The V-type H+-ATPase in Malpighian tubules of Aedes aegypti: localization and activity
1 Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853,
USA
2 Department of Biology, University of Osnabrück, D-49019
Osnabrück, Germany
* Author for correspondence (e-mail: kwb1{at}cornell.edu)
Accepted 17 March 2003
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Summary |
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Key words: V-type H+-ATPase, Na+/K+-ATPase, Malpighian tubule, electrolyte secretion, Na+/H+ exchange, polyclonal antibody, immunohistochemistry, western blot
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Introduction |
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Bafilomycin A1, a specific inhibitor of the V-type
H+-ATPase, completely inhibits transepithelial NaCl and KCl
secretion, and with it fluid secretion, in Malpighian tubules of Aedes
aegypti (Beyenbach et al.,
2000). Simultaneously, both apical and basolateral membrane
voltages and the transepithelial voltage decrease to zero. These studies
strongly suggest the H+ paradigm of epithelial transport for
Malpighian tubules.
In the present study, we used an antibody specific to the B subunit of the V-type H+-ATPase to localize the proton pump to the apical brush border membrane of principal cells, but not stellate cells, of Malpighian tubules of the yellow fever mosquito. We also measured enzyme activities of the V-type H+-ATPase and the Na+/K+-ATPase. We found much activity of the former and little of the latter.
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Materials and methods |
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Protein content of Malpighian tubules
Total protein in Malpighian tubules of Aedes aegypti was
determined with a BioRad DC Protein Assay kit (BioRad, Hercules, CA,
USA). The method is similar to the well-documented Lowry assay, which is based
on the reaction of proteins with an alkaline copper tartrate solution and
Folin reagent (Lowry et al.,
1951). In a typical protein determination, we prepared crude
tubule extracts (CTE) as described above using 100 Malpighian tubules from
female mosquitoes only. The CTE volume was 100 µl. To 20 µl of this CTE
and to 20 µl of bovine serum albumin (BSA) standards, 100 µl of reagent
A (alkaline copper tartrate solution; BioRad) and then 800 µl of reagent B
(Folin reagent; BioRad) were added. After 15 min, the absorbance was read at
750 nm using a Beckman spectrophotometer (DU-65) against the BSA standards
series (ranging from 0.25 mg ml-1 to 1.5 mg ml-1).
SDSPAGE and western blot
SDSPAGE was performed as described previously
(Wieczorek et al., 1990). In
brief, 10 µl of sample buffer (5-fold strength) was added to 40 µl of
CTE such that final concentrations of sample buffer were 125 mmol
l-1 Tris-HCl, 5% sucrose, 2% SDS, 0.005% bromophenol blue and 2%
ß-mercaptoethanol in a final volume of 50 µl at pH 6.8. After boiling
for 3 min on a hotplate and then cooling on ice, 7 µl aliquots
(approximately 11 µg protein) were loaded on each lane of the gel (BioRad
Mini Protean 3 chamber, T 17%/C 0.4%). The electrophoresis was started with a
current of 20 mA. After the sample had entered the stacking gel, the current
was increased to 45 mA. The proteins were transferred to nitrocellulose
membranes by semidry-blotting (60 min, 1 mA cm-2) using a
three-buffer system according to Kyhse Andersen
(1984
), modified by the
addition of 20% methanol. SDSPAGE lanes in the nitrocellulose membrane
were cut from the western blot lanes and stained with Ponceau S (Sigma, St
Louis, MO, USA).
The western blot membrane (nitrocellulose) was incubated for 60 min in
blocking solution consisting of TBSNT (20 mmol l-1 Tris-HCl, pH
7.5, 500 mmol l-1 NaCl, 0.02% NaN3, 0.05% Tween)
fortified with 3% fish gelatine. The membrane was then treated for 60 min with
three different primary antibodies diluted in TBSNT plus 1% fish gelatine
(1:1000). The antibodies were: (1) Ab 353-2 against the V1 complex
of the V-type H+-ATPase (Huss,
2001), (2) Ab 488-1 against the C subunit of the V1
complex of the V-type H+-ATPase
(Merzendorfer et al., 2000
)
and (3) Ab C23 against the B subunit of the V1 complex of the
V-type H+-ATPase (Huss,
2001
). In each case, the antigen was isolated from Manduca
sexta in the Wieczorek laboratory. Antibodies (antisera) were prepared in
guinea pigs by Charles River (Sulzfeld, Germany).
The western blot membrane was washed with TBSNT in a shaking bath for 3x5 min. The secondary antibody (anti-guinea-pig alkaline phosphatase conjugated, Sigma A-5062) was added after dilution in TBSNT plus 1% fish gelatine (1:30 000). Sixty minutes later, the membrane was washed again with TBSNT for 3x5 min. After rinsing with double-distilled water (ddH2O), the membrane was treated with 10 ml substrate-solution consisting of 50 mmol l-1 Tris-HCl, pH 9.5, 100 mmol l-1 NaCl, 50 mmol l-1 MgCl2, 0.34 mg ml-1 nitro blue tetrazolium (NBT; Sigma) and 0.17 mg ml-1 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP; Sigma). When protein bands became visible after 210 min, the membrane was rinsed with ddH2O and dried at room temperature.
Immunohistochemistry
One hundred Malpighian tubules were removed from female mosquitoes as
described above and collected in approximately 1 ml of mosquito Ringer
solution (Yu and Beyenbach,
2001). The Ringer solution was aspirated and the tubules were
transferred for fixation to 5 ml of 10% formaldehyde buffered with 33.3 mmol
l-1 NaH2PO4 and 45.8 mmol l-1
Na2HPO4 at pH 7.27.4. After 2.5 h of fixation,
the tubules were transferred to a stainless steel embedding mold, dehydrated
in a series of ethanol concentrations, ranging from 30% to 100% at 10%
increments, and embedded in paraffin wax. Serial sections were cut to a
thickness of 4 µm. The sections were deparaffinized in xylene, rehydrated
in a series of ethanol (100%, 95%, 70%) and washed in phosphate-buffered
saline (PBS: 145 mmol l-1 NaCl, 3.2 mmol l-1
NaH2PO4, 7.2 mmol l-1
Na2HPO4, pH 7.27.4). The sections were then
treated with 0.5% H2O2 for 10 min to suppress endogenous
peroxidase activity.
Slides treated in conventional ways showed little staining with the
antibody C23. By contrast, pre-treating slides for 5 min in 0.1 mol
l-1 citric buffer at pH 6.0 and 8090°C (microwave)
markedly improved the localization of antibody. The method is known as
heat-induced antigen retrieval (HIAR) and is frequently used to increase the
`antigenicity' of the antigens in formalin-fixed and paraffin-embedded
sections (Shi et al., 2001).
Although the mechanism of action of HIAR is not clear, it is believed that the
procedure loosens or breaks cross-linkages of antigen and fixative, freeing
epitopes for binding to antibody (Shi et
al., 2001
).
Unspecific binding was blocked with 10% normal rabbit serum (Zymed, San Francisco, CA, USA) for 20 min before the slides were treated with primary antibody at 37°C for 2 h. The primary antibody, Ab C23, was the same polyclonal antibody used in western blot analysis (diluted 1:2000 in PBS). The secondary antibody, biotinylated rabbit anti-guinea-pig IgG (Zymed) was 50-fold diluted in PBS and applied at room temperature for 20 min. Immunoreactivity was visualized by incubating the sections in streptavidin/peroxidase solution (prediluted; Zymed histostain® kit; Zymed) for 15 min and then in aminoethyl carbazole (AEC) chromogen substrate solution (Zymed) for 2 min Finally, the sections were counterstained with hematoxylin stain Gill's Formation #2 (Fisher, Fair Lawn, NJ, USA) for 10 s at room temperature.
ATPase activity measurements
Total ATPase activity was measured spectrophotometrically as the oxidation
of NADH, which was coupled to ATP hydrolysis as described by Scharschmidt et
al. (1979). The activity of
the Na+/K+-ATPase was measured as the ouabain- or
vanadate-sensitive ATPase activity, and the V-type H+-ATPase
activity was measured as the bafilomycin- or nitrate-sensitive ATPase
activity, as described by Lin and Randall
(1993
).
To free ATPase, we lysed Malpighian tubules in hypo-osmotic lysis buffer (20 mmol l-1 Tris-HCl, 2 mmol l-1 EGTA, pH 7.1). In a typical experiment, 225 Malpighian tubules from 45 female mosquitoes were homogenized on ice with a Teflon-coated pestle in 130 µl of lysis buffer. After ultrasonication for 1 min, the tubule extract was divided into six aliquots of 20 µl each and stored at -20°C.
On the day of the assay, the reaction buffer (125 mmol l-1 Tris buffer, 1 mmol l-1 EGTA, 120 mmol l-1 NaCl, 12.5 mmol l-1 KCl, 5 mmol l-1 NaN3, 5 mmol l-1 MgCl2, 5 mmol l-1 ATP, 2.5 mmol l-1 phosphoenolpyruvate, with or without ATPase inhibitor) was preincubated with 0.125 mmol l-1 NADH (Sigma) and 10 units each of L-lactic dehydrogenase (LDH, type XI; Sigma) and pyruvate kinase (PK; Sigma) for 30 min at room temperature. The ATPase reaction was started by adding 20 µl of tubule extract in a cuvette. The closed cuvette was quickly inverted for mixing and inserted into the spectrophotometer (General purpose UV/VIS DU520; Beckman).
The oxidation of NADH was measured as a function of time at 340 nm, the
wavelength of NADH absorption. The linear portion of this function [the slope
optical density (OD) per hour] was divided by the NADH extinction coefficient
(6.22 OD mmol-1) and normalized to protein concentration to obtain
the ATPase activity in tubule extracts
(Scharschmidt et al.,
1979).
The V-type H+-ATPase was calculated as the bafilomycin- or
NO3--suppressible portion of the total ATPase activity,
while the Na+/K+-ATPase activity was determined as the
ouabain- or vanadate-suppressible portion. Bafilomycin was used at a
concentration of 0.025 mmol l-1, NO3- at 100
mmol l-1, ouabain (Sigma) at 1 mmol l-1, and vanadate at
0.1 mmol l-1 (Lin and Randall,
1993).
Statistical treatment of the data
ATPase activity data are presented as means ± S.E.M. The
paired Student's t-test was used for the significant difference
(P<0.05) between control and experimental groups.
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Results |
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SDSPAGE and western blot
SDSPAGE of the extract of Aedes Malpighian tubules reveals
protein bands that co-localize with proteins of the V-type
H+-ATPase purified from the tobacco hornworm Manduca
sexta, suggesting that the tubule extract contains proteins of the V-type
H+-ATPase (Fig. 1A).
Western blot analysis confirms the presence of the V-type H+-ATPase
in Aedes Malpighian tubules (Fig.
1B). The mixture of antibodies (Ab 353-2) raised against proteins
of the cytoplasmic V1 complex identified more than 15 proteins in
the purified V-type H+-ATPase of Manduca sexta (lane 1,
Fig. 1B). Five protein bands,
with molecular masses of 16 kDa, 27 kDa, 43kDa, 56 kDa and 67 kDa, stained
prominently (lane 1, Fig. 1B).
Three of these proteins, with molecular masses of 16 kDa, 56 kDa and 67 kDa,
were also recognized by the antibody mixture in the extract of Aedes
Malpighian tubules (lane 2). The 67 kDa protein is most likely the A subunit
of the V1 complex (Wieczorek et
al., 1999b).
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Ab 488-1 is a polyclonal antibody raised against the C subunit of the V1 complex. The antibody clearly recognized subunit C in both the holoenzyme from Manduca sexta (lane 3) and the crude extract of Aedes Malpighian tubules (lane 4, Fig. 1B). Likewise, the polyclonal antibody Ab C23, raised against the B subunit of the V1 complex, identified this subunit in the holoenzyme of Manduca (lane 5, Fig. 1B) and in the crude extract of Aedes Malpighian tubules (lane 6, Fig. 1B).
Immunolabeling performed with preimmuno-serum (control) exhibited no labeling (not shown). Thus, Fig. 1 confirms the presence of the V-type H+-ATPase in Malpighian tubules of Aedes aegypti on the basis of antibodies raised against proteins of the V-type H+-ATPase purified from Manduca sexta.
Immunohistochemistry
Fig. 2 illustrates two
sequential microtome sections from the same paraffin block of Malpighian
tubules of Aedes aegypti. The two sections received the same
experimental treatment, including heat-induced antigen retrieval, exposure to
secondary antibody and staining with hematoxylin. The only difference is the
additional exposure of the section shown in
Fig. 2B to Ab C23, the primary
antibody specific to the B subunit of the V-type H+-ATPase. The
section shown in Fig. 2A was
exposed to the preimmuno-serum.
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Tubules appear in a mix of longitudinal and oblique sections
(Fig. 2). The tubule lumen
occasionally comes into view as the unstained clear space enclosed by the
apical brush border of mostly principal cells. The single principal cell of
the tubule reveals a large nucleus (18 µm diameter) when the cut has passed
through the center. Principal cells are further characterized by a tall dense
brush border. Most cells of the tubule are principal cells (86%); the
remainder are stellate cells (Satmary and
Bradley, 1984).
The obvious difference between the sections shown in Fig. 2A and Fig. 2B is the positive stain of the antibody specific to the V-type H+-ATPase in Fig. 2B. The antibody C23 recognized the B subunit of the V-type H+-ATPase most prominently in the brush border of principal cells, which is consistent with a high density of this proton pump at the apical membrane. Light staining of the cytoplasm of principal cells suggests the presence of the V-type H+-ATPase (or parts thereof containing the B subunit) associated with cytoplasmic structures. However, the antibody did not stain stellate cells, neither plasma membrane nor cytoplasm.
ATPase activities
Fig. 3 illustrates the
enzyme activities of the V-type H+-ATPase and the
Na+/K+-ATPase in extracts of Aedes Malpighian
tubules. Total ATPase activity was 3.14±0.35 µmol
h-1mg-1 protein in 10 control determinations. The
bafilomycin A1-sensitive ATPase activity was 1.58±0.30
µmol h-1 mg-1 protein in 10 determinations, which is
significantly different from zero (P=2.5x10-4). The
KNO3-sensitive ATPase activity was 1.88±0.20 µmol
h-1mg-1 protein in seven determinations, which is also
significantly different from zero (P=4.8x10-5). The
two V-type H+-ATPase activities measured with two different pump
inhibitors, bafilomycin A1 and NO3-, were not
significantly different, consistent with the complete inhibition of the V-type
H+-ATPase.
|
Inhibitors of the Na+/K+-ATPase had no significant effect on the total ATPase activity. The ouabain-sensitive ATPase activity was 0.26±0.12 µmol h-1 mg-1 protein in 10 determinations, which is not significantly different from zero. Likewise, the nonsensical negative ATPase activity (0.61±0.70 µmol h-1 mg-1 protein) measured in the presence of vanadate is not significantly different from zero. Again, the use of two different inhibitors, ouabain and vanadate, yielded similar activities of the Na+/K+-ATPase, consistent with complete inhibition.
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Discussion |
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The two polyclonal antibodies, C23 and 488-1, raised against the B and C
subunits, respectively, of the proton pump in the midgut of Manduca
sexta, identified their intended targets in the purified V-type
H+-ATPase and in crude extract of Aedes Malpighian tubules
(Fig. 1B, lanes 36),
thereby confirming the presence of the two subunits and hence the presence of
the V-type H+-ATPase in Malpighian tubules of the yellow fever
mosquito. Other laboratories have successfully used antibodies raised against
various subunits of the V-type H+-ATPase to identify this proton
pump in Malpighian tubules of the ant (Formica polyctena), moth
(Heliothis virescens) and locust (Locusta migratoria)
(Garayoa et al., 1995;
Lezaun et al., 1994
;
Pietrantonio and Gill,
1995
).
Localization of the V-type H+-ATPase in the brush border
membrane of principal cells
The V-type H+-ATPase was first identified as an enzyme
associated with endosomal membranes of lysosomes, clathrin-coated vesicles and
vacuoles of yeast and plants (Nelson,
1992; Stevens and Forgac,
1997
). In endosomal membranes, the V1 complex faces the
cytoplasm and the V0 complex points into the endosomal compartment.
Thus, protons are moved from the cytoplasm to the endosomal compartment,
raising endosomal H+ concentrations above cytoplasmic
concentrations and generating an endosomal membrane potential that is positive
inside. Both H+ and voltage gradients serve a variety of functions.
Voltage may drive the entry of Cl- into the endosome, acidifying
the endosomal compartment with HCl, when the endosomal membrane houses
Cl- channels next to the V-type H+-ATPase
(Marshansky and Vinay, 1996
).
The presence of malate channels serves electrogenic uptake of malate ions
(Pantoja and Smith, 2002
).
In epithelial membranes of animal cells, the V-type H+-ATPase is
often located at the apical side, where again voltage and H+
gradients can serve activities ranging from signal transduction
(Camello et al., 2000) to
nutrient uptake (Zhuang et al.,
1999
) and electrolyte transport
(Wieczorek et al., 1999a
). The
present study shows that the V-type H+-ATPase is densely expressed
in the brush border of principal cells of Malpighian tubules of Aedes
aegypti (Fig. 2). The
brush border is also densely populated by mitochondria
(Beyenbach, 2001
). Virtually
every microvillus is home to a mitochondrion
(Beyenbach, 2001
). The close
spatial relationship between ATP synthesis (mitochondria) and ATP utilization
(the V-type H+-ATPase) suggests a close temporal relationship
between metabolism and transepithelial transport
(Fig. 4A). Indeed, the
inhibition of ATP synthesis by dinitrophenol depolarizes the apical membrane
voltage from 111 mV to 9 mV within 1 min, consistent with the rapid inhibition
of transepithelial transport (Pannabecker
et al., 1992
). More recent studies confirm that intracellular ATP
concentration and electrogenesis by the V-type H+-ATPase in the
apical, microvillar plasma membrane are intimately coupled
(Wu and Beyenbach, 2003
).
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In Aedes Malpighian tubules, the V-type H+-ATPase
energizes transport not only across the apical membrane but also across the
epithelial shunt and across the basolateral membrane on the other side of the
cell (Beyenbach, 2001). As
illustrated in Fig. 4, the
transport of protons from the microvillar cytoplasm to the extracellular space
of the brush border constitutes a pump current that must return to the
cytoplasmic side of the V-type H+-ATPase
(Beyenbach, 2001
). Current
passing through the epithelial shunt is carried by Cl- passing from
hemolymph to tubule lumen as the mechanism of transepithelial Cl-
secretion (Masia et al.,
2000
). Current passing from the hemolymph into principal cells is
carried by K+ as the major mechanism of secretory K+
entry into the cell (Beyenbach and Masia,
2002
).
Although we expected to find the V-type H+-ATPase at the apical
membrane, we cannot be sure about the immunohistochemical evidence for the
presence of this proton pump in the cytoplasm of principal cells
(Fig. 2). The staining of the
cytoplasm may reflect antibody binding to parts of the holoenzyme such as the
V1 complex. The reversible dissociation of the V1
complex from the holoenzyme is known as a mechanism for regulating the
transport activity of the V-type H+-ATPase
(Wieczorek et al., 1999a). But
this dissociation should leave the V1 complex in close proximity to
the site of its dissociation from the holoenzyme, namely in microvilli of the
brush border. It should not yield the diffuse distribution of the B subunit in
the cytoplasm. For these reasons, we believe that the cytoplasmic signal
reflects various aspects of holoenzyme synthesis, sorting and trafficking,
although the association of the V-type H+-ATPase with endosomal
membranes of principal cells cannot be excluded.
The V-type H+-ATPase is absent from stellate cells in Malpighian
tubules of Aedes aegypti (Fig.
2). Stellate cells in Malpighian tubules of Drosophila
melanogaster do not express the V-type H+-ATPase according to
enhancer trapping studies in Dow's lab
(Sözen et al., 1997).
Moreover, stellate cells are thought to provide the transepithelial
Cl- shunt pathway in Drosophila Malpighian tubules
(O'Donnell et al., 1998
). Such
a transport role also fits our finding of Cl- channels in apical
membrane patches of Aedes Malpighian tubules
(O'Connor and Beyenbach,
2001
). Mediating passive transepithelial Cl- transport,
stellate cells may not need the V-type H+-ATPase.
ATPase activities
Between 50% and 60% of the total ATPase activity in extracts of
Aedes Malpighian tubules can be attributed to a nitrate- and
bafilomycin-sensitive component that reflects the activity of the V-type
H+-ATPase. The remaining ATPase activity is probably due to protein
kinases, nucleotide cyclases, myosin, DNA helicases and other ATP-consuming
processes. Bafilomycin A1 is known to inhibit the free V-type
H+-ATPase with an I50 of 0.4 nmol
mg-1 protein (Bowman et al.,
1988). Our use of a concentration 6000 times (2500 nmol
mg-1) that much should have completely inhibited the V-type
H+-ATPase in the tubule extracts used in the present study.
Consistent with the complete inhibition by bafilomycin is a similar V-type
H+-ATPase activity measured in Malpighian tubules with a maximal
dose of nitrate (Fig. 3).
Nitrate inhibits the free V-type H+-ATPase with an
I50 of 50 mmol l-1
(Dschida and Bowman, 1995
).
Bafilomycin is thought to block the proton channel of the V-type
H+-ATPase (Zhang et al.,
1994a). By contrast, there are two mechanisms for inhibiting the
V-type H+-ATPase by nitrate: (1) via oxidation of the
cystine residue on the A subunit, with the effect of preventing ATP
hydrolysis, and (2) via dissociation of the V1 complex
from the V0 complex (Dschida and
Bowman, 1995
).
Even though the V-type H+-ATPase activity was measured in a
crude extract of Malpighian tubules, the activity most probably reflects the
activity of the intact proton pump, i.e. the holoenzyme. The cytosolic
V1 complex is capable of hydrolyzing ATP in a
Ca2+-dependent manner, but this hydrolysis is blocked by
Mg2+ concentrations as low as 0.1 mmol l-1
(Gräf et al., 1996). The
Mg2+ concentration in our ATPase assays was 5 mmol l-1,
which should have inhibited any ATP hydrolysis by dissociated V1
complexes. When the V1 complex is attached to the V0
complex and in membrane-bound form, the hydrolysis of ATP is
Mg2+-rather than Ca2+-dependent
(Gräf et al., 1996
). It
is believed that the hydrophobic environment of the membrane changes the
conformation of the V1 complex, thereby switching the metal
requirement to Mg2+ (Gräf
et al., 1996
). Accordingly, the presence of 5 mmol l-1
Mg2+ in our ATP assay should have maximized V-type
H+-ATPase activity of the holoenzyme in apical membrane fragments
but inhibited ATP hydrolysis by the dissociated V1 complexes.
In the present study, we measured no detectable ouabain- or
vanadate-sensitive Na+/K+-ATPase activity in crude
extracts of Aedes Malpighian tubules
(Fig. 3). Likewise, we observed
no immediate effects of 1 mmol l-1 ouabain on the transepithelial
voltage and resistance of isolated Aedes Malpighian tubules
(Williams and Beyenbach,
1984). By contrast, significant effects of ouabain, such as the
partial (approximately 50%) inhibition of transepithelial fluid secretion,
were observed after ouabain treatment for more than 30 min
(Hegarty et al., 1991
). In
view of the strong expression of the V-type H+-ATPase detected by
immunostaining (Fig. 2) and by
biochemical (Fig. 3) and
electrophysiological (Beyenbach,
2001
; Wu and Beyenbach,
2003
) assays, it appears that the V-type H+-ATPase
first and foremost serves epithelial transport mechanisms, and that the
Na+/K+-ATPase, if it is present, may perhaps serve cell
housekeeping functions. Accordingly, ouabain will have no immediate effect on
transepithelial transport but it may compromise transepithelial transport
after normal cell housekeeping functions have been impaired. Further
experiments using a more sensitive assay for the
Na+/K+-ATPase activity, or immunostaining methods, may
help to reveal the presence of the Na+/K+ pump in
Malpighian tubules of Aedes aegypti.
Kinetics and thermodynamics of transport across the apical
membrane
Fig. 4 illustrates the
present model of transepithelial NaCl and KCl secretion by Malpighian tubules
of the yellow fever mosquito dependent on the central role of the V-type
H+-ATPase located in the apical membrane of principal cells. Since
proton-translocating ATPases can transport as many as three protons per ATP
molecule hydrolyzed (Tomashek and
Brusilow, 2000), the hydrolysis of 1.7 µmol ATP h-1
mg-1 protein is equivalent to H+ transport across the
apical membrane of the tubule at a rate of 5.1 µmol h-1
mg-1 protein. A single Malpighian tubule of the yellow fever
mosquito contains approximately 0.77 µg of protein. Hence, a proton
transport rate of 65 pmol min-1 can be estimated for the whole
tubule under control conditions. The estimated proton transport rate comes
close to 74 pmol min-1, the rate of transepithelial secretion of
Na+ and K+
(Beyenbach, 2001
). The
approximation suggests electroneutral Na+/H+ and
K+/H+ exchange transport across the apical membrane with
a stoichiometry of 1:1.
Since electroneutral Na+/H+ and
K+/H+ cannot be driven by voltage, the question arises
of whether the pH difference across the apical membrane has the thermodynamic
strength to drive Na+ and K+ from cell to lumen in
exchange for H+. A proton transport rate of 65 pmol
min-1 across the apical membrane is expected to decrease the pH in
the tubule lumen to values less than 1 in the time of only 1 min, providing a
driving force far greater than needed. However, measurements of the pH in the
tubule lumen of Malpighian tubules are invariably close to 7, and measures of
cytoplasmic pH are not that far off
(Bertram and Wessing, 1994;
Petzel et al., 1999
;
Zhang et al., 1994b
). What
then is the minimum pH difference across the apical membrane that is needed to
drive Na+/H+ and K+/H+ exchange
transport in Aedes Malpighian tubules? The answer to this question
requires knowledge of cytoplasmic cation concentrations in principal
cells.
In Malpighian tubules of ants, the intracellular K+
concentration (67 mmol l-1) is near electrochemical equilibrium
with the K+ concentration in the hemolymph
(Leyssens et al., 1994). The
equilibrium distribution of K+ probably also holds true in
Malpighian tubules of Aedes aegypti in view of a K+
conductance as large as 64% of the basolateral membrane conductance
(Beyenbach and Masia, 2002
).
Accordingly, in the presence of a peritubular K+ concentration of
3.4 mmol l-1 and a basolateral membrane voltage of -58 mV, the
intracellular K+ concentration can be estimated as 31.5 mmol
l-1 under control conditions, when the K+ concentration
in secreted (luminal) fluid is 91 mmol l-1
(Beyenbach, 2001
). Thus, the
movement of K+ from 31.5 mmol l-1 in the cell to 91 mmol
l-1 in the lumen requires a nearly 3-fold H+
concentration difference, or a pH difference of 0.5 across the apical
membrane. If the intracellular pH is 7.2, then a lumen pH of 6.7 would be
sufficient to drive K+/H+ exchange across the apical
membrane with a transport stoichiometry of 1:1.
Since fluid secreted into the tubule lumen is not sufficiently acidic to
drive cation/H+ antiport, the pH of an unstirred layer in the brush
border might be low enough, as in amphibian skin
(Larsen et al., 1996). In
addition, large negative surface charges of the apical membrane due to fixed
negative charges of glycosylated proteins, glycolipids and adsorbed proteins
may support a surface pH considerably lower than that in the aqueous solution
of the tubule lumen (Aronson and Giebisch,
1997
). Finally, the glycocalyx of the brush border may limit the
diffusion of H+, generating an acid microenvironment akin to that
in the intestinal brush border (Shimada,
1987
). These considerations of the kinetics and thermodynamics of
transport suggest, but do not prove, that the transport of
K+/H+ and Na+/H+ across the apical
membrane of principal cells of the Aedes Malpighian tubule can be
electroneutral, as previously suggested for Malpighian tubules of the ant
Formica (Leyssens et al.,
1993
; Zhang et al.,
1994b
). Additional experiments are needed to resolve the
mechanism, stoichiometry and regulation of cation/H+ exchange
across the apical membrane.
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Acknowledgments |
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References |
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