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

Abstract

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.

ANG II, ANG-(1-7), ALDO and AVP biphasic effects on Na+/H+ transport: the role of cellular calcium

“…In addition, the results of in vivo experiments on rat proximal convoluted tubules [191] were compatible with the notion that the NHE3 exchanger is stimulated by a moderate increase in [Ca 2+ ]i in the presence of a high dose of ANG-(1-7) (10 −6 M) and that the inhibition of this exchanger largely increases the [Ca 2+ ]i that was induced by low doses of ANG-(1-7) (10 −12 or 10 −9 M). These findings are in opposition to those described in previous studies of ANG II [113,198]. Additionally, the experiments in the in vivo rat proximal convoluted tubules study used BAPTA or thapsigargin [191] and confirmed the role of cytosolic calcium in the regulation of NHE3.…”

“…In research performed using MDCK cells (a permanent cell line with morphological and physiological similarities to cells in the collecting duct), we demonstrated that ANG II has a dose-dependent biphasic effect on Na + /H + in the distal nephron [113]. In this study, we used fluorescent probes to measure the recovery rate of intracellular pH [(pHi)r] after inducing the acidification of the pHi via an NH 4 Cl pulse, and we also monitored the [Ca 2+ ]i.…”

“…Hence, the activation/inhibition of Na + / H + exchanger by ALDO needed to be more clearly defined, because it was possibly that similar to ANG II [83,113,198], ALDO had a dosedependent biphasic effect on the Na + /H + exchanger (with low doses stimulating and high doses inhibiting the exchanger). In addition, it was also shown that an elevation in [Ca 2+ ]i has the following effects: i) it serves as a second messenger in the ALDO-mediated initiation of nongenomic Na + /H + exchanger activation [271], and ii) it is a prerequisite for the genomic action of ALDO, and strong evidence indicates that pregenomic hormonal responses can influence genomic processes [284].…”

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H<sup>+</sup>-ATPase subcellular vesicle trafficking

“…The cells were harvested using trypsin ethyleneglycol-bis (b-aminoethyl ether)-N, N'-tetraacetic acid (EGTA, 0.02%), seeded on sterile glass coverslips and incubated for 72 h in the DMEM medium until confluent. At the time of the experiment, the mean pHi was 7.16  0.04 (n = 97), measured by the exposure of the cells to 10 M BCECF-AM in the external solution, which was 3 HCO  -free and at pH 7.4 [22]; therefore, these MDCK cells are subtype C11 [23].…”

Human kidney anion exchanger 1 localisation in MDCK cells is controlled by the phosphorylation status of two critical tyrosines

“…Both aldosterone and angiotensin II treatment increase intracellular Ca 2+ levels (Gekle et al, 2002;Oliveira-Souza and De Mello-Aires, 2000) in MDCK cells, whereas vasopressin can increase cAMP levels or Ca 2+ by V1 and V2 receptors, respectively (Oliveira-Souza et al, 2004). All three hormones appear to increase proton transport and bicarbonate reabsorption (Wagner et al, 2004) implicating Ca 2+ and cAMP as potential signals for acidosis.…”