Differential activities of H+ extrusion systems in MDCK cells due to extracellular osmolality and pH

Elisabeth Feifel1, Markus Krall1, John P. Geibel2, and Walter Pfaller1

1 Institute of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria; and 2 Department of Surgery and Cellular and Molecular Physiology, Yale University, Medical School, New Haven, Connecticut 06510-8026

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The aim of the present study was to obtain detailed information on MDCK cell proton secretion characteristics under various growth conditions. Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4; 300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8) corresponding to the luminal fluid composition of the collecting duct segments found in the in renal cortex, the outer stripe of outer medulla and inner medulla. Proton fluxes were determined from the recovery of intracellular pH following an acid load induced by an NH4Cl pulse times the corresponding intrinsic buffering power (beta i). The intracellular buffering power was found to change only with culture medium osmolality but not with culture medium pH. In addition to an amiloride and Hoe-694-sensitive Na+/H+ exchange, Madin-Darby canine kidney (MDCK) cells possess a Sch-28080-sensitive, K+-dependent H+ extrusion mechanism that is increased upon adaptation of monolayers to hyperosmotic-acidic culture conditions. A significant contribution of the bafilomycin A1-sensitive vacuolar H+-ATPase could be found only in cells adapted to hyposmotic culture conditions. Exposure of MDCK cells to 10-5 or 10-7 M aldosterone for either 1 or 18 h did not alter the H+ extrusion characteristics significantly. The results obtained show that different extracellular osmolality and pH induce different MDCK phenotypes with respect to their H+-secreting systems.

proton-adenosinetriphosphatase; potassium-proton-adenosinetriphosphatase; Sch-28080; bafilomycin A1; intracellular pH; buffering capacity

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MADIN-DARBY CANINE KIDNEY (MDCK) cells, a widely used renal epithelial cell line, were originally isolated from an unknown site along the dog nephron. They form epithelial monolayers and, when grown on impermeable supports, develop domes resulting from unidirectional transport of solutes and water. These phenomena have led to the postulation that the cells originate from distal nephron cells (7). However, morphological as well as functional data exist [the expression of a carbonic anhydrase activity (19) and the ability to either secrete bicarbonate or protons (17)] which indicate that MDCK cells could be of collecting duct origin (27, 19). This idea is supported by the distribution of binding pattern (8) of peanut agglutinin (PNA) and wheat germ agglutinin, described as specific markers for intercalated (IC) and principal cells (PC) of the rabbit collecting duct, respectively (11, 13). When cultivated using standard culture conditions (300 mosmol/kgH2O and pH 7.4), PNA binding predominates in MDCK monolayers and is especially pronounced in dome-forming cells, which have been demonstrated to secrete bicarbonate (17). Morphologically these cells appeared as "dark," mitochondria-enriched cells (20). In subsequent investigations involving hyperosmotic-acidic growth conditions, mimicking the environment of the outer medullary collecting duct, PNA binding is abolished (8), but the number of electron microscopically "dark," mitochondria-enriched cells increases (20). From these observations, it was hypothesized that the dark, PNA-positive cells may resemble beta -IC cells, and the dark, PNA-negative MDCK cells may be related to H+-secreting alpha -IC-like cells. Surface pH measurements on these two respective dark cell types using pH microelectrodes appear to support this hypothesis (20). MDCK cells also exhibit an amiloride-sensitive Na+/H+ and a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange, particularly in dome-forming cells (17). Although the Na+/H+ exchange (NHE) plays an important role in MDCK cell H+ secretion (17, 28), additional evidence exists for Na+-independent H+ extrusion mechanisms (17, 18). Therefore, an active, rheogenic V-type H+-adenosinetriphosphatase (H+-ATPase) and/or an electrically silent, K+-dependent P-type ATPase previously found in the collecting duct (1, 10) may be potentially involved in MDCK proton extrusion.

The present study was designed to discriminate between the various mechanisms of acid secretion in MDCK monolayers and to functionally prove the existence of a K+-H+-ATPase and/or a rheogenic H+-ATPase and to further dissect the contribution of these systems to MDCK proton extrusion. For this purpose cells were adapted to hyposmotic and isosmotic growth conditions at pH 7.4, indicative of the primarily HCO<SUP>−</SUP><SUB>3</SUB>-secreting cortical collecting duct (CCD) regions, or to hyperosmotic and acidic growth conditions typical of the collecting duct transition segment from the outer to inner medulla.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Wild-type MDCK cells (American Type Culture Collection, CCL-34) from passage 66-95 were used for all experiments. Cultures were grown on glass coverslips in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin. After reaching confluence, cultures were adapted for a further 48 h to either isosmotic (300 mosmol/kgH2O, pH 7.4), hyperosmotic-acidic (600 mosmol/kgH2O, pH 6.8), or hyposmotic (200 mosmol/kgH2O, pH 7.4) culture conditions. In an additional series, MDCK cells were exposed to either hyperosmotic culture conditions (600 mosmol/kgH2O) at pH 7.4 or acidic culture conditions (300 mosmol/kgH2O) at pH 6.8. Bicarbonate concentration and resulting pH of the culture media were calculated for a constant, humidified incubator atmosphere at 5% CO2 (PCO2 of 35 mmHg; total CO2 of 1.05 mM). Adjustment of medium pH was achieved by addition or omission of NaCl and NaHCO3, making up 44 mM in total, corresponding to the concentration of the original formulation of DMEM (for pH 7.4, 21 mM NaHCO3 and 23 mM NaCl; for pH 6.8, 5 mM NaHCO3 and 39 mM NaCl). Osmolality was corrected by addition or omission of NaCl. In a second series of experiments, cells were exposed to either FCS-free medium supplemented with 10-5 or 10-7 M aldosterone for 1 h or 18 h, respectively, or only to FCS-free medium as a control.

Fluorescence measurements of intracellular pH. All experiments were performed in the absence of bicarbonate to avoid the participation of the HCO<SUP>−</SUP><SUB>3</SUB>/Cl- exchanger or other potentially bicarbonate-dependent processes. All intracellular pH (pHi) measurements were carried out in isosmotic solutions. Glass coverslip-grown cells were loaded with 3 µM of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes) for 15 min. Coverslips were washed twice in control buffer (solution 1) and mounted into a quartz cuvette at an angle of 60°. Ratiometric BCECF fluorescence (excitation 505/439 nm, emission 535 nm) measurements were continuously taken with a spectrofluorometer (F-4500, Hitachi) equipped with a thermostatically controlled cuvette holder (37°C) over a period of 20 min. H+ extrusion was obtained by determining the pHi recovery rate using the NH4Cl prepulse technique (solution 2, for 2 min) (2). The pHi recovery was then monitored in different solutions (Table 1) over a 12-min period. To discriminate between Na+- and/or K+-dependent and Na+- and K+-independent proton extrusion mechanisms, pHi changes were measured under standard conditions (solution 1) or under Na+-free (solution 3) or K+-free conditions (solution 4), respectively. To further delimit the contribution of a putative K+-H+-ATPase and/or H+-ATPase, measurements were carried out in the presence of Sch-28080 (1-3 × 10-4 M, Schering), a known specific, competitive inhibitor of the gastric K+-H+-ATPase (29), and bafilomycin A1 (1 × 10-6 M, Sigma), an inhibitor of the V-type H+-ATPase (3). Hoe-694 (1 × 10-4 M, Hoechst) (21) and amiloride (1 × 10-4 to 2 × 10-3, Sigma), both inhibitors of Na+/H+ exchangers, were used to inhibit the NHEs. To block Na+-K+-ATPase and to exclude potentially involved, ouabain-sensitive isoforms of the K+-H+-ATPase, all solutions were prepared with 1 × 10-3 M ouabain (Sigma). At the end of each measurement, BCECF calibration curves were obtained using the nigericin and high-K+ method (26). The calibration solutions were composed of 140 mM KCl, 10 mM NaCl, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and pH was adjusted to 7.8 and 6.8, respectively. Nigericin (Sigma) was prepared as a stock solution and added just before use in a final concentration of 10-5 M. Following withdrawal of NH4Cl, the pHi recovery rates (Delta pHi/Delta t, pH units/s) were measured once from the nadir pHi and from the steepest slope, if a further increase in Delta pHi/Delta t could be observed during pHi recovery.

                              
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Table 1.   Composition of solutions used for determination of pHi recovery

Determination of buffering capacity and H+ fluxes. The NH4Cl prepulse technique was also used to determine the intrinsic buffering power (beta i) (mM/pHiU) for all adaptation protocols listed above (2). The cells were exposed to a Na+-free bath (solution 3) containing Sch-28080, bafilomycin A1 and including 20 mM NH4Cl, which was then stepwise reduced to 0 mM (10, 5, 2.5, 1, 0.5 mM). Calculation of beta i was performed according to the formula beta i = &Dgr;[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB>/Delta pHi, where intracellular NH+4 concentration (&Dgr;[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB>) was calculated from the Henderson-Hasselbalch equation on the assumption that [NH3]i = [NH3]o. H+ fluxes (JH+, in mmol · min-1 · l-1) were determined as the product of recovery rate and the respective buffer capacity as JH+ = Delta pHi/Delta t × beta i.

With respect to variations in experimental results using cells from different passage number and day, paired experiments were performed on the same day with identically treated cells on separate coverslips. The data of JH+ changes per minute obtained from the same adaptation protocol and measurement condition were compared with the Wilcoxon two-tailed rank sum test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Buffer capacity dependence on osmolalities and pH. As shown in Fig. 1 a decrease of pHi induced by a stepwise reduction of extracellular NH4Cl increases beta i for all culture conditions as expected. The slope of this beta i increment depends on the osmolality but not on adaptation to a lowered pH in the culture media. Hyposmotic culture conditions induce an increase of beta i at low pHi, whereas hyperosmotic culture media induce a decrease of beta i, when pHi determinations were performed in isosmotic measurement solutions. The beta i values were utilized to determine the proton fluxes (JH+) as described above.


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Fig. 1.   Intracellular pH (pHi) dependence of buffering capacity in MDCK cells under different growth conditions. Plots of intrinsic buffering power (beta i) were obtained by stepwise lowering of the extracellular NH4Cl (20-0 mM). Solid line, regression of second order through originally calculated beta i values. Dashed lines, 95% confidence interval. Values of beta i were obtained for of MDCK cells adapted to growth culture conditions that were hyposmotic (200 mosmol/kgH2O, pH 7.4; A), isosmotic (300 mosmol/kgH2O, pH 7.4; B), isosmotic-acidic (300 mosmol/kgH2O, pH 6.8; C), hyperosmotic (600 mosmol/kgH2O, pH 7.4; D), or hyperosmotic-acidic (600 mosmol/kgH2O, pH 6.8; E).

The recovery rates proceeding from the nadir pHi after removal of NH4Cl in Na+-free measuring solution 3 as well as in solutions containing the various blockers differed from maximal recovery rates. The respective H+ fluxes, however, are nearly identical due to the increment in pHi over the recovery period, which is paralleled by a decrease of beta i.

Adaptation to different osmolality and pH. Adapting confluent monolayers of MDCK cells to hyperosmotic-acidic (600 mosmol/kgH2O, pH 6.8), isosmotic (300 mosmol/kgH2O, pH 7.4), and hyposmotic (200 mosmol/kgH2O, pH 7.4) media for 48 h leads to different intracellular resting pHi values. After reexposure of the cells to the isosmotic extracellular solutions at a pH of 7.4 for fluorescence measurements (solution 1), MDCK cells grown under isosmotic conditions display a pHi of 7.48 ± 0.01 (±SE; n = 43 coverslips) (23, 28), whereas monolayers adapted to hyperosmotic-acidic conditions show a pHi of 7.37 ± 0.015 (n = 43) and cells adapted to hyposmotic conditions display a resting pHi of 7.54 ± 0.02 (n = 43). Furthermore, these differences in pHi values are maintained following removal of NH4Cl. It should be noted that the cells adapted to hyperosmotic-acidic culture conditions approach a minimal pHi of 6.56 ± 0.02 SE after NH4Cl removal. The response to acidification of cells kept under isosmotic conditions result in a slightly higher pHi (6.65 ± 0.022). In hyposmotically adapted cells, a pHi of only 6.83 ± 0.026 is achieved by NH4Cl prepulsing (Fig. 2).


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Fig. 2.   Three representative tracings of ratiometric fluorescence measurements: full pHi recovery to baseline following an acid prepulse (20 mM, NH4Cl) in presence of 5 mM K+ and 135 mM Na+ (solution 1). MDCK cell monolayers are grown under 3 different culture conditions: 1) hyposmotic-pH 7.4, 2) isosmotic-pH 7.4, and 3) hyperosmotic-pH 6.8. Trace 1: highest resting pHi, highest alkalinization during NH4Cl load, weakest acidification, and most shallow recovery. Trace 2: slightly lower resting pHi, alkalinization during NH4Cl load, and acidification, but a more pronounced recovery. Trace 3: lowest resting pHi and alkalinization during NH4Cl load, but a stronger acidification, and the most pronounced recovery.

Complete recovery after NH4Cl prepulse to the initial resting pHi is achieved in solution 1 within 5 min in all types of adaptation. Return to initial pHi values is accelerated in hyperosmotically cultured cells compared with isosmotically and hyposmotically grown cells (Fig. 2).

JH+ in response to different extracellular Na+ and K+ concentrations. Omission of K+ from the measuring solution (solution 4) results in consistent reduction of proton fluxes. Removal of extracellular Na+ (solution 3) results in a significant (P <=  0.05) reduction of proton extrusion rates to less than 2 mmol/min for all adaptation protocols examined (Fig. 3).


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Fig. 3.   H+ fluxes of MDCK monolayers grown on glass coverslips after NH4Cl prepulse. Measurements were performed either in control solution (solution 1, Table 1, solid bars) or under K+-free (solution 4, Table 1, open bars) or Na+-free (solution 3, Table 1, cross-hatched bars) measurement conditions. H+ fluxes are of MDCK monolayers adapted to hyposmotic (A), isosmotic (B), or hyperosmotic-acidic (C) culture conditions. Bar means of original data ± SE. * Statistically significant differences from the corresponding control (P <=  0.05).

JH+ in the presence of inhibitors. The addition of ouabain does not influence H+ fluxes (data not shown). To discriminate between the contribution of Na+/H+ exchangers, K+-H+-ATPase, and a rheogenic H+-ATPase, different inhibitors were applied during the pHi recovery period. In experiments where the H+ extrusion driven by the NHEs is eliminated (solution 3, Na+ free), a clearly recognizable inhibition of H+ extrusion by Sch-28080 (1 × 10-4 M) can be detected for all adaptation protocols investigated (Fig. 4, left). However, for hyperosmotically grown cells, a significant reduction can be achieved only in the presence of higher Sch-28080 concentrations (3 × 10-4 M) (Fig. 4, left). Sch-28080 also reduces H+ fluxes significantly when applied under conditions where NHE-activity is normal (solution 1, including Na+) (Fig. 4, right). The competitive mode of action is clearly shown by the return of proton extrusion upon removal of Sch-28080 from the measuring solution (Fig. 5).


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Fig. 4.   H+ fluxes of MDCK monolayers after NH4Cl prepulsing: H+ fluxes of cells adapted to hyposmotic (A), isosmotic (B), or hyperosmotic-acidic (C) growth conditions. Left: remaining proton fluxes after inhibiting NHEs by removing of Na+ from measuring solution (solid bars) are further diminished by either addition of bafilomycin A1 (10-7 M, open bars) or Sch-28080 (1 × 10-4 M, dashed bars, 3 × 10-4 M, coarsed bar). The higher concentration of Sch-28080 did not further reduce proton fluxes in A or B. * Statistically significant differences in H+ fluxes induced by the added inhibitors corresponding to fluxes recorded under 0 mM Na+ conditions (P <=  0.05). Middle: proton fluxes recorded either after removal of Na+ from measuring solution (solid bars) or in presence of the specific NHE inhibitors amiloride (10-4 M) and/or Hoe-694 (10-4 M). * Statistically significant differences of proton fluxes determined under 0 mM Na+ conditions compared with the effect of the inhibitors (P <=  0.05). Right: H+ fluxes in solution 1 (solid bars) compared with H+ fluxes in solution 1 containing Sch-28080 (1 × 10-4 M), where the Na/H exchange is normal (P <=  0.05).


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Fig. 5.   Recordings of ratiometric fluorescence measurement obtained from hyperosmotically adapted MDCK monolayers showing the contribution of NHEs, H+-ATPase, and K+-H+-ATPase to the total H+ extrusion. Trace 1: complete pHi recovery in presence of 5 mM K+ and 135 mM Na+ (solution 1). Trace 2: pHi recovery in absence of Na+. Trace 3: competitive effect of Sch-28080 (3 × 10-4 M). Washout of Sch-28080 again allows pHi recovery via Na+-independent systems. Trace 4: inhibition of Na+-independent H+ extrusion when Sch-28080 is applied after prepulsing.

In contrast to the competitive inhibition of Sch-28080, the addition of the H+-ATPase inhibitor bafilomycin A1, in the absence of extracellular Na+, reveals a profound reduction of the proton extrusion only in MDCK cells adapted to hyposmotic culture conditions. No statistically significant effect of bafilomycin A1 is observed for cells adapted to isosmotic or hyperosmotic-acidic culture media (Fig. 4, left).

To further elucidate the level of contribution of NHEs to H+ secretion, NHE inhibitors Hoe-694 and amiloride were applied during the pHi recovery period. The H+ fluxes in the presence of these blockers are markedly different from those obtained in Na+-free solution only with respect to hyperosmotic-acidic and isosmotic cells. Absence of Na+ in the measurement solution leads to lower proton fluxes compared with those achieved under Hoe-694 (10-4 M) and amiloride (2 × 10-3 M) (Fig. 4, middle, and Fig. 6). Only the hyposmotic adaptation of MDCK cells results in H+ fluxes similar to that found in Na+-free measuring solution (Fig. 4, middle). The effects of the two NHE inhibitors, however, do not differ significantly. Furthermore, when Hoe-694 and amiloride were applied in combination, no additive effect can be observed with respect to a reduction in H+ extrusion (Fig. 4, middle).


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Fig. 6.   Original recordings of fluorescence measurements obtained from isosmotically adapted MDCK monolayers showing pHi recoveries under 5 mM K+ and 135 mM Na+ (trace 1) and 5 mM K+ and 135 mM Na+ including 1 × 10-4 M Hoe-694 (trace 2) in comparison to pHi recovery under Na+-independent measuring conditions (tracing 3). Hoe-694 does not inhibit pHi recovery to an extent as achieved by elimination of Na+-dependent H+ extrusion in Na+-free solution.

Culture medium osmolality and pH have adverse effects on MDCK H+ extrusion (Fig. 7). Increasing culture medium osmolality enhances H+ extrusion, whereas lowering medium pH during adaptation diminishes proton extrusion (in Fig. 7, compare A to B, B to C, and D to E).


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Fig. 7.   Proton fluxes of MDCK monolayers adapted to 5 different culture conditions. A: 200 mosmol/kgH2O, pH 7.4. B: 300 mosmol/kgH2O, pH 7.4. C: 300 mosmol/kgH2O, pH 6.8. D: 600 mosmol/kgH2O, pH 7.4. E: 600 mosmol/kgH2O, pH 6.8. For each measurement protocol, 3 paired experiments are presented to differentiate whether the changes of H+ fluxes seen under E are caused by either hyperosmotic (D) or acidic (C) adaptation. Presented are recovery rates after NH4Cl prepulsing under control (solid bars), after inhibition of NHEs (open bars) with further blockade of V-type H+-ATPase (dashed bars) or K+-H+-ATPase (coarsed bars). * Statistically significant differences between control and Na+-free proton extrusion. # Significant differences between recovery rates under Na+-free conditions and Na+-free conditions plus a specific inhibitor (P <=  0.05).

The mineralocorticoid aldosterone applied in two different concentrations (10-5, 10-7 M) and for two different time intervals (1 h and 18 h) did not markedly affect the proton extrusion of MDCK monolayers. A marginal tendency toward an elevation in H+ flux rates was found (data not shown).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the collecting duct of the mammalian kidney, a potential source of the MDCK cell line, at least two morphologically discernible cell types, namely, PC cells and IC cells, which differ in structure and function (11, 15), have been identified. Three different proton extrusion mechanisms have been described (for review see Refs. 22 and 25) for the IC cells, whose primary function is urinary acidification; Na+/H+ exchanger(s) (14, 16), a bafilomycin A1-sensitive H+ pump, as well as a Sch-28080-sensitive K+-H+-ATPase (1, 10, 24, 30) have all been functionally identified in this cell type. The present study reports functional evidence for the presence of all of these proton extrusion mechanisms in MDCK monolayers. Furthermore, the data presented suggest that the activities of these H+ secretion mechanisms differ depending on the composition of the extracellular culture media to which the MDCK cells were exposed.

We chose hypo-, iso-, and hyperosmotic-acidic adaptations in an effort to create culture conditions mimicking the environments of the following tubules: the superficial renal distal convoluted and CCD segment, the outer stripe of outer medulla collecting duct segment, as well as that of the collecting duct segments contained in the outer-inner medullary transition region. Previous experiments (20) have shown that these maneuvers induce changes in MDCK phenotype and thus should result in differences of H+ secretion. Hyposmotically adapted cells were expected to differentiate toward cells with functional characteristics similar to beta -IC cells in the CCD, whereas hyperosmotic-acidic MDCK cultures should result in a phenotype similar to the acid-secreting alpha -IC-like cells (15). In addition, isosmotic-acidic and hyperosmotic-pH 7.4 adaptations were investigated to differentiate between changes in H+ secretion observed following exposure of cells to hyperosmotic-acidic culture conditions. With these modifications in the extracellular growth solution, we felt confident that the question of whether acidification and/or changes of medium osmolality are responsible for the alterations of H+ extrusion could be answered.

Since net H+ extrusion can be related to pHi recovery only if intracellular buffering power stays constant, we have experimentally assessed changes in beta i under the various culture conditions used in the present study. The results obtained (Fig. 1) imply that the differences found must be attributed primarily to changes in extracellular medium osmolality.

From the results depicted in Fig. 3, it is evident that NHEs represent the major mechanism for MDCK cell H+ secretion. The contribution of the other acid extruders, the vacuolar H+-ATPase and the K+-H+-ATPase depends predominantly on the form of adaptation imposed on the confluent MDCK monolayers.

Omission of Na+ from the measuring solution was chosen to maximally suppress all Na+-driven H+ extrusion mechanisms. From the data shown in Fig. 6 it could be suggested that, in addition to the amiloride and Hoe-964-sensitive "housekeeping" NHE-1 (17, 23, 27), MDCK cells may also contain other Na+/H+ exchanger isoforms. This was supported by the less-pronounced effect of Hoe-694 and/or amiloride on the pHi recovery compared with that under Na+-free measurement conditions. Further support for participation of other NHEs is the finding that these differences were only found following hyperosmotic-acidic and isosmotic pretreatment (Fig. 4, middle). In contrast to other adaptations, the hyposmotic pretreatment may reduce the activity of NHE isoforms to an extent, that the inhibitor concentrations used were sufficient to inhibit all NHEs. Alternatively, hyposmotic pretreatment may suppress the expression of NHE-3.

Participation of a bafilomycin A1-sensitive vacuolar H+-ATPase could unambiguously be recovered only for MDCK monolayers adapted to hyposmotic growth conditions comparable to the apical cellular environment given in the outermost CCD portions (Fig. 4, left). It could be assumed that an active H+ secretion driven by a V-type H+-ATPase in the model presented is confined only to cells with functional characteristics similar to beta -IC-like cells, which have an increased frequency in the monolayer population following this type of adaptation (20). Indeed, a V-type H+-ATPase localization in beta -IC cells at the basolateral membrane domain of CCDs has been previously described (6, 12). However, attempts to localize this enzyme immunocytochemically in MDCK cells using antibodies against the 70- and 56-kDa subunits of bovine V-type H+-ATPase have failed (20), probably due to poor cross-reactivity between the bovine antibodies and MDCK cell protein.

If one assumes that MDCK cells resemble IC of the collecting duct cells, then these should potentially contain a K+-H+-ATPase as another H+ extruder in addition to NHEs and vacuolar H+-ATPase as reported for the IC cells of the mammalian collecting duct (1, 10, 30). The first evidence for the possible existence of such a K+-H+ pump was previously reported, showing an omeprazole-induced inhibition of net H+ current in dome-forming cells of MDCK monolayers (18). According to observations on the mammalian outer/inner medullary collecting duct, K+-dependent mechanisms significantly contribute to H+ secretion (10, 30). The maneuver of hyperosmotic-acidic adaptation, outlined above, should thus result in MDCK phenotypes with a clearly discernible K+-dependent H+ extrusion. Figure 3 shows that removal of K+ induced a reduction of H+ extrusion in MDCK cell acid secretion. Since, after blockade of all NHEs using a Na+-free extracellular measuring solution, a considerable H+ secretion capacity remains, the data suggest that other extrusion mechanisms such as the K+-H+-ATPase and/or H+-ATPase are activated or unmasked. That hyperosmotic-acidic cells indeed possess a higher K+-H+-ATPase activity can be concluded from the fact that Sch-28080 concenrations of  3 × 10-4 M were needed to maximally reduce JH+. Following iso- and hyposmotic exposure, maximum reduction was already achieved with 1 × 10-4 M Sch-28080 (Fig. 4, left). Such a Sch-28080-sensitive JH+ flux upon an acid load in the absence of extracellular Na+ strongly corroborates the view of MDCK cells being related to cells provided with IC cells cell properties. Support for this interpretation is given by the recent observation of Silver and Frindt (24), who reported a Sch-28080-sensitive, Na+ independent recovery of pHi after an acid load only for collecting duct IC cells and not for PCs.

The investigations performed show that MDCK monolayers adapted to hyperosmotic-acidic (pH 6.8) culture media display the steepest slope of pHi recovery followed by cells adapted to isosmotic and hyposmotic culture conditions (Fig. 2). This behavior is also given for H+ fluxes driven by the amiloride and/or Hoe-694-sensitive Na+/H+ exchange and the Sch-28080-sensitive H+ extrusion.

For the hyposmotically adapted cells, Na+-independent H+ secretion appears largely to be the result of a bafilomycin A1-sensitive V-type H+-ATPase, which obviously does not contribute to acid secretion to the same extent in the other two experimental protocols investigated (Fig. 3 and Fig. 4, left and middle).

The counteracting effects of increasing medium osmolality and decreasing pH on H+ extrusion (Fig. 7) cannot yet be explained conclusively. Potentially, part of the further decrease of culture medium pH may be caused by the cells themselves during the adaptation period to an acid media pH resulting in values below 6.8 (the initial value of the culture medium).

Previous investigations on MDCK cells have shown a stimulation of the NHEs upon exposure to aldosterone (17). In the present investigation no prominent effect subsequent to aldosterone administration could be observed, regardless of whether or not aldosterone was applied over different periods of time (1 h or 18 h) or at different concentrations (10-5M or 10-7 M). This would agree with a recent report claiming the absence of aldosterone receptors in IC cells (4, 5, 9) and thus support the view of MDCK cells resembling IC cell equivalents or alternatively, that MDCK cells do not possess the required cellular machinery necessary to process aldosterone. Furthermore, it cannot be excluded that in contrast to earlier investigations (18) MDCK cells used in this study did not respond to aldosterone due to passage number and/or altered state of differentiation.

From the results obtained in this study, it can be concluded that the major component of H+ secretion in MDCK monolayer cultures is mediated by NHEs. A K+-dependent Sch-28080-sensitive H+ extrusion contributes to acid secretion in MDCK cells grown under all culture conditions with a clear enhancement after adaptation to hyperosmotic-acidic media. The contribution of a bafilomycin A1-sensitive V-type H+-ATPase, considered to be the major extrusion system in the mammalian collecting duct, seems to play a significant role for acid secretion in MDCK cultures only when they are adapted to hyposmotic culture conditions.

    ACKNOWLEDGEMENTS

We thank Drs. P. Dietl, F. Lang, and N. P. Curthoys for critical comments.

    FOOTNOTES

This work was supported by the Austrian Science Foundation Grants P-7968-MED and P-9259-MED.

Address for reprint requests: W. Pfaller, Fritz-Preglstr. 3, Institute of Physiology, Univ. of Innsbruck, A-6010 Innsbruck, Austria.

Received 16 January 1996; accepted in final form 18 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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