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Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na⁺/H⁺ exchanger in proximal S3 segment: role of cytosolic calcium

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

Submitted 28 January 2008 ; accepted in final form 15 August 2008

	ABSTRACT

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The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na⁺/H⁺ exchanger and on the [Ca²⁺]_i were investigated in isolated rat S3 segment. Aldosterone [10^–12, 10^–10, or 10^–8 M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10^–6 M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca²⁺]_i and after 6-min pi there was a new increase of [Ca²⁺]_i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca²⁺]_i. RU 486 prevented the stimulatory effect of aldosterone (10^–12 M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10^–6 M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca²⁺]_i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca²⁺]_i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca²⁺]_i in the lower range (at 10^–12 M aldosterone) and inhibition by increases at high levels (at 10^–6 M aldosterone) or decreases in [Ca²⁺]_i (at 10^–6 M aldosterone plus RU 486).

mineralocorticoid stimulatory/inhibitory action; NHE1; proximal tubule; intracellular pH; intracellular Ca²⁺

Aldosterone is the chief mineralocorticoid of the body and has an important role in the maintenance of Na⁺, K⁺, and acid-base balance of distal nephron segments, through its effects on renal electrolyte excretion (13, 25, 27, 33). The effects of aldosterone on its target cells have long time been considered to be mediated exclusively through the genomic pathway and were characterized by an 45-min lag period; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms (11–13, 17, 25, 43). On the other hand, in a series of in vitro studies, aldosterone showed to have a half maximal effect on both rapid (15 min) and delayed (120 min) Na⁺ flux (10, 25).

The Na⁺/H⁺ exchanger has been identified as a target for nongenomic regulation by this mineralocorticoid. Aldosterone rapidly increases Na⁺/H⁺ exchanger activity in a variety of cells, including distal colon and renal epithelial cell lines (9, 27), but aldosterone rapidly inhibits apical NHE3 and HCO₃^– reabsorption in medullary thick ascending limb (5). So, the activation/inhibition of Na⁺/H⁺ exchanger by aldosterone remains to be defined, since it is possibly that similar to angiotensin II (18, 26, 28), aldosterone has a dose-dependent biphasic effect on the Na⁺/H⁺ exchanger (low doses stimulate and high doses inhibit it).

In addition, an elevation in cytosolic Ca²⁺ serves as a second messenger in the nongenomic Na⁺/H⁺ exchanger activation initiated by aldosterone (11) and is a prerequisite for the genomic action of aldosterone, with strong evidence that the pregenomic hormone response can influence the genomic processes (34).

Therefore, considering that the physiological doses of aldosterone in blood are 10^–10–10^–9 M and that they can increase or decrease in conditions of extracellular volume modification, the purpose of this work was to investigate the genomic and nongenomic effects of aldosterone (10^–12, 10^–10, 10^–8, and 10^–6 M) on the Na⁺/H⁺ exchanger and the role of intracellular calcium in these processes. The experiments were done in isolated proximal S3 segment of rat, a proximal tubular portion that is less studied. The activity of the Na⁺/H⁺ exchanger was evaluated by the cellular pH recovery after the acidification of the pH_i by the NH₄Cl pulse.

	MATERIALS AND METHODS

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Tubule Preparation

Male Wistar rats (80 g) were anesthetized by tiletamine/zolazepam (Zoletil-Virbac). Their kidneys were removed and slices 2 mm in thickness were prepared and transferred to ice-cold normal Ringer solution. In continuation, proximal tubule S3 segments were isolated by microdissection from the outer stripe of the outer medulla, using a modification of the method originally described by Burg et al. (6). S3 segments were then transferred to glass coverslips prepared with poly-D-lysine (for tubule adhesion). The coverslips were mounted on an inverted microscope (Olympus IX70) in a thermostatically regulated perfusion chamber whose solutions were changed by means of valves. After the experiments, the isolated tubules were removed from the coverslips and submitted to histological analysis.

Measurement of pH_i by Fluorescence Microscopy

pH_i was monitored using the fluorescent probe 2',7'- bicarboxyethyl-5,6-carboxyfluorescein acetoxy-methyl ester (BCECF-AM). The S3 segments were loaded for 10 min with 10 µM BCECF-AM in solution 1 (Table 1). After the loading period, the perfusion chamber was rinsed with solution 1 to remove the BCECF-containing solution. During the experiments, the images were acquired with an intensified ICCD-350F camera. The dye-loaded tubule cells were alternately excited at 440 and 490 nm, and the emission was monitored at 530 nm every 5 s. The fluorescence excitation ratio, I₄₉₀/I₄₄₀, was displayed in pseudo-color on the monitor, and a maximum of six areas per tubule was defined for measurement. The pH_i was standardized by the high K⁺/nigericin (solution 2) technique (36).

The S3 segments were first superfused with 140 mM Na⁺ control solution (solution 1), exhibiting the basal pH_i. Cell pH recovery was examined after the acidification of pH_i with the NH₄Cl pulse technique (3) after 1.5-min exposure to 20 mM NH₄Cl (solution 3). The rate of recovery was measured in the control situation [with external 140 mM Na⁺ (140 Na⁺_e), solution 1], in the absence of external Na⁺ (0 Na⁺_e; solution 4) or in the presence of the following agents: N,N-hexamethylene amiloride [HMA; an Na⁺/H⁺ exchanger inhibitor (10^–4 M)], ethanol alone (in the similar concentrations used to prepare the solutions of aldosterone 10^–12 or 10^–6 M), or aldosterone (10^–12, 10^–10, 10^–8, or 10^–6 M). The rate of recovery was studied in the presence of preincubation (pi) with aldosterone alone (for 1-h, 15- or 2-min pi) or plus spironolactone [a mineralocorticoid receptor (MR) antagonist (10^–6 M)], actinomycin D [an inhibitor of gene transcription (10^–6 M)], cycloheximide [an inhibitor of protein synthesis (40 mM)], mifepristone [RU 486; a glucocorticoid receptor (GR) antagonist (10^–6 M)], HOE 694 [a specific inhibitor of basolateral NHE1 (500 nM)], or S3226 [a specific inhibitor of apical NHE3 (1 µM)]. These drugs were added 15 min before the application of aldosterone. In all the experiments, we calculated the rate of pH_i recovery (dpH_i/dt, pH U/min) from the first 2 min after the start of the pH_i recovery curve, by linear regression analysis. Calculations and drawings were performed by the Excel program after import of the results by a data-acquisition program.

Measurement of [Ca²⁺]_i by Fluorescence Microscopy

Changes in [Ca²⁺]_i were monitored fluorometrically by using the calcium-sensitive probe FLUO-4-AM (28) in the presence of external Na⁺. The S3 segments were loaded for 15 min with 10 µM FLUO-4-AM at 37°C and rinsed in Tyrode's solution (solution 5). FLUO-4 intensity emitted above 505 nm was imaged by using laser excitation at 488 nm on a Zeiss LSM 510 confocal microscope. The images were continuously acquired (at time intervals of 2 s) before and after substitution of experimental solutions. For each tubular Ca²⁺ measurement, the maximum fluorescent signal for 10 cells of the same S3 segment was averaged and then used to calculate the [Ca²⁺]_i of this S3 segment. Transformation of the fluorescence signal to [Ca²⁺]_i was performed by calibration with ionomycin (5 µM; maximum Ca²⁺ concentration) followed by EGTA (2.5 mM; minimum Ca²⁺ concentration) according to the Grynkiewicz equation (15). This equation was originally used for FURA-2 fluorescence, but in previous studies we have results indicating that the basal and both low and high levels of [Ca²⁺]_i measured from single-wavelength FLUO-4 were similar to those measured from dual-wavelength FURA-2 (29, 30). To know the integrity of the cells used in these experiments, after the [Ca²⁺]_i measurements the tubules were submitted to histological analysis.

RT-PCR to MR and GR Receptors in Proximal S3 Segment

For reverse transcriptase-PCR (RT-PCR), the isolated S3 segments were lysed and homogenized and total RNA was extracted. cDNA was synthetized from 250 ng of total RNA, using 200 U/µl of Superscript II (Invitrogen), 50 ng/µl random hexamers as primer, 0.5 mM dNTPs (Invitrogen), and 0.5 mM MgCl₂. Before the addition of RT, dNTPs, and MgCl₂, reaction mixture was incubated at 65°C for 5 min to allow the primers annealing with the poly A tail of the mRNA. cDNA was then synthetized at 42°C for 1 h and used in several PCR reactions. β-Actin, GR, and MR fragments were amplified by PCR for 30 cycles in 25 µl of total volume that contained 2.5 U of TaqDNA Polymerase (Invitrogen), 0.5 mM dNTPs, and 10 pmol of specific primers. Annealing temperature was 55°C. The primers used were: β-actin (980 bp) 5'-GTC CAT CGT GGG CCG CCC GC-3' (sense bp 102–121) and 5'-CTC TTG CTT GCT AAT CCA CAT-3' (antisense 1069–1089) (Database gi: 46411161), MR (380 bp) 5'-AGA AGA TGC ATC AGT CTG CC-3' (sense bp 2504–2523) and 5'-GTG ATG ATC TCC ACC AGC AT-3' (antisense bp 2865–2884) (Database gi: 205340), and GR (530 bp) 5'-TGC AGC AGT GAA ATG GGC AA-3' (sense bp 1719–1738) and 5'-GGG AAT TCA ATA CTC ATG GTC-3' (antisense bp 2232–2252) (Database gi: 66528676).

Solutions and Reagents

The solutions utilized are described in Table 1. These solutions had an osmolality of 300 mosmol/kgH₂O and pH 7.4. BCECF-AM and FLUO-4-AM were obtained from Molecular Probes (Eugene, OR), Superscript II and Polymerase from Invitrogen, and the other reagents from Sigma (St. Louis, MO).

Statistic

The results are presented as means ± SE; N/n is the number of tubules/number of tubular areas. Data were analyzed statistically by ANOVA followed by the Bonferroni's contrast test. Differences were considered significant if P < 0.05.

This study was approved by the Biomedical Sciences Institute/USP-Ethical Committee for Animal Research (CEEA).

	RESULTS

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Acid Extrusion in S3 Segment of Rat Proximal Tubule

Figure 1A shows a representative experiment in which S3 segments were first bathed with 140 mM Na⁺ solution, exhibiting the basal pH_i. After 2-min exposure to NH₄Cl, during which cell pH_i increased transiently, the removal of NH₄Cl caused a rapid acidification of pH_i as a result of NH₃ efflux. In the presence of external 140 mM Na⁺, the initial fall in pH_i was followed by a rapid recovery toward the basal value. Figure 1B shows that in the absence of external Na⁺, the pH_i recovery rate was markedly decreased (and the final pH_i was different from the basal value). This effect is reversed with the return of Na⁺ to the bathing solution (and the final pH_i was not significantly different from the basal value). Figure 1C indicates that in the presence of external 140 mM Na⁺ plus HMA [a Na⁺/H⁺ exchanger inhibitor (10^–4 M)] the pH_i recovery rate also was markedly decreased (and the final pH_i was different from the basal value).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Intracellular pH (pHi) recovery after cellular acidification with the NH4Cl pulse technique in proximal S3 segment of rat. A: in the presence of 140 mM Na+ in the bath, the initial fall in pHi is followed by a recovery of pHi toward the basal value. B: in the absence of external Na+, the pHi recovery rate was markedly decreased (and the final pHi was different from the basal value). This effect is reversed with the return of Na+ to the bathing solution. C: in the presence of external 140 mM Na+ plus N,N-hexamethylene amiloride [HMA; an Na+/H+ exchanger inhibitor (10–4 M)], the pHi recovery rate also was decreased (and the final pHi was different from the basal value).

Figure 2 indicates that in the control situation (140 mM Na⁺_e) the mean of pH_i recovery rate in the first 2 min after cellular acidification with the NH₄Cl pulse was 0.176 ± 0.007 pH U/min (N/n = 23/115), and in the absence of external Na⁺ or in the presence of HMA it signicantly decreased to 0.064 ± 0.005 pH U/min (N/n = 15/68) and 0.056 ± 0.003 pH U/min (N/n = 7/24), respectively. These behaviors indicate that in S3 segment pH_i recovery is mostly dependent on the Na⁺/H⁺ exchanger. However, even in the absence of Na⁺ or in the presence of HMA, a significant slow rate of pH_i recovery is still observed, due to a Na⁺-independent H⁺ extrusion mechanism.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Initial rate of pHi recovery of proximal S3 segment of rat after cellular acidification with NH4Cl, in the presence of 140 mM Na+ external solution (control), 0 Na+ external solution, or 140 mM Na+ external solution plus HMA (10–4 M). Values are means ± SE. N/n = Number of tubules/number of areas. *P < 0.001 vs. control.

1 h of hormonal pi. The main values of pH_i responses found in the principal experimental groups with 1 h of aldosterone pi are in Table 2.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effect of aldosterone [Aldo; 10–12, 10–10, 10–8, and 10–6 M, 1-h preincubation (pi)] or ethanol (16.7 mM, 1-h pi) on the initial rate of pHi recovery after acute acidification in S3 segment of proximal tubule. Values are means ± SE. N/n = Number of tubules/number of areas. *P < 0.001 vs. control or ethanol. &P < 0.001 vs. 10–12 M Aldo. #P < 0.0001 vs. 10–10 M Aldo. P < 0.001 vs. 10–8 M Aldo.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Initial rate of pHi recovery after cellular acidification with NH4Cl (A) and [Ca2+]i (B) on proximal S3 segment of rat, in the presence of aldosterone (10–12 and 10–6 M, 1-h pi) alone or plus spironolactone (10 µM), actinomycin D (10–6 M), or cycloheximide (40 mM). Values are means ± SE. N/n = Number of tubules (each tubule is the average of 10 cells)/number of areas. *P < 0.001 vs. respective control. &P < 0.001 vs. 10–12 M Aldo. #P < 0.05 vs. 10–6 M Aldo. P < 0.001 vs. respective control and 10–12 M Aldo. P < 0.001 vs. respective control and 10–6 M Aldo.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Effect of aldosterone (10–12, 10–10, 10–8, and 10–6 M, 15-min pi) on the initial rate of pHi recovery after acute acidification on proximal S3 segment of rat. Values are means ± SE. N/n = Number of tubules/number of areas. *P < 0.05 vs. control. &P < 0.05 vs. 10–12 M Aldo. #P < 0.001 vs. 10–10 M Aldo. P < 0.001 vs. 10–8 M Aldo.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Initial rate of pHi recovery after cellular acidification with NH4Cl (A) and [Ca2+]i (B) on proximal S3 segment of rat, in the presence of aldosterone (10–12 and 10–6 M, 15-min pi) alone or plus spironolactone (10 µM), actinomycin D (10–6 M), cycloheximide (40 mM), or RU 486 (10–6 M). Values are means ± SE. N/n = Number of tubules (each tubule is the average of 10 cells)/number of areas. *P < 0.05 vs. respective control. &P < 0.05 vs. 10–12 M Aldo. P < 0.001 vs. 10–6 M Aldo.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Initial rate of pHi recovery after cellular acidification with NH4Cl (A) and [Ca2+]i (B) on proximal S3 segment of rat, in the presence of aldosterone (10–12 and 10–6 M, 2-min pi) alone or plus spironolactone (10 µM), actinomycin D (10–6 M), cycloheximide (40 mM), or RU 486 (10–6 M). Values are means ± SE. N/n = Number of tubules (each tubule is the average of 10 cells)/number of areas. *P < 0.05 vs. respective control. &P < 0.001 vs. 10–12 M Aldo. P < 0.001 vs. 10–6 M Aldo.

Effect of Aldosterone on [Ca²⁺]_i of Proximal S3 Segment

The present data show that proximal S3 segment exhibited a mean baseline [Ca²⁺]_i of 100 ± 2 nM (n = 87). This value did not change after 1 min, 15 min, or 1 h of pi with Tyrode^,s solution. However, after 1 min of addition of aldosterone (10^–12 or 10^–6 M) to the bath, there was a transient (1.5 min) and dose-dependent increase in [Ca²⁺]_i [of 56 and 133% of the control value, respectively (Fig. 7B)], followed by a recovery toward the basal value. After 6 min of pi with aldosterone (10^–12 or 10^–6 M), the [Ca²⁺]_i begins to increase and after 10 min it was 127 and 227% of the control value, respectively, and after 1 h of hormonal pi these values did not significantly change (Figs. 6B and 4B, respectively). In the presence of spironolactone, actinomycin D, or cycloheximide alone, the [Ca²⁺]_i was not different from the control value. Also, the stimulatoty effect of aldosterone (after 2 or 15 min of pi) on [Ca²⁺]_i was not modified by these drugs (Figs. 7B and 6B, respectively). However, after 1 h of hormonal pi, the [Ca²⁺]_i was significantly reduced by these inhibitors of the genomic effects (Fig. 4B). In addition, RU 486 alone caused a significant decrease in the [Ca²⁺]_i (of 28% of the control value), prevented the stimulatory effect of aldosterone (10^–12 M, 2- or 15-min pi), and reversed the stimulatory effect of aldosterone (10^–6 M, 2- or 15-min pi) on the [Ca²⁺]_i to an inhibitory effect (Figs. 7B and 6B, respectively).

Figure 8 indicates the detection by RT-PCR of MR and GR in proximal S3 segments.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Detection by RT-PCR of mineralocorticoid receptor (MR; 380 bp) and glucocorticoid receptor (GR; 530 bp) in proximal S3 segments of rat. β-Actin (980 bp) is a positive control. M = 1 kb plus DNA ladder.

	DISCUSSION

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Our data demonstrate that S3 segment cells in pH 7.4 HCO₃^–-free solution maintain a mean baseline pH_i of 7.09 ± 0.006 (N/n = 338/1.670), a value compatible with the data found in the best-characterized renal epithelial Madin-Darby canine kidney (MDCK) cells (28). Our results indicating that in S3 segment cells in the control situation (140 mM Na_e⁺) the mean pH_i recovery rate in the first 2 min after cellular acidification with the NH₄Cl pulse is 0.176 ± 0.007 pH U/min (N/n = 23/115) are in accordance with the values found by us in renal (28) and colon (26) epithelial cells.

The present data show that removal of extracellular Na⁺ resulted in a significant inhibition of the pH_i recovery that is subsequently reversed with the return of Na⁺ to the bathing solution, indicating that the pH_i recovery in the rat kidney proximal S3 segment is mostly dependent on Na⁺/H⁺ exchanger (Fig. 1). However, even in the absence of Na⁺ or in the presence of HMA (a Na⁺/H⁺ exchanger inhibitor), a significant rate of pH_i recovery was still observed (Fig. 2). Preliminary studies from our laboratory indicate that in rat S3 segment the vacuolar H⁺-ATPase is responsible for the Na⁺-independent H⁺ extrusion mechanism (data not shown), as observed in rabbit kidney proximal S3 segment (22). However, this Na⁺-independent H⁺ extrusion mechanism initiates 2.5 min after cellular acidification with the NH₄Cl pulse (Fig. 1, B and C) and does not interfere in the present evaluation of the rate of pH_i recovery dependent on the Na⁺/H⁺ exchanger (since it is calculated from the first 2 min after cellular acidification).

For a long time it has been known that the mineralocorticoids, including aldosterone, act on acid excretion of distal nephron segments such as the cortical and medullary collecting ducts (33, 41). In addition, some authors found evidence that aldosterone acts on proximal tubule cells. Drumm et al. (7) postulated that aldosterone stimulates activity and surface expression of NHE3 in human renal proximal tubule epithelial cell (RPTEC). Krug et al. (21) and Zallocchi et al. (42) demonstrated that aldosterone induces expression of Na⁺/H⁺ exchanger isoforms in brush-border membrane. Pinto et al. (32) indicate that aldosterone increases NHE activity, in immortalized proximal tubular epithelial cells from spontaneously hypertensive rats (SHR). However, our results indicate, for the first time, that aldosterone modulates the mechanism of pH_i regulation via Na⁺/H⁺ exchanger in isolated proximal S3 segment.

Our data show that aldosterone at low concentrations (10^–12–10^–8 M, 1-h, 15- or 2-min pi) causes a significant increase in the velocity of pH_i recovery; however, at high concentration (10^–6 M; 1-h, 15- or 2-min pi), aldosterone decreases it. Our present results are in accordance with the data of Braun et al. (4) that observed a rapid dose-dependent stimulatory effect of aldosterone on Src kinase in the kidney cell line M-1. Our data also are in accordance with the data indicating that in proximal cells of SHR aldosterone stimulates the Na⁺/H⁺ exchanger in time- and concentration-dependent manner (32). However, the present study is the first demonstration, to our knowledge, that aldosterone has a dose-dependent biphasic (stimulatory/inhibitory) effect on the Na⁺/H⁺ exchanger. A similar dose-dependent biphasic effect of angiotensin II on the Na⁺/H⁺ exchanger has been reported in the proximal tubule (18) and in MDCK (28) and colon (26) epithelial cells. Thus, it is possible that, similar to angiotensin II, the dose-dependent biphasic effect of aldosterone on the Na⁺/H⁺ exchanger corresponds to an action on the regulation of extracellular volume. In this manner, it is interesting to consider 1) the physiological level of aldosterone in blood is 10^–10–10^–9 M but it may increase or decrease in conditions of extracellular volume modification, 2) cardiovascular tissues produce aldosterone, with the result that this hormone is more concentrated in vascular tissue than in the circulation (2), 3) high salt intake increases aldosterone production in cardiovascular tissues (35), and 4) at the present time, we do not have information about the aldosterone levels in the tubular luminal compartment or in the peritubular capillary, but it is necessary to remember that it is probable that, similar to angiotensin II, aldosterone may concentrate in the tubular lumen due to fluid reabsorption.

Our results show that the dose-dependent biphasic effect we observed on the Na⁺/H⁺ exchanger is only due to aldosterone since in the presence of ethanol alone (1-h pi), in the similar concentrations used to prepare the solutions of aldosterone 10^–12 or 10^–6 M, the basal pH_i and the pH_i recovery rate were not different from the control values (Table 2 and Fig. 3).

Our data show that with HOE 694 (a specific inhibitor of basolateral NHE1) alone or plus aldosterone (10^–12 or 10^–6 M, 2-min pi), there was a complete inhibition of the pH_i recovery rate, indicating that this process is only due to the NHE1 isoform. Additionally, S3226 (a specific inhibitor of apical NHE3) alone did not change the pH_i recovery rate and also did not prevent the stimulatory effect of aldosterone (10^–12 M, 2-min pi) or the inhibitory effect of aldosterone (10^–6 M, 2-min pi) on this parameter. These results show that the apical NHE3 isoform of the Na⁺/H⁺ exchanger has no participation on the biphasic effect of aldosterone on the pH_i recovery rate in S3 segment, confirming that this process is only due the basolateral NHE1 isoform. These data are in accordance with studies in proximal cells of SHR (32) indicating that NHE1 is the most likely NHE isoform involved in the aldosterone-induced increase in NHE activity.

Several studies identify the Na⁺/H⁺ exchanger as a target for genomic and nongenomic regulation by aldosterone and show that this mineralocorticoid can control the absorptive function of epithelial tissues through regulation of this exchanger (8, 11–13, 23, 24, 27). The present results indicate that spironolactone (a MR antagonist), actinomycin D (an inhibitor of gene transcription), or cycloheximide (an inhibitor of protein synthesis) alone did not show effects on the pH_i recovery rate and on the [Ca²⁺]_i and failed to prevent the short-term effects of aldosterone (10^–12 and 10^–6 M, 15- or 2-min pi) on these parameters (Figs. 6 and 7) but not the long-term effects of aldosterone (10^–12 and 10^–6 M, 1-h pi; Fig. 4) on these parameters. So, our study indicates that aldosterone has a dose-dependent biphasic effect on the NHE1 exchanger via a nongenomic (15- or 2-min pi) and genomic (1-h pi) pathway. The genomic aldosterone-induced regulation of the NHE1 exchanger seems to be a mineralocorticoid-specific effect, since the aldosterone receptor antagonist spironolactone significantly inhibited the activity of the exchanger, as had been observed in RPTEC (7) or from SHR (32). The present results are in accordance with our finding of MRs in the proximal S3 segments by RT-PCR (Fig. 8). Our data are also consistent with the studies of Krug et al. (20) and Todd-Turla et al. (37) that had reported mRNA and protein expression of the MR in the proximal tubule, and with the results of Pinto et al. (32) that confirmed the presence of the MR transcript by PCR in SHR proximal tubular cells.

The direct nongenomic aldosterone-induced effects, many in second messengers, may occur via the classical MR. The possible involvement of MR in mediating some rapid responses has been suggested for steroid hormones (9) and Grossmann et al. (14) demonstrated that MR contributes to rapid aldosterone activation of the ERK 1/2 pathway in Chinese hamster ovary cells. However, the receptor for the nongenomic aldosterone-induced mechanism is still unknown. The rapid increase in nongenomic activity of the Na⁺/H⁺ exchanger we observed seems not to occur by MR, because spironolactone failed to prevent this increase (Figs. 6 and 7). To improve our knowledge of this mechanism, we used RU 486 (a GR antagonist), since it is known that aldosterone-induced effects may also occur via GR receptors, although the affinity of GR for aldosterone is lower. Our results showed that RU 486 alone caused a significant decrease in the velocity of pH_i recovery and in the [Ca²⁺]_i (both of 28% of the control value), prevented the stimulatory effect of aldosterone (10^–12 M, 15- or 2-min pi) on both parameters, and maintained the inhibitory effect of aldosterone (10^–6 M, 15- or 2-min pi) on the velocity of pH_i recovery but reversed its stimulatory effect on [Ca²⁺]_i to an inhibitory effect. So, our results indicate that probably the GRs participate in the direct nongenomic (2 or 15 min) dose-dependent biphasic effect of aldosterone on the NHE1 exchanger. These results are also in accordance with the studies of Todd-Turla et al. (37) in proximal tubules and with our data indicating the presence of these receptors in S3 segments by RT-PCR (Fig. 8). However, the possible physiological role of GR action should also be considered since the GR antagonist alone reduces the velocity of pH_i recovery and the [Ca²⁺]_i below the basal levels (Figs. 6 and 7). This could be due to an unspecific inhibitory action of this GR antagonist, but it might also indicate that, in basal conditions, a basal level of aldosterone or some glucocorticoid binding to GR receptors may exist, causing some tonic activation of the Na⁺/H⁺ exchanger and the [Ca²⁺]_i. This question remains to be determined.

The present data indicate that 1 min after addition of aldosterone (10^–12 or 10^–6 M) to the bath, there was a transient and dose-dependent increase of the [Ca²⁺]_i. After 10 min of hormonal addition the [Ca²⁺]_i was significantly higher, mainly for aldosterone 10^–6 M. After 1 h of hormonal addition, the [Ca²⁺]_i remained high and not different from the values found with hormonal pi for 10 min. These results are in accordance with several authors who found an aldosterone-induced increase in [Ca²⁺]_i (11, 14, 17, 19, 31, 39), indicating that [Ca²⁺]_i is a prerequisite for aldosterone action. Nongenomic and pregenomic actions usually involve second messengers, such as inorganic ions, cAMP, or various protein kinases. Calcium is one of the second messengers and its increase is the first intracellular signal in response to aldosterone (34).

Our results show that the dose-dependent biphasic effect of aldosterone on the Na⁺/H⁺ exchanger is associated with an increase in [Ca²⁺]_i, similar to that observed with angiotensin II (26, 28). It has been shown that the NHE1 exchanger (the major basolateral isoform of the Na⁺/H⁺ exchanger in polarized epithelial cells 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 Ca²⁺/calmodulin, thus suppressing the inhibition, that is, stimulating the exchanger at low Ca²⁺/calmodulin levels. A low-affinity site, however, binds with calcium and calmodulin only at high concentrations, and under these conditions inhibits the exchanger activity (26, 28, 38). This behavior is compatible with our present findings indicating stimulation of NHE1 exchanger by increases of [Ca²⁺]_i in the lower range (at 10^–12 M aldosterone, 1-h, 15- or 2-min pi) and inhibition of NHE1 exchanger at high [Ca²⁺]_i levels (at 10^–6 M aldosterone, 1-h, 15- or 2-min pi; Figs. 4, 6, and 7). This behavior is also compatible with our results showing that spironolactone, actinomycin, or cycloheximide did not affect the effects of aldosterone (10^–12 or 10^–6 M, 15- or 2-min pi) on [Ca²⁺]_i and on the biphasic action of the NHE1 exchanger, but these agents impair the effects of aldosterone (10^–12 or 10^–6 M, 1-h pi) on [Ca²⁺]_i and on the NHE1 exchanger (Figs. 4, 6, and 7). Our results with RU 486 also confirm this behavior (Figs. 6 and 7) since 1) this GR antagonist alone caused a significant decrease in the velocity of pH_i recovery and in the [Ca²⁺]_i (both of 28% of the control value), 2) RU 486 prevented the stimulatory effect of aldosterone (10^–12 M, 15- or 2-min pi) in both parameters, and 3) RU 486 maintains the inhibitory effect of aldosterone (10^–6 M, 15- or 2-min pi) on the velocity of pH_i recovery because it reversed the stimulatory effect of aldosterone (10^–6 M, 15- or 2-min pi) on [Ca²⁺]_i to an inhibitory effect. So, our present results are also compatible with inhibition of the NHE1 exchanger at low [Ca²⁺]_i levels (at 10^–6 M aldosterone plus RU 486).

On the other hand, our present data showing a nongenomic effect of aldosterone on [Ca²⁺]_i in proximal S3 segment are in agreement with results found in M-1 cortical collecting duct cells (17). Additionally, our findings indicating a genomic effect of aldosterone (10^–6 M, 1-h pi) in increasing [Ca²⁺]_i to 254% of the control value in proximal S3 segment are similar to data showing that exposure of A₆D₂ cells to aldosterone (10^–6 M) promotes an increase in [Ca²⁺]_i to 230% of the control value, 60–70 min after addition of the hormone, and this rise in [Ca²⁺]_i was inhibited in magnitude by actinomycin and cycloheximide, suggesting that aldosterone induces an increase in [Ca²⁺]_i via a process dependent on mRNA and protein synthesis (31). However, the effect of a long-term increase in [Ca²⁺]_i is still not adequately explained. Numerous reports suggest that a chronic increase in [Ca²⁺]_i downregulates apical sodium channels and reduces transepithelial movement of sodium in epithelial cells (40).

In summary, the present study on the proximal S3 segment suggests a role for cell calcium in regulating the process of pH_i recovery after the acid load induced by NH₄Cl, mostly mediated by the basolateral NHE1, stimulated by aldosterone (10^–12, 10^–10, and 10^–8 M) and impaired by aldosterone (10^–6 M). The data indicate a hormonal genomic (1 h) and nongenomic (15 or 2 min) action on [Ca²⁺]_i and on the NHE1. They are compatible with stimulation of the NHE1 by increases in cell calcium in the lower range (at 10^–12 M aldosterone) and inhibition by increases in cell calcium at high levels (at 10^–6 M aldosterone) or decreases in cell calcium (at 10^–6 M aldosterone plus RU 486). The present results indicating that MR and probably GR participate, respectively, in genomic and nongenomic effects of aldosterone on [Ca²⁺]_i and on the NHE1 are in accordance with our findings indicating the presence of these receptors in the proximal S3 segments by RT-PCR. These aldosterone effects may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Pesquisas. Portions of this work were presented, as poster communication, at the Annual Meeting of the American Society of Nephrology, San Francisco, CA, 2004, and, as free communication, at the World Congress of Nephrology, Rio de Janeiro, Brazil, 2007 and were published in abstract form (J Am Soc Nephrol 15: 78A, 2004 and WCN 2007 Book of Abstracts, T-FC-103).

	FOOTNOTES

 

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

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