The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pH(i)) and cytosolic free calcium concentration ([Ca(2+)](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. pH(i) recovery rate was examined in the first 2 min after the acidification of pH(i) with a NH(4)Cl pulse. In the control situation, the pH(i) 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 microM) 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 [Ca(2+)](i) progressively from 99 +/- 10 (n = 20) to 234 +/- 7 mM (n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca(2+)](i), and the subsequent addition of ANG II caused a recovery of [Ca(2+)](i) but without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca(2+)](i) in regulating the process of pH(i) 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.

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Leite-Dellova DC, Oliveira-Souza M, Malnic G, Mello-Aires M. Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na

Bicarbonate reabsorption was evaluated by stationary microperfusion "in vivo" early distal (ED) and late distal (LD) segments of at kidney. Intratubular pH was recorded by double-barreled of H+ exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of angiotensin II (ANG II) (10(-12) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.930 +/- 0.060 to 2.64 +/- 0.210 nmol.cm-2.s-1 in luminally perfused tubules and from 0.850 +/- 0.040 to 2.03 +/- 0.210 nmol.cm-2.s-1 during capillary perfusion) and LD segments from 0.310 +/- 0.130 to 2.16 +/- 0.151 nmol.cm-2.s-1 during luminal perfusion and from 0.530 +/- 0.031 to 2.16 +/- 0.211 nmol.cm-2.s-1 with capillary perfusion). The addition of the AT1-receptor antagonist losartan (10(-6) M) to luminal perfusion blocked luminal ANG II-mediated stimulation in ED and LD segments. 5-(N,N-hexamethylene)amiloride (10(-4) M) added to luminal perfusion inhibited luminal ANG II-mediated stimulation in ED (by 81%) and LD (by 54%) segments. The addition of bafilomycin A1 (2 x 10(-7) M) to luminal perfusion does not affect luminal ANG II-mediated stimulation in ED segments but reduces it in LD segments (by 33%). During the addition of atrial natriuretic peptide (ANP) (10(-6) M) or ANG II plus ANP in both segments, no significant differences in HCO3- reabsorption were observed. Our results indicate that luminal ANG II acts to stimulate Na+/H+ exchange in ED and LD segments via activation of AT1 receptors, as well as the vacuolar H(+)-adenosinetriphosphatase in LD segments. ANP does not affect HCO3- reabsorption in either ED or LD segments and does not impair the stimulation caused by ANG II.

Bicarbonate reabsorption was evaluated by stationary microperfusion of in vivo early distal (ED) and late distal (LD) segments of rat kidney. Intratubular pH was recorded by double-barreled H ion-exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of luminal arginine vasopressin (AVP, 10(-9) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.931 +/- 0.061 to 2.12 +/- 0.171 nmol.cm-2.s-1] and LD segments [from 0.542 +/- 0.086 to 1.67 +/- 0.111 nmol.cm-2.s-1]. The addition of the V1-receptor antagonist [(d (CH2)5, Tyr (Et)2) arginine vasopressin] (10(-5) M) to luminal perfusion blocked luminal AVP mediated stimulation in ED and LD segments. 5-(N, N-hexamethylene) amiloride (10(-4) M) added to luminal perfusion inhibited luminal AVP-mediated stimulation in ED (by 63.7%) and LD (by 34.1%) segments. The addition of Bafilomycin A1 (2 x 10(-7) M) to the luminal perfusion did not affect luminal AVP-mediated stimulation in ED segments, but reduced it (by 31.7%) in LD segments. Our results indicate that luminal AVP acts to stimulate the Na(+)-H+ exchange in ED and LD segments via activation of V1 receptors, as well as the vacuolar H(+)-ATPase in LD segments.

The acute direct action of angiotensin-(1-7) [ANG-(1-7)] on bicarbonate reabsorption (JHCO(3)(-)) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO(3)(-) is 2.82 ± 0.078 nmol·cm(-2)·s(-1) (50). ANG-(1-7) (10(-12) or 10(-9) M) in luminally perfused tubules decreases JHCO(3)(-) (36 or 60%, respectively), but ANG-(1-7) (10(-6) M) increases it (80%). A779 increases JHCO(3)(-) (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1-7) on it. S3226 decreases JHCO(3)(-) (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 JHCO(3)(-) and that the biphasic dose-dependent effect of ANG-(1-7) on JHCO(3)(-) is mediated by the Mas receptors via the Na(+)/H(+) exchanger 3 (NHE3). The control value of intracellular Ca(2+) concentration ([Ca(2+)](i)), as monitored using fura-2 AM, is 101 ± 2 nM (6), and ANG-(1-7) (10(-12), 10(-9), or 10(-6)M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca(2+)](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 [Ca(2+)](i) and JHCO(3)(-). Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1-7) on [Ca(2+)](i) and JHCO(3)(-) may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca(2+)](i) modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

Background/Aims: To assess the possible contribution of the β-adrenergic overstimulation in early stages of renal injury, the present study evaluated, in rats, the effects of the β-adrenoceptor agonist isoproterenol (ISO) on renal function and morphology, as well as the renal mRNA and protein expression of the NADPH oxidase isoform 4 (Nox 4) and subunit p22^phox, endoplasmic reticulum (ER) stress, pro-inflammatory, pro-apoptotic and renin-angiotensin system (RAS) components. Methods: Wistar rats received ISO (0.3 mg.kg^-1.day^-1 s.c.) or vehicle (control) for eight days. At the end of the treatment, food and water intake, urine output and body weight gain were evaluated and renal function studies were performed. Renal tissue was used for the morphological, quantitative PCR and immunohistochemical studies. Results: ISO did not change metabolic parameters or urine output. However it induced a decrease in renal blood flow and an increase in the filtration fraction. These changes were accompanied by increased cortical mRNA and protein expression for the renal oxidative stress components including Nox 4 and p22^phox; ER stress, pro-inflamatory, pro-apoptotic as well as RAS components. ISO also induced a significant increase in medullar renin protein expression. Conclusion: These findings support relevant information regarding the contribution of specific β-adrenergic hyperactivity in early stage of renal injury, indicating the reactive oxygen species, ER stress and intrarenal RAS as important factors in this process.

The results are compatible with stimulation of the Na+/H+ exchanger by increases of [Ca2+]i in the lower range (at 10(-12) or 10(-9) mol/L AVP, via basolateral V1 receptors) and inhibition at high [Ca2+]i levels (at 10(-6) mol/L AVP, via basolateral V1 and V2 receptors). ANP, by impairing the path causing the increase in [Ca2+]i, blocks both the stimulatory and inhibitory effects of AVP on Na+-dependent pHi recovery.