Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo 05508-900, Brazil
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ABSTRACT |
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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 (1012
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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INTRODUCTION |
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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 1012
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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MATERIALS AND METHODS |
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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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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
(1012, 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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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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RESULTS |
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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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[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
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DISCUSSION |
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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 · cm2) and strain
II (from later passages, 100-110, with 100
· 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 1012
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 (106 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 (107 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 1012 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 109 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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