The maintenance of intracellular pH (pHi) is accomplished by a complex mechanism and involves several ion transporters in the plasma membrane (Na+/H+, H+-ATPase, H+/K+-ATPase, Cl-/HCO3- and Na+-HCO3-) as well as intracellular buffers.
A large number of investigations have indicated that in proximal tubules, low concentrations of angiotensin II (Ang II) stimulate, whereas high concentrations inhibit, the Na+/H+ exchanger1,2. This dual hormonal regulation was also recently demonstrated in our previous study in Madin-Darby canine kidney (MDCK) cells, a cell line with many morphological and physiological similarities with the mammalian distal nephron3. In contrast, the effect of Ang II on H+-ATPase has been the subject of some controversy. In proximal tubules4 and late distal segments5 of rat kidney, low concentrations of Ang II stimulate H+-ATPase. However, in rat cortical collecting duct, a segment that contains several structural components similar to those of the late distal tubule, Ang II (10-10 to 10-5 mol/L) causes a dose-dependent decrease in H+-ATPase activity6.
On the other hand, recent results suggest that an increase of cytosolic calcium ([Ca2+]i) might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H+-ATPase7. There is also considerable evidence showing that cell acidification stimulates a calcium-mediated exocytotic insertion of proton pumps, and that this process is important in regulating pHi8,9,10. Furthermore, atrial natriuretic peptide (ANP) has been shown to inhibit [Ca2+]i increases produced by Ang II in cultured mesangial cells11 and MDCK cells3, suggesting that there may be some interaction between these two vasoactive peptide hormones in the regulation of pHi by H+-ATPase. In addition, recently we found that in MDCK cells ANP impairs both stimulatory and inhibitory effects of Ang II on pHi regulation via the Na+/H+ exchanger3.
The purpose of the present investigation was to explore further the effects of Ang II (10-12, 10-9 or 10-7 mol/L) in regulating the process of pHi recovery mediated by H+-ATPase. pHi recovery was monitored in MDCK cells by using the fluorescent probe BCECF. The experiments were done in nominally HCO3-/CO2–free medium, after an acid load induced by NH4Cl, in presence of zero Na+ plus Schering 28080 (a specific inhibitor of H+/K+-ATPase), an experimental condition in which the H+-ATPase is the only mechanism of pHi recovery in activity12. Since the role of vesicle trafficking and exocytosis in the regulation of H+ transport in MDCK cells has been documented13,14, we also examined the effect of Ang II on [Ca2+]i. In addition, we studied the interaction of Ang II plus ANP (10-6 mol/L) or dimethyl-BAPTA/AM (5 
10-5 mol/L, an intracellular calcium chelator15) on pHi recovery and [Ca2+]i.
Our present results suggest a role of the increase of [Ca2+]i in regulating the dose-dependent stimulatory effect of Ang II on H+-ATPase. ANP or dimethyl-BAPTA/AM, by impairing the path causing the increase in [Ca2+]i, blocks this stimulatory effect of Ang II. This hormonal interaction observed in MDCK cells may represent a relevant mechanism of pHi regulation in the intact animal.
METHODS
Cell culture
Serial cultures of wild-type MDCK cells (passages 66-75; American Type Culture Collection, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle's medium [DMEM; Gibco, Grand Island, NY, USA; supplemented with 2 mmol/L glutamine, 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 
g/mL streptomycin] at 37°C, 95% humidified air (5% CO2, pH 7.4) in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL, USA). The cells were harvested with trypsin ethyleneglycol-bis (b-aminoethyl ether)-N, N'-tetraacetic acid (EGTA, 0.02%), seeded on sterile glass coverslips and then incubated again for 72 hours in the same medium to become confluent.
Measurement of pHi by fluorescence microscopy
Intracellular pH was monitored using the fluorescent probe 2',7'-biscarboxyethyl-5, 6-carboxyfluorescein (BCECF)3. Briefly, cells grown to confluence on glass coverslips were loaded by exposure for 20 minutes to 10 mol/L BCECF-AM in the control solution (solution 1, Table 1). After the loading period, the glass coverslips were placed into a thermo-regulated chamber mounted on an inverted epifluorescence microscope (Nikon, TMD). The measured area under the microscope had a diameter of 260 m and contained of the order of 40 cells. The coverslips remained in a fixed position, so that the same cells were studied throughout the experiment. All experiments were performed at 37°C. The cells were alternately excited at 440 or 490 nm with a 150 W xenon lamp and the fluorescence emission was monitored at 520 nm by a photomultiplier-based fluorescence system (Georgia Instruments, PMT-400) at time intervals of 5 seconds. The 490/440 excitation ratio corresponds to a specific pHi. At the end of each experiment, calibration of the BCECF signal was achieved by exposing the cells for 15 minutes to a K+-HEPES buffer solution containing 10 mol/L nigericin (solution 2; Table 1), at pH 6.5, 7.0 or 7.5.
Cell pH recovery
After the acidification of pHi by two minutes of exposure to 20 mmol/L NH4Cl (solution 3; Table 1)16, cell pH recovery was examined in presence of zero Na+ (solution 4; Table 1) plus Schering 28080 (1 10-5 mol/L), in the following situations: control, in presence of 10-12, 10-9 or 10-7 mol/L Ang II and/or 10-6 mol/L ANP or 5 10-5 mol/L dimethyl-BAPTA/AM. Since the rate of pH recovery depends on the value of cell pH achieved by the acid load17, we used experiments in which these values were not significantly different between the studied groups Table 2. None of the experiments showed a change in pH during the initial one to two minute period, and pH recovery started thereafter. We calculated the rate of pHi recovery (dpHi/dt, pH units per min) from the first two minutes after the start of the pHi recovery curve by linear regression analysis Figure 1.
Figure 1.
Intracellular pH (pHi) recovery after cellular acidification with the NH4Cl pulse technique in MDCK cells. (A) In the presence of 145 mmol/L Na+ external solution, the initial fall in pHi is followed by a recovery of pHi toward the basal value. (B) In the presence of zero Na+ external solution plus Schering 28080 (1 10-5 mol/L; a specific inhibitor of H+/K+-ATPase), the pHi recovery rate is markedly decreased and the final pHi was significantly different from the basal value; this effect is subsequently reversed with the return of 145 mmol/L Na+ solution to the bath. (C) In the presence of zero Na+ external solution plus Schering 28080, the addition of Ang II (10-12 mol/L) causes a significant increase in the velocity of pHi recovery, but the pHi recovery was not complete; with the return of 145 mmol/L Na+ solution to the bathing medium, the pHi recovery rate increases and the final pHi was not significantly different from the basal value. Abbreviations are: B, basal pHi; Sch, Schering 28080 (see text).
Table 2 - Summary of pHi responses in MDCK cells to the addition of different agents after an acute acid load.
Full table Measurement of [Ca2+]i by fluorescence confocal microscopy
Changes in [Ca2+]i were monitored fluorometrically by using the calcium-sensitive probe fluo 4-AM3. Briefly, confluent cultures were loaded with 10 mol/L fluo 4-AM at 37°C for 40 minutes and rinsed in Tyrode solution (solution 5; Table 1) containing 0.2% bovine serum albumin (BSA; pH 7.4). Fluo 4 fluorescence intensity emitted above 505 nm was imaged in vivo by using ultraviolet laser excitation at 488 nm on a Zeiss LSM 510 confocal microscope. The images were continuously acquired before and after substitution of experimental solutions, at time intervals of 10 seconds, for a total of 200 seconds. For each experiment the maximum fluorescent signal for 10 cells was averaged and then used for analysis. Transformation of the fluorescent signal to [Ca2+]i was performed by calibration with ionomycin (30 mol/L; the maximum Ca2+ concentration) followed by EGTA (2.5 mmol/L; the minimum Ca2+ concentration) according to the Grynkiewicz equation18.
Solutions and reagents
The composition of the solutions utilized is described in Table 1. These solutions had an osmolality of about 300 mOsm/kg H2O, which is the value found in the culture medium used for these cells. This osmolality was used to avoid changes of volume when the cells were transferred from the culture medium to the experimental solutions. Twenty-eight amino acid ANP was purchased from Bachem Fine Chemicals (New Haven, CT, USA) and Fluo-4/AM, BCECF-AM and dimethyl-BAPTA/AM from Molecular Probes (Eugene, OR, USA). Ang II (1046 molecular weight) and all other applied chemicals were obtained from Sigma Chemical Company (St. Louis, MO, USA).
Statistics
The results are presented as means 
SEM; (N) is the number of experiments. Data were analyzed statistically by analysis of variance (ANOVA) followed by the Bonferroni contrast test. Differences were considered significant if P < 0.05.
RESULTS
Intracellular pH
Figure 1a shows a representative experiment in which MDCK cells were first bathed with 145 mmol/L Na+ solution, exhibiting the basal pHi. After two minutes of exposure to NH4Cl, during which the cell pHi increased transiently, NH4Cl removal caused a rapid acidification of pHi as a result of NH3 efflux. In the presence of external 145 mmol/L Na+ the initial fall in pHi was followed by a recovery of pHi toward the basal value. This behavior has been shown before in MDCK cells, and evidence was presented suggesting that it was due to the activity of the Na+/H+ exchanger, H+/K+-ATPase and H+-ATPase12. Figure 1b indicates that, in presence of external zero Na+ plus Schering 28080, the pHi recovery rate was markedly decreased and the final pHi was significantly different from the basal value. This behavior was due to the inhibition of the activity of the Na+/H+ exchanger (by the removal of extracellular Na+) and of the H+/K+-ATPase (by the specific inhibitor, Schering 28080), the H+-ATPase being the only mechanism of pH recovery in activity12. Figure 1b also shows that this effect is subsequently reversed with the return of 145 mmol/L Na+ solution to the bathing medium. Thus, since the purpose of the present investigation was to study the effect of Ang II in regulating the process of pHi recovery mediated by H+-ATPase, in all our experiments the cell pH recovery was examined following the acidification of pHi with the NH4Cl pulse technique, in presence of zero Na+ plus Schering 28080. Figure 1c indicates that the addition of Ang II (10-12 mol/L) to the bath causes a significant increase in the velocity of pHi recovery via H+-ATPase, but during this experimental situation the pHi recovery was not complete. With the return of 145 mmol/L Na+ solution to the bathing medium, the pHi recovery rate increases and the final pHi was not significantly different from the basal value.
Table 2 summarizes the main values of pHi responses found in all the studied experimental groups. Our results indicate that MDCK cells in pH 7.4 HCO3--free solution had a mean baseline pHi of 7.16 0.03 (N = 83).
Figure 2 demonstrates the effect of Ang II (10-12, 10-9 or 10-7 mol/L) on the pHi recovery by H+-ATPase. In the control situation, the pHi recovery rate was 0.024 0.002 pH units/min (N = 16) and the final pHi was significantly different from the basal value (6.64 0.06 vs. 7.19 0.04; Table 2). The addition of Ang II (10-12, 10-9 or 10-7 mol/L) to the bath causes a dose-dependent significant increase of the velocity of pHi recovery (of about 63%, 108% and 163% of the control value, respectively) and during these situations the final pHi was still significantly different from the basal value Table 2.
Figure 2.
Effects of 10-12, 10-9 or 10-7 mol/L angiotensin II (Ang II) on the initial rate of pHi recovery mediated by H+-ATPase following acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na+ external solution plus Schering 28080. Symbols are: (
) Control; (
) Ang II. #P < 0.05 vs. Control.
The effects of ANP (10-6 mol/L) or Ang II (10-12 or 10-7 mol/L) plus ANP on the rate of pHi recovery are shown in Figure 3. With ANP alone the pHi recovery rate was not significantly different from the control value; however, ANP impairs the stimulatory effects of Ang II (10-12 or 10-7 mol/L).
Figure 3.
Effects of 10-6 mol/L ANP, and 10-12 or 10-7 mol/L Ang II plus 10-6 mol/L ANP on the initial rate of pHi recovery mediated by H+-ATPase following acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na+ external solution plus Schering 28080. Symbols are: () Control; (
) ANP; () Ang II; (
) Ang II + ANP. #P < 0.05 vs. Control; *P < 0.05 vs. 10-12 or 10-7 mol/L Ang II.
Figure 4 shows that with dimethyl-BAPTA/AM (5 10-5 mol/L) the pHi recovery rate was not significantly different from the control value. However, dimethyl-BAPTA/AM decreases significantly the stimulatory effects of Ang II (10-12 or 10-7 mol/L) on this process. A statistically significant difference was not encountered between the pHi recovery values measured during addition of ANP or dimethyl-BAPTA/AM Table 2.
Figure 4.
Effects of 5 10-5 mol/L dimethyl-BAPTA/AM, and 10-12 and 10-7 mol/L Ang II plus dimethyl-BAPTA/AM on the initial rate of pHi recovery mediated by H+-ATPase following acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na+ external solution plus Schering 28080. Symbols are: () Control; (
) dimethyl-BAPTA-AM; () Ang II; () Ang II + dimethyl-BAPTA/AM. #P < 0.05 vs. Control; *P < 0.05 vs. 10-12 or 10-7 mol/L Ang II.
Cytosolic calcium
Madin-Darby canine kidney cells exhibited a mean baseline [Ca2+]i of 100 10 nmol/L (N = 40; Table 3). Ang II (10-12, 10-9 or 10-7 mol/L) increased [Ca2+]i in a dose-dependent manner (by 45%, 80% or 134% of the control value, respectively). ANP 10-6 mol/L led to a significant decrease in [Ca2+]i of 60% of control value. In the presence of ANP, Ang II caused a recovery of [Ca2+]i without exceeding normal baseline values even at Ang II 10-7 mol/L. Table 3 also shows that dimethyl-BAPTA/AM (5 10-5 mol/L) led to a significant decrease in [Ca2+]i (of 51% of control value). In the presence of dimethyl-BAPTA/AM, the addition of Ang II caused only a partial recovery of [Ca2+]i.
Table 3 - Cytosolic free calcium concentration in MDCK cells subjected to 10-12, 10-9 or 10-7 mol/L Ang II alone or plus 10-6 mol/L ANP or 50 
mol/L dimethyl-BAPTA/AM.
Full table DISCUSSION
The aim of this study was to clarify the effects of 10-12, 10-9 and 10-7 mol/L Ang II in regulating the process of pHi recovery mediated by H+-ATPase. The experiments were done in MDCK cells, a permanent cell line originated from the renal collecting duct. According to the classification of Richardson et al19, there are two strains of this cell line: strain I, derived from early passages, from 60 to 70, with resistance over 300 ohm 
cm2; and the strain II from later passages, 100 to 110, with about 100 ohm cm2. In our present study, the MDCK cells were from passages 66 to 75 and exhibited a resistance of 320 ohm cm2; thus, our MDCK cells were from cell strain I according to Richardson et al19.
Valentich identified two cell types of MDCK cells strain I20. One type is ciliated, with light cytoplasm and few apical vesicles, showing similarities with the principal cells. The other is non-ciliated, with dark cytoplasm, apical vesicles and carbonic anhydrase activity, corresponding to the intercalated cells of the collecting duct. Devuyst et al also documented the heterogeneity of MDCK cells strain I by using immunocytochemical markers21. They found that 30% of these cells had peanut lectin binding capacity, two-thirds showed carbonic anhydrase activity, and 5% displayed band 3 immunoreactivity. These immunoreactive cells may correspond to intercalated cells, and would be the same cells where the H+-ATPase studied in the present work was localized. The heterogeneity of the strain I was confirmed by Gekle et al, who cloned two MDCK cell subtypes, C7 and C11, with different morphology and function22. The C7 subtype resembles principal cells of the renal collecting duct and exhibits a pHi of 7.39. The C11 subtype establishes a transepithelial Cl- and pH gradient; two-thirds of these cells exhibit peanut lectin binding capacity, thus resembling intercalated cells of the renal collecting duct, and they maintain a pHi at 7.16. Our present results demonstrate that MDCK cells in pH 7.4 HCO3--free solution maintain a mean baseline pHi of 7.16 0.03 (N = 83), a value compatible with the MDCK cell subtype C1122. However, we did not distinguish between the two cell subtypes present in our preparation.
According to Fernández and Malnic, the more important of the mechanisms of pHi recovery after an acid load in MDCK cells strain I is the Na+/H+ exchanger, since the removal of extracellular Na+ led to a 40% reduction in the rate of pHi recovery12. However, the reduction of extracellular K+ or the addition of Schering 28080 caused a significant reduction of Na+-independent pHi recovery, confirming the presence of a H+/K+-ATPase in these cells12, as had been shown earlier23,24. In addition, when using a low K+ extracellular solution plus concanamycin (a specific inhibitor of the vacuolar H+-ATPase) the Na+-independent pHi recovery was almost completely inhibited, showing the presence of a vacuolar H+-ATPase in these cells12. This H+-ATPase also was found in clone C11 of MDCK cells25. The two Na+-independent proton secretion mechanisms, the H+/K+-ATPase and the vacuolar H+-ATPase, are mechanisms similar to those found in the intercalated cells of mammalian collecting duct.
Angiotensin II
We studied the effect of Ang II in regulating the process of pHi recovery in presence of zero Na+ plus Schering 28080, a situation in which the H+-ATPase is the only mechanism of pHi recovery in activity, according to the aforementioned authors. Our results indicate, to our knowledge for the first time in MDCK cells, a dose-dependent stimulatory effect of Ang II on H+-ATPase Figure 2. This hormonal effect is via angiotensin II subtype 1 (AT1) receptor, since in previous studies we demonstrated that, in MDCK cells, losartan (an AT1 receptor antagonist) totally prevents the stimulatory effect of Ang II on the net rate of pHi recovery3.
Our results show that [Ca2+]i increases progressively as Ang II concentrations increase from 10-12 to 10-7 mol/L Table 3. These results are in accordance with data from the literature. It has been proposed that low doses of Ang II increase cell calcium via AT1B receptors, which activate phospholipase C (PLC), causing the stimulation of inositol triphosphate (IP3) and diacylglycerol, which in turn elevate cell calcium by its liberation from cell stores26. At high concentrations, Ang II is known to interact with AT1A receptors, causing the liberation of arachidonic acid, which is part of a path that elevates cell calcium by activating voltage sensitive calcium channels of the plasma membrane26,27. The rise in [Ca2+]i may enhance active proton transport by two separate pathways: (1) an increased exocytotic insertion of proton pumps into the apical membrane (as occurs in the turtle urinary bladder, proximal tubule and cortical collecting duct28,29), and (2) depolarization of the cell secondary to a rise in [Ca2+]i10. This latter pathway may increase the pump rate by reducing the adverse electrical gradient against which the electrogenic proton pump must work. These behaviors are compatible with our data showing that Ang II stimulates the velocity of pHi recovery mediated by H+-ATPase Figure 2.
Our results are compatible with Wagner et al's study of isolated rat proximal tubule fragments4, which indicated that 10-9 mol/L Ang II stimulates proton extrusion via H+-ATPase by a process involving brush border insertion of vesicles. Our present data also agree with our previous studies showing that in late distal segments of rat kidney 10-12 mol/L Ang II stimulates H+-ATPase5. In addition, our results are in accordance with recent studies suggesting that a rise of [Ca2+]i might represent part of a physiological mechanism to stimulate H+-ATPase-mediated protein export under acid conditions7. However, our results are at variance with previous data by Tojo, Tisher and Madsen6, who showed that in rat cortical collecting duct (a segment with which MDCK cells have many morphological and physiological similarities), Ang II 10-10 to 10-5 mol/L causes a dose-dependent decrease in H+-ATPase activity (measured as the bafilomycin-sensitive ATPase activity by a fluorometric method), with a maximum inhibition at 10-8 mol/L. Besides species and method differences, this discrepancy may be explained by the fact that in their study the tubule segments were incubated in presence of Ang II for up to 60 minutes before determining the H+-ATPase activity, against two to five minutes in our present experiments. According to those authors, the most likely explanation in their experimental conditions for the parabolic dose-response curve is the ability of Ang II to activate multiple signaling pathways, including additional messengers besides those acting in our experiments, such as cyclic guanosine monophosphate (cGMP)30. It is unlikely, however, that this signaling pathway should account for the effects observed in the present study, since we have previously demonstrated that the stimulatory effect of Ang II on the rate of pHi recovery in MDCK cells is completely abolished by losartan, a specific AT1-receptor antagonist3, and there is no evidence that AT1 receptors are coupled to the cGMP pathway.
Ang II plus ANP
Our current study found that during the addition of 10-6 mol/L ANP alone the pHi recovery rate is not significantly different from the control value; however, ANP impairs the stimulatory effect of Ang II 10-12 and 10-7 mol/L on the velocity of pHi recovery Figure 3. These data are compatible with our results concerning the effect of this hormone on [Ca2+]i Table 3. ANP alone does not affect the velocity of pHi recovery since it causes a decrease of cytosolic free calcium, which by itself does not impair cellular H+ secretion. However, ANP impairs the effect of Ang II on the velocity of pHi recovery because it impairs the increase of [Ca2+]i in response to Ang II, thus modulating the cellular action of Ang II. It is possible that this is a general mechanism responsible for the apparently antagonistic interaction between ANP and Ang II observed in MDCK cells, mesangial cells and proximal tubules3,11,31.
Ang II plus dimethyl-BAPTA/AM
Since recent results suggested that an increase of [Ca2+]i might initiate events that lead to activation of cellular H+ secretion7, we studied the effect of dimethyl-BAPTA/AM, an intracellular calcium chelator15, on cellular pH recovery mediated by H+-ATPase.
Similar to ANP, dimethyl-BAPTA/AM alone does not affect the rate of pHi recovery since, like ANP, it causes a decrease of cytosolic free calcium. On the other hand, also like ANP, dimethyl-BAPTA/AM impairs the stimulatory effect of Ang II on the velocity of pHi recovery because it impairs the increase of [Ca2+]i in response to Ang II (Figure 4 and Table 3).
In conclusion, our study results suggest a role of [Ca2+]i in regulating the process of pHi recovery after the acid load induced by NH4Cl, mediated by H+-ATPase and stimulated by Ang II. These findings are compatible with a dose dependent stimulation of H+-ATPase by increases of [Ca2+]i at 10-12, 10-9 and 10-7 mol/L Ang II. In agreement with these results, ANP and dimethyl-BAPTA/AM, by decreasing cytosolic free calcium to about 40% and 49% of the control value, respectively, do not affect the pHi recovery but, by impairing the path causing the increase in cell calcium, block the stimulatory effects of Ang II on this process. This hormonal interaction observed in MDCK cells, which is a cell line with many morphological and physiological similarities as the mammalian collecting duct, may represent a relevant mechanism of pHi regulation in the intact animal.
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Acknowledgments
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Programa de Apoio a Núcleos de Excelência (Pronex, no. 66.1029/1998-0) and Conselho Nacional de Pesquisas (CNPq).
