A (PKA) exclusively phosphorylates subunit C
as the molecular switch that joins V1 and V0 complexes
to activate ATP hydrolysis and proton transport
(Rein et al., 2008;
Voss et al., 2007).
Immunohistochemical studies show further that, upon serotonin stimulation,
subunits C and B of the V1 complex, PKA and actin all localize to
the apical membrane of the salivary gland epithelial cell
(Rein et al., 2008;
Voss et al., 2007). Actin
might be recruited to the apical membrane in an apparent fortification of the
cytoskeleton for anchoring the V1 complex and the holoenzyme in
place (Vitavska et al., 2005;
Vitavska et al., 2003;
Wieczorek et al., 2003).
In the present study, the role for PKA in the kinin-stimulated diuresis is
supported by (1) the decrease of subunits A and B of the V-type H+
ATPase in the cytosol (Table
1), (2) the decrease in the regulatory subunit type II of PKA
(Table 1) and (3) the reversal
of the effect of aedeskinin-III on transepithelial fluid secretion by H89, an
inhibitor of protein kinase A (Fig.
4). However, inconsistent with a role for PKA is the lack of
effect of any aedeskinin on intracellular cAMP levels after 2 min of
stimulation (Fig. 5), which
confirms previous observations of cAMP-independent diuretic effects of (1)
aedeskinins in Aedes Malpighian tubules
(Cady and Hagedorn, 1999) and
(2) drosokinin in Drosophila Malpighian tubules
(Terhzaz et al., 1999).
The dilemma posed by the above conflicting observations would be solved by
a stimulation of PKA that is independent of cAMP
(Fig. 7C). Such a mechanism has
been observed in mammalian cells. It is the NF-
B signaling pathway that
targets regulated proteolysis (Zhong et
al., 1997). In brief, stimulation of the NF-B signaling
pathway leads to the proteolytic degradation of IKB, an inhibitory protein of
not only the transcription factor NF-B but also of the catalytic
subunit of PKA (PKAcat). Thus, activation of the NF-B
signaling pathway could activate PKAcat, bringing about the
assembly of the V-type H+ ATPase without an increase in
intracellular cAMP (Fig. 7). Of
interest is that protein kinase C (PKC) can activate the NF-B signaling
pathway (McAllister-Lucas et al.,
2001; Steffan et al.,
1995). The NF-B signaling pathway occurs in insects, where
one of the insect IKB proteins is known as the Drosophila cactus
protein (Belvin et al., 1995).
Alternatively, it is possible that PKC phosphorylates subunit C directly,
which leaves open the question of whether PKA or PKC mediates the assembly and
activation of the V-type H+ ATPase in Aedes Malpighian
tubules (Fig. 7C).
Hypothesis 2: aedeskinin-III signals to the paracellular pathway
The kinins are known to increase the transepithelial Cl–
conductance of Malpighian tubules as part of the diuretic mechanism for
increasing the transepithelial secretion of NaCl, KCl and water
(Fig. 1). Stellate cells
provide the route for Cl– secretion in Drosophila
Malpighian tubules (O'Donnell et al.,
1996; O'Donnell et al.,
1998). Stellate cells might also do so in Malpighian tubules of
Aedes aegypti under resting, control conditions
(O'Connor and Beyenbach,
2001). However, upon stimulation with kinin diuretic peptides, the
major route for transepithelial Cl– secretion is between
epithelial cells (Beyenbach,
2003; Pannabecker et al.,
1993; Yu and Beyenbach,
2001; Yu and Beyenbach,
2004).
Ca2+ plays a major role in mediating the effects of kinin
peptides on paracellular Cl– conductance because the effects
of kinins can be duplicated by A23187, an ionophore that allows
Ca2+ to enter cells from the peritubular Ringer solution
(Clark et al., 1998). Our
proteomic study is consistent with the signaling role of Ca2+
because three Ca2+-binding proteins are affected by aedeskinin-III:
calreticulin, endoplasmin and regucalcin
(Table 1).
Possessing as many as 50 Ca2+-binding sites, calreticulin is a
major Ca2+-binding protein and therefore an important regulator of
Ca2+ signaling (Coppolino and
Dedhar, 1998; Yamaguchi,
2005), including signaling to cell-adhesion molecules
(Coppolino and Dedhar, 1999;
Coppolino et al., 1997). The
departure of calreticulin and endoplasmin from the cytosol is expected to
increase the concentration of free Ca2+ in the cytoplasm, thereby
enhancing the effects of Ca2+
(Table 1). By contrast, the
arrival of regucalcin in the cytosol is expected to decrease the free
[Ca2+] in the cytoplasm through the activation of
Ca2+-uptake pumps in the endoplasmic/sarcoplasmic reticulum
(intracellular Ca2+ stores) and mitochondria
(Yamaguchi, 2005).
Extracellular Ca2+ is needed to sustain the stimulatory effects
of the kinins in Malpighian tubules (Yu
and Beyenbach, 2002). Detailed studies in Malpighian tubules of
Aedes aegypti have elucidated the following signaling pathway that
leads to the elevation of intracellular [Ca2+], as illustrated in
Fig. 8. The binding of
aedeskinin to its G-protein-coupled receptor activates phospholipase C, which
converts phosphatidylinositol (4,5)-bisphosphate into two intracellular
messengers: membrane-associated diacylglycerol (DAG) and cytoplasmic inositol
(1,4,5)-trisphosphate [Ins(1,4,5)P3]. The binding of
Ins(1,4,5)P3 to receptors at the endoplasmic reticulum
opens Ca2+ channels, allowing the entry of Ca2+ into the
cytoplasm. As the [Ca2+] in the cytoplasm rapidly rises, the rapid
fall of the [Ca2+] in the endoplasmic reticulum triggers the
opening of Ca2+ channels in the plasma membrane by means of a
`store-depletion mechanism'. Ca2+ enters the cell, raising the
intracellular [Ca2+] even more
(Fig. 8). Ca2+
binding to inactive PKC allows PKC to interact with DAG in the plasma
membrane, thus targeting PKC to the membrane. When PKC binds to DAG, the
kinase is activated. As the active PKC is now membrane bound, phosphorylations
are limited to substrates in close proximity to the membrane and cytoskeleton
(Fig. 8).
|
One known substrate of PKC is adducin
(Matsuoka et al., 1998).
Adducin is a membrane-skeletal protein found at the junctions of actin and
spectrin that colocalize at sites of cell–cell contact
(Kaiser et al., 1993;
Kaiser et al., 1989). The
phosphorylation of adducin by rho kinase promotes the association of F-actin
and spectrin to form a spectrin–actin meshwork beneath the plasma
membrane (Fukata et al., 1999;
Gardner and Bennett, 1987;
Kimura et al., 1998;
Li et al., 1998).
Phosphorylation by PKC inhibits the actin-capping and spectrin-recruiting
activities of adducin, thereby destabilizing the cytoskeleton
(Matsuoka et al., 1998). The
appearance of phosphorylated adducin in the cytosol after aedeskinin treatment
is consistent with the destabilization and remodeling of the cytoskeleton
(Table 1;
Fig. 8).
The reorganization of the cytoskeleton is further supported by the
appearance in the cytosol of (1) ADF and (2) actin
(Table 1;
Fig. 8). If this cytoskeletal
remodeling takes place along the paracellular pathway, it might mediate the
switch-like increase in paracellular Cl– conductance when
kinin diuretic peptides trigger diuresis in Malpighian tubules
(Fig. 8). The cytoskeletal
remodeling of the paracellular pathway might include vesicular traffic to and
from the paracellular pathway, as indicated by the decrease in cytoplasmic rab
GDI in the presence of aedeskinin-III
(Table 1). GDI mediates in part
the trafficking of vesicles between donor membranes and target membranes
(Stein et al., 2003). Rab is a
small G protein that is found at tight junctions in polarized cells
(Zahraoui et al., 1994).
Accordingly, the aedeskinin-triggered diuresis might involve the trafficking
of proteins between the plasma membrane and submembrane vesicles in regions of
the paracellular pathway. The above discussion makes clear that the sudden
increase in paracellular Cl– conductance triggered by kinin
diuretic peptides requires the paracellular pathway functioning as a continuum
between extracellular and intracellular components.
The paracellular pathway in Malpighian tubules of Aedes aegypti
Studies of vertebrate epithelia have taken the lead in our understanding of
the physiological regulation of the paracellular transport pathway
(Anderson and Van Itallie,
1995; Cereijido,
1991; Hopkins et al.,
2000; Madara,
1998). In particular, the regulation of the paracellular pathway
has been localized to the tight junction
(Hopkins et al., 2003;
Kahle et al., 2004;
Schneeberger and Lynch, 2004;
Shen et al., 2008). The
regulation of the paracellular pathway in invertebrates has been researched
far less, even though Malpighian tubules display the most dynamic and dramatic
changes in a paracellular transport activity
(Beyenbach, 2003;
Pannabecker et al., 1993;
Yu and Beyenbach, 2002;
Yu and Beyenbach, 2004). It
raises questions about the differences between tight and septate junctions
that enable septate junctions to respond to signaling with switch-like speed
and with a wide dynamic range of paracellular electrical conductance.
In electron micrographs, tight junctions in vertebrates and septate
junctions in invertebrates have very different geometries. Tight junctions are
located at the most apical region of epithelial cells, and they extend into
the paracellular pathway for only a short distance, less than 1 µm.
Moreover, tight junctions are points of contact between neighboring cells that
are formed by the interaction of the extracellular loops of the integral
membrane proteins claudin and occludin
(Furuse et al., 1998;
Ikenouchi et al., 2005).
Interactions of these extracellular loops obliterate the extracellular space
and give the appearance of the focal fusion of the plasma membranes of
neighboring cells. Beyond the less than 1 µm region of the vertebrate tight
junction, the lateral interstitial space accounts for more than 95% of the
length of the paracellular pathway, given a conservative epithelial cell
height of 20 µm.
In marked contrast, septate junctions span the whole height of the epithelial cell from apical to basal poles of epithelial cells over the length of many microns (Fig. 6). Ladder-like septa appear to make sure that the lateral plasma membranes of adjacent cells do not fuse. They are kept approximately 160 Å apart (Fig. 6C). What forms the rungs of the paracellular ladder is unknown. The constitution of the electron-dense material in the space between septa is also unknown.
In view of the wide paracellular space (160 Å) and the small molecular diameters of Na+, K+, Cl– and water (<5 Å), it follows that the usual solutes of hemolymph (including glucose, 70 Å) can occupy the intercellular septate junctional space – unless septa prevent access. Preventing access might account for the barrier function of septate junctions. By contrast, a reversible, zipper-like opening of septa would provide access, thereby increasing the paracellular conductance/permeability. Such an increase would be expected to be substantial in view of the width –160 Å – of the paracellular path now open.
In vertebrate tight junctions, claudins and occludins provide the barrier
and permselectivity properties of the tight junction. Moreover, claudins
display a stunning spontaneous breaking and resealing
(Sasaki et al., 2003). Using
GFP-tagged claudin in living cells it was possible to observe claudin strands
breaking, migrating and reconnecting in minutes, demonstrating what
physiologically could be open and closed routes for paracellular transport.
Similarly, the opening and closing of septa-forming proteins could regulate
the permeability and conductance of septate junctions. The finding of
claudin-like proteins, such as sinuous and megatrachea in septate junctions
(Behr et al., 2003;
Wu et al., 2004), raises the
intriguing question of whether septate junctions also undergo spontaneous
turnover and remodeling.
What the present proteomic study urges is therefore the identification of (1) the proteins that form the ladder of septate junctions and (2) their scaffolding proteins on the cytoplasmic side. These interactions might mediate the switch-like on/off changes in paracellular Cl– conductance that meet the diuretic and antidiuretic needs of the insect.
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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,β,
, subunits of G protein; GTP, guanosine triphosphate;
PLC, phospholipase C; PIP2, phosphatidylinositol
(4,5)-bisphosphate; DAG, diacylglycerol; PKC, protein kinase C; SERCA,
sarcoplasmic and endoplasmic reticulum calcium ATPase.