Interaction of angiotensin II and atrial natriuretic peptide on pHi regulation in MDCK cells

M. Oliveira-Souza and M. De Mello-Aires

Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo 05508-900, Brazil


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The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pHi) and cytosolic free calcium concentration ([Ca2+]i) was investigated in Madin-Darby canine kidney cells by using the fluorescent probes 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (AM) and fura 2-AM or fluo 4-AM. pHi recovery rate was examined in the first 2 min after the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.088 ± 0.014 pH units/min (n = 14); in the absence of external Na+, this value was decreased. ANG II (10-12 or 10-9 M) caused an increase in this value, but ANG II (10-7 M) decreased it. ANP (10-6 M) or dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50 µM) alone did not affect this value but impaired both stimulatory and inhibitory effects of ANG II. ANG II (10-12, 10-9, or 10-7 M) increased [Ca2+]i progressively from 99 ± 10 (n = 20) to 234 ± 7 mM (n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca2+]i, and the subsequent addition of ANG II caused a recovery of [Ca2+]i but without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca2+]i in regulating the process of pHi recovery mediated by the Na+/H+ exchanger, stimulated/impaired by ANG II, and not affected by ANP or ANG II plus ANP. This hormonal interaction may represent physiologically relevant regulation in conditions of volume alterations in the intact animal.

intracellular pH; Madin-Darby canine kidney cells


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THE MAINTENANCE OF INTRACELLULAR pH (pHi) is essential for cellular physiology and proliferation. Its regulation is accomplished by a complex and not uniform mechanism, depending on the cell type being analyzed, and involves various 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 in renal tubules have indicated that H+ secretion and HCO3- reabsorption are subject to ANG II action. In renal proximal tubules, picomolar concentrations of ANG II stimulate, whereas micromolar concentrations inhibit, basolateral Na+-HCO3- cotransport (8, 19, 27). However, we have found that atrial natriuretic peptide (ANP) (10-6 M) alone does not affect HCO3- reabsorption in the proximal nephron but impairs the stimulation caused by ANG II (10-12 M) by 50% (15). More recently, we have demonstrated that luminal ANG II (10-12 M) stimulates Na+/H+ exchange in early distal and late distal segments of rat kidney, as well as the vacuolar H+-ATPase in late distal segments; ANP does not affect HCO3- reabsorption in either early distal or late distal segments and, as opposed to what was seen in proximal tubule, does not impair the stimulation caused by ANG II (5).

On the other hand, studies exploring the mechanisms that control H+ secretion by acid-secreting epithelia have emphasized the importance of cytosolic free calcium concentration ([Ca2+]i) in this process (30). Nevertheless, ANP has been shown to inhibit [Ca2+] i rises produced by ANG II in cultured mesangial cells (4) and Madin-Darby canine kidney (MDCK) cells (22), suggesting that there may be some interaction between these two vasoactive peptide hormones in the regulation of pHi.

The purpose of the present investigation is to clarify the mechanism of interaction between ANG II and ANP in the modulation of pHi. We used MDCK cells, a permanent cell line that is among the best characterized renal epithelial cells and that is known to be the site of Na+/H+ exchange (24, 29, 34). pHi and [Ca2+]i were determined with the fluorescent probes 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and fura 2 or fluo 4, respectively.

Our studies indicate a role for cell calcium in regulating the process of pHi recovery after the acid load induced by NH4Cl, mediated by a basolateral Na+/H+ exchanger and stimulated/impaired by ANG II via activation of AT1 receptors. The results are compatible with stimulation of Na+/H+ exchange by increases in cell calcium in the lower range (at 10-12 or 10-9 M ANG II) and inhibition at high cell calcium levels (at 10-7 M ANG II). ANP or dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), decreasing cytosolic free calcium, respectively, to ~35 or 48% of control value, does not affect the pHi recovery but, in impairing the path causing the increase in cell calcium, blocks both stimulatory and inhibitory effects of ANG II on this process. In agreement with these results, EGTA, a calcium chelator that decreases cytosolic free calcium to 15% of control value, significantly decreases the velocity of pHi recovery and impairs the stimulatory effect of ANG II on this process but does not affect the inhibitory effect of ANG II. This hormonal interaction that we observed in MDCK cells (a cell line with many morphological and physiological similarities to the mammalian collecting duct) may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.


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Cell culture. Wild-type MDCK cells obtained from the American Type Culture Collection (ATCC, Rockville, MD) were used for all experiments (passages 60-63). Serial cultures were maintained in DMEM (GIBCO, Grand Island, NY) supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were grown at 37°C, 95% humidified air-5% CO2 (pH 7.4) in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL). The cells were harvested with trypsin EGTA (0.02%), seeded on sterile glass coverslips, and incubated again for 72 h in the same medium to become confluent.

Fluorescent measurement of pHi. pHi was monitored by using the fluorescent probe BCECF. Cells grown to confluence on glass coverslips were loaded with the dye by exposure for 20 min to 10 µM BCECF-AM in the control solution (solution 1, Table 1). BCECF-AM enters the cells and is rapidly converted to the anionic-free acid form by intracellular esterases. After the loading period, the glass coverslips were rinsed with the control solution to remove the BCECF-containing solution and placed into a thermoregulated chamber mounted on an inverted epifluorescent microscope (TMD, Nikon). The measured area under the microscope had a diameter of 260 µm and contained on the order of 42 cells. The coverslips remained in a fixed position, so that the same cells were studied throughout the experiment. Bathing solutions were rapidly exchanged without disturbing the position of the coverslips. All experiments were performed at 37°C. The cells were alternately excited at 455 or 505 nm with a 150-W xenon lamp, and the fluorescence emission was monitored at 530 nm by a photomultiplier-based fluorescence system (PMT-400, Georgia Instruments) at time intervals of 5 s. The 505/455 excitation ratio corresponds to a specific pHi. At the end of each experiment, calibration of the BCECF signal was achieved by the high-K+-nigericin method (32), exposing the cells for 15 min to a K+-HEPES buffer solution containing 10 µM nigericin (solution 2, Table 1) at pH 6.5, 7.0, or 7.5. 

                              
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Table 1.   Composition of solutions

Cell pH recovery. Cell pH recovery was examined after the acidification of pHi with the NH4Cl pulse technique (7) after 2-min exposure to 20 mM NH4Cl (solution 3, Table 1), in the following situations: control (in the presence of external 145 mM Na+, solution 1, Table 1); in the absence of external Na+ (solution 4, Table 1); or in the presence of ANG II (10-12, 10-9, or 10-7 M) and/or losartan (10-6 M), ANP (10-6 M), dimethyl-BAPTA-AM (50 µM), or EGTA (2.5 mM). Because the rate of pH recovery depends on the value of cell pH achieved by the acid load (37), we used experiments in which these values were not significantly different among the studied groups (Table 2). In all the experiments, we calculated the initial rate of pHi recovery (dpHi/dt, pH units/min) from the first 2 min of the recovery curve by linear regression analysis.

                              
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Table 2.   Summary of pHi responses in MDCK cells to addition of different agents after an acute acid load

Fluorescent measurement of [Ca2+]i. Changes in [Ca2+]i were monitored fluorometrically by using the calcium-sensitive probe fura 2-AM as previously described (33). The loading period for fura 2-AM (2.5 µM) was ~1 h in cells suspended in Tyrode solution (solution 5, Table 1) containing 0.2% bovine serum albumin. Fura 2 fluorescence was measured in 2.5-ml aliquots of the cell suspensions (106 cells/ml) with a Perkin-Elmer model LS-5 fluorescence spectrophotometer set at 520-nm emission wavelength and excitation wavelengths alternating between 340 and 380 nm, with slit widths of 3 nm for excitation and 10 nm for emission. The cell suspensions were maintained at 37°C and continuously stirred. A calibration procedure was performed at the end of each experiment.

To study the effects of dimethyl-BAPTA-AM on the regulation of [Ca2+]i, we performed a series of experiments in which changes in [Ca2+]i were monitored fluorometrically by using the calcium-sensitive probe fluo 4-AM. MDCK cells were grown to confluence on uncoated glass-bottom microwells (Mat-Tek, Ashland, MA) at a density of 2.5 × 105 cells/ml. Twenty-four hours after plating, confluent cultures were loaded with 10 µM fluo 4-AM at 37°C for 40 min and rinsed in Tyrode solution (solution 5, Table 1) containing 0.2% bovine serum albumin (pH 7.4). Cells were placed at room temperature, and fluo 4 fluorescence intensity emitted above 505 nm was imaged by using ultraviolet laser excitation at 488 nm on a Zeiss LSM 510 real-time confocal microscope. The images were continuously acquired before and after addition of experimental solutions, at time intervals of 10 s, for a total of 200 s. 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 µM; maximum concentration) followed by EGTA (2.5 mM; minimum concentration) according to the Grynkiewicz equation (17), using the dissociation constant of 345 nM (according to the Molecular Probes catalog). Under these conditions, mean control [Ca2+]i for MDCK cells was 99.0 ± 1.5 nM (n = 19), a value not significantly different from the basal value of [Ca2+]i monitored with the fluorescent probe fura 2 in these cells in suspension, as previously described [99.0 ± 10 nM (n = 20)].

Solutions and reagents. The composition of the solutions utilized is described in Table 1. These solutions had an osmolality between 325 and 330 mosmol/kgH2O, which is the value found in the culture medium used for these cells. This osmolality was used to avoid changes when the cells were transferred from the culture medium to the experimental solutions. ANG II (1,046 molecular weight) was generously provided by the Department of Biophysics of University Federal do Estado de São Paulo (São Paulo, Brazil). Twenty-eight-amino acid ANP was purchased from Bachem Fine Chemicals (New Haven, CT), fura 2-AM, fluo 4-AM, BCECF-AM, and dimethyl-BAPTA-AM from Molecular Probes and losartan (DuP-753) from DuPont Merck (Wilmington, DE). All other applied chemicals were of analytic grade and obtained from Sigma.

Statistics. The results are presented as means ± SE; (n) is the number of experiments. Data were analyzed statistically by analysis of variance followed by Bonferroni's contrast test. Differences were considered significant if P < 0.05.


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pHi. In all experiments, the cell pH recovery was examined after the acidification of pHi with the NH4Cl pulse technique. Figure 1 shows two representative experiments. Cells were first bathed with 145 mM Na+ solution, exhibiting the basal pHi. After 2-min exposure to 20 mM NH4Cl, during which cell pHi increased transiently, NH4Cl removal caused a rapid acidification of pHi as a result of NH3 efflux. In the presence of extracellular 145 mM Na+, the initial fall in pHi is followed by a recovery of pHi toward the basal value (Fig. 1A). Removal of extracellular Na+ resulted in a significant inhibition of the pHi recovery that is subsequently reversed with the return of Na+ to the extracellular solution (Fig. 1B).


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Fig. 1.   Intracellular pH (pHi) recovery after cellular acidification with the NH4Cl pulse technique. A: the initial fall in pHi is followed by a recovery of pHi toward the basal value in the presence of 145 mM extracellular Na+. B: removal of extracellular Na+ resulted in a significant inhibition of the pHi recovery that is subsequently reversed with the return of Na+ to the extracellular solution. C, basal pHi.

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 have a mean baseline pHi of 7.17 ± 0.04 (n = 173).

Figure 2 indicates the effect of the absence of external Na+ on the main pHi recovery rate. In the control situation (in presence of external 145 mM Na+), the main pHi recovery rate in the first 2 min was 0.088 ± 0.014 pH units/min (n = 14; and the final pHi was not significantly different from the basal value, 7.17 ± 0.02 vs. 7.15 ± 0.07, Table 2). In the absence of external Na+, the pHi recovery rate was reduced to 40% of control value (and pHi recovery was not complete, Table 2); however, even in the absence of Na+, a significant rate of pHi recovery was still observed. This effect is reversed with the return of Na+ to the bathing solution (and final pHi was not significantly different from the basal value, Table 2), indicating that the pHi recovery is mostly dependent on Na+/H+ exchange.


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Fig. 2.   Effect of ANG II (10-12, 10-9, and 10-7 M) on the initial rate of pHi recovery after acute intracellular acidification in Madin-Darby canine kidney (MDCK) cells. The experiments were done in the presence or absence of extracellular Na+ (Nae+; 145 mM). R, recovery with the return of Na+ to the bathing solution; n, No. of experiments. *P < 0.05 vs. control (C). +P < 0.05 vs. ANG II (10-12 M). #P < 0.05 vs. ANG II (10-9 M).

Figure 2 also shows that the addition of ANG II (10-12 M) to the bath causes a significant increase (38% of control value) in the velocity of pHi recovery, whereas in the presence of ANG II (10-9 M) this increase is still more significant (123% of control value; during both situations the final pHi was not significantly different from the basal value, Table 2). However, the addition of ANG II (10-7 M) significantly decreases (77% of control value) the velocity of pHi recovery (and pHi recovery was not complete, Table 2). In the absence of external Na+, both stimulatory effects of ANG II are significantly inhibited (and pHi recovery was not complete, Table 2). With the return of Na+ to the bathing solution, both stimulatory effects of ANG II are subsequently partly recovered, indicating that they are mostly dependent on Na+/H+ exchange.

Figure 3 shows the effect of addition of losartan (10-6 M) or losartan (10-6 M) plus ANG II (10-9 or 10-7M) to the bath on the rate of pHi recovery. With losartan alone, the pHi recovery rate was not significantly different from the control value (and the final pHi was not significantly different from the basal value, Table 2). These data demonstrate that losartan has no intrinsic effects on pHi responses. However, losartan impairs the stimulatory effect of ANG II (10-9M) and the inhibitory effect of ANG II (10-7M). These results indicate that both stimulatory and inhibitory effects of ANG II on the net rate of pHi recovery are via activation of AT1 receptors.


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Fig. 3.   Effect of losartan (10-6 M) or losartan (10-6 M) plus ANG II (10-9 or 10-7 M) on the initial rate of pHi recovery after acute intracellular acidification in MDCK cells. n, No. of experiments. *P < 0.05 vs. control (C). +P < 0.05 vs. ANG II (10-9 M). #P < 0.05 vs. ANG II (10-7 M).

Figure 4 gives the effect of addition of ANP (10-6M) or ANP (10-6M) plus ANG II (10-12, 10-9, or 10-7 M) to the bath on the rate of pHi recovery. With ANP alone, the pHi recovery rate was not significantly different from the control value (and the final pHi was not significantly different from the basal value, Table 2). However, ANP impairs both stimulatory effects of ANG II (10-12 and 10-9 M) on the velocity of pHi recovery (and during both situations, the final pHi was not significantly different from the basal value, Table 2). ANP also impairs the inhibitory effect of ANG II (10-7 M) on the net rate of pHi recovery (but during this situation pHi recovery was not complete, Table 2). These results indicate that ANP alone does not affect cellular pH recovery but impairs both stimulatory and inhibitory effects of ANG II.


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Fig. 4.   Effect of atrial natriuretic peptide (ANP; 10-6 M) or ANP (10-6 M) plus ANG II (10-12, 10-9, or 10-7 M) on the initial rate of pHi recovery after acute intracellular acidification in MDCK cells. n, No. of experiments. * P < 0.05 vs. control (C). +P < 0.05 vs. ANG II (10-12 M). #P < 0.05 vs. ANG II (10-9 M). &P < 0.05 vs. ANG II 10-7 M).

Because several studies show the importance of [Ca2+]i in cellular H+ secretion (35, 38), we studied the effect of addition of dimethyl-BAPTA-AM [50 µM; an intracellular calcium chelator (30)] or EGTA [2.5 mM; a calcium chelator that decreases cytosolic free calcium to a minimum value (22, 38)] to the medium on cellular pH recovery. Figure 5 shows that with dimethyl-BAPTA-AM alone the pHi recovery rate was not significantly different from control value (and the final pHi was not significantly different from the basal value, Table 2). Dimethyl-BAPTA-AM impairs both stimulatory effects of ANG II (10-12 and 10-9 M) on the velocity of pHi recovery (and during both situations, the final pHi was not significantly different from the basal value, Table 2). Dimethyl-BAPTA-AM also impairs the inhibitory effect of ANG II (10-7 M) on the net rate of pHi recovery (but during this situation, pHi recovery was not complete, Table 2). Figure 6 summarizes that, with the addition of EGTA alone, the pHi recovery decreases significantly (73%) from control value (and the final pHi was significantly different from the basal value, Table 2). EGTA also significantly decreases (71%) the stimulatory effect of ANG II (10-9 M) on the velocity of pHi recovery (and pHi recovery was not complete, Table 2). However, a statistically significant difference was encountered between the pHi recovery values measured during addition of EGTA and EGTA plus ANG II (10-9 M), indicating that EGTA does not block the entire stimulatory effect of ANG II (10-9 M). On the other hand, EGTA does not affect the inhibitory effect of ANG II (10-7 M) on the net rate of pHi recovery (and the final pHi was significantly different from the basal value, Table 2). Taken together, these results suggest a role of cytosolic free calcium in regulating the net rate of pHi recovery, mediated by Na+/H+ exchange and stimulated/impaired by ANG II.


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Fig. 5.   Effect of dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 50 µM) or dimethyl-BAPTA-AM (50 µM) plus ANG II (10-12, 10-9, or 10-7 M) on the initial rate of pHi recovery after acute intracellular acidification in MDCK cells. n, No. of experiments. *P < 0.05 vs. control (C). +P < 0.05 vs. ANG II (10-12 M). #P < 0.05 vs. ANG II (10-9 M). &P < 0.05 vs. ANG II (10-7 M). $P < 0.05 vs. dimethyl-BAPTA-AM.



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Fig. 6.   Effect of EGTA (2.5 mM) or EGTA (2.5 mM) plus ANG II (10-9 or 10-7 M) on the initial rate of pHi recovery after acute intracellular acidification in MDCK cells. n, No. of experiments. *P < 0.05 vs. control (C). +P < 0.05 vs. ANG II (10-9 M). #P < 0.05 vs. EGTA.

To functionally define the apical or basolateral Na+/H+ exchanger membrane localization, we performed a series of experiments in which MDCK cells were grown on permeant filter supports (Transwell 3.0 µm pore size, 12 mm diameter; Costar, Cambridge, MA), making it possible to independently measure the effect of the absence of external Na+ on either the apical or basolateral surface on pHi recovery. Figure 7 summarizes these results. In the control situation, in presence of external 145 mM Na+ on both the apical and basolateral membrane surfaces, the pHi recovery rate in the first 2 min was 0.099 ± 0.01 pH units/min [n = 12; a value not significantly different from 0.088 ± 0.014 pH units/min (n = 14), the control value found when the cells were grown on coverslips in presence of external 145 mM Na+]. In the absence of external Na+ on both membrane surfaces, the pHi recovery rate was markedly decreased. This effect is subsequently reversed with the return of Na+ to the bathing solution on both membrane surfaces, confirming that pHi recovery is mostly dependent on Na+/H+ exchange. In the absence of external Na+ on the apical membrane surface, the pHi recovery rate was not significantly different from the control value. However, in the absence of external Na+ on the basolateral membrane surface, the pHi recovery rate was significantly decreased and, with the return of Na+ to the basolateral surface, the pHi recovery rate increased to a value not significantly different from the control value. On the basis of these data, we may conclude that the Na+/H+ exchanger responsible for the Na+-dependent pHi recovery observed in the present study is located on the basolateral membrane.


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Fig. 7.   Effect of extracellular Na+ on the initial rate of pHi recovery after acute intracellular acidification in MDCK cells. These experiments were done in cells growing on permeant filter supports in the presence (145 mM) or absence of external Na+ at the apical and/or basolateral membrane surface. AM, apical membrane surface; BLM, basolateral membrane surface; n, No. of experiments. *P < 0.05 vs. control (presence 145 mM of external Na+ at the apical and basolateral membrane surface).

[Ca2+]i. To obtain more information about the mechanism of interaction of ANG II and ANP on the modulation of pHi, we also studied the effects of ANG II, ANP, dimethyl-BAPTA-AM, and EGTA on the regulation of [Ca2+]i.

Figure 8 shows that MDCK cells exhibited a mean baseline [Ca2+]i of 99 ± 10 nM (n = 20). The subsequent addition of ANG II (10-12, 10-9, and 10-7 M) increased [Ca2+]i progressively from control values to 234 ± 7 nM (n = 10) in a dose-dependent manner. The addition of ANP (10-6 M) to the bathing solution leads to a rapid and significant decrease in [Ca2+]i from control values to 35 ± 8 nM (n = 10). In the presence of ANP, the subsequent addition of ANG II (10-12, 10-9, and 10-7 M) caused a recovery of [Ca2+]i that reached 63 ± 9 nM (n = 10), thus without exceeding normal baseline values even at ANG II (10-7 M). Figure 8 also shows that the addition of dimethyl-BAPTA-AM (50 µM) to the bathing solution leads to a significant decrease in [Ca2+]i from control values to 48 ± 3 nM (n = 12). In the presence of dimethyl-BAPTA-AM, the subsequent addition of ANG II (10-12, 10-9, and 10-7 M) caused a recovery of [Ca2+]i to 86 ± 3 (n = 9), 100 ± 3 (n = 11), and 147 ± 5 nM (n = 9), respectively. However, the addition of EGTA (2.5 mM) to the cell suspension leads to a significant decrease in [Ca2+]i from control values to 15 ± 7 nM (n = 8). In the presence of EGTA, the subsequent addition of ANG II (10-12, 10-9, and 10-7 M) did not cause a recovery of [Ca2+]i.


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Fig. 8.   Effect of ANG II (10-12, 10-9, and 10-7 M), and/or ANP (10-6 M), and/or dimethyl-BAPTA-AM (50 µM), and/or EGTA (2.5 mM) on free calcium concentration in the cytosol ([Ca2+]i) of MDCK cells. n, No. of experiments. *P < 0.05 vs. control (C). +P < 0.05 vs. ANP. #P < 0.05 vs. dimethyl-BAPTA-AM.


    DISCUSSION
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ABSTRACT
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The purpose of this study was to clarify the mechanism of interaction between ANG II and ANP in the modulation of pHi in MDCK cells, a permanent cell line originated from the renal collecting duct. According to the classification of Richardson et al. (28), there are two strains of this cell line: strain I (derived from an early passage, 60-70, with resistance over 3,000 Omega  · cm2) and strain II (from later passages, 100-110, with 100 Omega  · cm2). In the present study the MDCK cells were from passage 60 to passage 63, thus from cell strain I according to the aforementioned authors. The heterogeneity of the strain I of MDCK cells was confirmed by Gekle et al. (13), who cloned two MDCK cells subtypes designated C7 and C11, with different morphologies and functions. The C7 subtype resembles principal cells of the renal collecting duct and exhibits an intracellular pH of 7.39 ± 0.05 (n = 7), whereas the C11 subtype resembles intercalated cells of the renal collecting duct and maintains intracellular pH at 7.16 ± 0.05 (n = 8). Our data demonstrate that MDCK cells in pH 7.4 HCO3--free solution maintain a mean baseline pHi of 7.17 ± 0.02 (n = 173), a value compatible with the MDCK cell subtype C11 (13). However, we did not distinguish between the two cell types present in this preparation. Our data are in accordance with the studies of Wiegmann et al. (38), who have shown by both fluorometry and video microscopy that MDCK cells had a mean pHi of 7.12 ± 0.01 (n = 50). Our present results also agree with the value of 7.17 ± 0.01 (n = 23) found by Fernández and Malnic (11) in MDCK cells, strain I.

Our data show that in the absence of external Na+ the net rate of pHi recovery after an NH4Cl prepulse was reduced to 40% of control value (Fig. 2). The relationship between Na+ transport and pHi changes in MDCK cells was described by several authors (11, 14). However, even in the absence of Na+ a significant rate of pHi recovery was still observed, due to Na+- independent H+ extrusion mechanisms. Our results indicating that pHi recovery is mostly dependent on Na+/H+ exchange are in accordance with Fernández and Malnic (11), who found three different mechanisms of pHi recovery in MDCK cells: the Na+/H+ exchanger, the H+/K+-ATPase, and the vacuolar H+-ATPase. According to these authors, the more important of these mechanisms is the Na+/H+ exchanger, because the removal of extracellular Na+ led to a 43% reduction in the rate of pHi recovery. Most studies localized the exchanger to the basolateral membrane (29, 34). More recently, it was shown that the isoform NHE1 of the Na+/H+ exchanger (the only isoform expressed spontaneously in these cells) was expressed at both sides of the polarized MDCK cells, with a preference for the apical side (24). In the present studies performed on permeant filter supports, it was possible to define that the Na+/H+ exchanger accounting for the Na+-dependent pHi recovery is located on the basolateral membrane (Fig. 7).

Our results indicate, for the first time in MDCK cells, that low concentrations of ANG II stimulate and high concentrations of ANG II inhibit the velocity of Na+-dependent pHi recovery (Fig. 2). This dose-dependent biphasic effect of ANG II on Na+/H+ exchange has been observed before in rat proximal tubules (19, 27). Studies in renal tubules have demonstrated a dose-dependent biphasic response to ANG II also in a variety of other physiological mechanisms: volume and HCO3- absorption (8, 16), regulation of apical membrane K+ channels (21), 86Rb uptake (12), and Na+-K+-2Cl- cotransport activity (1). Importantly, concentrations of ANG II measured in proximal tubule fluid and star vessel plasma in the rat kidney cortex in vivo ranged from 10 to 40 nM, values several orders of magnitude higher than concentrations in systemic plasma (23). Furthermore, ANG II levels in the renal medulla are even higher than those in the cortex (23). Thus the concentrations of ANG II in the medullary collecting duct may be similar to ANG II levels measured in the renal medulla in vivo, suggesting that the transport effects we observed in MDCK cells (a cell line having many morphological and physiological similarities to the mammalian collecting duct) may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.

The results of the present study indicate that both stimulatory and inhibitory effects of ANG II on the net rate of pHi recovery were prevented by simultaneous addition of losartan, an AT1-receptor antagonist (Fig. 3). AT1 is the predominant receptor type in the kidney and is thought to mediate most of the effects of ANG II on tubular transport (2). In previous studies, we confirmed that ANG II acts to stimulate Na+/H+ exchange in early and late distal segments of rat kidney via activation of the AT1 receptor (5).

In the present experiments in MDCK cells, similar to findings in in vivo proximal tubules (15) but opposed to what we demonstrated in in vivo cortical distal tubule (5), ANP counteracted both the stimulatory and the inhibitory effect of ANG II (Fig. 4). Our present data are compatible with the identification of ANP receptors in MDCK cells (25). Although only few ANP receptors have been found in cortical distal tubule, such receptors are widely distributed in renal tissue, their mRNA having been detected in cortical and especially in medullary collecting duct (31). It is thus possible that MDCK cells present properties more akin to medullary collecting duct with respect to these receptors. In addition, an interaction between ANP and ANG II has been observed in a variety of tissues: ANP inhibits the vasoconstrictor effect of ANG II in vitro (20), as well as the systemic pressor action of ANG II (9), ANG II-stimulated aldosterone synthesis (3), and ANG II-stimulated proximal tubular Na+ transport (15).

To obtain information on the mechanism of the interaction of these hormones on pHi regulation, we studied their effects on the regulation of [Ca2+]i. Our results indicate that MDCK cells exhibited a mean baseline [Ca2+]i of 99 ± 10 nM (n = 20). These data agree with the value of 120 ± 29 nM (n = 6) found by Borle and Bender (6) or of 125 ± 7 nM (n = 50) found by Wiegmann et al. (38) in MDCK cells.

Our results show that [Ca2+]i increases progressively as ANG II concentrations increase from 10-12 to 10-9 and 10-7 M (Fig. 8). 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 stores. The activation of protein kinase C (PKC), via phosphorylation, may stimulate the Na+/H+ exchanger (10). This behavior is compatible with our data showing that low concentrations of ANG II stimulate the velocity of Na+-dependent pHi recovery (Fig. 2). 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 membrane (10, 16). At high cytosolic concentrations, calcium may inhibit Na+/H+ exchange by activating Na+/Ca2+ exchange at the cell membrane and thereby increasing cell sodium, which decreases the gradient responsible for H+ extrusion by the exchanger. However, this mechanism is somewhat questionable considering the large discrepancy between [Ca2+]i and extracellular sodium concentrations. On the other hand, it has been shown that the NHE1 exchanger [the major basolateral form of the Na+/H+ exchanger in polarized epithelial cells (36), as in the present situation] has calmodulin binding sites at the cytoplasmatic regulatory domain, which modulate its activity. A high-affinity site, which is tonically inhibitory, binds to low calcium/calmodulin, thus suppressing the inhibition, that is, stimulating the exchanger at low calcium/calmodulin levels. A low-affinity site, however, binds with calcium and calmodulin only at high concentrations, and, under these conditions, inhibits the exchanger activity (35, 36). This behavior is compatible with our present findings indicating stimulation of Na+/H+ exchanger by increases of [Ca2+]i in the lower range (at 10-12 or 10-9 M ANG II) and inhibition at high [Ca2+]i levels (at 10-7 M ANG II). This behavior is also compatible with our results showing the effect of addition of EGTA [a calcium chelator that decreases cytosolic free calcium to 15% of control value (Fig. 8)] to the medium on cellular pH recovery (Fig. 6). With the addition of EGTA alone, the velocity of pHi recovery decreases significantly (73%) from the control value. EGTA also impairs (71%) the stimulatory effect of ANG II (10-9 M) on the velocity of pHi recovery. This behavior is also in agreement with our data showing that EGTA does not affect the inhibitory effect of ANG II (10-7 M) on the velocity of Na+-dependent pHi recovery (Fig. 6), but in this situation the decrease in the rate of cellular H+ secretion is due to a marked decrease of cytosolic free calcium (Fig. 8) and not to a pronounced [Ca2+]i increase, as in the presence of ANG II (10-7 M) alone in the medium. However, EGTA did not entirely block the stimulatory effect of ANG II (10-9 M). A possible explanation would be the existence of another mechanism for low-dose ANG II action that might follow a cellular calcium-independent signaling path, involving activation of an inhibitory G protein and inhibition of adenyl cyclase, causing the fall of cell levels of cAMP and of the catalytic activity of protein kinase A. This path may activate the Na+/H+ exchanger (26).

Our results show that when ANP (10-6 M) is added to the cell suspension, [Ca2+]i decreases to ~35% of control value. In the presence of ANP, the subsequent addition of ANG II (10-12, 10-9 and 10-7M) caused a recovery of [Ca2+]i but without exceeding normal baseline values even at ANG II (10-7 M) (Fig. 8). These data are compatible with our results concerning the effect of this hormone on the velocity of Na+-dependent pHi recovery. In contrast with EGTA, ANP alone does not affect the velocity of Na+-dependent pHi recovery because it causes only a moderate decrease in cytosolic free calcium compared with the minimal [Ca2+]i values found in the presence of EGTA. On the other hand, ANP impairs both stimulatory and inhibitory effects of ANG II on the velocity of Na+-dependent pHi recovery because it impairs the increase in [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 a variety of other tissues (3, 4, 9, 18, 20) and in proximal tubule (15).

This behavior is also in agreement with the results concerning the effect of dimethyl-BAPTA-AM on the velocity of pHi recovery. In contrast with EGTA, but similar to ANP, dimethyl-BAPTA-AM alone does not affect the rate of pHi recovery since it causes, like ANP, only a moderate decrease in cytosolic free calcium (Fig. 8). On the other hand, like ANP, dimethyl-BAPTA-AM impairs both stimulatory and inhibitory effects of ANG II on the velocity of pHi recovery because it impairs, like ANP, the increase in [Ca2+]i in response to ANG II (Fig. 8). However, the pHi recovery values measured in the presence of ANP plus ANG II (10-7 M) are significantly smaller than the values found in the presence of dimethyl-BAPTA-AM plus ANG II (10-7 M) (Table 2) because with ANP plus ANG II (10-7 M), the [Ca2+]i is 63 ± 9 nM (n = 10), and with dimethyl-BAPTA-AM plus ANG II (10-7 M) the [Ca2+]i reaches 147 ± 5 nM (n = 9) (Fig. 8).

In conclusion, the results obtained in our studies suggest a role for cell calcium in regulating the process of pHi recovery after the acid load induced by NH4Cl, mediated by the basolateral Na+/H+ exchanger, and stimulated/impaired by ANG II via activation of AT1 receptors. They are compatible with stimulation of Na+/H+ exchange by increases in cell calcium in the lower range (at 10-12 or 10-9 M ANG II) and inhibition at high cell calcium levels (at 10-7 M ANG II). This finding is also compatible with the demonstration of two sites on the COOH terminal of the Na+/H+ exchanger, one stimulating Na+/H+ activity at low [Ca2+]i levels and the other inhibiting this activity at high [Ca2+]i (35, 36). ANP and dimethyl-BAPTA-AM, decreasing cytosolic free calcium, respectively, to ~35 and 48% of control value, do not affect the pHi recovery but, impairing the path causing the increase in cell calcium, block both stimulatory and inhibitory effects of ANG II on this process. In agreement with these results, EGTA (a calcium chelator that decreases cytosolic free calcium to 15% of control value) significantly decreases the velocity of pHi recovery and impairs the stimulatory effect of ANG II on this process. On the other hand, EGTA does not affect the inhibitory effect of ANG II because, in this situation, the decrease in the rate of cellular H+ secretion is due to a marked decrease in cytosolic free calcium and not to a pronounced [Ca2+]i increase, as in the presence of ANG II (10-7 M) alone in the medium.

Because the superfusion with EGTA does not block 10-9 M ANG II action entirely, it is possible that signaling mechanisms besides [Ca2+]i contribute to the action of ANG II on Na+/H+ exchange. Actually, although we have shown extensive evidence for the importance of [Ca2+]i, not all data are unequivocal in this respect, e.g., the finding that the marked reduction in cell calcium by EGTA impairs action of the exchanger, but the more moderate fall in [Ca2+]i caused by ANP and dimethyl-BAPTA-AM does not, and that high levels of ANG II increase [Ca2+]i but reduce exchanger activity. It is possible that [Ca2+]i is regulatory in a certain range of concentration, other pathways contributing to exchanger regulation outside this range. Our results thus show that there is a relationship between ANG II action and cytosolic calcium levels. [Ca2+]i increases when ANG II concentrations increase, although this relationship is not always linear. However, this increase in [Ca2+]i is not always related to an enhanced activation of Na+/H+ exchange, because very high (10-7 M) ANG II levels lead to high [Ca2+]i but cause an inhibition of Na+/H+ exchange activity. Therefore, the relationship between Na+/H+ exchange and [Ca2+]i is not straightforward. This might be due to two mechanisms; the first, is that there might be really an activation of Na+/H+ exchange by [Ca2+]i (directly or via PKC) in a certain limited range of [Ca2+]i, as suggested by Douglas and Hopfer (10). The other possibility would be that the relationship between [Ca2+]i and Na+/H+ exchange is not causal but indirect, the main activating pathway being, e.g., PLC-IP3-PKC or via arachidonic acid, or other signaling pathways, [Ca2+]i being a consequence of the activation of these pathways but not a direct cause for Na+/H+ exchange activation. Whether [Ca2+]i modification represents an important direct mechanism for exchanger activation or a side effect of other signaling pathways must await additional studies. However, the hormonal interaction we observed in MDCK cells (a cell line with many morphological and physiological similarities to the mammalian collecting duct) may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.


    ACKNOWLEDGEMENTS

The authors thank Dr. Gerhard Malnic for careful reading of the manuscript. They also thank Drs. Alice Teixeira Ferreira and Maria Etsuko Miamoto Oshiro for help with the measurement of cytosolic free calcium technique with fura 2-AM.


    FOOTNOTES

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisas (CNPq).

Address for reprint requests and other correspondence: M. de Mello-Aires, Dept. of Physiology and Biophysics, Instituto de Ciências Biomédicas, Univ. of São Paulo, SP 05508-900, Brazil (E-mail: mmaires{at}fisio.icb.usp.br).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 September 1999; accepted in final form 19 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amlal, H, Vernimmen C, Legoff C, Paillard M, and Bichara M. Control of Na+-K+-(NH4+)-2Cl- cotransport by angiotensin II-activated transduction pathways in medullary thick ascending limb cells. J Am Soc Nephrol 4: 862, 1993.

2.   Ardaillou, R. Angiotensin II receptors. J Am Soc Nephrol 10: S30-S39, 1999[ISI][Medline].

3.   Atarashi, K, Mulrow PJ, Fanco-Saenz R, Snajdar R, and Rapp J. Inhibition of aldosterone production by an atrial extract. Science 224: 992-994, 1984[ISI][Medline].

4.   Barnett, R, Ortiz PA, Blaufox S, Singer S, Nord EP, and Ramsammy L. Atrial natriuretic factor alters phospholipid metabolism in mesangial cells. Am J Physiol Cell Physiol 258: C37-C45, 1990[Abstract/Free Full Text].

5.   Barreto-Chaves, MLM, and de Mello-Aires M. Effect of luminal angiotensin II and ANP on early and late cortical distal tubule HCO3- reabsorption. Am J Physiol Renal Fluid Electrolyte Physiol 271: F977-F984, 1996[Abstract/Free Full Text].

6.   Borle, AB, and Bender C. Effects of pH on Cai2+, Nai+, and pHi of MDCK cells: Na+-Ca2+ and Na+-H+ antiporter interactions. Am J Physiol Cell Physiol 261: C482-C489, 1991[Abstract/Free Full Text].

7.   Boron, WF, and De Weer P. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 67: 91-112, 1976[Abstract].

8.   Coppola, S, and Frömter E. An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K+ conductance in renal proximal tubules. II. Effect of micromolar concentrations. Pflügers Arch 427: 151-156, 1994[ISI][Medline].

9.   Di Nicolantonio, R, Stevens J, Weaver D, and Morgan TO. Captopril antagonizes the hypotensive action of atrial natriuretic peptide in anaesthetized rat. Clin Exp Pharmacol Physiol 13: 311-314, 1986[ISI][Medline].

10.   Douglas, JG, and Hopfer U. Novel aspect of angiotensin receptors and signal transduction in the kidney. Annu Rev Physiol 56: 649-669, 1994[ISI][Medline].

11.   Fernández, R, and Malnic G. H+ ATPase and Cl- interaction in regulation of MDCK cell pH. J Membr Biol 163: 137-145, 1998[ISI][Medline].

12.   Ferreri, NR, Escalante BA, Zhao Y, An SJ, and McGiff JC. Angiotensin II induces TNF production by the thick ascending limb: functional implications. Am J Physiol Renal Physiol 274: F148-F155, 1998[Abstract/Free Full Text].

13.   Gekle, M, Wünsch S, Oberleithner H, and Silbernagl S. Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties. Pflügers Arch 428: 157-162, 1994[ISI][Medline].

14.   Goldfarb, D, and Nord EP. Asymmetric affinity of Na+-H+ antiporter for Na+ at the cytoplasmic vs. external transport site. Am J Physiol Renal Fluid Electrolyte Physiol 253: F959-F968, 1987[Abstract/Free Full Text].

15.   Gomes, NG, and de Mello Aires M. Interaction of atrial natriuretic factor and angiotensin II in proximal HCO3- reabsorption. Am J Physiol Renal Fluid Electrolyte Physiol 262: F303-F308, 1992[Abstract/Free Full Text].

16.   Good, DW, George T, and Wang DH. Angiotensin II inhibits HCO3- absorption via a cytochrome P-450-dependent pathway in MTAL. Am J Physiol Renal Physiol 276: F726-F736, 1999[Abstract/Free Full Text].

17.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

18.   Hirata, M, Kohse KP, Chang CH, Ikebe T, and Murad F. Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells. J Biol Chem 265: 1268-1273, 1990[Abstract/Free Full Text].

19.   Houillier, P, Chambrey R, Achard JM, Froissart M, Poggioli J, and Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na+/H+ antiport activity in proximal tubule. Kidney Int 50: 1496-1505, 1996[ISI][Medline].

20.   Kleinert, HD, Maack T, Atlas SA, Januszewicz A, Sealey JE, and Laragh JH. Atrial natriuretic factor inhibits angiotensin-, norepinephrine-, and potassium-induced vascular contractility. Hypertension 6, Suppl1: I-141-I-147, 1984.

21.   Lu, M, Zhu Y, Balazy M, Reddy KM, Falck JR, and Wang W. Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney. J Gen Physiol 108: 537-547, 1996[Abstract].

22.   Nascimento-Gomes, G, Souza MO, Vecchia MG, Oshiro MEM, Ferreira AT, and Mello-Aires M. Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Madin-Darby canine kidney cells. Braz J Med Biol Res 28: 609-613, 1995[ISI][Medline].

23.   Navar, LG, Imig JD, Zou L, and Wang CT. Intrarenal production of angiotensin II. Semin Nephrol 17: 412-422, 1997[ISI][Medline].

24.   Noel, J, Roux D, and Pouysségur J. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines. J Cell Sci 109: 929-939, 1996[Abstract/Free Full Text].

25.   Pandey, KN, Inagami T, and Misono KS. Three distinct forms of atrial natriuretic factor receptors: kidney tubular epithelium cells and vascular smooth muscle cells contain different types of receptors. Biochem Biophys Res Commun 147: 1146-1152, 1987[ISI][Medline].

26.   Pouysségur, J. Molecular biology and hormonal regulation of vertebrate Na+/H+ exchanger isoforms. Renal Physiol Biochem 17: 190-193, 1994[Medline].

27.   Reilly, AM, Harris PJ, and Williams DA. Biphasic effect of angiotensin II on intracellular sodium concentration in rat proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 269: F374-F380, 1995[Abstract/Free Full Text].

28.   Richardson, J, Scalera V, and Simmons NL. Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochim Biophys Acta 673: 26-36, 1981[ISI][Medline].

29.   Rosenberg, SO, Berkowitz PA, Li L, and Schuster VL. Imaging of filter-grown epithelial cells: MDCK Na+-H+ exchanger is basolateral. Am J Physiol Cell Physiol 260: C868-C876, 1991[Abstract/Free Full Text].

30.   Stuart, RO, Sun A, Panichas M, Hebert SC, Brenner BM, and Nigam SK. Critical role for intracellular calcium in tight junction biogenesis. J Cell Physiol 159: 423-433, 1994[ISI][Medline].

31.   Terada, Y, Tomita K, Nonoguchi H, Yang T, and Marumo F. PCR localization of C-type natriuretic peptide and B-type receptor mRNAs in rat nephron segments. Am J Physiol Renal Fluid Electrolyte Physiol 267: F215-F222, 1994[Abstract/Free Full Text].

32.   Thomas, J, Buchsbaum R, Zimniak A, and Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218, 1979[ISI][Medline].

33.   Tsien, RY. Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci 11: 419-424, 1988[ISI][Medline].

34.   Vilella, S, Guerra L, Helmle-Kolb C, and Murer H. Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells. Pflügers Arch 420: 275-281, 1992[ISI][Medline].

35.   Wakabayashi, S, Bertrand B, Ikeda T, Pouysségur J, and Shigekawa M. Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H+-sensitive and Ca2+ regulation-defective. J Biol Chem 269: 13710-13715, 1994[Abstract/Free Full Text].

36.   Wakabayashi, S, Shigekawa M, and Pouysségur J. Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77: 51-74, 1997[Abstract/Free Full Text].

37.   Weintraub, WH, and Machen TE. pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 257: G317-G327, 1989[Abstract/Free Full Text].

38.   Wiegmann, TB, Welling LW, Beatty DM, Howard DE, Vamos S, and Morris SJ. Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells. Am J Physiol Cell Physiol 265: C1184-C1190, 1993[Abstract/Free Full Text].


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