Am J Physiol Cell Physiol Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 279: C1319-C1326, 2000;
0363-6143/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Codina, J.
Right arrow Articles by Dubose, T. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Codina, J.
Right arrow Articles by Dubose, T. D., Jr.
Vol. 279, Issue 5, C1319-C1326, November 2000

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-Na+-K+-ATPase (3, 23) but not on the gastric H+-K+-ATPase (HKalpha 1). In contrast, specific Sch-28080 binding sites have been identified on HKalpha 1 that are conspicuously absent in the alpha 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 HKalpha 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 HKalpha 1, both Northern and immunoblot analyses did not reveal changes in HKalpha 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 (HKalpha 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 HKalpha 1 in renal transport during respiratory acidosis and respiratory alkalosis (13). In that study, the ATPase activity of alpha 1-Na+-K+-ATPase was very similar to the level of activity of HKalpha 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 HKalpha 1 or HKalpha 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 HKalpha 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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (9K):
[in this window]
[in a new window]
  		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.

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.


View larger version (36K):
[in this window]
[in a new window]
  		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.

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.


View larger version (33K):
[in this window]
[in a new window]
  		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.

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.


View larger version (26K):
[in this window]
[in a new window]
  		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.

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.


View larger version (26K):
[in this window]
[in a new window]
  		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.

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).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6.   Sch-28080 depletes intracellular ATP in mIMCD-3 cells. The experiments were performed as described in MATERIALS AND METHODS.



View larger version (26K):
[in this window]
[in a new window]
  		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.



View larger version (33K):
[in this window]
[in a new window]
  		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.



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 9.   Intracellular ATP in DDT1MF-2 cells is not affected by Sch-28080. The experiment was performed as described in Fig. 7.

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 HKalpha 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 HKalpha 1 was defined as inhibition of pHi recovery after a NH4Cl load. In studies in isolated inner medullary collecting ducts perfused in vitro, HKalpha 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.


View larger version (22K):
[in this window]
[in a new window]
  		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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha -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 HKalpha 1 activity by binding directly to the alpha -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 HKalpha 1 activity. However, a parallel increase in HKalpha 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 HKalpha 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 alpha 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 HKalpha 2, not HKalpha 1, mRNA or protein was upregulated in the renal medulla by this condition. In addition, when expressed heterologously, HKalpha 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 HKalpha 2, such findings could then be reconciled. Moreover, it would be unnecessary to invocate the emergence of a Sch-28080-sensitive variant of HKalpha 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amlal, H, Nakamura S, Galla JH, and Soleimani A. Colonic H,K-ATPase replaces gastric H,K-ATPase in inner medullary collecting duct (IMCDt) in potassium depletion and mediates NH4+ secretion (Abstract). J Am Soc Nephrol 9: 2, 1998.

2.   Amlal, H, Wang Z, and Soleimani M. Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol 273: C1194-C1205, 1997[Abstract/Free Full Text].

3.   Asano, S, Matsuda S, Hoshina S, Sakamoto S, and Takeguchi N. A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH-28080. J Biol Chem 274: 6848-6854, 1999[Abstract/Free Full Text].

4.   Bengele, HH, Schwartz JH, McNamara ER, and Alexander EA. Chronic metabolic acidosis augments acidification along the inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 250: F690-F694, 1986.

5.   Buffin-Meyer, B, Younes-Ibrahim M, Barlet-Bas C, Cheval L, Marsy S, and Doucet A. K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct. Am J Physiol Renal Physiol 272: F124-F131, 1997[Abstract/Free Full Text].

6.   Campbell, WG, Weiner ID, Wingo CS, and Cain BD. H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line. Am J Physiol Renal Physiol 276: F237-F245, 1999[Abstract/Free Full Text].

7.   Canessa, CM, Horisberger JD, and Rossier BC. Mutation of a tyrosine in the H3-H4 ectodomain of Na,K-ATPase alpha  subunit confers ouabain resistance. J Biol Chem 268: 17722-17726, 1993[Abstract/Free Full Text].

8.   Cheval, L, Elalouf JM, and Doucet A. Re-evaluation of the expression of the gastric H,K-ATPase alpha  subunit along the rat nephron. Pflügers Arch 433: 539-541, 1997[Web of Science][Medline].

9.   Codina, J, Delmas-Mata JT, and DuBose TD, Jr. Expression of Halpha 2 protein is increased selectively in renal medulla by chronic hypokalemia. Am J Physiol Renal Physiol 275: F433-F440, 1998[Abstract/Free Full Text].

10.   Codina, J, Kone BC, Delmas-Mata JT, and DuBose TD, Jr. Functional expression of the colonic H+,K+-ATPase alpha -subunit. Pharmacologic properties and assembly with X+, K+-ATPase beta -subunits. J Biol Chem 271: 29759-29763, 1996[Abstract/Free Full Text].

11.   Codina, J, Pressley TA, and DuBose TD, Jr. The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon. J Biol Chem 274: 19693-19698, 1999[Abstract/Free Full Text].

12.   Cougnon, M, Planelles G, Crowson MS, Shull GE, Rossier BC, and Jaisser F. The rat distal colon P-ATPase alpha  subunit encodes a ouabain-sensitive H+, K+-ATPase. J Biol Chem 271: 7277-7280, 1996[Abstract/Free Full Text].

13.   Eiam-ong, S, Laski ME, Kurtzman NA, and Sabatini S. Effect of respiratory acidosis and respiratory alkalosis on renal transport enzymes. Am J Physiol Renal Fluid Electrolyte Physiol 267: F390-F399, 1994[Abstract/Free Full Text].

14.   Grishin, AV, Bevensee MO, Modyanov NN, Rajendran V, Boron WF, and Caplan MJ. Functional expression of the cDNA encoded by the human ATP1AL1 gene. Am J Physiol Renal Fluid Electrolyte Physiol 271: F539-F551, 1996[Abstract/Free Full Text].

15.   Guntupalli, J, Onuigbo M, Wall S, Alpern RJ, and DuBose TD, Jr. Adaptation to low-K+ media increases H+-K+-ATPase but not H+-ATPase-mediated pHi recovery in OMCD1 cells. Am J Physiol Cell Physiol 273: C558-C571, 1997[Abstract/Free Full Text].

16.   Kone, BC, and Higham SC. A novel N-terminal splice variant of the rat H+-K+-ATPase alpha 2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia. J Biol Chem 273: 2543-2552, 1998[Abstract/Free Full Text].

17.   Kraut, JA, Hiura J, Besancon M, Smolka A, Sachs G, and Scott D. Effect of hypokalemia on the abundance of HKalpha 1 and HKalpha 2 protein in the rat kidney. Am J Physiol Renal Physiol 272: F744-F750, 1997[Abstract/Free Full Text].

18.   Mendlein, J, and Sachs G. Interaction of a K+-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K+-ATPase. J Biol Chem 265: 5030-5036, 1990[Abstract/Free Full Text].

19.   Nakamura, S, Wang Z, Galla JH, and Soleimani M. K+ depletion increases HCO3- reabsorption in OMCD by activation of colonic H+-K+-ATPase. Am J Physiol Renal Physiol 274: F687-F692, 1998[Abstract/Free Full Text].

20.   Ono, S, Guntupalli J, and DuBose TD, Jr. Role of H+-K+-ATPase in pHi regulation in inner medullary collecting duct cells in culture. Am J Physiol Renal Fluid Electrolyte Physiol 270: F852-F861, 1996[Abstract/Free Full Text].

21.   Palasis, M, Kuntzweiler TA, Arguello JM, and Lingrel JB. Ouabain interactions with the H5-H6 hairpin of the Na,K-ATPase reveal a possible inhibition mechanism via the cation binding domain. J Biol Chem 271: 14176-14182, 1996[Abstract/Free Full Text].

22.   Pohl, SL, Krans HM, Kozyreff V, Birnbaumer L, and Rodbell M. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. VI. Evidence for a role of membrane lipids. J Biol Chem 246: 4447-4454, 1971[Abstract/Free Full Text].

23.   Price, EM, Rice DA, and Lingrel JB. Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha  subunit. J Biol Chem 265: 6638-6641, 1990[Abstract/Free Full Text].

24.   Sabolic, I, Brown D, Verbavatz JM, and Kleinman J. H+-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole. Am J Physiol Renal Fluid Electrolyte Physiol 266: F868-F877, 1994[Abstract/Free Full Text].

25.   Sangan, P, Rajendran VM, Mann AS, Kashgarian M, and Binder HJ. Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am J Physiol Cell Physiol 272: C685-C696, 1997[Abstract/Free Full Text].

26.   Schultheis, PJ, Wallick ET, and Lingrel JB. Kinetic analysis of ouabain binding to native and mutated forms of Na,K-ATPase and identification of a new region involved in cardiac glycoside interactions. J Biol Chem 268: 22686-22694, 1993[Abstract/Free Full Text].

27.   Wall, SM, and Koger LM. NH4+ transport mediated by Na+-K+-ATPase in rat inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 267: F660-F670, 1994[Abstract/Free Full Text].

28.   Wall, SM, Mehta P, and DuBose TD, Jr. Dietary K+ restriction upregulates total and Sch-28080-sensitive bicarbonate absorption in rat tIMCD. Am J Physiol Renal Physiol 275: F543-F549, 1998[Abstract/Free Full Text].

29.   Wall, SM, Trinh HN, and Woodward KE. Heterogeneity of NH4+ transport in mouse inner medullary collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 269: F536-F544, 1995[Abstract/Free Full Text].

30.   Wallmark, B, Briving C, Fryklund J, Munson K, Jackson R, Mendlein J, Rabon E, and Sachs G. Inhibition of gastric H+,K+-ATPase and acid secretion by SCH-28080, a substituted pyridyl(1,2a)imidazole. J Biol Chem 262: 2077-2084, 1987[Abstract/Free Full Text].

31.   Wang, D, Buja LM, and McMillin JB. Acetyl coenzyme A carboxylase activity in neonatal rat cardiac myocytes in culture: citrate dependence and effects of hypoxia. Arch Biochem Biophys 325: 249-255, 1996[Medline].

32.   Younes-Ibrahim, M, Barlet-Bas C, Buffin-Meyer B, Cheval L, Rajerison R, and Doucet A. Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1141-F1147, 1995[Abstract/Free Full Text].

Am J Physiol Cell Physiol 279(5):C1319-C1326
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Codina, J. Li, and T. D. DuBose Jr.
CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake
Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1279 - C1286.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
X. Xu, W. Zhang, and B. C. Kone
CREB trans-activates the murine H+-K+-ATPase {alpha}2-subunit gene
Am J Physiol Cell Physiol, October 1, 2004; 287(4): C903 - C911.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S.-L. Xia, L. Wang, M. N. Cash, X. Teng, R. A. Schwalbe, and C. S. Wingo
Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors
Am J Physiol Renal Physiol, August 1, 2004; 287(2): F204 - F214.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
A. K. Rajasekaran and S. A. Rajasekaran
Role of Na-K-ATPase in the assembly of tight junctions
Am J Physiol Renal Physiol, September 1, 2003; 285(3): F388 - F396.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. J. Bek, H. C. Reinhardt, K.-G. Fischer, J. R. Hirsch, C. Hupfer, E. Dayal, and H. Pavenstadt
Up-Regulation of Early Growth Response Gene-1 Via the CXCR3 Receptor Induces Reactive Oxygen Species and Inhibits Na+/K+-ATPase Activity in an Immortalized Human Proximal Tubule Cell Line
J. Immunol., January 15, 2003; 170(2): 931 - 940.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. E. FRANK and I. D. WEINER
Effects of Ammonia on Acid-Base Transport by the B-Type Intercalated Cell
J. Am. Soc. Nephrol., August 1, 2001; 12(8): 1607 - 1614.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Petrovic, Z. Spicer, T. Greeley, G. E Shull, and M. Soleimani
Novel Schering and ouabain-insensitive potassium-dependent proton secretion in the mouse cortical collecting duct
Am J Physiol Renal Physiol, January 1, 2002; 282(1): F133 - F143.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Codina, J.
Right arrow Articles by Dubose, T. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Codina, J.
Right arrow Articles by Dubose, T. D., Jr.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online