AJP - Renal Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 275: F487-F501, 1998;
0363-6127/98 $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 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 Google Scholar
Google Scholar
Right arrow Articles by Amlal, H.
Right arrow Articles by Soleimani, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amlal, H.
Right arrow Articles by Soleimani, M.
Vol. 275, Issue 4, F487-F501, October 1998

Activation of H+-ATPase by hypotonicity: a novel regulatory mechanism for H+ secretion in IMCD cells

Hassane Amlal, Akhil Goel, and Manoocher Soleimani

Department of Medicine, University of Cincinnati School of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effect of hypotonicity on H+-ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H+-ATPase activity measured as the Na+- and K+-independent intracellular pH (pHi) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH2O) increased pHi recovery by ~350% (P < 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H+-ATPase) and was not dependent on external K+, indicating lack of involvement of H+-K+-ATPase. H+-ATPase activation was acute, independent of cell calcium, and was not secondary to Cl- channel activation. The magnitude of H+-ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH2O. H+-ATPase upregulation in hypotonicity was significantly blocked in the presence of staurosporine or calphostin C or in cells pretreated with phorbol 12-myristate 13-acetate (PMA), indicating involvement of protein kinase C. Hypotonicity inhibited the Na+/H+ exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H+-ATPase. In conclusion, a novel regulatory mechanism of H+-ATPase by hypotonicity is described. The increased H+-ATPase activity in hypotonicity may be responsible for increased HCO-3 reabsorption and maintained acid-base homeostasis in hyposmolar states.

proton-adenosinetriphosphatase; inner medulla; hypotonicity; sodium/proton exchange; acid-base balance

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE INNER MEDULLARY collecting duct (IMCD) plays an important role in plasma volume regulation and acid-base homeostasis. Plasma volume regulation is predominantly attained through electrolyte and water reabsorption (39, 40), whereas acid-base homeostasis is mainly achieved via H+ secretion and HCO-3 reabsorption (7, 8, 35). The ability of IMCD to regulate sodium and water reabsorption is dependent upon the hydration state of the body and plasma antidiuretic hormone (ADH) concentration (39, 40). IMCD is constantly subjected to varying osmolar states. It has been shown that Na+/H+ exchange is stimulated by hypertonicity in IMCD (37). However, the effect of a decrease in osmotic pressure (hypotonicity) on H+-ATPase or Na+/H+ exchange has not been studied in the IMCD or other nephron segments.

Cultured IMCD cells (mIMCD-3) express Na+/H+ exchanger isoforms NHE-2 and NHE-1 on their basolateral membrane (38), and an ATP-dependent, Na+-independent H+-translocating pump, probably on the luminal membrane (4). mIMCD-3 cells grown in hypertonic medium show upregulation of their Na+/H+ exchange activity, predominantly via enhanced expression of NHE-2 (37). The purpose of the present experiments was to study the effect of hypotonicity on H+-ATPase and Na+/H+ exchange activity in mIMCD-3 cells. Our results demonstrate that acute hypotonicity activates H+-ATPase and that this effect is predominantly mediated via protein kinase C (PKC). Increased H+-ATPase activity in response to hypotonicity is also observed in LLC-PK1 cells. Hypotonicity inhibits the basolateral NHE-2 and/or NHE-1 isoforms (in mIMCD-3 cells) and has no effect on the luminal NHE-3 (in LLC-PK1 cells). The increased H+-ATPase activity may be responsible for increased renal HCO-3 reabsorption that is observed in hyposmolar states (15, 29).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture Procedures

mIMCD-3 cells were cultured in a 1:1 mixture of Ham's F-12 and DMEM (DMEM-F12) containing 2.5 mM L-glutamine and 2.438 g/l sodium bicarbonate (GIBCO-BRL) supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, and 10% fetal bovine serum. LLC-PK1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cultured cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every third day.

Intracellular pH Measurement

Changes in intracellular pH (pHi) were monitored using the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) as described (3, 4). mIMCD-3 or LLC-PK1 cells were grown to confluence on coverslips and incubated in the presence of 5 µM BCECF in a solution consisting of 140 mM NaCl, 0.8 mM K2HPO4, 0.2 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 5 mM glucose at pH 7.4 (solution A, Table 1). To measure pHi, each coverslip was positioned diagonally in a cuvette that was placed in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual-excitation spectrofluorometer (PTI, double-beam fluorometer; Photon Technology International, South Brunswick, NJ). The monolayer was then perfused with the appropriate solutions (Table 1). The fluorescence ratio at excitation wavelengths of 500 and 450 nm was utilized to determine pHi values in the experimental groups by comparison to the calibration curve that was generated by KCl/nigericin technique. The emission wavelength was recorded at 525 nm. The H+-ATPase activity was determined as the initial rate of pHi recovery (dpHi/dt, pH/min) from an acid load induced by NH3/NH+4 withdrawal in an Na+-free solution. The dpHi/dt was calculated by fitting to a linear equation the first 3 min of the time course of pHi recovery. Correlation coefficients for these linear fits averaged 0.982 ± 0.004. For Na+/H+ exchange activity measurement, the rate of recovery from an acid load was determined in the presence of a sodium-containing solution (Table 1) and diethylstilbestrol (DES; to inhibit H+-ATPase activity).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Composition of experimental solutions

Buffering Power

The intrinsic intracellular buffering power (beta i; mM per pHi unit) was measured in mIMCD-3 cells using the NH+4 pulse method as described (10, 35)
&bgr;<SUB>i</SUB> = &Dgr;[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB>/&Dgr;pH<SUB>i</SUB>
The beta i was measured in solutions that were CO2/HCO-3 free (to block the Na+-HCO-3 cotransporter) and Na+ free (to block the Na+/H+ exchanger). In addition, 50 µM DES (to inhibit H+-ATPase) (4) and 1 mM verapamil (which blocks NH+4 entry into the cells via K+/NH+4 antiport) (3) were present. All monolayers were initially incubated in isotonic or hypotonic solution and monitored for pHi. At steady-state pHi, addition of 40 mM NH+4/NH3 [NH+4 replacing tetramethylammonium (TMA+)] caused a rapid initial increase in cell pH due to the influx of NH3 and subsequent generation of NH+4. Extracellular NH+4 concentration ([NH+4]) was then reduced in a stepwise manner to 0 mM (20, 10, 5, 2.5, 0) in either isotonic (300 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) solutions. The beta i was determined in both solutions with paired experiments performed on the same day with identically treated cells on separate coverslips.

Materials

DMEM-F12 medium was purchased from GIBCO-BRL. BCECF-AM was purchased from Molecular Probes. DES, N-ethylmaleimide (NEM), nigericin, staurosporine, diphenylamine-2-carboxylate (DPC), DIDS, phorbol 12-myristate 13-acetate (PMA), and other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Statistics

Results are expressed as means ± SE. Statistical significance between experimental groups was determined by Student's t-test or by one-way analysis of variance.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Acute Hypotonicity on Na+-Independent H+ Extrusion

mIMCD-3 cells grown to confluence were incubated in an HCO-3-free, HEPES/Tris-buffered medium [extracellular pH (pHo) of 7.40] and gassed with 100% O2. Cells were loaded with BCECF in an Na+-containing solution (solution A, Table 1) and then switched to an Na+-free medium (solution B, Table 1) 5 min before pHi monitoring. In Na+-free solution, the resting pHi was 7.20 ± 0.004 (n = 9). mIMCD-3 cells were pulsed with 20 mM NH4Cl (replacing 20 mM TMA-Cl in solution B, Table 1) and acid loaded by replacing NH4Cl solution with a sodium-free solution that was either isotonic (300 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) (solutions B and C, respectively, Table 1). The pHi in acid-loaded cells decreased to 6.120 ± 0.014 in isotonic (n = 4) vs. 6.128 ± 0.010 in hypotonic (n = 5) solution (P > 0.05, Fig. 1A). The rate of pHi recovery from an acid load (dpHi/dt) was greater than threefold higher in hypotonic vs. isotonic media (0.061 ± 0.005 and 0.017 ± 0.003 pH units/min in hypotonic and isotonic media, respectively; P < 0.01, Fig. 1, A and B). These results indicate that acute hypotonicity increases the rate of Na+-independent pHi recovery.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of hypotonicity on Na+-independent intracellular pH (pHi) recovery in mIMCD-3 inner medullary collecting duct cells. A: representative pHi tracings of cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), incubated in Na+-free solution [Na+ replaced with tetramethylammonium (TMA+)] and monitored for baseline pHi in isotonic solution. Cells were then pulsed with 20 mM NH4Cl and acid loaded by exposure to an Na+- and NH+4-free solution that was either isotonic or hypotonic. B: initial rates of pHi recovery (dpHi/dt) in mIMCD-3 cells in isotonic (n = 4) or hypotonic solution (n = 5). C: the monolayer was acid loaded in isotonic solution and then exposed to hypotonic media during pHi recovery (n = 5). D: pHi recovery from acid load in cells that were incubated in hypotonic media 30 min before pHi monitoring (n = 5).

In the above experiments, cells were exposed to either a hypotonic or isotonic solution during NH+4 withdrawal, and the rate of pHi recovery from the acid load was then measured in the same solution. The purpose of the next series of experiments was to compare the effect of hypotonicity and isotonicity on Na+-independent pHi recovery in the same experiment so that each monolayer serves as its own control. Accordingly, mIMCD-3 cells were incubated and acid loaded in an Na+-free isotonic solution (solution B, Table 1), and the rate of pHi recovery was monitored in the same isotonic solution. After several minutes, cells were switched to the hypotonic solution and further monitored. As indicated in Fig. 1C, switching to hypotonic solution significantly increased the rate of H+ extrusion in mIMCD-3 cells (the rate of pHi recovery increased from 0.0195 ± 0.006 in isotonic to 0.068 ± 0.004 pH units/min in hypotonic media; n = 5, P < 0.001, Fig. 1C).

Acute exposure of mIMCD-3 cells to hypotonic media can increase the cell volume, decrease the buffering capacity, and indirectly affect the rate of pHi recovery from an acid load.1 To avoid any changes in cell volume secondary to acute hypotonicity, mIMCD-3 cells were preincubated in hypotonic media (solution C, Table 1) for 30 min before pHi monitoring for cell volume to return to normal. Figure 1D demonstrates that cells preincubated and assayed in hypotonic medium showed greater than threefold increase in the rate of pHi recovery from an acid load vs. cells assayed in isotonic solution (pHi recovery rate was 0.058 ± 0.004 in hypotonic and 0.017 ± 0.003 pH units/min in isotonic solution, n = 5, P < 0.001).

Effect of Hypotonicity on Buffering Power in mIMCD-3 Cells

We have measured cell buffering capacity in hypotonic or isotonic medium (See MATERIALS AND METHODS). The results (summarized in Table 2) illustrate that at baseline pHi, beta i of cells incubated in hypotonic solution is decreased [16.575 ± 1.4 (n = 5) in hypotonic vs. 17.985 ± 1.75 mM/pHi unit in isotonic solution (n = 4)]; however, this difference is not statistically significant (P > 0.05). Similar results were obtained at acidic pH (beta i = 30.173 ± 1.06 vs. 32.165 ± 1.07 mM/pHi unit for hypotonic and isotonic solution, respectively; n = 5 for each, P > 0.05). The lack of effect of hypotonicity on beta i is likely due to rapid correction of cell volume, as regulatory volume decrease (RVD) in IMCD cells has been shown to be completed within 5 min (21). The buffering capacity, however, increased at acidic pHi in both isotonic and hypotonic solutions (Table 2), which is in agreement with published reports (10, 35).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Intracellular buffering power of mIMCD-3 cells in isotonic or hypotonic solution at 37°C

Effect of Acute Hypotonicity on Na+-Independent pHi Recovery in CO2/HCO-3-Buffered Medium

In these sets of experiments, the effect of acute hypotonicity on Na+-independent pHi recovery was studied in a physiological solution containing CO2/HCO-3. Accordingly, mIMCD-3 cells were incubated in isotonic CO2/HCO-3-buffered medium (25 mM K-HCO3 replacing TMA-Cl), gassed with 5% CO2-95% O2 at pHo 7.40 (solution B, Table 1), acid loaded by NH+4 withdrawal, and monitored for pHi recovery in isotonic (300 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) solution in the same monolayer. A representative pHi tracing is shown in Fig. 2A and indicates that acute hypotonicity increased the rate of Na+-independent pHi recovery in HCO-3-containing solution (dpHi/dt = 0.025 ± 0.004 in isotonic vs. 0.069 ± 0.005 in hypotonic; n = 5 for each group, P < 0.001, Fig. 2B). Thus these results indicate that hypotonicity increases the rate of Na+-independent pHi recovery in the presence or absence of CO2/HCO-3.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of hypotonicity on Na+-independent pHi recovery in mIMCD-3 cells in CO2/HCO3 buffered solution. A: representative pHi tracing of cells incubated in Na+-free solution containing 25 mM HCO-3 (25 mM K-HCO3 replacing TMA-Cl in solution B, Table 1) and monitored for baseline pHi in isotonic solution. Cells were first acid loaded in isotonic solution, pulsed with 20 mM NH4Cl, and acid loaded in hypotonic solution that contained 25 mM HCO-3 (25 mM K-HCO3 replacing TMA-Cl in solution C, Table 1). B: initial rates of pHi recovery (dpHi/dt) in mIMCD-3 cells in isotonic (n = 5) or hypotonic solution (n = 5).

Effect of Extracellular K+ or Ca2+ on H+ Extrusion in Hypotonicity

mIMCD-3 cells express an Na+-and K+-independent, ATP-dependent, H+-extruding transporter (4). However, in addition to this pump, mIMCD-3 cells also express mRNAs encoding two potassium-dependent H+-K+-ATPases (gastric and colonic isoforms) (32). To determine whether hypotonicity-induced stimulation of H+ extrusion is mediated via H+-K+-ATPase transporters, mIMCD-3 cells were assayed in an Na+-free medium in the absence or presence of 5 mM K+ (solutions D and E, Table 1). Cells were incubated in K+-containing or K+-deficient solution, acid loaded, and monitored for pHi recovery. Figure 3A demonstrates that hypotonicity-induced upregulation of the Na+-independent H+ extrusion was also observed in K+-deficient solution (0.053 ± 0.004 vs. 0.048 ± 0.004 pH units/min, in the presence or absence of K+ in the media; respectively, P > 0.05, n = 4). These results indicate that H+-K+-ATPase transporters were not responsible for hypotonicity-induced stimulation of H+ extrusion.2 The rate of pHi recovery from an acid load in isotonic solution was also not affected by the presence or absence of K+ in the perfusion solution (Fig. 3A).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3.   A: effect of extracellular K+ ([K+]o) on Na+-independent pHi recovery in mIMCD-3 cells. Cells were pulsed with NH+4 in isotonic, Na+- and K+-free solution and then acidified by ammonium withdrawal in either the same isotonic solution or in hypotonic solution. The experiment was repeated in the presence of 5 mM K+. B: effect of extracellular Ca2+ ([Ca2+]o) on Na+-independent pHi recovery in mIMCD-3 cells. Initial rate of pHi recovery was measured in isotonic or hypotonic solution in the absence or presence of 1 mM Ca2+ in the solutions.

In the next series of experiments, the effect of extracellular Ca2+ on hypotonicity-induced H+ secretion stimulation was examined. mIMCD-3 cells were incubated in an isotonic Na+-free solution that either contained 2 mM Ca2+ (solution B supplemented with 1 mM CaCl2) or no Ca2+ (the 0 mM Ca2+ solution in addition contained 5 mM EGTA to chelate any residual extracellular Ca2+; solution F, Table 1). Acute exposure of mIMCD-3 cells to hypotonic solution caused a rapid increase in the rate of pHi recovery from an acid load both in the presence or absence of Ca2+ [0.053 ± 0.005 (n = 3) with Ca2+ vs. 0.051 ± 0.003 pH/min with no Ca2+ (n = 5); P > 0.05, Fig. 3B]. The rate of pHi recovery from an acid load in isotonic solution was the same in the presence or absence of Ca2+ [0.019 ± 0.004 (n = 4) vs. 0.021 ± 0.003 pH/min (n = 4); P > 0.05; Fig. 3B]. These results indicate that the presence or absence of Ca2+ in the media (and by inference a Ca2+/nH+ exchange) does not play any role in mediating the effect of hypotonicity on pHi recovery in mIMCD-3 cells.

Effect of H+-ATPase Inhibitors on Hypotonicity-Induced pHi Recovery

To examine the mechanism of the Na+-independent pHi recovery further, the effect of DES, an inhibitor of the vacuolar H+-ATPase (4, 17, 24), and NEM were examined. DES and NEM were added to the cells during NH+4 withdrawal. Figure 4A shows that the rate of pHi recovery from an acid load in hypotonic medium was almost abolished in the presence of 50 µM DES (0.006 ± 0.002 and 0.063 ± 0.005 pH units/min for DES and its vehicle respectively; n = 4, P < 0.0001, Fig. 4A). Figure 4B shows that DES inhibits the Na+-independent pHi recovery in both isotonic as well as hypotonic media (n = 4). NEM, an inhibitor of V-type H+-ATPase, also inhibited the pHi recovery from an acid load (n = 4, Fig. 4C). Taken together, the results of the above studies indicate that the DES-sensitive, Na+- and K+-independent H+ extrusion mechanism that is stimulated by hypotonicity represents H+-ATPase.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of H+-ATPase inhibitors on hypotonicity-induced pHi recovery. A: mIMCD-3 cells were acid loaded in hypotonic solution in absence (vehicle) or presence of 50 µM diethylstilbestrol (DES) (n = 4). B and C: mIMCD-3 cells were acid loaded in isotonic solution and then switched to hypotonic solution in presence of 50 µM DES (B) (n = 4) or 200 µM N-ethylmaleimide (NEM; C) (n = 4).

Hypotonicity Activates the H+-ATPase in a Concentration-Dependent Manner

The purpose of the next series of experiments was to examine the tonicity dependence of H+-ATPase stimulation by hypotonic media. Cells were incubated in Na+-free isotonic solution (solution B, Table 1) and were acid loaded by replacing NH+4-containing solution with either isotonic or various hypotonic solutions [the concentration of TMA-Cl in solution B (Table 1) was appropriately decreased to achieve desired osmolarities]. As shown in Table 3, H+-ATPase activity increased with progressive reduction in the osmolarity of the solution. Table 2 shows the baseline pHi (prior to NH4Cl addition) and the nadir pHi (after NH+4 withdrawal).

                              
View this table:
[in this window]
[in a new window]
  		Table 3.   pHi values and effect of various levels of osmolality on H +-ATPase activity in mIMCD-3 cells

Effect of Relative Hypotonicity on H+-ATPase Activity

The purpose of the next series of experiments was to determine whether activation of H+-ATPase also occurs in cells that are grown in hypertonic medium and then exposed to an isotonic solution (relative hypotonicity). Cells were grown to confluence on coverslips in isotonic media and were either switched to hypertonic medium or remained in isotonic medium (100 mM NaCl was added to the cultured medium to achieve an osmolality of 500 mosmol/kgH2O) for 48 h. mIMCD-3 cells are osmotically tolerant and can survive in highly osmotic media (~900 mosmol/kgH2O) by accumulating organic osmolytes (33). No evidence of increased cell death was apparent by microscope. For experiments, media was removed and cells were exposed to either an Na+-free hypertonic solution (solution G, Table 1) or an Na+-free isotonic solution (solution B, Table 1) for 30 min before pHi monitoring. A representative experiment depicted in Fig. 5 demonstrates that resting pHi in cells grown in hypertonic medium and assayed in isotonic solution (hyper-iso) was more alkaline than that of cells grown and assayed in hypertonic medium (hyper-hyper) (pHi was 7.28 ± 0.006 and 7.13 ± 0.008 for hyper-iso and hyper-hyper group, respectively; P < 0.002, n = 4 for each group, Fig. 5A). The rate of Na+-independent pHi recovery (dpHi/dt) in cells grown in hypertonic medium and assayed in isotonic solution was approximately sevenfold greater than that of cells incubated and assayed in hypertonic solution (0.046 ± 0.004 in hyper-iso and 0.006 ± 0.003 pH units/min in hyper-hyper group; P < 0.0001, n = 5 for each, Fig. 5B). As shown, the rate of pHi recovery in cells grown and assayed in isotonic media was greater than that of cells grown and assayed in hypertonic media (0.023 ± 0.005 in iso-iso vs. 0.006 ± 0.003 pH units/min in hyper-hyper group; P < 0.001, n = 5, Fig. 5B). Last, when cells were grown in isotonic medium and assayed in hypertonic or isotonic solution, the rate of pHi recovery from acidosis was decreased in hypertonic solution (solution G, Table 1) compared with isotonic solution [dpHi/dt was 0.011 ± 0.005 pH units/min in hypertonic solution (n = 5) vs. 0.021 ± 0.004 pH units/min in isotonic solution (n = 4); P < 0.001]. These results demonstrate that relative hypotonicity increases, whereas hypertonicity decreases H+-ATPase activity.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of relative hypotonicity on H+-ATPase activity in mIMCD-3 cells. Cells were incubated in hypertonic media (500 mosmol/kgH2O) for 48 h and then were either exposed to hypertonic or isotonic solution for 30 min before pHi monitoring. A: representative pHi tracings showing that in isotonic solution the resting pH is higher and the recovery from acid load is faster (n = 5). B: initial rate of pHi recovery (dpHi/dt) of cells incubated and assayed in hypertonic media (hyper-hyper, b) is inhibited compared with the cells incubated and assayed in isotonic (iso-iso, a; n = 4); however, dpHi/dt of cells incubated in hypertonic and assayed in isotonic solution (hyper-iso; c) is activated (n = 5). ** P < 0.001 compared with a. $ P < 0.0001 compared with b. @ P < 0.001 compared with a.

Hypotonicity-Induced H+-ATPase Stimulation is not Secondary to Cl- Channel or Cl-/Base Exchange Activation

A recent report suggested that IMCD cells undergo RVD after hypotonic shock by activating the Cl- channel (21). Activation of Cl- channel by hypotonic stress in mIMCD-3 cells can lead to membrane potential depolarization, which in turn can result in H+-ATPase activation. To investigate this possibility, we have studied the effect of hypotonicity on H+-ATPase activity in the presence of DPC, a Cl- channel inhibitor (12, 19). A representative experiment shown in Fig. 6A indicates that the presence of DPC, at 100 µM, did not block the stimulatory effect of hypotonicity on H+-ATPase (the rate of pHi recovery from an acid load in hypotonic medium was 0.050 ± 0.004 and 0.052 ± 0.004 pH units/min in the presence or absence of DPC, respectively; P > 0.05, n = 4; Fig. 6, A and B). Last, we found that hypotonicity-induced stimulation of pHi recovery from an acid load was not blocked in the presence of 100 µM DIDS (recovery rate was 0.046 ± 0.005 and 0.049 ± 0.003 pH units/min for DIDS and its vehicle, respectively; P > 0.05, n = 4, Fig. 6B), indicating that Cl-/OH- exchange was not responsible for enhanced rate of recovery from an acid load.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of diphenylamine-2-carboxylate (DPC) and DIDS on hypotonicity-induced H+-ATPase activation in mIMCD-3 cells. A: representative pHi tracing of cells incubated in iosotonic solution and then acid loaded in hypotonic solution in absence (vehicle) or presence of 100 µM DPC (n = 4). B: initial rate of Na+-independent pHi recovery in hypotonic solution in presence or absence of 100 µM DPC (n = 4). B: the same protocol described in A was used to test the effect of 100 µM DIDS (n = 4).

Taken together, the above results indicate that hypotonicity-induced H+-ATPase stimulation in mIMCD-3 cells was not mediated via changes in cell volume, was not dependent on extracellular K+ or Ca2+, and was not secondary to activation of Cl- channel or Cl-/base exchange or changes in intracellular buffering capacity.

Mechanism of Hypotonicity-Induced H+-ATPase Activation

Role of exocytosis in hypotonicity-induced H+-ATPase activation. We first tested whether enhanced H+-ATPase activity by hypotonicity was mediated via insertion of new transport proteins into plasma membrane (exocytosis). Na+-independent pHi recovery was assayed in the presence of colchicine at 20 µM for the entire duration of the experiment. A representative experiment is shown in Fig. 7 and demonstrates that hypotonicity-induced H+-ATPase activation was not affected by colchicine (the rate of pHi recovery increased from 0.018 ± 0.004 in isotonic to 0.061 ± 0.007 pH units/min in hypotonic media; n = 5, P < 0.001, Fig. 7). Colchicine (20 µM) had no effect on baseline H+-ATPase activity in isotonic solution [the rate of pHi recovery was 0.017 ± 0.003 for colchicine (n = 4) vs. 0.018 ± 0.004 pH units/min (n = 5) for control, P > 0.05].


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of colchicine on hypotonicity-induced H+-ATPase activation. mIMCD-3 cells were incubated and acid loaded in isotonic solution in the presence of 20 µM colchicine. pHi recovery from acid load was then recorded in hypotonic solution (n = 5).

Hypotonicity-induced H+-ATPase activation is mediated via a phosphorylation-dependent mechanism. Several studies have suggested that PKC can activate H+-ATPase (31). We next sought to determine the role of PKC in mediating the stimulatory effect of hypotonicity on H+-ATPase in mIMCD-3 cells. mIMCD-3 cells were incubated in an isotonic solution (solution B, Table 1) and then acid loaded in a hypotonic medium (solution C, Table 1) in the presence of either 100 nM staurosporine (a PKC inhibitor) (42, 44) or its vehicle (the inhibitor or its vehicle were added to the cells 5 min before the acid load). As shown in Fig. 8A, H+-ATPase activity in hypotonic solution decreased by ~56% (pHi recovery was 0.078 ± 0.004 and 0.035 ± 0.005 pH units/min in the absence or presence of staurosporine, respectively; P < 0.002, n = 4 for each, Fig. 8B). Interestingly, incubation of mIMCD-3 cells with 100 nM staurosporine in isotonic solution had no effect on the rate of pHi recovery from an acid load (pHi recovery rate was 0.017 ± 0.005 and 0.020 ± 0.006 pH units/min in the absence or presence of staurosporine, respectively; P > 0.05, n = 4 for each, Fig. 8B). We have also examined the effect of calphostin C, a more specific inhibitor of PKC (26), on hypotonicity-induced stimulation of H+-ATPase. As shown in Fig. 8C, incubation of mIMCD-3 cells with 2 µM calphostin C for 7 min decreased the effect of hypotonicity on H+-ATPase activity by ~57% (Fig. 8C), with pHi recovery decreasing from 0.066 ± 0.004 (n = 4) to 0.029 ± 0.003 pH units/min (n = 5) in the presence of calphostin C (P < 0.001, Fig. 8D). Calphostin C had no effect on H+-ATPase activity in isotonic medium (dpHi/dt = 0.019 ± 0.005 and 0.017 ± 0.007 pH units/min for calphostin C or its vehicle, respectively; P > 0.05, n = 4 for each, Fig. 8D).


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of staurosporine and calphostin C (PKC inhibitors) on hypotonicity-induced H+-ATPase activation. A and C: representative pHi tracings of cells incubated in isotonic solution and then acid loaded in a hypotonic solution. A: in presence of 100 nM staurosporine (n = 4) or its vehicle (n = 4). C: in presence of 2 µM calphostin C (n = 5) or its vehicle (n = 4). B and D: effect of 100 nM staurosporine (stauro) or its vehicle (B) and 2 µM calphostin C (calph) or its vehicle (D) on initial rate of pHi recovery (dpHi/dt) in hypotonic or isotonic solution. NS, not significant.

To examine the role of PKC in mediating the effect of hypotonicity on H+ pump further, mIMCD-3 cells were depleted of their PKC by overnight preincubation with 0.1 µM PMA (11). The results shown in Fig. 9A demonstrate that downregulation of PKC significantly blocked the stimulatory effect of hypotonicity on H+-ATPase activity (pH recovery rate in hypotonic medium was 0.076 ± 0.003 and 0.037 ± 0.005 pH units/min in cells incubated with either the vehicle or PMA, respectively; P < 0.002, n = 5, Fig. 9B). Incubation of mIMCD-3 cells with PMA had no effect on pHi recovery from an acid load in isotonic medium (recovery rate was 0.021 ± 0.004 and 0.024 ± 0.05 pH units/min for vehicle and PMA respectively; P > 0.05, n = 5, Fig. 9B). Taken together, these results indicate that hypotonicity stimulates H+-ATPase through a signal transduction pathway involving PKC.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 9.   Effect of overnight incubation with phorbol 12-myristate 13-acetate (PMA) on hypotonicity-induced H+-ATPase activation. A: representative pHi tracings showing the inhibitory effect of overnight PMA treatment on hypotonicity-induced H+-ATPase activation (n = 5). B: overnight PMA treatment decreases the effect of hypotonicity on dpHi/dt without affecting the baseline H+-ATPase activity in isotonic media.

Whether PKC mediation of H+-ATPase activation by hypotonicity is direct (by phosphorylating one or more of the H+-ATPase subunits) or indirect (via phosphorylation of an intermediary protein) remains unanswered at the present. Differentiating between these two possibilities, however, is very difficult, as no antibodies are available that could recognize the H+-ATPase subunits in the renal medulla. To address the mechanism of hypotonicity-induced, PKC-mediated stimulation of H+-ATPase further, we studied the effect of acute PKC activation on H+-ATPase in an isotonic medium. The rational for these experiments was that, if PKC mediation of H+-ATPase activation by hypotonicity was direct, then PKC activation in an isotonic medium should also stimulate H+-ATPase. The results are summarized in Fig. 10 and show that incubation of mIMCD-3 cells with 0.1 to 5 µM PMA in isotonic solution (solution B, Table 1) did not increase the rate of the Na+-independent pHi recovery from an acid load [0.023 ± 0.008 (n = 5) vs. 0.021 ± 0.004 (n = 4) pH units/min for PMA and its vehicle, respectively; P > 0.05, Fig. 10, A and B]. These results suggest that PKC mediation of H+-ATPase stimulation by hypotonicity is dependent on the hypotonic milieu and is likely via involvement of intermediary elements, since direct activation of PKC in isotonic medium did not stimulate H+-ATPase.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 10.   Acute effect of PMA, cAMP, H-89, or genistein on the rate of Na+-independent pHi recovery (H+-ATPase) in mIMCD-3 cells. A: representative pHi tracings of cells incubated and acid loaded in isotonic solution in presence of 0.1-5 µM PMA or its vehicle (added 5 min before acid loading). B: acute effect of PMA on dpHi/dt (n = 5 compared with its vehicle, n = 4). C: effect of exogenous 8-bromo-cAMP (500 µM, isotonic media), N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89; 5 µM, hypotonic media), or genistein (10-40 µM, hypotonic solution) on dpHi/dt.

Role of other kinases (PKA and PTK) in hypotonicity-induced H+-ATPase activation. To test whether protein kinase A (PKA) regulates H+-ATPase activity, cells were incubated with 500 µM 8-bromo-cAMP (a PKA activator) for 5-8 min in an isotonic Na+-free solution (solution B, Table 1), and the pHi recovery from acid load was then monitored in the same solution. The rate of pHi recovery was not significantly affected by the presence of cAMP (dpHi/dt was 0.022 ± 0.008 and 0.024 ± 0.006 pH units/min in the absence or presence of cAMP, respectively; n = 5 for each, P > 0.05, Fig. 10C). We next examined whether PKA plays any role in the transduction of hypotonicity effect on H+-ATPase. Accordingly, mIMCD-3 cells were incubated for 5-8 min with 5 µM N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a PKA inhibitor (2, 14), in an isotonic Na+-free solution (solution B, Table 1) and then acid loaded in hypotonic Na+-free solution (solution C, Table 1). As indicated in Fig. 10C, the rate of pHi recovery in hypotonic medium was not affected by the presence of the PKA inhibitor (dpHi/dt was 0.061 ± 0.006 and 0.065 ± 0.003 pH units/min in the absence or presence of cAMP, respectively; n = 5 for each group, P > 0.05, Fig. 10C).

The protein tyrosine kinase (PTK) has been reported to mediate the effect of osmotic stress on Na+/H+ exchange in perfused medullary thick ascending limb tubule (20). The purpose of the next series of experiments was to determine whether PTK plays any role in H+-ATPase activation by hypotonicity. Using the same protocol as described above for H-89, we found that incubation of mIMCD-3 cells with 10 to 40 µM genistein, a PTK inhibitor (1), had no effect on hypotonicity-induced H+-ATPase activation (dpHi/dt was 0.072 ± 0.005 and 0.068 ± 0.007 pH units/min for genistein or its vehicle, respectively; n = 5 for each, P > 0.05, Fig. 10C). Taken together, the results of the above experiments indicate that the effect of hypotonicity on H+-ATPase activity in mIMCD-3 cells was not mediated via PKA- or PTK-dependent pathways.

Effect of Acute Hypotonicity on H+-ATPase in LLC-PK1 Cells

To determine whether hypotonicity-induced stimulation of H+-ATPase is unique to mIMCD-3 cells, cultured proximal tubule (LLC-PK1) cells were exposed to hypotonic media (150 mosmol/kgH2O) and assayed for H+-ATPase activity in a manner similar to that in Fig. 1. As shown in Fig. 11A, exposure of LLC-PK1 cells to a hypotonic media (solution C, Table 1) significantly increased H+-ATPase activity (the recovery rate from an acid load was 0.018 ± 0.003 in isotonic vs. 0.053 ± 0.005 pH/min in hypotonic medium; n = 5 for each, P < 0.001, Fig. 11B). These results indicate that H+-ATPase activation by hypotonicity may be widespread among epithelial cells.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 11.   Effect of hypotonicity on H+-ATPase in LLC-PK1 cells. A: representative pHi tracings of cells incubated in Na+-free isotonic solution and then acid loaded either in the same solution or in hypotonic media. B: dpHi/dt is increased by 3-fold in hypotonic (n = 5) solution compared with isotonic (n = 5) solution.

Effect of Hypotonicity on Na+/H+ Exchange

In the last series of experiments, the effect of hypotonicity on Na+/H+exchange activity was examined. mIMCD-3 or LLC-PK1 cells were acid loaded in Na+-free, isotonic solution (solution B, Table 1) and then exposed to either an Na+-containing hypotonic solution (65 mM TMA-Cl in solution C was replaced with 65 mM NaCl) or an Na+-containing isotonic solution (65 mM of TMA-Cl in solution B was replaced with 65 mM NaCl). Both Na+-containing solutions contained 50 µM DES to inhibit H+-ATPase. As shown in Fig. 12, the Na+/H+ exchanger activity in mIMCD-3 cells (mediated via NHE-1 and NHE-2 isoforms) (38) was significantly decreased in hypotonic solution [the Na+-dependent pHi recovery from an acid load was 0.215 ± 0.008 in isotonic (n = 4) vs. 0.126 ± 0.011 pH/min in hypotonic medium (n = 5); P < 0.001, Fig. 12A]. Interestingly, the Na+/H+ exchanger activity in LLC-PK1 cells (mediated via NHE-3 isoform) (37) remained unaltered in hypotonicity (the Na+-dependent pHi recovery from an acid load was 0.219 ± 0.005 in isotonic vs. 0.217 ± 0.009 pH/min in hypotonic medium; P > 0.005, n = 5, Fig. 12B).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 12.   Effect of hypotonicity on Na+/H+ exchanger isoforms (NHE-1 and NHE-2 in mIMCD-3 cells and NHE-3 in LLC-PK1 cells). A: mIMCD-3 cells were acid loaded in Na+-free isotonic solution in the presence of 50 µM DES; cells were then exposed either to isotonic (n = 4) or hypotonic (n = 5) solution that contained 65 mM Na+. B: LLC-PK1 cells were acid loaded in Na+-free isotonic solution in presence of 50 µM DES and then exposed to isotonic solution that contained 65 mM Na+. The same monolayer was acid loaded in Na+-free isotonic solution in presence of 50 µM DES and then exposed to hypotonic solution that contained 65 mM Na+ (n = 5).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The renal inner medulla, which is predominantly comprised of IMCD, is exposed to a high osmotic environment and is constantly subjected to varying osmolar states, depending on plasma osmolarity and level of plasma ADH (39, 40). However, the effect of altered tonicity on H+ transport pathways in kidney cells has not been studied. The results of current experiments indicate that hypotonicity activates H+-ATPase in cultured IMCD cells in the presence or absence of CO2/HCO3 in the media (Figs. 1 and 2). This stimulatory effect was acute, not dependent on external K+ or Ca2+, and was not secondary to Cl- channel activation (Figs. 3 and 7). The hypotonicity-induced H+-ATPase activation was significantly blocked by inhibitors of PKC, consistent with the possibility that phosphorylation of a regulatory protein or the pump itself might be responsible for upregulation of the transporter (Figs. 8 and 9). Stimulation of H+-ATPase by hypotonicity was also observed in cultured proximal tubule (LLC-PK1) cells, indicating that this phenomenon may be widespread among epithelial cells (Fig. 11). Hypotonicity inhibited the Na+/H+ exchanger isoforms (NHE-1 and/or NHE-2) in mIMCD-3 cells (Fig. 12A), indicating that its stimulatory effect is specific to H+-ATPase. Hypotonicity had no effect on the Na+/H+ exchanger (NHE-3) in LLC-PK1 cells (Fig. 12B).

The data indicate that hypotonicity increases the H+-ATPase activity. It is generally accepted that acute exposure of cells to hypotonic solution causes a rapid and transient increase in cell volume (cell swelling), which would return to normal through an RVD mechanism (13). The increase in cell volume can lead to a decrease in intracellular buffering capacity and an increase in H+ distribution space. However, it is unlikely that changes in cell volume are responsible for increased Na+-independent pHi recovery in hypotonic states. First, increased H+-ATPase activity was evident even when the cells were in hypotonic medium for 40 min (Fig. 1D) and had normalized their volume, as RVD in IMCD cells is completed within 5 min (21). Second, acute hypotonic stress had opposite effects on Na+/H+ exchange and H+-ATPase activities in mIMCD-3 cells (Fig. 12A). The effect of acute hypotonicity on Na+/H+ exchange isoforms described above is consistent with the results reported in Chinese hamster ovary cells transfected with Na+/H+ exchange isoforms NHE-1, NHE-2, and NHE-3 (24).

The effect of acute hypotonicity on the rate of pHi recovery from an acid load was independent of extracellular K+ (Fig. 3A) and extracellular Ca2+ (Fig. 3B), indicating lack of involvement of H+-K+-ATPase or Ca2+/nH+ exchanger. Indeed, it has been reported that hypotonicity inhibits H+-K+-ATPase (27). Although our experiments were performed in the nominal absence of HCO-3/CO2, possible activation of Cl-/base exchange (i.e., Cl-/OH-) by hypotonicity could potentially explain the cell alkalinization in hypotonicity. However, presence of 100 µM DIDS, an inhibitor of Cl-/base exchange, had no effect on the rate of pHi recovery in hypotonic solution (Fig. 6B). As reported previously from this laboratory, H+-ATPase activity in mIMCD-3 cells was not inhibited by bafilomycin A1 (4); however, it was completely abolished by other H+-ATPase inhibitors such as N,N'-dicyclohexylcarbodiimide (DCCD), NEM, and DES (4). The current results indicate that hypotonicity-induced pHi recovery was prevented in the presence of 50 µM DES or 200 µM NEM (Fig. 4), two known inhibitors of H+-ATPase (4, 17, 24). At low concentration (50 µM), DES does not affect Na+/H+ exchange (Fig. 12A and data not shown). Moreover, 50 µM DES does not block gastric H+-K+-ATPase in microperfused mouse terminal IMCD (IMCDt) (41). Further experiments are needed to explore the specificity of DES at higher concentrations.

RVD in many cells types includes activation of Cl- channel (16, 21, 29, 37, 45). Crowe et al. (16) have reported that during osmotic swelling, electrogenic Na+ and Cl- influx were increased in renal epithelial A6 cells (16). A Cl- channel is expressed in the apical membrane domain of IMCD cells (25). Activation of Cl- channel by hypotonicity can result in Cl- efflux and indirect activation of the electrogenic H+-ATPase due to membrane potential depolarization. However, inhibition of Cl- channel with 100 µM DPC (19) did not prevent the hypotonicity-induced H+-ATPase activation (Fig. 6, A, and B). Kizer et al. (24) have shown that DPC inhibits Cl- secretion through the apical Cl- channel in IMCD cells (mIMCD-K2). In their study, Volk et al. (45) did not use DPC to block the hypotonicity-activated Cl- current; however, they (45) and others (5, 18, 28) have shown that 100 µM DIDS strongly blocks the hypotonicity-induced Cl- channel activation. Our results (Fig. 6B) clearly indicate that 100 µM DIDS does not have any effect on hypotonicity-induced H+-ATPase activation.

Whether hypotonicity affects the Na+ channel in mIMCD-3 cells remains unknown. Our experiments were performed in the absence of Na+ in the media, and therefore, Na+ channel remained inactive for the duration of the experiment. Good (20) has reported that hyperosmotic stress can regulate Na+/H+ exchanger (NHE-3) in medullary thick ascending limb of rat kidney via PTK (20), but whether such mechanism can operate on NHE-2 and NHE-1 in response to hyposmotic stress is not known. More studies are needed to investigate the cellular mechanism underlying the effect of hypotonicity on Na+/H+ exchanger in mIMCD-3 cells.

The cellular mechanism underlying the effect of hypotonicity on vacuolar H+-ATPase activation remains to be fully determined. Our results demonstrate that activation of PKA by exogenous cAMP or its inhibition by H-89 does not affect the activity of H+-ATPase in mIMCD-3 cells in either isotonic or hypotonic solutions (Fig. 10C). Inhibition of PTK by genistein also had no effect on hypotonicity-induced H+-ATPase activation (Fig. 10C). However, depletion of PKC by preincubation with PMA (11, 37) significantly reduced the effect of hypotonicity on H+-ATPase activation (Fig. 9, A and B). Furthermore, presence of calphostin C or staurosporine, at concentrations specific for PKC inhibition, decreased H+-ATPase upregulation (Fig. 8). The effect of PKC inhibition (staurosporine and calphostin C) or PKC depletion (incubation with PMA) were specific to hypotonicity-induced H+-ATPase activation, as these maneuvers had no effect on H+-ATPase activity in isotonic medium (Figs. 8, B and D, and 9B). Furthermore, acute activation of PKC by exposure of mIMCD-3 cells to 0.1 or 5 µM PMA had no effect on H+-ATPase activity (Fig. 10), suggesting that a hypotonic milieu was essential for PKC effect. PMA had no effect on baseline pHi in hypotonic solution (solution C, Table 1, data not shown). Although these results are consistent with phosphorylation of an intermediary protein rather than the H+-ATPase itself, the definite answer should come from immunoprecipitation studies using specific antibodies that could recognize the inner medulla H+-ATPase. At the present, available antibodies do not recognize the H+-ATPase in the inner medulla. Alternatively, quantitation of membrane-bound or cytosolic PKC isoforms with the use of specific antibodies would be helpful to determine whether translocation of this kinase precedes hypotonicity-induced H+-ATPase stimulation. Such a process could lead to activation of organellar motility with resultant fusion of intracellular acidic organelles with the plasma membrane (43). Lack of effect of colchicine at either low (20 µM) (Fig. 7) or high concentration (200 µM) (data not shown) on pHi recovery, however, makes this possibility unlikely. Alternatively, increased V-H+-ATPase-mediated proton extrusion in response to hypotonicity may be attributed to alterations in function of existing plasma membrane pumps. Phosphorylation of pump subunits or recently described activator protein (46) may serve to modulate V-ATPase function. The role of PKC will likely be additive to other intermediary regulatory processes activated by hypotonicity. The transduction pathways mediating the effect of hypotonicity on H+ secretion remain complex and need further and full exploration.

Stimulation of V-H+-ATPase by hypotonicity was not associated with increased intracellular Ca2+ concentration ([Ca2+]i), as no detectable increase in [Ca2+]i in response to hypotonicity was observed (data not shown). The failure to detect an increase in cell Ca2+in response to hypotonicity in mIMCD-3 cells is in agreement with recent studies in IMCD cells showing that reduction in extracellular osmolality had no effect on [Ca2+]i (45).

The activation of V-H+-ATPase by acute hypotonicity must be integrated into the control of urinary acidification during antidiuresis. On the basis of the above experiments indicating that switching from a hypertonic medium to an isotonic solution (relative hypotonicity) increases H+-ATPase activity (see RESULTS), it is intriguing to postulate that similar alterations in the osmolality of inner medulla can affect H+-ATPase activity. This is specifically relevant to in vivo situation, as inner medulla is constantly subjected to varying osmolar states, depending on plasma osmolarity and level of plasma ADH (39, 40). It has been reported previously that an ADH-induced antidiuresis in rat is associated with an increase in urinary net acid excretion (9), which could in part be explained by direct stimulation of tubular fluid acidification in distal nephron by ADH (9). However, our results show that reduction of the extracellular osmolarity, independent of ADH concentration (there was no ADH in the experimental solutions), increased H+-ATPase activity (Table 3). Moreover, exogenous cAMP (the principal messenger of ADH) or PKA inhibition do not affect the activity of V-H+-ATPase in mIMCD-3 cells (Fig. 10C). On the basis of these observations, we propose that part of the effect of ADH on net acid excretion or bicarbonate absorption in distal nephron could be indirect and mediated via alterations in the osmolarity of the immediate interstitium surrounding the collecting duct. This could explain the maintenance of plasma bicarbonate concentration within or slightly below the normal range despite water retention and significant reduction in serum sodium in patients with the syndrome of inappropriate secretion of ADH (8). It has been reported that chronic hypotonic volume expansion by ADH administration and increased water load is associated with increased distal nephron acidification (15, 29). Although the identity of this acidification process was not examined in those studies, it is likely that, on the basis of cellular transporters identified in distal nephron, H+-ATPase was the transporter that was activated in hypotonic state. The tonicity of the medullary interstitium was not measured in those studies; however, it is reasonable to conclude that, on the basis of published reports in similar conditions, hypotonic volume expansion is associated with decreased medullary interstitial osmolality. Stimulation of acid secretion restoring plasma bicarbonate concentration to normal values during prolonged ADH administration in dogs was ascribed to a measured increase in the aldosterone secretion (29). However, aldosterone cannot account for increased H+-ATPase in mIMCD-3 cells, as no aldosterone was present the experimental solutions.

In conclusion, acute exposure of mIMCD-3 cells to hypotonic medium is associated with marked stimulation of H+ secretion via V-H+-ATPase. The effect of hypotonicity on V-H+-ATPase activity is mediated to a large extent through a calcium-independent, PKC-dependent pathways. The increased H+-ATPase activity in hypotonicity may be responsible for increased HCO-3 reabsorption and maintained acid-base homeostasis in hyposmolar states.

    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789 and DK-52821 and by a grant from Dialysis Clinic Incorporated.

    FOOTNOTES

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. §1734 solely to indicate this fact.

1 The increase in dpHi/dt in the hyposmotic mIMCD-3 cells could, in theory, be the result of a decrease in cell buffering capacity secondary to cell swelling rather than a true increase in H+-ATPase activity. This possibility is very unlikely for several reasons. First, in cells incubated in hypotonic solution for 45 min, H+-ATPase activity was increased, which indicates that stimulatory effect of hypotonicity on H+-ATPase is present even after full regulatory volume adjustment in cells. Second, the volume of the hyposmotic cells would have to be more than fourfold that of the control cells to decrease the buffering capacity by fourfold and account for the increase in dpHi/dt. Buffering capacity, however, remained the same in hypotonic solution compared with isotonic solution. Third, a decrease in cell buffering capacity should have affected the Na+/H+ exchanger activity in the same direction. The Na+/H+ exchanger activity in mIMCD-3 cells, however, decreased in hypotonic medium. These results indicate that stimulatory response of the H+-ATPase to hypotonicity is a true adaptive regulation of the transporter.

2 Measured K+ concentration in collected K+-free solutions was undetectable (<0.3 meq/l), whereas K+ concentration in control solutions was ~5.2 meq/l. These results indicate that possible K+ leak from the cells to the lumen with subsequent K+-H+-ATPase activation is unlikely.

Address for reprint requests: H. Amlal, Univ. of Cincinnati Hospital, 231 Bethesda Ave, MSB (Rm. 5502), Cincinnati, OH 45267-0585.

Received 3 March 1998; accepted in final form 26 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shiuya, and Y. Fukami. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262: 5592-5595, 1987[Abstract/Free Full Text].

2.   Amlal, H., C. Legoff, C. Vernimmen, M. Paillard, and M. Bichara. Na+-K+ (NH+4)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PC, and 20-HETE. Am. J. Physiol. 271 (Cell Physiol. 40): C455-C463, 1996[Abstract/Free Full Text].

3.   Amlal, H., and M. Soleimani. K+/NH+4 antiporter: a unique ammonium carrying transporter in the kidney inner medulla. Biochim. Biophys. Acta 1323: 319-333, 1997[Medline].

4.   Amlal, H., W. Zhaohui, and M. Soleimani. Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells. Am. J. Physiol. 273 (Cell Physiol. 42): C1194-C1205, 1997[Abstract/Free Full Text].

5.   Bakhramov, A, C. Fenech, and T. B. Bolton. Chloride current activated by hypotonicity in cultured human astrocytoma cells. Exp. Physiol. 80: 373-389, 1995[Abstract].

6.   Bartter, F. C., and W. B. Schwartz. The syndrome of inappropriate secretion of antidiuretic hormone. Am. J. Med. 42: 790-806, 1976.

7.   Bengele, H. H., E. R. McNamara, J. H. Schwartz, and E. A. Alexander. Acidification adaptation along the inner medullary collecting duct. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F1155-F1159, 1988[Abstract/Free Full Text].

8.   Bengele, H. H., J. H. Schwartz, E. R. McNamara, and E. A. Alexander. Chronic metabolic acidosis augments acidification along the inner medullary collecting duct. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F690-F694, 1986.

9.   Bichara, M., O. Mercier, P. Houillier, M. Paillard, and F. Leviel. Effects of antidiuretic hormone on urinary acidification and on tubular handling of bicarbonate in the rat. J. Clin. Invest. 80: 621-630, 1987.

10.   Boyarski, G., M. B. Ganz, R. B. Sterzel, and W. F. Boron. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO-3. Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988[Abstract/Free Full Text].

11.   Burns, K. D., T. Homma, and R. C. Harris. Regulation of Na+/H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F607-F616, 1991[Abstract/Free Full Text].

12.   Cabantchik, Z. I., and R. Greger. Chemical probes for anion transporters of mammalian cell membranes. Am. J. Physiol. 262 (Cell Physiol. 31): C803-C827, 1992[Abstract/Free Full Text].

13.   Chamberlin, M. E., and K. Strange. Anisosmotic cell volume regulation: a comparative view. Am. J. Physiol. 257 (Cell Physiol. 26): C159-C173, 1989[Abstract/Free Full Text].

14.   Chijiwa, T., A. Mishima, M. Hagiwara, M. Sano, and K. Hayashi. Inhibition of forskolin-induced neurite out-growth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP- dependent protein kinase. N-[2-(p-bromocyannamyl-amino)ethyl]-5-isoquinolinesulfonamide (H-89) of PC12D pheochromocytoma cells. J. Biol. Chem. 265: 5267-5272, 1990[Abstract/Free Full Text].

15.   Cohen, J. J., H. N. Hulter, N. Smithline, J. C. Melby, and W. B. Schwartz. The critical role of the adrenal gland in the renal regulation of acid-base equilibrium during chronic hypotonic expansion. J. Clin. Invest. 58: 1201-1208, 1976.

16.   Crowe, W. E., J. Ehrenfeld, E. Brochiero, and N. K. Wills. Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells. J. Membr. Biol. 144: 81-91, 1995[Medline].

17.   Cuppolatti, J. D., F. Aures, and G. Sachs. The lysosomal H+ pump; 8-azido-ATP inhibition and the role of chloride in H+ transport. Biochim. Biophys. Acta 899: 276-284, 1987[Medline].

18.   Diaz, M., M. A. Valverde, C. F. Higgins, C. Rucareanu, and F. V. Sepulveda. Volume-activated chloride channels in Hela cells are blocked by verapamil and dideoxyforskolin. Pflügers Arch. 422: 347-353, 1993[Medline].

19.  Di Stefano, A., M. Wittner, E. Schlatter, H. J. Lang, H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker of the Cl- conductive pathway in Cl-- transporting epithelia. Pflügers Arch. 405, Suppl.: S95-S100, 1985.

20.   Good, D. W. Hyperosmolality inhibits bicarbonate absorption in rat medullary thick ascending limb via a protein-tyrosine kinase-dependent pathway. J. Biol. Chem. 270: 9883-9889, 1995[Abstract/Free Full Text].

21.   Grunewald, J. M., R. W. Grunewald, and R. K. H. Kinne. Ion content and cell volume in isolated collecting duct cells: effect of hypotonicity. Kidney Int. 44: 509-517, 1993[Medline].

22.   Hilden, S. A., C. A. Johns, and N. E. Madias. Cl--dependent ATP-driven transport in rabbit renal cortical endosomes. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F885-F897, 1988[Abstract/Free Full Text].

23.   Husted, R. F., K. A. Volk, R. D. Sigmund, and J. B. Stokes. Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator. J. Clin. Invest. 95: 644-650, 1995.

24.   Kapus, A., S. Grinstein, S. Wasan, R. Kandasamy, and J. Orlowski. Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation. J. Biol. Chem. 269: 23544-23552, 1994[Abstract/Free Full Text].

25.   Kizer, N. L., B. Lewis, and B. A. Stanton. 1995. Electrogenic sodium absorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2). Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995[Abstract/Free Full Text].

26.  Kobayashi, E., H. Nakano, M. Morimoto, and T. Tamaoki. Calphostin C (UCN-1028C), a novel microbial compound is a highly potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 15: 159, 548-553, 1989.

27.   Krall, M., E. Feifel, J. P. Geibel, and W. Pfaller. Functional expression of K+/H+-ATPase in MDCK cells (Abstract). J. Am. Soc. Nephrol. 6: 310, 1995.

28.   Kubitz, R., R. Warth, N. Allert, K. Kunzelmann, and R. Greger. Small- conductance chloride channels induced by cAMP, Ca2+, and hypotonicity in HT29 cells: ion selectivity, additivity and stilbene sensitivity. Pflügers Arch. 421: 447-454, 1992[Medline].

29.   Lowance, D. C., and W. B. Schwartz. The effect of chronic hypotonic volume expansion on the renal regulation of acid-base equilibrium. J. Clin. Invest. 51: 2928-2940, 1972.

30.   Nilius, B., J. Sehrer, F. Viana, C. De Greef, L. Raeymaekers, and J. Eggermont. Volume-activated Cl- currents in different mammalian nonexcitable cell types. Pflügers Arch. 428: 364-371, 1994[Medline].

31.   Nördstrom, T., S. Grinstein, G. F. Brisseau, M. F. Manolson, and O. D. Rotstein. Protein kinase C activation accelerates proton extrusion by vacuolar-type H+-ATPases in murine peritoneal macrophages. FEBS Lett. 350: 82-86, 1994[Medline].

32.   Ono, S., J. Guntupalli, and T. D. Dubose. Role of H+-K+-ATPase in pHi regulation in inner medullary collecting duct cells in culture. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F852-F861, 1996[Abstract/Free Full Text].

33.   Rauchman, M. I., S. K. Nigam, E. Delpire, and S. R. Gullans. An osmotically tolerant medullary collecting duct cell line from an SV40 transgenic mouse. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F416-F424, 1993[Abstract/Free Full Text].

34.   Richardson, R. M. A., and R. T. Kunau. Bicarbonate reabsorption in the papillary collecting duct: effect of acetazolamide. Am. J. Physiol. 243 (Renal Fluid Electrolyte Physiol. 12): F74-F80, 1982.

35.   Roos, A., and W. F. Boron. Intracellular pH. Physiol. Rev. 61: 296-434, 1981[Free Full Text].

36.   Schwiebert, E. M., J. W. Mills, and B. A. Stanton. Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line. J. Biol. Chem. 269: 7081-7089, 1994[Abstract/Free Full Text].

37.   Soleimani, M., C. Bookstein, J. A. McAteer, Y. J. Hattabaugh, G. L. Bizal, M. W. Musch, M. Villereal, M. C. Rao, R. L. Howard, and E. B. Chang. Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells. J. Biol. Chem. 269: 15613-15168, 1994[Abstract/Free Full Text].

38.   Soleimani, M., G. Singh, G. L. Bizal, R. S. Gullans, and J. A. McAteer. Na+/H+ exchangers isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization and differential regulation. J. Biol. Chem. 269: 27973-27978, 1994[Abstract/Free Full Text].

39.   Sonnenberg, H. Secretion of salt and water into the medullary collecting duct of Ringer-infused rats. Am. J. Physiol. 228: 565-568, 1975.

40.   Sonnenberg, H. Medullary collecting-duct function in antidiuretic and in salt- or water-diuretic rats. Am. J. Physiol. 226: 501-506, 1974.

41.   Suguru, N., H. Amlal, M. Soleimani, and J. H. Galla. Pathways for HCO-3 reabsorption in mouse medullary collecting duct segments (Abstract). J. Am. Soc. Nephrol. 8: 8, 1997.

42.   Tamaoki, T., H. Nomoto, I. Takahashi, M. Morimoto, and F. Tomita. Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402, 1986[Medline].

43.   Trifaro, J., M. Vatate, and A. R. del Castil. Cytoskeleton and molecular mechanisms in neurotransmitter release by neurosecretory cells. Eur. J. Pharmacol. 225: 83-104, 1992[Medline].

44.   Verme, T. B., R. T. Velarde, R. M. Cunningham, and S. R. Hootman. Effects of staurosporine on protein kinase C and amylase secretion from pancreatic acini. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G548-G553, 1989[Abstract/Free Full Text].

45.   Volk, K. A., Z. Chong, R. F. Husted, and J. B. Stokes. Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F552-F559, 1996[Abstract/Free Full Text].

46.   Zhang, K., Z. Wang, and S. Gluck. Identification and partial purification of a cytosolic activator of vacuolar H+-ATPases from mammalian kidney. J. Biol. Chem. 267: 9701-9705, 1992[Abstract/Free Full Text].

Am J Physiol Renal Physiol 275(4):F487-F501


This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
M. E. Handlogten, S.-P. Hong, C. M. Westhoff, and I. D. Weiner
Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)
Am J Physiol Renal Physiol, August 1, 2005; 289(2): F347 - F358.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. E. Handlogten, S.-P. Hong, C. M. Westhoff, and I. D. Weiner
Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3)
Am J Physiol Renal Physiol, October 1, 2004; 287(4): F628 - F638.
[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. Cell Physiol.Home page
S. Nakamura
Glucose activates H+-ATPase in kidney epithelial cells
Am J Physiol Cell Physiol, July 1, 2004; 287(1): C97 - C105.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
Y.-H. Kim, J. Kim, A. S. Verkman, and K. M. Madsen
Increased expression of H+-ATPase in inner medullary collecting duct of aquaporin-1-deficient mice
Am J Physiol Renal Physiol, September 1, 2003; 285(3): F550 - F557.
[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 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 Google Scholar
Google Scholar
Right arrow Articles by Amlal, H.
Right arrow Articles by Soleimani, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amlal, H.
Right arrow Articles by Soleimani, M.

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