BAFILOMYCIN A1 AT NANOMOLAR CONCENTRATIONS SATURABLY INHIBITS A PORTION OF TURTLE BLADDER ACIDIFICATION CURRENT
Department of Physiology, New York College of Osteopathic Medicine,
New York Institute of Technology, Old Westbury, Long Island, NY 11568-8000,
USA
*
Author for correspondence (e-mail:
syoumans{at}iris.nyit.edu
)
Accepted May 17, 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: turtle, Pseudemys scripta, urinary bladder, acid secretion, bafilomycin A1, vacuolar H+ ATPase, Sch-28080
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In every case tested to date, the antibiotic bafilomycin A1 acts
as a potent and, at suitable concentrations, specific inhibitor of eucaryotic
vacuolar H+-ATPases (Bowman et al.,
1988;
Dröse et al.,
1993
; White,
1994
;
Dröse and Altendorf,
1997
; Finbow and Harrison,
1997
; Stevens and Forgac,
1997
). The potency of the
inhibitor is such that inhibition seen at concentrations of 1 to a few nmol
l-1, or less, is often taken as indicative of the presence of a
vacuolar H+-ATPase (Dröse and
Altendorf, 1997
). In various
intact or fractionated eucaryotic systems in which molar units were reported,
and the criterion evaluated was inhibition of H+ transport or
H+-ATPase activity, 50%-inhibitory concentrations (IC50)
have ranged from a low of 0.2 nmol l-1 (Crider et al.,
1994
) to a high of 3 nmol
l-1 (Sundquist et al.,
1990
; Nanda et al.,
1992
), with essentially
complete inhibition occurring between 1 (Crider et al.,
1994
) and no more than 20 nmol
l-1 (Sundquist et al.,
1990
; Nanda et al.,
1992
).
In contrast, it has been reported that acid secretion by intact turtle
bladders is inhibited by bafilomycin, but a concentration of more than 100
nmol l-1 is needed to cause 50 % inhibition and 500 nmol
l-1 or more for essentially complete inhibition, which are levels
some 25- to 500-fold higher (Kohn et al.,
1993). The results have been
interpreted to mean that in turtle bladders, acid secretion is driven by a
single active transporter, a V-type H+-ATPase with unusually low
sensitivity to bafilomycin A1 (Kohn et al.,
1993
). However, the
concentration profile is important, for three reasons. First, such relative
insensitivity to bafilomycin, if confirmed, would be an unusual finding, and
might well define a new class or subclass of vacuolar ATPase. Second, it is
well-known that bafilomycin A1 at these higher concentrations is an
effective inhibitor of P-type ion-transporting ATPases, a group that includes
Na+/K+-ATPases, H+/K+-ATPases, the
H+-ATPase of Neurospora plasma membrane, and the CaATPase
of sarcoplasmic reticulum (Bowman et al.,
1988
;
Dröse et al.,
1993
;
Dröse and Altendorf,
1997
). Balifomycin only shows
specificity for vacuolar ATPases at suitably low concentrations (Bowman et
al., 1988
;
Dröse et al.,
1993
;
Dröse and Altendorf,
1997
), so an action observed
at higher concentrations must be evaluated with care. Consequently, the
possibility cannot be excluded that the inhibition seen with high bafilomycin
concentrations in the turtle bladder could have been due, at least in part, to
effects on other transport processes. Third, these findings, if true, would
seem to indicate that the turtle bladder has different active acid-secretory
mechanisms than the renal collecting duct, which is currently thought to
utilize a typical vacuolar H+-ATPase and one or more
H+/K+ exchange ATPases (Brown et al.,
1988
; Wingo and Smolka,
1995
; Caviston et al.,
1999
; DuBose et al.,
1999
; Jaisser and Beggah,
1999
).
In preliminary studies with turtle bladders, however, we noted substantial inhibition at low, subnanomolar concentrations. In view of the discrepancy of this observation with the published literature, and considering the importance of bafilomycin A1 as a characteristic inhibitor of V-type H+-ATPases, we decided to reinvestigate its action on intact turtle bladders. The purpose of the present study then was to determine: (i) the concentration dependence of bafilomycin's inhibitory effect on acid secretion by turtle bladders over a range known to be specific for other V-type H+-ATPases; and (ii) whether this range of concentrations inhibits none, a portion, or essentially all of such acid secretion.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Solutions
The luminal and serosal tissue surfaces in vitro were bathed in
identical reptilian Ringer's solution, which contained, in mmol
l-1: NaCl, 80; NaHCO3, 20; KCl, 3.5; MgSO4,
1.0; CaCl2, 1.8; Na2HPO4, 1.25; and glucose,
11. The final pH after equilibrating with 95 % O2:5 %
CO2 was 7.3-7.4. Inhibitor stock solutions were prepared as
follows: bafilomycin A1, 2 µg per 100 µl (32.1 µmol
l-1) in dimethyl sulphoxide (DMSO); Sch-28080, 100 mmol
l-1 in DMSO. Bafilomycin A1 concentrations were
determined photometrically by the method of Bowman et al. (Bowman et al.,
1988). The stocks were prepared
ahead of time and stored in portions at -20 °C (bafilomycin) or +4 °C
(Sch-28080). We found that stocks of either compound had to be used within 6-8
weeks. It was also found necessary to minimize the time during which working
samples of bafilomycin A1 were kept at temperatures of 0 °C or
higher during an experiment. The inhibitory potency deteriorated markedly
beyond a period of 4-5 h at ice temperature. The final DMSO vehicle
concentration in experiments never exceeded 0.3 %. In pilot studies, no
vehicle effects were seen up to at least that concentration.
Data analysis
In general, data are presented as the mean ± S.E.M. for the number
of experiments indicated. Figs
1 and
3 were prepared by scanning and
digitizing the appropriate chart recorder tracing and importing the files into
Sigma Plot. In the experiments in which the half-maximal inhibitory
concentration (IC50) was determined, IC50 was determined
for each individual experiment and is expressed here as the mean ±
S.E.M. The curve in Fig. 2 was
calculated from the mean ± S.E.M. for the response to bafilomycin
A1 at each indicated concentration, using Sigma Plot. Statistical
significance was determined, as appropriate, with a paired Student's
t-test.
|
|
|
Materials
Turtles were obtained from Kons Scientific, Germantown, WI, USA and
Carolina Biological, Burlington, NC, USA. Sch-28080 was a gift from Dr James
Kaminski of Schering-Plough. All other reagents were obtained from Sigma.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mucosal addition
In contrast, bafilomycin A1 added to the mucosal solution alone
did inhibit the acidification current. The results of one experiment are
depicted in Fig. 1B, which
shows the effects of a graded sequence of concentrations of bafilomycin.
Inhibition was pronounced at the lowest concentration tested, 0.1 nmol
l-1, and saturation was reached by 5-10 nmol l-1. The
concentration dependence observed in four such experiments is summarized by
the doseresponse curve in Fig.
2. When the half-maximal inhibition was determined for each
experiment individually, the average was 0.47±0.17 nmol l-1.
The inhibition again reached a plateau at 5-10 nmol l-1, with
65±4 % and 67±5 % of the original acid secretion inhibited and
35±4 and 33±5 % remaining, respectively. The H+
secretion remaining was significantly different from zero (P<0.03
at both 5 and 10 nmol l-1). No significant difference was found
between these two highest doses (P>0.1). In subsequent experiments
therefore, a concentration of 5nmol l-1 bafilomycin A1
was used, 10 times the IC50, a multiple used commonly in transport
or enzyme studies.
Effect of Sch-28080 on bafilomycin A1-insensitive acid
secretion
Bafilomycin A1 inhibited the majority of the acidification
current, but there was a portion that clearly was not affected, at
concentrations that are specific for V-type H+-ATPases in other
preparations. Fig. 3 shows an
experiment in which the bafilomycin-resistant component was tested for
sensitivity to a different inhibitor of transport-ATPases, Sch-28080. In this
representative experiment, acidification that was probably due to vacuolar
H+-ATPase activity was first inhibited by 5 nmol l-1
bafilomycin in the mucosal medium. The bolus addition of bafilomycin decreased
the magnitude of the short-circuit current in this experiment from -6.1 µA
to -2.1 µA, with a half-time (t1/2) of 4.1 min. A
stable plateau was reached within 50 min. At this point, 30 µmol
l-1 Sch-28080 was added to the mucosal solution. Sch-28080
inhibited the bafilomycin A1-resistant acidification current,
reducing it from -2.1 µA to +0.1 µA with a rapid onset,
t1/2=1.5 min. The current stabilized near zero within 15
min.
Fig. 4 summarizes the results of seven such experiments, here expressed as the percentage of total H+ transport (acidification current) that was inhibited by each agent. 5 nmol l-1 bafilomycin caused acid secretion to decline by 71±4 % (P<0.01), a decrement similar to that seen in the experiments in which the concentration dependence (dose-response) was determined. The acid secretion that remained at this point, in the presence of bafilomycin only, while reduced remained significantly different from zero (P<0.03), as before. This residual bafilomycin-resistant acid transport was inhibited completely by 30 µmol l-1 Sch-28080, which caused a decline of 29±4 % of the original acid secretion (P<0.01 versus null). In the presence of both 5 nmol l-1 bafilomycin and 30 µmol l-1 Sch-28080, the acidification current was not statistically distinguishable from zero (+0.21±0.11 µA, P>0.1). Thus, the results indicate that the two inhibitors, used at concentrations at which each is known in other systems to act specifically, together inhibit essentially all the acid secretion in these bladders. Further, the results are consistent with the concentrations used, 5 nmol l-1 bafilomycin and 30 µmol l-1 Sch-28080, being the maximally inhibiting level of each inhibitor for its respective transport process.
|
Reversal of the sequence of inhibitor addition
If bafilomycin A1 and Sch-28080 exert independent effects on two
different transport systems, then the order in which they are added should
have little or no effect on the degree of inhibition produced by each. To test
this, we ran a group of eight experiments, in each of which one hemibladder
was exposed on the mucosal side first to 5 nmol l-1 bafilomycin
A1 and subsequently to 30 µmol l-1 Sch-28080, while
the mated hemibladder (from the same animal) received the reverse sequence of
inhibitors. The results are summarized in
Table 1. When bafilomycin was
given first, it produced a decrement in the acidification current of
2.64±0.85 µA (initial baseline, -3.19±1.31 µA (per 1.43
cm2), which amounted to 69.9 % of the total effect that would
ultimately be produced by both inhibitors. Sch-28080 subsequently caused a
further decrement of 1.29±0.55 µA, the remaining 31±9 % of
the combined inhibitor effect. With both inhibitors present, the SCC
(acidification current) did not differ statistically from zero
(+0.74±0.94 µA, P>0.45; N=8), as before. In the
paired hemibladders, where Sch-28080 was given first, the SCC was decreased by
1.15±0.45 µA (baseline -6.10±2.86 µA (per 1.43
cm2), which was 28±8 % of the ultimate combined inhibitor
effects on the acidification current in this group of hemibladders.
Bafilomycin subsequently caused a further decrement of 2.73±0.70 µA,
the remaining 72±8 % of the combined inhibitor effect. Again, when both
inhibitors were present the acidification current did not differ significantly
from zero (-2.22±2.08 µA per 1.43 cm), P>0.3).
Table 2 shows that the order of
addition had no influence on the effect produced by bafilomycin, either in
terms of absolute decrement of the short circuit current (P>0.8,
bafilomycin added first versus added second) or the percentage
decrement (P>0.65 for addition first versus second).
Likewise, the order of addition had no effect on the decrement produced by
Sch-28080, either in terms of absolute current (P>0.7; Sch-28080
added first versus added second) or percentage decrement
(P>0.65 for addition first versus second). These results
indicate that the order of addition had no effect on the magnitude of
inhibition produced by either inhibitor. The results are consistent with the
hypothesis that bafilomycin A1 and Sch-28080 exert independent
effects on independent transport processes in this tissue.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results of the present experiments
We made use of this specificity in the present experiments. We find that
bafilomycin A1 inhibits with high sensitivity approximately 70 % of
the baseline acid secretion of intact turtle bladders, with inhibition
developing over the range of 0.1 to 10 nmol l-1, giving a sigmoidal
doseresponse curve, and exhibiting an IC50 of 0.47 nmol
l-1 (Fig. 2). This
is the first demonstration that acid secretion in intact turtle bladders is
inhibited by bafilomycin A1 at concentrations known to be specific
for V-type H+-ATPases in other tissues. The remaining 30 % of acid
secretion is more resistant to bafilomycin.
The apparent sigmoidal shape of the bafilomycin doseresponse curve
is not necessarily an expected result. The simplest hypothetical model would
be one in which the inhibitory ligand binds reversibly to a uniform population
of unhindered binding sites located, one each, on a uniform population of
protein molecules. If the binding is one to one, does not induce
oligomerization of the binding sites or protein molecules, and does not induce
secondary conformational states of the binding site with different affinities
for the receptor, then a hyperbolic doseresponse relationship would be
expected (Segel, 1975). The
fact that this is not seen in the present case appears to indicate that at
least one of these assumptions is violated. A sigmoidal curve clearly raises
the possibility of cooperativity in the binding of bafilomycin A1,
and hence the presence of multiple binding sites (Segel,
1975
). However, there are
other possible interpretations and with the information available it is not
feasible to distinguish between them.
Previous results with the turtle bladder
Our results differ from those of an earlier study, which reported that
turtle bladder acid secretion is inhibited by bafilomycin A1, but
only at far higher concentrations than we report here (Kohn et al.,
1993). The results led the
authors to conclude that acid secretion in the turtle bladder is driven by a
single active transport process, involving a vacuolar H+-ATPase
with unusually low sensitivity to bafilomycin A1. This is, to date,
a unique finding among eucaryotic cells, and the known features of bafilomycin
interaction with V-ATPases in other eucaryotic systems, discussed below, argue
for caution in interpreting the claim. It is not entirely clear why lower
bafilomycin sensitivity was found in the earlier study, but potential sources
of discrepancy can be identified. The concentrations of bafilomycin used in
that study, 50 nmol l-1 to >600 nmol l-1, were much
greater than those presently employed. The reported IC50 (approx.
100 nmol l-1) was 200-fold greater and the lowest concentration
used in the study, 50 nmol l-1, was tenfold higher than required to
produce saturation in our experiments. Hence, the earlier study used
concentrations considerably above the range where we observed a sigmoidal
doseresponse to bafilomycin. Since we find that the turtle bladder
displays both a highly bafilomycin-sensitive component of acid secretion and a
second, less-sensitive or insensitive, component, the lower overall tissue
sensitivity reported previously appears to be, at least in part, a composite
effect resulting from determining a single IC50 in the presence of
both high- and low-sensitivity transport systems. In addition, bafilomycin is
a somewhat labile compound and storage conditions can impinge on its
biological activity (Dröse et al.,
1993
; Farina and Gagliardi,
1999
; see also Materials and
methods). Finally, the possibility of unappreciated differences in the
metabolic states of the animals cannot be entirely ruled out as a contributing
factor.
Comparison to other systems
It is of interest to compare these two sets of findings in the turtle
bladder with what is known of bafilomycin's action in other systems. The
discovery by Bowman et al. (Bowman et al.,
1988) that bafilomycin
A1 inhibited vacuolar H+-ATPases at markedly low
concentrations suggested a specific interaction. However, they also reported
that the IC50 in molar units was strictly dependent on the amount
of protein present in homogenized preparations. This raised the possibility
that bafilomycin interacted nonspecifically with one or more proteins or that,
as a lipophilic compound, it partitioned extensively into the lipid phase of
membranes. Hanada et al. (Hanada et al.,
1990
) confirmed the dependence
on endogenous protein concentration but also reported that neither exogenously
added protein (albumin) nor lipid (phospholipids) had a detectable effect on
the IC50. Hanada et al. further determined, by kinetic analysis,
that the V-ATPase of chromaffin granules contained a single type of
bafilomycin interaction site, that it had high affinity for the inhibitor, and
that 50 % inhibition occurred at a ratio of 1 mole bafilomycin per mole of
enzyme (Hanada et al., 1990
).
Essentially 100 % inhibition was reached at 7-8 moles bafilomycin. From these
considerations, it can be concluded that bafilomycin interaction in these
studies occurred at a distinct binding site, and that the apparent dependence
on protein quantity was in fact a dependence on the number or density of that
binding site in the preparation.
In coated vesicles and in osteoclasts, the binding site has been localized
to V0, the membrane-bound portion of the V-ATPase assembly,
although there is controversy as to which peptide subunit or subunits of
V0 contain the site (Hanada et al.,
1990; Crider et al.,
1994
; Zhang et al.,
1994
; Mattsson and Keeling,
1996
; Forgac,
2000
). While the structure of
the binding site is not yet known, its specificity for bafilomycin has been
characterized in some detail. High-affinity binding has been shown to depend
on specific structural elements contained in a 16- to 18-member macrolactone
ring, which characterizes bafilomycin (16-member) and related macrolide
antibiotics, including the concanamycins (Dröse
et al., 1993
; Farina and
Gagliardi, 1999
; Gagliardi et
al., 1999
). Artificial
constructs have been prepared that contain these structures, but are otherwise
structurally unrelated to bafilomycin, and a number of these also inhibit
V-ATPases with high affinity (Farina and Gagliardi,
1999
; Gagliardi et al.,
1999
). Thus, the available
evidence indicates that bafilomycin interacts with eucaryotic vacuolar-ATPases
through a binding site that is characterized by a high affinity and high
specificity for the inhibitor.
Perhaps not surprisingly, there is a striking consistency in the threshold
concentrations that have been reported to produce inhibition. In a variety of
experimental preparations (intact tissue, suspended cells, subcellular
fractions or isolated molecules) in which a molar doseresponse to
bafilomycin was determined using inhibition of H+-ATPase activity
or H+ transport as the criterion, IC50 values ranging
only from 0.2 to 3 nmol l-1, with maximal inhibition achieved by 1
to no more than 20 nmol l-1, have been reported, (Bowman et al.,
1988; Moriyama and Nelson,
1989
; Moriyama and Futai,
1990
; Sundquist et al.,
1990
; Mattsson et al.,
1991
; Mattsson et al.,
1993
; Nanda et al.,
1992
; Armitage and Wingo,
1994
; Crider et al.,
1994
; Keeling et al.,
1997
). Exceptions to this
pattern of concentration dependence have been demonstrated only in procaryotes
(Chen and Konisky, 1993
;
Yokoyama et al., 1994
). Thus,
our present findings in the turtle bladder are consistent with results
reported for a variety of eucaryotic cells, in which the structural
requirements for bafilomycin's inhibitory effect on V-ATPases appear to be
highly conserved.
Comparison to results in the mammalian kidney
In the mammalian kidney, acid secretion in the cortical and outer medullary
collecting duct segments (CCD and OMCD, respectively) has also been shown to
be driven in part, but not wholly, by V-ATPase activity (Khadouri et al.,
1991; Armitage and Wingo,
1994
; Wingo and Smolka,
1995
; Tsuruoka and Schwartz,
1997
). Immunological and
biochemical evidence has demonstrated a vacuolar-ATPase in the intercalated
cells of the CCD and OMCD (Brown et al.,
1988
; Kim et al.,
1999
). Furthermore, a
vacuolar-ATPase has been shown to contribute to H+ secretion in
intact tubule segments from the OMCD and to be maximally inhibited by 5 nmol
l-1 luminal bafilomycin (Armitage and Wingo,
1994
; Tsuruoka and Schwartz,
1997
). Extensive evidence also
indicates that at least two H+/K+-ATPase isoforms are
present in the CCD and OMCD, that these contribute to acid secretion, and that
their activity is unaffected by nanomolar concentrations of bafilomycin (Wingo
et al., 1990
; Armitage and
Wingo, 1994
; Zhou and Wingo,
1994
; Tsuruoka and Schwartz,
1997
; Caviston et al.,
1999
; DuBose et al.,
1999
; Jaisser and Beggah,
1999
). The activity of
H+/K+-ATPase in the renal tubule is however inhibited by
Sch-28080, with apparently full inhibition reached at 10 µmol
l-1 (Armitage and Wingo,
1994
; Tsuruoka and Schwartz,
1997
). Furthermore, in intact
CCD and OMCD tubule segments, it has been reported that H+
secretion attributable to a V-ATPase and to an
H+/K+-ATPase or ATPases together account for all acid
secreted (Armitage and Wingo,
1994
; Tsuruoka and Schwartz,
1997
). The portion
attributable to bafilomycin-sensitive H+ transport has been
reported variously to be 30-65 % of the total in the OMCD with the remainder
being inhibitable by Sch-28080 (Armitage and Wingo,
1994
; Tsuruoka and Schwartz,
1997
). Thus, our findings
indicate that the turtle bladder resembles the CCD and OMCD of the mammalian
tubule in that all three tissues utilize two distinct active transport
mechanisms to secrete acid, one of which is very likely driven by a vacuolar
H+-ATPase. In the present experiments we find that
bafilomycin-sensitive H+ secretion amounts to approximately 70 % of
the baseline acidification current.
Bafilomycin-resistant H+ secretion in turtle bladder
The question arises as to what the remaining 30 %, the
bafilomycin-resistant component, is in turtle bladders. In principle it could
be due either to residual H+ transport by a V-ATPase or it could
arise from a different transport process. However, such a
bafilomycin-resistant `residual' is not a characteristic of V-type
H+-ATPases in other systems (Bowman et al.,
1988; Moriyama and Nelson,
1989
; Moriyama and Futai,
1990
; Sundquist et al.,
1990
; Mattsson et al.,
1991
; Mattsson et al.,
1993
; Nanda et al.,
1992
; Armitage and Wingo,
1994
; Crider et al.,
1994
; Keeling et al.,
1997
). It seems more likely
then that the bafilomycin-resistant component seen in the turtle bladder
represents a different transport process.
Work with isolated membranes also suggests this. Cell membranes isolated
from the epithelium of the turtle bladder display ATP-dependent H+
transport, and we have shown elsewhere that part of the acid transport is
highly sensitive to inhibition by vanadate ions, and is probably due to a
P-type ATPase (Youmans and Barry,
1991a; Youmans and Barry,
1991b
). The activity of this
transporter was found to be eliminated when the donor animals were
alkali-loaded, suggesting strongly that it is involved in or linked to urinary
acidification (Youmans and Barry,
1991b
). Furthermore, in a
sodium-free medium, the vanadate-sensitive transport of H+ was
found to depend absolutely on the presence of potassium and valinomycin. These
findings, taken together with the direction of H+ transport,
suggested that the P-type ATPase was in fact a K+/H+
exchange ATPase (Youmans and Barry,
1991a
).
Results obtained with isolated membranes thus raise the possibility that
the bafilomycin A1-resistant portion of acid secretion seen
presently with intact tissues could be due to an
H+/K+-exchange ATPase, as is the case in the renal
tubule. Hence, we chose to test the susceptibility of this transport to an
inhibitor of the gastric isoform of H+/K+-ATPase,
Sch-28080 (Scott et al., 1987;
Wallmark et al., 1987
; Briving
et al., 1988
). We found that
the residual H+ secretion remaining in the presence of 5 nmol
l-1 bafilomycin A1 was completely inhibited by Sch-28080
at a concentration of 30 µmol l-1
(Fig. 3 and
Table 1). In gastric
preparations, the IC50 for Sch-28080 ranges from 0.1 to 3.0 µmol
l-1 and the full inhibitory concentration from 3.0 to 30 µmol
l-1, depending on the preparation, pH and ambient K+
concentration (Scott et al.,
1987
; Wallmark et al.,
1987
; Briving et al.,
1988
). Sch-28080 at 30 µmol
l-1 thus is sufficient to maximally inhibit the gastric
H+/K+-ATPase under a variety of experimental conditions.
On the other hand, it is known that Sch-28080 does not inhibit vacuolar
H+-ATPases, including that of the renal collecting duct, at
concentrations up to at least 50-100 µmol l-1 (Cheval et al.,
1991
; Sabolic et al.,
1994
; Wingo and Smolka,
1995
). Hence, the effect that
we see at the lower concentration, 30 µmol l-1, is consistent
with essentially full inhibition of an H+/K+-ATPase.
When the sequence of inhibitor addition was reversed (i.e. Sch-28080 given
first), there was no change in either the absolute or percentage inhibition
caused by either inhibitor and the two together again reduced the
acidification current to zero (Table
1, Table 2). This
is consistent with the two inhibitors acting independently on separate
transport processes. We must point out that our findings with Sch-28080, while
consistent with, do not in themselves establish the presence, of an
H+/K+-ATPase in the intact turtle bladder. They do,
however, seem to argue strongly against a vacuolar H+-ATPase being
the source of bafilomycin-resistant acid secretion.
In summary, this is the first study to show: (i) that acid secretion by intact turtle bladders is highly sensitive to bafilomycin A1, at concentrations specific for vacuolar H+-ATPases in other systems; (ii) that a portion of the acid transport is unaffected by these low concentrations of bafilomycin; and (iii) that the bafilomycin A1-resistant portion is eliminated by Sch-28080 at a concentration similar to those which inhibit the gastric H+/K+-ATPase. Our results are consistent with the presence of two distinct acid-secretory processes in the intact turtle urinary bladder, one of which appears to be due to a `typical' vacuolar H+-ATPase. The nature of the second process, its presence previously unrecognized in this tissue, awaits further definition.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armitage, F. E. and Wingo, C. S. (1994).
Luminal acidification in K-replete OMCDi: contributions of H-K-ATPase and
bafilomycin-A1-sensitive H-ATPase. Am. J.
Physiol. 267,F450
-F458.
Bowman, E. J. and Bowman, B. J. (2000). Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A. J. Exp. Biol. 203,97 -106.[Abstract]
Bowman, E. J., Siebers, A. and Altendorf, K. (1988). Bafilomycins: a class of inhibitors of membrane-ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA 85,7972 -7976.[Abstract]
Briving, C., Andersson, B.-M., Nordberg, P. and Wallmark, B. (1988). Inhibition of gastric H+/K+-ATPase by substituted imidazo[1,2a]pyridines. Biochim. Biophys. Acta 946,185 -192.[Medline]
Brown, D., Hirsch, S. and Gluck, S. (1988). Localization of a proton-pumping-ATPase in rat kidney. J. Clin. Invest. 82,2114 -2126.[Medline]
Caviston, T. L., Campbell, W. G., Wingo, C. S. and Cain, B. D. (1999). Molecular identification of the renal H+,K+-ATPases. Semin. Nephrol. 19,431 -437.[Medline]
Chen, W. and Konisky, J. (1993). Characterization of a membrane-associated-ATPase from Methanococcus voltae, a methanogenic member of the Archaea. J. Bacteriol. 175,5677 -5682.[Abstract]
Cheval, L., Barlet-Bas, C., Khadouri, C., Feraille, E., Marsy,
S. and Doucet, A. (1991). K(+)-ATPase-mediated
Rb+ transport in rat collecting tubule: modulation during
K+ deprivation. Am. J. Physiol.
260,F800
-F805.
Crider, B. P., Xie, X. S. and Stone, D. K.
(1994). Bafilomycin inhibits proton flow through the
H+ channel of vacuolar proton pumps. J. Biol.
Chem. 269,17379
-17381.
Dröse, S. and Altendorf, K.
(1997). Bafilomycins and concanamycins as inhibitors of V-ATPases
and P-ATPases. J. Exp. Biol.
200, 1-8.
Dröse, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A. and Altendorf, K. (1993). Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32,3902 -3906.[Medline]
DuBose, T. D., Jr., Gitomer, J. and Codina, J. (1999). H+,K+-ATPase. Curr. Opin. Nephrol. Hypertens. 8,597 -602.[Medline]
Durham, J. H., Matons, C. and Brodsky, W. A.
(1987). Vasoactive intestinal peptide stimulates alkali excretion
in turtle urinary bladder. Am. J. Physiol.
252,C428
-C435.
Farina, C. and Gagliardi, S. (1999). Selective inhibitors of the osteoclast vacuolar proton-ATPase as novel bone antiresorptive agents. Drug Discov. Today 4, 163-172.[Medline]
Finbow, M. E. and Harrison, M. A. (1997). The vacuolar H+-ATPase: a universal proton pump of eukaryotes. Biochem. J. 324,697 -712.[Medline]
Forgac, M. (2000). Structure, mechanism and regulation of the clathrincoated vesicle and yeast vacuolar H+-ATPases. J. Exp. Biol. 203, 71-80.[Abstract]
Gagliardi, S., Rees, M. and Farina, C. (1999). Chemistry and structure activity relationships of bafilomycin A1, a potent and selective inhibitor of the vacuolar H+-ATPase. Curr. Med. Chem. 6,1197 -1212.[Medline]
Graber, M. L. and Devine, P. (1993). Omeprazole and SCH 28080 inhibit acid secretion by the turtle urinary bladder. Ren. Physiol. Biochem. 16,257 -267.[Medline]
Hanada, H., Moriyama, Y., Maeda, M. and Futai, M. (1990). Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1. Biochem. Biophys. Res. Commun. 170,873 -878.[Medline]
Jaisser, F. and Beggah, A. T. (1999). The nongastric H+-K+-ATPases: molecular and functional properties. Am. J. Physiol. 276,F812 -F824.[Medline]
Keeling, D. J., Herslöf, M., Ryberg, B., Sjögren, S. and Sölvell, L. (1997). Vacuolar H(+)-ATPases. Targets for drug discovery? Ann. NY Acad. Sci. 834,600 -608.[Medline]
Khadouri, C., Cheval, L., Marsy, S., Barlet-Bas, C. and Doucet, A. (1991). Characterization and control of proton-ATPase along the nephron. Kidney. Int. Suppl. 33,S71 -S78.[Medline]
Kim, J., Kim, Y. H., Cha, J. H., Tisher, C. C. and Madsen, K.
M. (1999). Intercalated cell subtypes in connecting tubule
and cortical collecting duct of rat and mouse. J. Am. Soc.
Nephrol. 10,1
-12.
Kniaz, D. and Arruda, J. A. (1991). Adaptation to metabolic alkalosis by the turtle urinary bladder. Proc. Soc. Exp. Biol. Med. 196,444 -450.[Abstract]
Kohn, O. F., Mitchell, P. P. and Steinmetz, P. R.
(1993). Sch-28080 inhibits bafilomycin-sensitive H+
secretion in turtle bladder independently of luminal [K+].
Am. J. Physiol. 265,F174
-F179.
Kohn, O. F., Hand, A. R., Mitchell, P. P. and Steinmetz, P.
R. (1997). Intra- and submembrane particle densities during
CO2 stimulation of H+ secretion in turtle bladder.
Am. J. Physiol. 272,F491
-F497.
Mattsson, J. P. and Keeling, D. J. (1996). [3H]Bafilomycin as a probe for the transmembrane proton channel of the osteoclast vacuolar H(+)-ATPase. Biochim. Biophys. Acta 1280,98 -106.[Medline]
Mattsson, J. P., Väänänen, K., Wallmark, B. and Lorentzon, P. (1991). Omeprazole and bafilomycin, two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H(+)-translocating-ATPases. Biochim. Biophys. Acta 1065,261 -268.[Medline]
Mattsson, J. P., Lorentzon, P., Wallmark, B. and Keeling, D. J. (1993). Characterization of proton transport in bone-derived membrane vesicles. Biochim. Biophys. Acta 1146,106 -112.[Medline]
Moriyama, Y. and Futai, M. (1990). Presence of
5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type
H(+)-ATPase in neurosecretory granules from bovine posterior
pituitary. J. Biol. Chem.
265,9165
-9169.
Moriyama, Y. and Nelson, N. (1989).
H+-translocating-ATPase in Golgi apparatus. Characterization as
vacuolar H+-ATPase and its subunit structures. J. Biol.
Chem. 264,18445
-18450.
Nanda, A., Gukovskaya, A., Tseng, J. and Grinstein, S.
(1992). Activation of vacuolar-type proton pumps by protein
kinase C. Role in neutrophil pH regulation. J. Biol.
Chem. 267,22740
-22746.
Nelson, N. and Harvey, W. R. (1999). Vacuolar
and plasma membrane proton-adenosinetriphosphatases. Physiol.
Rev. 79,361
-385.
Sabolic, I., Brown, D., Verbavatz, J.-M. and Kleinman, J.
(1994). H+-ATPases of renal cortical and medullary
endosomes are differentially sensitive to Sch-28080 and omeprazole.
Am. J. Physiol. 266,F868
-F877.
Scheffey, C., Shipley, A. M. and Durham, J. H.
(1991). Localization and regulation of acidbase secretory
currents from individual epithelial cells. Am. J.
Physiol. 261,F963
-F974.
Scott, C. K., Sundell, E. and Castrovilly, L. (1987). Studies on the mechanism of action of the gastric microsomal (H++K+)-ATPase inhibitors SCH 32651 and SCH 28080. Biochem. Pharmacol. 36, 97-104.[Medline]
Segel, I. H. (1975). Enzyme Kinetics. New York, Chichester, Weinheim, Brisbane, Singapore, Toronto: John Wiley & Sons.
Steinmetz, P. R., Omachi, R. S. and Frazier, H. S. (1967). Independence of hydrogen ion secretion and transport of other electrolytes in turtle bladder. J. Clin. Invest. 46,1541 -1548.[Medline]
Stetson, D. L. and Steinmetz, P. R. (1985). Alpha and beta types of carbonic anhydrase-rich cells in turtle bladder. Am. J. Physiol. 249,F553 -F565.[Medline]
Stevens, T. H. and Forgac, M. (1997). Structure, function and regulation of the vacuolar (H+)-ATPase. Annu. Rev. Cell. Dev. Biol. 13,779 -808.[Medline]
Sundquist, K., Lakkakorpi, P., Wallmark, B. and Väänänen, K. (1990). Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption. Biochem. Biophys. Res. Commun. 168,309 -313.[Medline]
Tsuruoka, S. and Schwartz, G. J. (1997).
Metabolic acidosis stimulates H+ secretion in the rabbit outer
medullary collecting duct (inner stripe) of the kidney. J. Clin.
Invest. 99,1420
-1431.
Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson,
R., Mendlein, J., Rabon, E. and Sachs, G. (1987). Inhibition
of gastric H+,K+-ATPase and acid secretion by SCH 28080,
a substituted pyridyl(1,2a)imidazole. J. Biol. Chem.
262,2077
-2084.
White, P. J. (1994). Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles. J. Exp. Bot. 45,1397 -1402.
Wieczorek, H., Brown, D., Grinstein, S., Ehrenfeld, J. and Harvey, W. R. (1999). Animal plasma membrane energization by proton-motive V-ATPases. BioEssays 21,637 -648.[Medline]
Wingo, C. S. and Smolka, A. J. (1995). Function
and structure of H-K-ATPase in the kidney. Am. J.
Physiol. 269,F1
-F16.
Wingo, C. S., Madsen, K. M., Smolka, A. and Tisher, C. C. (1990). H-K-ATPase immunoreactivity in cortical and outer medullary collecting duct. Kidney Int. 38,985 -990.[Medline]
Yokoyama, K., Akabane, Y., Ishii, N. and Yoshida, M.
(1994). Isolation of prokaryotic
V0V1ATPase from a thermophilic eubacterium
Thermus thermophilus. J. Biol. Chem.
269,12248
-12253.
Youmans, S. J. and Barry, C. R. (1989). ATP-dependent H+ transport by the turtle bladder: NBD-C1 preferentially inhibits the vanadate-insensitive component in isolated membranes. Biochem. Biophys. Res. Commun. 161,312 -319.[Medline]
Youmans, S. J. and Barry, C. R. (1991a). Effects of valinomycin on vanadate-sensitive and vanadate-resistant H+ transport in vesicles from turtle bladder epithelium: evidence for a K+/H+ exchanger. Biochem. Biophys. Res. Commun. 176,1285 -1290.[Medline]
Youmans, S. J. and Barry, C. R. (1991b). Physiological role for vanadate-inhibitable active H+ transport: a new model for distal urinary acidification. Biochem. Biophys. Res. Commun. 180,1505 -1512.[Medline]
Zhang, J., Feng, Y. and Forgac, M. (1994).
Proton conduction and bafilomycin binding by the V0 domain of the
coated vesicle V-ATPase. J. Biol. Chem.
269,23518
-23523.
Zhou, X. and Wingo, C. S. (1994). Stimulation
of total CO2 flux by 10 % CO2 in rabbit CCD: role of an
apical Sch-28080- and Ba-sensitive mechanism. Am. J.
Physiol. 267,F114
-F120.