The acute direct action of angiotensin-(1–7) [ANG-(1–7)] on bicarbonate reabsorption ( JHCO₃⁻) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO₃⁻is 2.82 ± 0.078 nmol·cm⁻²·s⁻¹(50). ANG-(1–7) (10⁻¹²or 10⁻⁹M) in luminally perfused tubules decreases JHCO₃⁻(36 or 60%, respectively), but ANG-(1–7) (10⁻⁶M) increases it (80%). A779 increases JHCO₃⁻(30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1–7) on it. S3226 decreases JHCO₃⁻(45%) and changes the stimulatory effect of ANG-(1–7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1–7). Our results indicate that in the basal condition endogenous ANG-(1–7) inhibits JHCO₃⁻and that the biphasic dose-dependent effect of ANG-(1–7) on JHCO₃⁻is mediated by the Mas receptors via the Na⁺/H⁺exchanger 3 (NHE3). The control value of intracellular Ca²⁺concentration ([Ca²⁺]_i), as monitored using fura-2 AM, is 101 ± 2 nM ( 6 ), and ANG-(1–7) (10⁻¹², 10⁻⁹, or 10⁻⁶M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca²⁺]_i(25%) but impairs the stimulatory effect of all doses of ANG-(1–7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1–7) dose-dependent effects on [Ca²⁺]_iand JHCO₃⁻. Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1–7) on [Ca²⁺]_iand JHCO₃⁻may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca²⁺]_imodification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na⁺/H⁺exchanger and on the [Ca²⁺]_iwere investigated in isolated rat S3 segment. Aldosterone [10⁻¹², 10⁻¹⁰, or 10⁻⁸M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10⁻⁶M 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⁺]_iand after 6-min pi there was a new increase of [Ca²⁺]_ithat 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 (10⁻¹²M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10⁻⁶M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca²⁺]_ito 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⁺]_iand on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca²⁺]_iin the lower range (at 10⁻¹²M aldosterone) and inhibition by increases at high levels (at 10⁻⁶M aldosterone) or decreases in [Ca²⁺]_i(at 10⁻⁶M aldosterone plus RU 486).

The direct action of aldosterone (10⁻¹²M) on net bicarbonate reabsorption ( JHCO₃⁻) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in JHCO₃⁻from a mean control value of 2.84 ± 0.08 [49/19 ( n° of measurements/ n° of tubules)] to 4.20 ± 0.15 nmol·cm⁻²·s⁻¹(58/10). Aldosterone perfused into peritubular capillaries also increased JHCO₃⁻, compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca²⁺]_i), monitored fluorometrically. In the presence of ethanol (in similar concentration used to prepare the hormonal solution), spironolactone (10⁻⁶M, a mineralocorticoid receptor antagonist), actinomycin D (10⁻⁶M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the JHCO₃⁻and the [Ca²⁺]_iwere not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on JHCO₃⁻and on [Ca²⁺]_i. However, in the presence of RU 486 alone [10⁻⁶M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on JHCO₃⁻and on [Ca²⁺]_iwas observed; this antagonist also inhibited the stimulatory effect of aldosterone on JHCO₃⁻and on [Ca²⁺]_i. These studies indicate that luminal or peritubular aldosterone (10⁻¹²M) has a direct nongenomic stimulatory effect on JHCO₃⁻and on [Ca²⁺]_iin proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates JHCO₃⁻in middle proximal tubule.

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H+-ATPase and on cytosolic free calcium concentration ([Ca2+]i) were investigated in isolated proximal S3 segments of rats during superfusion with an Na+-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH4Cl pulse, was 0.064 ± 0.003 pH units/min ( n = 17/74) and was abolished with concanamycin. Aldosterone (10−12, 10−10, 10−8, or 10−6 M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca2+]i was 103 ± 2 nM ( n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca2+]i and after 6-min preincubation there was a new increase in [Ca2+]i that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca2+]i but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca2+ chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H+-ATPase and on [Ca2+]i. The results are compatible with stimulation of the H+-ATPase by increases in [Ca2+]i (at 10−12-10−6 M aldosterone) and inhibition of the H+-ATPase by decreases in [Ca2+]i (at 10−12 or 10−6 M aldosterone plus RU 486).

The acute effects of angiotensin-1–7 [ANG-(1–7)] on the reabsorptive bicarbonate flow (J[ ]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H⁺. In WKY rats, the control J[ ] was 2.40 ± 0.10 nmol·cm⁻²·s⁻¹( n = 120); losartan (10⁻⁷M) or A779 (10⁻⁶M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[ ]. ANG-(1–7) had biphasic effects on J[ ]: at 10⁻⁹M, it inhibited, and at 10⁻⁶, it stimulated the flow. S3226 [10⁻⁶M, a specific Na⁺-H⁺exchanger 3 (NHE3) antagonist] decreased J[ ] and changed the stimulatory effect of ANG-(1–7) to an inhibitory one but did not alter the inhibitory action of ANG-(1–7). In SHR, the control J[ ] was 2.04 ± 0.13 nmol·cm⁻²·s⁻¹( n = 56), and A779 and/or losartan reduced the flow. ANG-(1–7) at 10⁻⁹M increased J[ ], and ANG-(1–7) at 10⁻⁶M reduced it. The effects of A779, losartan, and S3226 on the J[ ] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1–7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1–7) at 10⁻⁹or 10⁻⁶M. In hypertensive animals, a high plasma level of ANG-(1–7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.