Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowfly Calliphora vicina
1 Institut für Biochemie und Biologie, Zoophysiologie, Universität
Potsdam, Lennéstr. 7a, D-14471 Potsdam, Germany
2 Carl Zeiss Jena GmbH, Advanced Imaging Microscopy, Carl-Zeiss-Promenade
10, D-07745 Jena, Germany
* Author for correspondence (e-mail: obaumann{at}rz.uni-potsdam.de)
Accepted 14 March 2003
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Summary |
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These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical `potassium pump' that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K+/nH+ antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V0V1 holoenzymes might constitute a regulatory mechanism for the control of pump activity.
Key words: vacuolar ATPase, V-ATPase, portasome, bafilomycin, Na+/K+-ATPase, serotonin, 5-hydroxytryptamine, regulation, assembly, immunocytochemistry, insect, blowfly, Calliphora vicina.
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Introduction |
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Secretion in the abdominal portion of the salivary glands is stimulated by
serotonin [5-hydroxytryptamine (5-HT)], which acts as a hormone in this system
(Berridge and Patel, 1968).
Exposure to 5-HT results in the activation of two intracellular messenger
pathways, namely the cyclic AMP (cAMP) system and the
inositol(1,4,5)-trisphosphate/Ca2+ system
(Heslop and Berridge, 1980
;
Berridge and Heslop, 1981
;
Berridge et al., 1983
;
Litosch et al., 1985
;
Zimmermann and Walz, 1997
,
1999
). The 5-HT-induced
increase in cAMP activates an electrogenic active K+ transport
mechanism (Berridge et al.,
1976
; Berridge,
1977
). Cl movement across the epithelial layer
occurs passively through Cl conductances that open in
response to the 5-HT-dependent rise in cytosolic Ca2+
(Berridge et al., 1976
;
Berridge, 1977
). The resulting
transepithelial KCl transport then provides the osmotic driving force for
fluid secretion.
The molecular correlate of the active K+ transport mechanism in
blowfly salivary glands has not been determined so far. For other insect
epithelia that secrete a K+-rich fluid into a luminal space, such
as the midgut, Malpighian tubules and antennal sensilla, evidence has been
presented that a vacuolar-type H+-ATPase (V-ATPase) is localized in
the apical membrane of the epithelial cells and is involved with the extrusion
of K+ (Schweikl et al.,
1989; Wieczorek et al.,
1989
; Klein and Zimmermann,
1991
; Klein, 1992
;
Maddrell and O'Donnell, 1992
;
Harvey and Wieczorek, 1997
).
V-ATPase is a multi-subunit transporter composed of a catalytic ATP-binding
V1 component that resides on the cytoplasmic side of the membrane
and a membrane-bound V0 component that forms an H+
channel (Stevens and Forgac,
1997
; Wieczorek et al.,
1999
; Nishi and Forgac,
2002
). The electrochemical H+ gradient generated by the
V-ATPase may then drive a secondary K+ transport via a
K+/nH+ antiporter, resulting in a net extrusion
of K+ (Wieczorek et al.,
1991
; Lepier et al.,
1994
; Harvey and Wieczorek,
1997
).
The present study examines the hypothesis that, as in other insect epithelia, K+ secretion in blowfly salivary glands is powered by a V-ATPase. Ultrastructural methods, ATPase assays and immunotechniques in conjunction with a panel of antibodies against various subunits of V-ATPase have been used to demonstrate that V-ATPase is present within the secretory cells and that it is highly enriched on their apical membranes. Stimulation of the salivary glands with 5-HT results in an activation of V-ATPase activity, whereas Na+/K+-ATPase, residing on the basolateral membranes of the secretory cells, is unaffected by hormone treatment. Furthermore, we present the results of experiments that indicate the way in which the V-ATPase is activated upon 5-HT stimulation.
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Materials and methods |
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Electron microscopy
For high-pressure freezing, glands were placed in the 50-µm-deep cavity
of aluminium platelets, surrounded with 1-hexadecene and covered with a second
platelet. The samples were transferred to a high-pressure freezing apparatus
(HPM 010, Balzers Union, Liechtenstein), frozen in liquid nitrogen at a
hydrostatic pressure of 2x108 Pa
(Moor et al., 1980) and
freeze-substituted in dry acetone containing 2% OsO4 as described
in detail previously (Klein and
Zimmermann, 1991
; Zimmermann,
2000
). After being washed in dry acetone at room temperature,
specimens were embedded in Epon resin. Ultrathin sections were stained with
uranyl acetate and lead citrate and examined in a CM100 electron microscope
(Philips, Eindhofen, The Netherlands) operated at 80 kV or 100 kV.
ATPase activity assays
After dissection, glands were incubated for 3 min in Ringer solution or
stimulated for 3 min with 1 µmol l1 5-HT. Subsequently,
glands were homogenized on ice in 0.3 mol l1 sucrose, 0.1
mmol l1 EGTA, 0.1 mol l1 imidazol (pH
7.2), 0.1% ß-mercaptoethanol and proteinase inhibitors (leupeptin, 10
µg ml1; pepstatin A, 10 µg ml1;
Pefabloc SC, 1 mg ml1). ATPase activity was probed in
aliquots of 10 µl, representing the equivalent of four glands. The aliquots
were adjusted to 112 mmol l1 NaCl, 30 mmol
l1 KCl, 5 mmol l1 MgCl2, 0.1
mmol l1 EGTA, 0.1 mol l1 imidazol (pH
7.2), 30 mmol l1 sucrose, 5 mmol l1
Na2ATP, 0.01% ß-mercaptoethanol and 4% dimethyl sulfoxide
(DMSO; carrier solution for bafilomycin A1), resulting in a final
volume of 100 µl. In the case of 5-HT-stimulated tissue, 1 µmol
l1 5-HT was added to the homogenizing medium and the assay
mixtures. To resolve bafilomycin A1-, ouabain-sensitive and the
remaining ATPase activity, 1 µmol l1 bafilomycin
A1 (Sigma, Deisenhofen, Germany) or 1 mmol l1
ouabain (Sigma) was added to the respective assay mixtures. After incubation
for 60 min at 30°C, reactions were stopped by adding 100 µl of 1 mol
l1 H2SO4. The preparations were then
centrifuged for 10 min at 18 000 g at 4°C; inorganic
phosphate (Pi) liberated during the experiment was determined in an
aliquot of the supernatants as a phosphomolybdate complex
(Bonting et al., 1961). The
concentration of phosphomolybdate was measured photometrically; 10 nmoles
Pi formed in the 100 µl assay led to a change in light
extinction (Ê) of 0.36 at 707 nm. Enzyme activity values were corrected
for background Pi levels by subtracting the extinction values of
homogenate-free controls.
Antibodies
Na+/K+-ATPase was detected with monoclonal mouse
antibody 5IgG (Takeyasu et
al., 1988
), which is known to cross-react with the
Na+/K+-ATPase
-subunit of various insects,
including dipteran flies (Lebovitz et al.,
1989
; Baumann,
1997
). Antibody a5 was applied at a concentration of 20 µg
ml1 for immunofluorescence labelling and at 1.5 µg
ml1 on western blots. Subunits A and E of V-ATPase were
identified by polyclonal antibodies made in rabbits against the C-terminus of
bovine V-ATPase subunits A and E (Roussa
et al., 1998
). The antigens exhibit 73% amino acid identity with
the corresponding regions in Drosophila homologues (Drosophila
melanogaster vacuolar ATPase subunit A gene, GenBank Acc. No. U19742;
Guo et al., 1996
). Both
antibodies were used at a dilution of 1:200 for immunofluorescence labelling
and at 1:2500 for western blotting. Subunit B of V-ATPase was identified on
western blots (dilution 1:10 000) by a polyclonal antibody raised in rabbits
against a 279-amino-acid region (residues 79357) of the V-ATPase
subunit B from the insect Culex quinquefasciatus
(Filippova et al., 1998
). This
region of the Culex V-ATPase subunit B shares 98% amino acid identity
with that published for Drosophila V-ATPase subunit B
(Davies et al., 1996
).
Subunits a and d of V-ATPase were detected with polyclonal antibodies that
were raised in guinea pigs by injection of the N-terminal 390-amino-acid-long
region of subunit a or complete subunit d of Manduca sexta V-ATPase
(H. Wieczorek, unpublished). Sequence identity between the Manduca
antigens and the corresponding Drosophila polypeptides amounts to
approximately 90% for each of the two subunits (Merzendorfer et al.,
1997
,
2000
;
Adams et al., 2000
). These
antibodies were applied at 1:5000 to 1:10 000 on western blots and at 1:1000
for immunofluorescence labelling. Monoclonal antibody DM 1A against
-tubulin was purchased from Sigma and diluted 1:4000 for western blot
analysis. Secondary antibodies conjugated to Cy3 or horseradish peroxidase
(HRP) were obtained from Rockland (Gilbertsville, PA, USA) and American Qualex
(La Mirada, CA, USA).
Fluorescence microscopy
Salivary glands were fixed for 2 h either in 3% paraformaldehyde and 0.2%
glutaraldehyde in 0.1 mol l1 phosphate buffer (PB), pH 7.4,
or in 3% paraformaldehyde in PB supplemented with 1 mmol l1
of the cross-linking reagent dithiobis(succinimidyl proprionate) (Pierce,
Rockland, IL, USA). After a 10-min wash in PB, specimens were labelled with
F-actin probe OregonGreenphalloidin and the DNA stain DAPI, both used
according to the manufacturers instructions (Molecular Probes, Eugene, OR,
USA). For indirect immunofluorescence staining, fixed specimens were washed in
PB, transferred to 10% (w/v) sucrose in PB for 1 h, incubated overnight in 25%
sucrose in PB and frozen in melting isopentane (165°C). Sections
(10 µm thick) were cut on a cryostat and placed on cover slips coated with
poly-L-lysine. The sections were successively incubated in (1) 0.01% Tween 20
in phosphate-buffered saline (PBS), (2) 50 mmol l1
NH4Cl in PBS, (3) PBS and (4) blocking solution containing 1%
normal goat serum, 0.8% bovine serum albumin (BSA), 0.1% fish gelatin and 0.5%
Triton X-100 in PBS, each step lasting 5 min at room temperature. The samples
were then incubated overnight at 4°C with primary antibodies diluted in
blocking solution, washed for 3x10 min in PBS and reacted with
fluorochrome-tagged secondary antibodies and OregonGreenphalloidin for
1 h at room temperature. Sections were washed again for 3x10 min in PBS
and mounted in Mowiol 4.88 (Farbwerke Hoechst, Frankfurt, Germany) containing
2% n-propyl gallate. Fluorescence images were recorded with a Zeiss LSM 510 or
a Zeiss LSM 5 PASCAL confocal laser-scanning microscope (Carl Zeiss, Jena,
Germany).
Immunoblot analysis
Salivary glands were homogenized on ice in reducing sample buffer (Carl
Roth, Karlsruhe, Germany). The preparations were heated for 5 min to 60°C
and centrifuged for 10 min at 18 000 g to remove
non-solubilized material. Proteins were separated on sodium dodecylsulphate
(SDS) 12% polyacrylamide gels in the Laemmli buffer system
(Laemmli, 1970) and
electrotransferred onto nitrocellulose sheets. After being incubated for 30
min with 5% low-fat dry milk in Tween buffer (0.1% Tween 20, 150 mmol
l1 NaCl, 10 mmol l1 Tris, pH 7.5), blots
were reacted for 216 h with primary antibodies diluted in Tween buffer,
washed several times with Tween buffer and an intermittent treatment in a
solution containing 2 mol l1 urea, 1% Triton X-100 and 0.1
mol l1 glycine (pH 7.5), reacted for 1 h with the
appropriate HRP-coupled secondary antibody and finally rinsed extensively with
Tween buffer. Bound antibody was visualized by enhanced chemiluminescence
(Pierce, Rockland, IL, USA).
Biochemical analysis of V-ATPase distribution
Non-stimulated or 5-HT-stimulated glands were homogenized in extraction
buffer containing 70 mmol l1 sucrose, 50 mmol
l1 KCl, 50 mmol l1 Tris (pH 7.5), 20 mmol
l1 dithiothreitol and a cocktail of protease inhibitors (see
above). Membranes and cytosolic proteins were then separated by centrifugation
for 30 min at 150 000 g at 4°C. Proteins within the
supernatant fraction were precipitated by the method of Wessel and Flügge
(1984), solubilized in sample
buffer and heated to 60°C for 3 min. The pellet fraction was washed twice
with extraction buffer, resuspended in sample buffer and also heated to
60°C. Western blots were carried out as described above. To determine the
relative amount of each protein within the pellet and the supernatant, images
of the western blots were recorded with a cooled CCD camera and analysed
densiometrically by use of the software program Metamorph (Universal Imaging
Corp., West Chester, PA, USA).
Statistical analysis
Data are presented as means ± S.D. Statistical comparisons were made
by an independent Student's t-test. P values of <0.001
were considered as highly significant; P values of >0.2 were
considered as nonsignificant.
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Results |
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High magnification views reveal that the cytoplasmic aspect of the apical
membrane is almost entirely covered by an electron-dense coat approximately 15
nm in thickness (Fig. 1B). This
coat appears to be composed of densely packed 10-nm-wide particles attached to
the membrane by a short stalk (arrowheads in
Fig. 1B). The particles thus
resemble portasomes, the structural correlate of V-ATPase
(Gupta and Berridge, 1966;
Harvey, 1980
;
Klein and Zimmermann, 1991
;
Klein, 1992
). No particles
have been detected on the basolateral domain of the plasma membrane
(Fig. 1C), suggesting that
portasomes are localized exclusively on the apical domain.
Identification of V-ATPase and Na+/K+-ATPase
within the salivary gland
Transepithelial electrolyte and water transport in animals is energized by
active ion transport mechanisms, such as the
Na+/K+-ATPase or the V-ATPase. Western blot analysis of
blowfly salivary gland with an antibody against the catalytic -subunit
of Na+/K+-ATPase and antibodies against various subunits
of the V0 portion and of the V1 portion of V-ATPase
suggests that both ion pumps are present within the secretory cells
(Fig. 2). The V-ATPase
antibodies used for these experiments were raised against V-ATPase subunits of
other insects or of vertebrates; the antigens, however, display a high
homology with the corresponding regions in the respective V-ATPase subunits of
Drosophila, a dipteran fly closely related to Calliphora
(see Materials and methods). Each of the antibodies intensely labelled a band
of the appropriate electrophoretic mobility on western blots of blowfly
salivary glands (anti-Na+/K+-ATPase
-subunit,
110 kDa; anti-V-ATPase subunit A,
74 kDa; anti-subunit B,
58
kDa; anti-subunit E,
32 kDa; anti-subunit a,
115 kDa; anti-subunit
d,
39 kDa).
|
To examine the expression of Na+/K+-ATPase and
V-ATPase within salivary glands by an alternative method, we measured the
ATPase activity of homogenized glands in the absence and presence of a
selective inhibitor for each transporter. When bafilomycin A1, a
highly specific inhibitor of V-ATPase
(Bowman et al., 1988;
Dröse and Altendorf,
1997
), was applied to homogenized salivary glands, it inhibited
ATPase activity with an IC50 of approximately 3 nmol
l1 (Fig. 3A).
At a saturating bafilomycin concentration of 1 µmol l1,
ATPase activity was reduced by 43.1±5.7%, suggesting that a relatively
large amount of the total ATPase activity was provided by the
bafilomycin-sensitive V-ATPase under our assay conditions. When ouabain, a
specific inhibitor of Na+/K+-ATPase, was applied at 1
mmol l1, a concentration easily within the saturating range
in other systems (e.g. Lebovitz et al.,
1989
), ATPase activity was reduced by 18.6±4.5%
(Fig. 3B). These findings
suggest that approximately 60% of the Pi detected within the assay
was due to the activity of V-ATPase and Na+/K+-ATPase,
supporting the conclusion that secretory cells are equipped with both ion
transporters. The remaining Pi may have been liberated by other
ATPases present within the homogenate and/or may have resulted from endogenous
Pi trapped within vesicles.
|
Distribution of V-ATPase and Na+/K+-ATPase
within the secretory cells
The localization of V-ATPase and Na+/K+-ATPase within
non-stimulated secretory cells was determined by immunofluorescence labelling
of cryostat sections (Fig. 4).
In order to provide a spatial reference for the position of the immunoreactive
structures, specimens were co-labelled with OregonGreenphalloidin, a
probe for filamentous actin. By this procedure, the apical membrane, including
the canaliculi, was visualized as a brightly fluorescent structure that
embraced the nuclei, whereas the basal surface of the secretory cells was
identified by weak reactivity with phalloidin
(Fig. 4A,E,I;
Zimmermann, 2000).
|
Anti-Na+/K+-ATPase stained the basal and lateral sides of the secretory cells, whereas the apical domain exhibited no immunoreactivity for Na+/K+-ATPase (Fig. 4B,F,J). By contrast, antibodies against V-ATPase subunit A (data not shown), subunit E (Fig. 4C,G,K) and subunit d (Fig. 4D,H,L) bound to the apical membrane including the invaginations. Staining of the invaginations with anti-V-ATPase antibodies was not homogeneous over the entire membrane domain but varied in intensity (Fig. 4C,D). This may have been due to the compact structure of the microvilli and cross-linkage of proteins within the microvilli, resulting in poor accessibility of the antibodies to the antigens. Anti-V-ATPase antibodies further bound to vesicular structures within the secretory cells (Fig. 4C,D,K,L), indicating the presence of V-ATPase on cell organelles.
These results suggest that Na+/K+-ATPase is restricted to the basolateral membrane. V-ATPase is concentrated at the apical domain of the plasma membrane and is present in smaller amounts on cell organelles. Staining patterns with Na+/K+-ATPase and V-ATPase antibodies and with phalloidin were similar in non-stimulated (Fig. 4) and 5-HT-stimulated glands (data not shown), indicating that exposure to 5-HT does not lead to dramatic reorganization of the membrane domains or redistribution of pump molecules within the cells.
Effect of 5-HT on ATPase activity
If any of the transporters identified above is involved in driving
5-HT-induced fluid secretion, the activity of the transporter(s) should
increase upon stimulation with 5-HT. To examine this possibility, the ATPase
activity of both transporters was determined in salivary glands treated with
5-HT at a concentration known to be saturating in physiological assays
(Berridge and Prince, 1972).
Under these conditions, total ATPase activity within the homogenate increased
from 22.3±6.0 nmol Pi gland1
h1 to 31.5±5.4 nmol Pi
gland1 h1
(Fig. 3B). Ouabain-sensitive
ATPase activity did not differ significantly between control and
5-HT-stimulated tissue, whereas bafilomycin-sensitive ATPase activity was
increased by a factor of approximately 2 after exposure to 5-HT. The increase
in bafilomycin-sensitive ATPase activity (8.8 nmol Pi
gland1 h1) compares well with the increase
in total ATPase activity (9.2 nmol Pi gland1
h1), suggesting that V-ATPase may be the main ATPase that is
activated in response to 5-HT in our experimental assay.
Effect of 5-HT on the assembly state of V-ATPase
It has been proposed that assembly/disassembly of the
V0V1 holoenzyme is involved in the regulation of
V-ATPase activity in various systems
(Sumner et al., 1995;
Kane, 1995
;
Kane and Parra, 2000
;
Wieczorek et al., 2000
). To
examine whether this mode of regulation also occurs in blowfly salivary
glands, non-stimulated and 5-HT-stimulated glands were homogenized, a crude
membrane preparation was isolated by high-speed centrifugation, and the
relative distribution of the respective proteins between the pellet and the
cytosolic supernatant was probed on western blots.
In control tissue, only a minor fraction of the V1 subunits A
(24.4±13.4%) and E (41.7±7.6%) was detected within the pellet
(Fig. 5). More than 90% of the
V0 subunits a and d and essentially all
Na+/K+-ATPase -subunits were retained in the
pellet, demonstrating that membranes were almost entirely recovered in this
fraction. Upon 5-HT stimulation, the relative amount of V1 proteins
was significantly increased within the pellet fraction (subunit A,
56.6±8.3%; subunit E, 63.6±7.3%), whereas no change could be
detected in the relative distribution of the V0 subunits or of the
Na+/K+-ATPase
-subunit. To exclude the
possibility that cytosolic proteins were generally enriched in the pellet
after 5-HT stimulation, we also determined the relative distribution of
-tubulin between both fractions. Moreover, we analysed the total amount
of protein in the pellet and the supernatant on SDS gels stained with
Coomassie Blue. Neither method showed any significant difference between the
pellet and supernatant in response to 5-HT stimulation. We thus conclude that
5-HT stimulation leads to a specific recruitment of V1 subunits to
the membranes.
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Discussion |
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Further support for the concept that V-ATPase-driven H+
transport energizes the apical membrane for K+ extrusion into the
lumen of the salivary glands could be provided by in vivo experiments
with bafilomycin A1. Application of bafilomycin A1 at
concentrations as high as 10 µmol l1, however, has no
obvious effect on the 5-HT-induced transepithelial potential changes of
isolated salivary glands, the electrophysiological response accompanying
secretion (B. Zimmermann, unpublished results). We suggest that this results
from the limited accessibility of bafilomycin A1 to V-ATPase in
vivo. Since the drug was applied from the bath side and since the
paracellular pathway is occluded by septate junctions
(Skaer et al., 1975;
Zimmermann, 2000
), bafilomycin
A1 must cross the basolateral membrane and the cytoplasm in order
to reach its target site. Because of its high lipophilicity
(Dröse and Altendorf,
1997
), however, bafilomycin A1 may accumulate in the
basolateral membrane and become entrapped in this membrane domain. Similar
problems have been reported for other systems with externally applied
bafilomycin A1 (Dröse and
Altendorf, 1997
; Beyenbach et
al., 2000
; Boudko et al.,
2001
).
In conclusion, active K+ transport across the apical membrane of
blowfly secretory cells may occur by a mechanism similar to that in other
insect epithelia. In the goblet cells of the midgut of Manduca sexta,
the system that has been studied most thoroughly in this respect, V-ATPase
establishes an electrochemical gradient across the apical membrane that is
then used for K+ transport via a
K+/nH+ exchanger
(Wieczorek et al., 2000). The
combined action of both transporters, V-ATPase and
K+/nH+ exchanger, results in a net flux of
K+ from the cytosol into the midgut lumen. Future studies of the
blowfly salivary gland should determine whether this system also has a
K+/nH+ exchanger.
Regulation of V-ATPase
Several mechanisms have been proposed to play a role in the control of
V-ATPase activity (Stevens and Forgac,
1997; Wieczorek et al.,
2000
; Nishi and Forgac,
2002
): (1) the density of V-ATPase molecules within the plasma
membrane may be changed by shuttling pump molecules between an internal
vesicular pool and the cell surface; (2) disulphide bond formation between
cysteine residues in subunit A may lead to reversible inhibition of V-ATPase
activity; (3) the coupling efficiency of proton transport and ATPase activity
may change; (4) V-ATPase may be activated or inhibited by regulatory proteins;
and (5) the active V-ATPase holoenzyme may reversibly dissociate into its
inactive V0 and V1 components.
Our observations suggest that the first of the above mechanisms, namely changes in pump density within the plasma membrane via exo/endocytotic pathways, does not play a prominent role in the regulation of V-ATPase activity in blowfly salivary glands. Electron microscopy has not provided evidence of any 5-HT-induced changes in cell morphology indicative of the shuttling of pump molecules between a vesicular pool and the cell surface. Moreover, immunofluorescence microscopy has not visualized a redistribution of V-ATPase-immunoreactive vesicles within the secretory cells upon exposure to 5-HT. Concerning mechanisms 24, there is no evidence so far that supports or undermines their occurrence in salivary glands. The finding that 5-HT treatment stimulates a recruitment of V1 sector proteins to the membranes suggests, however, that mechanism 5, an assembly/disassembly of the V0V1 holoenzyme, might be used to control V-ATPase activity within the secretory cells. Since both the amount of membrane-associated V1 sector subunits and V-ATPase activity roughly double upon 5-HT stimulation, it may be further proclaimed that the assembly of the V-ATPase holoenzyme is directly linked to the activation of V-ATPase.
Attempts to observe 5-HT-induced assembly of V-ATPase by electron
microscopy or immunofluorescence microscopy have failed. The reason for this
may be that the 5-HT-dependent change in the state of V-ATPase assembly is not
an all-or-nothing effect. As deduced from the biochemical assay, the amount of
membrane-bound or cytosolic V1 sectors changes by a factor of about
two, and neither of the above methods may be sensitive enough to detect this
shift. Alternatively, unassembled V1 sectors may not be freely
diffusible and able to spread throughout the cytoplasm but may remain in the
vicinity of the apical membrane. V1 sectors could be spatially
restrained in their mobility by binding to actin filaments within the
microvilli or by interaction with other membrane-associated proteins. Studies
on osteoclasts have demonstrated that V-ATPase subunit B interacts with actin
filaments in vitro and in vivo
(Lee et al., 1999;
Holliday et al., 2000
).
Moreover, an isoform of V-ATPase subunit B has been shown to contain a
C-terminal PDZ-binding motif and to associate with the PDZ protein
Na+/H+ exchanger regulatory factor
(Breton et al., 2000
).
A reversible assembly/disassembly of the V-ATPase holoenzyme has been
demonstrated previously in two other systems. In goblet cells of
Manduca midgut, V-ATPase dissociates into its components during
moulting or starvation (Sumner et al.,
1995; Gräf et al.,
1996
). Similarly, a rapid dissociation of the V1 sector
from the V0 sector occurs on the yeast vacuole upon glucose
deprivation (Kane, 1995
;
Parra and Kane, 1998
). In both
cases, the intracellular messenger system that regulates the status of
V-ATPase assembly has remained elusive. For yeast, conventional second
messenger systems, such as the cAMP and the protein kinase C pathways, have
been excluded (Parra and Kane,
1998
). Recent studies on yeast have identified a protein complex
involved with the regulation of V-ATPase assembly
(Seol et al., 2001
); the final
link between the glucose level in the medium and the assembly of the V-ATPase
holoenzyme, however, is still missing.
For the blowfly salivary gland, it is well documented that the active
K+ transport mechanism is regulated via the cAMP pathway
(Berridge et al., 1976;
Berridge, 1977
). It may thus be
presumed that the assembly state and activity of V-ATPase are controlled by
cAMP and protein kinase A. We have started to examine this hypothesis but
preliminary results indicate that the modes of V-ATPase regulation are far
more complex than expected.
Function of Na+/K+-ATPase in blowfly salivary
gland
In addition to the apical V-ATPase, the plasma membrane of the secretory
cells contains another ion pump, Na+/K+-ATPase, that
resides on the basolateral membrane. Stimulation with 5-HT does not affect
Na+/K+-ATPase activity, suggesting that
Na+/K+-ATPase does not have a fundamental role in
secretion. This conclusion is further supported by pharmacological experiments
on isolated salivary glands, demonstrating that the rate of fluid secretion
and the ionic composition of the saliva during 5-HT stimulation do not change
after addition of ouabain (Berridge and
Schlue, 1978). Na+/K+-ATPase may thus be
required only for maintaining the electrochemical Na+ and
K+ gradient across the basolateral membrane in resting glands, as
suggested by Berridge and Schlue
(1978
).
Conclusions
Our results implicate a V-ATPase as the ion pump that provides the driving
force for K+ extrusion into the lumen of blowfly salivary glands.
The apical membrane of blowfly salivary gland cells houses a massive number of
V-ATPase molecules, and V-ATPase activity is stimulated by 5-HT. The results
of biochemical assays suggest further that the 5-HT-dependent regulation of
pump activity involves the assembly of V0 and V1 sectors
to the V0V1 holoenzyme. Since there is extensive
knowledge on second messenger pathways activated upon 5-HT stimulation,
blowfly salivary glands might provide an attractive model system for analysing
the detailed sequence of events that occur between the arrival of an external
stimulus and the activation of V-ATPase within animal cells.
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Acknowledgments |
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References |
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