Sch-28080 depletes intracellular ATP
selectively in mIMCD-3 cells
Juan
Codina,
Joseph
Cardwell,
Jeremy J.
Gitomer,
Yan
Cui,
Bruce C.
Kone, and
Thomas D.
Dubose Jr.
Department of Internal Medicine, University of Kansas School of
Medicine, Kansas City, Kansas 66160-7350
 |
ABSTRACT |
Two H+-K+-ATPase isoforms are present
in kidney: the gastric, highly sensitive to Sch-28080, and the colonic,
partially sensitive to ouabain. Upregulation of Sch-28080-sensitive
H+-K+-ATPase, or "gastric"
H+-K+-ATPase, has been demonstrated in
hypokalemic rat inner medullary collecting duct cells (IMCDs).
Nevertheless, only colonic H+-K+-ATPase mRNA
and protein abundance increase in this condition. This study was
designed to determine whether Sch-28080 inhibits transporters other
than the gastric H+-K+-ATPase. In the presence
of bumetanide, Sch-28080 (200 µM) and ouabain (2 mM) inhibited
86Rb+ uptake (>90%). That
86Rb+ uptake was almost completely abolished by
Sch-28080 indicates an effect of this agent on the
Na+-K+-ATPase. ATPase assays in membranes, or
lysed cells, demonstrated sensitivity to ouabain but not Sch-28080.
Thus the inhibitory effect of Sch-28080 was dependent on cell
integrity. 86Rb+-uptake studies without
bumetanide demonstrated that ouabain inhibited activity by only
50%. Addition of Sch-28080 (200 µM) blocked all residual
activity. Intracellular ATP declined after Sch-28080 (200 µM) but
recovered after removal of this agent. In conclusion, high
concentrations of Sch-28080 inhibit K+-ATPase activity in
mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.
ouabain; inner medullary collecting duct; adenosine
5'-triphosphatase
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INTRODUCTION |
INHIBITION BY
OUABAIN of ATPase activity is a widely accepted marker of
Na+ pump activity in vitro. Conversely, inhibition by
Sch-28080 has been used to designate
H+-K+-ATPase activity (21, 30).
Specific binding sites for ouabain have been identified on the
1-Na+-K+-ATPase (3,
23) but not on the gastric H+-K+-ATPase
(HK
1). In contrast, specific Sch-28080 binding sites have been identified on HK
1 that are
conspicuously absent in the
1-Na+-K+-ATPase
(3). On the basis of such observations, ouabain and Sch-28080 have been widely used to delineate which
X+-K+-ATPase is responsible for either
K+ absorption or H+ secretion by the distal
nephron. Accordingly, by convention, functions that are blocked by
Sch-28080 have been assumed to be mediated by HK
1
(15, 19, 28). Nevertheless, this assumption has been
challenged in several experimental models. Chronic dietary K+ depletion increased the fraction of bicarbonate
absorption
(JtCO2) sensitive
to Sch-28080 in rat isolated perfused collecting duct segments
(19, 28). Whereas this increase in
JtCO2 could be assumed to be the result of
upregulation of HK
1, both Northern and immunoblot
analyses did not reveal changes in HK
1 mRNA or protein
abundance in rat renal medulla during chronic hypokalemia (9,
17). Rather, with hypokalemia, several groups have detected a
selective increase in abundance of colonic
H+-K+-ATPase (HK
2) mRNA and
protein that was site specific for the medullary collecting tubule
(9, 17, 25).
High concentrations of Sch-28080 (~100 µM) have been used to
delineate the role of HK
1 in renal transport during
respiratory acidosis and respiratory alkalosis (13). In
that study, the ATPase activity of
1-Na+-K+-ATPase was very similar
to the level of activity of HK
1, defined as
"Sch-28080-sensitive ATPase activity." High concentrations of
Sch-28080 (>100 µM) have also been used to identify three unique types (type I, type II, and type III) of K+-ATPase activity
(5, 32). Nevertheless, designation of HK
1 or HK
2 as the functional equivalent of any of these
ATPase activities has not been possible (8). Moreover,
Sabolic et al. (24) reported that high concentrations of
Sch-28080 and omeprazole (100 µM) inhibit the H+-ATPase
nonselectively. This transporter is not involved in K+
homeostasis but is regulated in response to metabolic acidosis (2, 4).
The purpose of this study was to evaluate the specificity of Sch-28080
by determining whether Sch-28080 inhibits K+ transporters
other than HK
1. Our data demonstrate that both Sch-28080
at high concentrations (200 µM) and ouabain (2 mM) block Na+ pump activity in an established renal inner medullary
cell line, mouse inner medullary collecting duct cells
(mIMCD-3), in culture. Moreover, we demonstrate, for the first
time, that in contrast to the direct inhibitory effect of ouabain on
the
-subunit of the Na+ pump, the inhibitory effect of
high concentrations of Sch-28080 was the result of depletion of
intracellular ATP.
 |
MATERIALS AND METHODS |
Reagents.
Dulbecco's modified Eagle's medium (DMEM), cat. no. D-8900; newborn
calf serum, cat. no. N-4637; gentamicin, cat. no. G-1272; Ham's F-12,
cat. no. N-3520; and soybean trypsin inhibitor, cat. no. T-9003, were
purchased from Sigma (St. Louis, MO). Twenty-four-well dishes were
purchased from Corning (Corning, NY; cat. no. 25820-24) or Nunc
(Nalge Nunc, Naperville, IL; cat no. 150628). Trypsin-EDTA was
purchased from Life Technologies (Gaithersburg, MD; cat. no. 25300-062). Sch-28080 (a gift from Dr. J. Kaminski at
Schering-Plough Research Institute) was dissolved at 50 mM in DMSO.
DDT1MF-2 and BEAS-2B cells were gifts from Dr. R. B. Clark at the University of Texas Health Science Center at Houston.
Plasma membranes and ATPases assays were performed as described
previously (11, 22).
Cell culture and 86Rb+ uptake.
mIMCD-3, mouse outer medullary collecting duct (mOMCD1),
human embryonic kidney (HEK-293), and DDT1MF-2 cells were
grown in the presence of DMEM supplemented with newborn calf serum
(10%) and gentamicin (50 µg/ml) and were adjusted to pH 7.4 by
addition of NaHCO3 (7.5%), as described previously by our
laboratory (15, 20). BEAS-2B cells were grown in the
presence of Ham's F-12 containing gentamicin and serum at the
concentrations described above. Cells were grown to confluency at
37°C in a humidified environment in 24-well dishes. Before the assay,
the cells were washed four times (1.5 ml/cell) with buffer A
(145 mM NaCl, 1 mM KCl, 1.2 mM MgSO4, 2 mM
Na2HPO4, 1 mM CaCl2, 200 µM
bumetanide, and 32 mM HEPES, pH 7.4) at 37°C and then calibrated for
15 min with the same buffer. The buffer was removed and replaced with fresh buffer A that contained either ouabain or Sch-28080 as
appropriate at different concentrations (see figure legends). After 15 min, the solution was aspirated and replaced by 250 µl of the
corresponding solution containing 86Rb+
(3-8 × 106 counts/min). The reaction was allowed
to proceed for 15 min at 37°C. The buffer was aspirated and washed
five times with 1.5 ml of buffer B (100 mM MgCl2
and 10 mM HEPES, pH 7.4) at 4°C. Cells were dissolved by addition of
400 µl of buffer C (0.1 M NaOH and 2% SDS) at 65°C for
30 min. Resuspended cells (400 µl) were used to determine
86Rb+ uptake (16, 27). When
experiments were performed using HEK-293 cells, Nunc dishes replaced
Corning dishes to facilitate cell adherence.
ATPase assays in cell lysates.
Cells were grown to confluency in 10-cm dishes, washed with saline,
lifted by scraping, and centrifuged at 3,000 rpm for 5 min at 4°C in
a top table centrifuge (Biofuge 17R). The cells were resuspended in
buffer D (5 mM Tris · HCl, pH 8.0, 1 mM EDTA-Tris, 100 µM phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml
soybean trypsin inhibitor) and were homogenized by passing the
suspension five to six times through a 28-gauge needle. The ATPase
assay was performed for 30 min at 37°C, as described previously by
our laboratory, in an excess concentration of ATP (11).
ATP assay.
ATP levels in the cells were determined using bioluminescence as
described by Wang et al. (31). Cells were grown to near confluency in 24-well dishes and incubated as described in the 86Rb+-uptake experiments, except the
86Rb+ was omitted from the incubation medium.
Somatic cell ATP-releasing agent (500 µl; Sigma, cat. no. FL-ASC) was
added to each well and swirled. Different dilutions of the sample were
performed with somatic cell ATP-releasing agent to ensure linearity of
the assay. The amount of light emitted was measured immediately using a
TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). A standard curve
was constructed using known concentrations of ATP over the linear range
of the assay (0-10 nM).
 |
RESULTS |
Sch-28080 and ouabain block 86Rb+ uptake in
mIMCD-3 cells in culture.
The results of a representative 86Rb+-uptake
experiment are displayed in Fig.
1. Figure 1A
demonstrates that ouabain inhibited 86Rb+
uptake in a dose-dependent manner (IC50 ~30 µM).
These results are consistent with the well-known inhibitory effect of
ouabain on the renal Na+ pump. Because our experiments were
performed in the presence of bumetanide (200 µM), our findings, in
agreement with previously published data (14, 16, 29),
substantiate that the Na+-K+-ATPase and the
Na+-K+-2Cl
cotransporter are the
major pathways for K+ entry to the cell. Figure
1B demonstrates that Sch-28080 at concentrations >10 µM
also inhibited 86Rb+ uptake in a similar
dose-dependent manner (IC50 ~ 60 µM). Because Sch-28080 (200 µM) inhibited
86Rb+ uptake (>90%), it seems
reasonable to conclude that Sch-28080 acted by blocking the
Na+-K+-ATPase.

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Fig. 1.
86Rb+ uptake by mouse inner
medullary collecting duct (mIMCD-3) cells. A: inhibitory
effect of ouabain (plus bumetanide). B: inhibitory effect of
Sch-28080 (plus bumetanide). Both ouabain and Sch-28080 inhibited
86Rb+ uptake (>90%). These experiments were
performed in the presence of 1 mM KCl, 145 mM NaCl, and 200 µM
bumetanide.
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To test whether the effects of either ouabain or Sch-28080 (to inhibit
K+-ATPase in mIMCD-3 cells) were reversible, we
used the 86Rb+-uptake assay during the
application of, and after removal of, either ouabain or Sch-28080. In
Fig. 2 (left), mIMCD-3 cells
were incubated with ouabain (2 mM) and 86Rb+
uptake was blocked dramatically (as shown in Fig. 1). Removal of
ouabain for 15 min at 37°C reestablished
86Rb+ uptake. Figure 2 (right)
demonstrates similarly that the inhibitory effect of Sch-28080 (200 µM) was also reversible.

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Fig. 2.
The inhibitory effect of ouabain and Sch-28080 on
86Rb+ uptake is reversible in mIMCD-3 cells.
Reversibility of ouabain (left) and Sch-28080
(right) on 86Rb+ uptake. All
experiments were performed as described in Fig. 1 and in the presence
of bumetanide. In the recovery experiment, after incubation for 30 min
with ouabain (2 mM) or Sch-28080 (200 µM), the inhibitors were
removed, incubation continued for an additional 15 min,
86Rb+ was added, and incubation continued for
an additional 15 min. The reaction was then stopped and
86Rb+ uptake was measured.
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We prepared plasma membranes, as described previously by our laboratory
(11, 22), and performed the experiment described in Fig.
3 to determine whether the
Na+ pump of mIMCD-3 cells displayed a predictable pattern
of response to either ouabain or Sch-28080. ATPase activity was
measured in the presence of ATP 1) under basal conditions
(no K+ or Na+ added), 2) in the
presence of 5 mM K+, 3) in the presence of 50 mM
Na+, or 4) in the presence of 5 mM
K+ and 50 mM Na+. The studies were performed in
the presence or absence of either 1 mM ouabain or 200 µM Sch-28080. A
representative experiment is displayed in Fig. 3. Basal activity was
not modified by addition of 5 mM K+ or 50 mM
Na+ to the assay. Basal ATPase activity and activity in the
presence of K+ or Na+ alone was not sensitive
to either ouabain or Sch-28080. However, addition of both
K+ and Na+ to the assay induced an increase in
ATP hydrolysis (Na+-K+-ATPase) that was
sensitive to 2 mM ouabain but insensitive to 200 µM Sch-28080. This
finding demonstrates that the mIMCD-3
Na+-K+-ATPase in broken cell preparations is
sensitive to high concentrations of ouabain but totally insensitive to
Sch-28080. However, in the 86Rb+-uptake
experiments in intact cells described above, a clear inhibitory effect
by 200 µM Sch-28080 on the Na+ pump (inhibition by
>90%) was observed.

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Fig. 3.
ATPase activity in plasma membranes prepared from mIMCD-3
cells. Plasma membranes were prepared as described previously
(11). The ATPase assay was performed in the absence (None)
or presence of 10 mM KCl, 50 mM NaCl, or 10 mM KCl + 50 mM NaCl.
The assay was performed in the absence (control) or in the presence of
2 mM ouabain or 200 µM Sch-28080.
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The ATPase assay was performed in the presence of 50 mM NaCl with or
without addition of KCl (10 mM; Fig. 4)
in mIMCD-3 cells that were lysed as described in MATERIALS AND
METHODS. The results demonstrate that on homogenization,
2 mM ouabain blocked ATPase (Na+-K+-ATPase)
activity in both groups, consistent with a direct effect of ouabain on
the Na+ pump. In contrast, addition of 200 µM Sch-28080
did not inhibit ATPase activity (in any group). The results from Figs.
1, 3, and 4 confirm that the effect of Sch-28080 on the Na+
pump was nonspecific. Moreover, the inhibitory effect of Sch-28080 on
the Na+ pump was dependent on cell integrity.

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Fig. 4.
ATPase activity in whole homogenates prepared from
mIMCD-3 cells. mIMCD-3 cells were homogenized as described in
MATERIALS AND METHODS. ATPase assay was performed in the
presence of 50 mM NaCl, in the absence or in the presence of 10 mM KCl.
The experiment was performed in the absence (control) or in the
presence of 2 mM ouabain or 200 µM Sch-28080.
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Next, we investigated whether Sch-28080 blocked only the
Na+ pump or if it blocked additional mechanisms of
K+ entry into cells. These studies were performed by
deleting bumetanide from the 86Rb+-uptake
experiments. A representative experiment is displayed in Fig.
5. Ouabain (2 mM) inhibited
86Rb+ uptake by 50-60% when bumetanide
was not present (solid bar). Addition of 200 µM Sch-28080 inhibited
86Rb+ uptake (hatched bar) by >90%. Addition
of 2 mM ouabain plus 200 µM Sch-28080 did not alter the inhibitory
effect of Sch-28080. These data, taken together, demonstrate that the
inhibitory effect of Sch-28080 is not specific for the Na+
pump but, rather, extends by a common mechanism to additional K+ transporters in mIMCD-3 cells in culture.

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Fig. 5.
Sch-28080 blocks K+ transporters in mIMCD-3
cells in addition to the Na+ pump. In the absence of
bumetanide, 200 µM Sch-28080 induced a greater inhibitory effect than
2 mM ouabain alone on 86Rb+ uptake. The
experiments were performed as described in MATERIALS AND
METHODS except that bumetanide was not added.
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ATP is required for active transport by cells and for the activity of
the Na+ pump. As displayed in Fig.
6, we measured total intracellular ATP
content in control and in mIMCD-3 cells treated with 2 mM ouabain or
200 µM Sch-28080. Ouabain alone (solid bar) did not alter
intracellular ATP content. However, incubation of cells with Sch-28080
caused a dramatic reduction in total ATP content. Removal of the
Sch-28080 from the bathing solution for 15 min at 37°C reestablished
both intracellular ATP (Fig. 7) and
86Rb+ uptake (see Fig. 2). In keeping with this
observation in mIMCD-3 cells, we have also observed inhibition of
86Rb+ uptake by Sch-28080 in mOMCD1
cells and in HEK-293 cells (data not shown). Nevertheless,
the inhibitory effect of Sch-28080 on 86Rb+
uptake described in these cell lines did not extend to all cell lines
studied. For example, 200 µM Sch-28080 did not inhibit
86Rb+ uptake (in the absence or presence of
bumetanide) in DDT1MF-2 cells (Fig.
8), a hamster smooth muscle cell line;
BEAS-2B cells, a human bronchial cell line; or in oocytes from
Xenopus laevis (data not shown). In addition, 200 µM
Sch-28080 did not decrease the content of ATP in DDT1MF-2
cells in culture (Fig. 9).

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Fig. 6.
Sch-28080 depletes intracellular ATP in mIMCD-3 cells.
The experiments were performed as described in MATERIALS AND
METHODS.
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Fig. 7.
ATP depletion by Sch-28080 is reversible in mIMCD-3
cells. In the control group, the cells were incubated with buffer
throughout. In the group treated with Sch-28080, cells were incubated
for 30 min with buffer and then for 30 min with 200 µM Sch-28080. In
the third group (recovery period), the cells were incubated for 30 min
with 200 µM Sch-28080 and then with buffer without Sch-28080 for 30 min. Placement of the 24-well dish on ice (see MATERIALS
AND METHODS) stopped the assay.
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Fig. 8.
Sch-28080 does not inhibit 86Rb+
uptake in DDT1MF-2 cells. The experiment was performed as
described in Fig. 1 and in the presence or absence of 200 µM
bumetanide.
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Fig. 9.
Intracellular ATP in DDT1MF-2 cells is not
affected by Sch-28080. The experiment was performed as described in
Fig. 7.
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Several laboratories, including our own (6, 15, 19, 20,
28), have employed low concentrations of Sch-28080 (10 µM) in
studies in medullary collecting duct cells in culture, or in inner
medullary collecting ducts perfused in vitro, to define the role of
HK
1 in pHi recovery and K+
absorption during either chronic hypokalemia or metabolic acidosis. In
experiments in inner and outer medullary collecting duct cells in
culture, the activity of HK
1 was defined as inhibition
of pHi recovery after a NH4Cl load. In studies
in isolated inner medullary collecting ducts perfused in vitro,
HK
1 activity was defined as Sch-28080-inhibitable
JtCO2. To simulate the effect of prolonged
exposure of cells in culture or in isolated tubules perfused in vitro,
we conducted the experiment displayed in Fig. 10 (left). In this study,
mIMCD-3 cells in culture were incubated for an extended period (45 min)
with either low (10 µM) or high concentrations (200 µM) of
Sch-28080. Preincubation with high concentrations of Sch-28080, as
demonstrated previously in Figs. 1, 2, and 5, resulted in a marked
reduction in 86Rb+ uptake (>90%). In
contrast, preincubation with low concentrations of Sch-28080 for 45 min
resulted in a reduction of 86Rb+ uptake of only
20%. The data displayed in Fig. 10 (right) reveal that high
concentrations of Sch-28080 (200 µM) decreased intracellular ATP
concentration ([ATP]i) by >90%. This finding
is in agreement with the data displayed in Figs. 6 and 7. In contrast,
preincubation with low concentrations of Sch-28080 (10 µM) decreased
[ATP]i by only 20%. Although the reductions in
86Rb+ uptake and in ATP concentrations were
significant, the observed decrease in these parameters with low
concentrations of Sch-28080 was significantly less marked than that
seen with prolonged exposure to higher concentrations.

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Fig. 10.
Low concentrations of Sch-28080 (10 µM) decrease
86Rb+ uptake and intracellular ATP with
prolonged incubation. Experiments were performed as described in Figs.
1, 2, and 5; however, the preincubation time was extended from 15 to 45 min at 37°C. The decrease in both 86Rb+
uptake and intracellular ATP was significant but much less dramatic
than the reduction achieved with higher concentrations (see Figs. 1, 2,
and 5).
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 |
DISCUSSION |
Our results demonstrate that not only ouabain but also Sch-28080
inhibits Na+-K+-ATPase-mediated
86Rb+ uptake in mIMCD-3 cells in culture (Fig.
1). The mechanism of inhibition by ouabain and Sch-28080 differ,
however. The inhibitory effect of ouabain was observed in intact cells
(Fig. 1), membrane preparations (Fig. 3), and cell lysates (Fig. 4).
These results are in agreement with the demonstration that the
1-Na+-K+-ATPase contains a
binding site for ouabain (7, 26). In contrast, Sch-28080
inhibited 86Rb+ uptake only in intact mIMCD-3
cells (Fig. 1). Moreover, this inhibitory effect on
K+-ATPase activity disappeared after cellular
homogenization when assays were performed in the presence of exogenous
ATP (Figs. 3 and 4). This observation suggests an "indirect" effect
by Sch-28080 on the Na+ pump through intracellular ATP
depletion. In addition, this observation is compatible with the absence
of a specific binding site for Sch-28080 on any of the known
-Na+-K+-ATPase subunits (18,
30). Furthermore, our data demonstrate that the inhibitory
effect of Sch-28080 on 86Rb+ uptake is mediated
by intracellular ATP depletion (Figs. 6 and 7). This interpretation is
in agreement with the observation that Sch-28080 does not decrease
Na+-dependent K+-ATPase (Na+ pump)
activity in cell lysates or membrane preparations.
It is interesting to note, however, that Sch-28080 did not block
86Rb+ uptake or affect ATP content in all cell
lines. Indeed, an effect of Sch-28080 was not demonstrated in
DDT1MF-2 or BEAS-2B cells or in oocytes from X. laevis. A possible explanation for such a selective effect of
Sch-28080 may be differences in cell or mitochondrial membrane
permeability to the agent. Namely, if Sch-28080 does not enter the cell
or mitochondria, it cannot decrease the intracellular ATP content and,
therefore, an effect on 86Rb+ uptake would not
be observed.
Our findings also reveal that ouabain decreased
86Rb+ uptake by 50% in mIMCD-3 cells (Fig. 5).
Nevertheless, on addition of bumetanide to the assay, this degree of
inhibition increased to almost 100% (Figs. 1 and 2). In contrast,
Sch-28080 reduced 86Rb+ uptake by >90% in the
presence or absence of bumetanide. It has been demonstrated previously
that low concentrations of Sch-28080 (<10 µM) inhibit
HK
1 activity by binding directly to the
-subunit (18, 30). In addition, however, Sabolic et al.
(24) have reported that Sch-28080 and omeprazole (100 µM) inhibit H+-ATPase activity in renal cortical and
medullary endosomes in the presence of ATP (1.5 mM). Our data
demonstrate that Sch-28080, in high concentrations (200 µM),
decreases the intracellular concentration of ATP. The predicted
sequalae of intracellular ATP depletion would be to limit activity of
the Na+ pump, which is entirely dependent on
[ATP]i. Depletion of [ATP]i may also
contribute to a decrease in activity of the
Na+-K+-2Cl
cotransporter by
increasing the intracellular Na+ concentration
and diminishing Na+ entry. Nevertheless, our data cannot
exclude a direct effect of Sch-28080 on the
Na+-K+-2Cl
cotransporter.
Total JtCO2 is increased by chronic hypokalemia
in collecting ducts perfused in vitro. This increase is inhibited by
low concentrations (~10 µM) of Sch-28080 (19, 28). In
addition, Campbell et al. (6) demonstrated that low
concentrations of Sch-28080 impaired intracellular pH recovery in
RCCT-28A cells after a NH4+ load. These results have
been interpreted as evidence for a direct effect of Sch-28080 on
HK
1 activity. However, a parallel increase in
HK
1 mRNA and protein during chronic hypokalemia has not
been observed (9, 17). On the basis of results from the
present study, an indirect effect of Sch-28080 on collecting duct
JtCO2 in chronic hypokalemia should be
considered a possibility in these experiments. In this regard, our
findings (Fig. 1) demonstrate that Sch-28080 at low concentrations
(~10 µM) does not block 86Rb+ uptake in
mIMCD-3. However, by extending the preincubation time from 15 to 45 min, low concentrations of Sch-28080 (10 µM) inhibited 86Rb+ uptake by 20% (Fig. 10). We do not know
if our observation using the 86Rb+-uptake assay
can be extrapolated to
JtCO2 or
pHi recovery experiments, where exposure of cells or
tubules to Sch-28080 extends to periods of at least 45 min. On the
basis of results obtained with prolonged incubation at low
concentrations of this agent (Fig. 10), it seems logical to speculate
that a portion of the inhibition attributed to a "specific" effect
of Sch-28080 on HK
1 in kidney might represent, in part,
a nonspecific response, attributable to a decrease in intracellular
ATP. Because we have not examined the effect of Sch-28080 on ATP
content in cells of stomach or colon origin, we concede that these
observations may be pertinent only to renal cell lines.
In summary, our data demonstrate that Sch-28080 inhibits
1-Na+-K+-ATPase activity in
mIMCD-3 cells by depletion of intracellular ATP. This nonspecific
effect by an agent widely assumed to be a specific inhibitor of the
gastric H+-K+-ATPase (30) now
requires reconsideration, which takes into account the concentration of
Sch-28080, as well as the setting and cell type in which these
observations have been made. With this view in mind, we can now offer a
possible explanation for the disparity among results obtained from in
vitro perfusion studies in the rat outer medullary collecting duct or
inner medullary collecting duct and in established mouse cell lines
from the same region of the nephron vs. results obtained in
heterologous expression systems (10, 12, 19, 28). In
isolated tubules and in cells in culture, the increase in
JtCO2 or the pHi recovery rate
induced by chronic hypokalemia has been reported uniformly to be
Sch-28080 sensitive (19, 28). Nevertheless, only
HK
2, not HK
1, mRNA or protein was
upregulated in the renal medulla by this condition. In addition, when
expressed heterologously, HK
2 has been shown uniformly
to be insensitive to Sch-28080. Accordingly, if the "effect" of
Sch-28080 observed in intact tubules or in these renal cell lines was
the result of a nonspecific effect of Sch-28080 on the
Na+-K+-ATPase or HK
2, such
findings could then be reconciled. Moreover, it would be unnecessary to
invocate the emergence of a Sch-28080-sensitive variant of
HK
2 (1).
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grant DK-30603 (to T. D. DuBose)
and an individual National Research Service Award (to J. J. Gitomer).
 |
FOOTNOTES |
B. C. Kone is an Established Investigator of the American Heart
Association and recipient of Grant DK-47981. J. Cardwell, an
undergraduate from the Univ. of Wyoming, was a participant in the
Summer Research Student Program of the Univ. of Texas Health Science
Center at Houston during the course of this study.
Address for reprint requests and other correspondence: T. D. DuBose, Jr., Dept. of Internal Medicine, Univ. of Kansas School of
Medicine, 3901 Rainbow Blvd., 4035 Delp, Kansas City, KS 66160-7350 (E-mail: tdubose{at}kumc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 March 2000; accepted in final form 19 May 2000.
 |
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