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. 2012 Mar 15;302(6):F679-87.
doi: 10.1152/ajprenal.00368.2011. Epub 2011 Dec 14.

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

Peng Sun  1 , Peng Yue, Wen-Hui Wang
Affiliations

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

Peng Sun et al. Am J Physiol Renal Physiol. .

Abstract

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.

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Figures

Fig. 1.
Fig. 1.
A: channel recording shows the effect of 50 nM ANG II on epithelial Na+ channel (ENaC) activity in the cortical collecting duct (CCD). The experiments were performed in a cell-attached patch and ANG II was directly added to the bath. The top 2 traces show the channel activity before ANG II and the bottom 2 traces demonstrate ENaC activity after addition of ANG II. The channel closed level is indicated by “C” and the holding potential was 60 mV (hyperpolarization). B: perforated whole cell recording shows the effect of ANG II (50 nM) on the amiloride-sensitive whole cell Na currents in a principal cell (PC) of the CCD. The bath solution contains (in mM) 135 Na methanesulfonate, 5 KCl, 2 CaCl2, 1 MgCl2, 2 glucose, 5 BaCl2, and 10 HEPES adjusted to pH 7.4 with NaOH. The pipette solution was composed of (in mM) 7 KCl, 123 aspartic acid, 20 CsOH, 20 TEAOH, 5 EGTA, 10 HEPES, 3 MgATP, and 0.3 NaGDPβS with the pH adjusted to 7.4 with KOH. The PC was clamped from −100 to 60 mV with 20-mV increment. The Na currents were determined by adding amiloride (10 μM) at the end of experiments. C: current-voltage (I-V) plot summarizes 6 experiments similar to those demonstrated in B. *Significant difference between ANG II's group and the corresponding control value.
Fig. 2.
Fig. 2.
A: perforated whole cell recording shows the effect of ANG II (50 nM) on the amiloride-sensitive Na currents of PC in the presence of losartan (10 μM). The PC was clamped from −100 to 60 mV with 20-mV increment. The Na currents were determined by adding amiloride at the end of experiments. B: I-V plot summarizes 5 experiments similar to those demonstrated in A.
Fig. 3.
Fig. 3.
A: whole cell recording shows the effect of ANG II (50 nM) on amiloride-sensitive Na currents at −100 mV in the presence of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-AM (50 μM). B: I-V plot summarizes results of the experiments in which the effect of ANG II on the whole cell Na currents was examined in the presence of BAPTA-AM. C: whole cell recording shows the effect of phorbol-12-myristate 13-acetate (PMA; 5 μM) on amiloride-sensitive Na currents at −100 mV in the presence of BAPTA-AM. D: I-V plot summarizes the results of experiments in which the effect of PMA on whole cell Na currents was examined in the presence of BAPTA-AM. The CCD was incubated with BAPTA-AM-containing solution for 10 min before adding ANG II or PMA. The whole cell Na currents in PC were measured with the perforated whole cell recording from −100 to 60 mV with 20-mV increment. The Na currents were determined by adding amiloride at the end of experiments. *Significant difference between control and experimental groups.
Fig. 4.
Fig. 4.
A: I-V plot summarizes experiments in which the effect of ANG II on the whole cell Na currents was examined in the presence of GF109203 (5 μM). The whole cell Na currents in the PC were measured with the perforated whole cell recording from −100 to 60 mV with 20-mV increment. The Na currents were determined by adding amiloride at the end of experiments. B: bar graph summarizes results of ANG II's effect on Na currents in the absence of or in the presence of PKC inhibitors (5 μM GF109203X, 100 nM calphostin C).
Fig. 5.
Fig. 5.
A: I-V curve summarizes experiments in which the effect of ANG II on the whole cell Na currents was examined in the presence of diphenyleneiodonium chloride (DPI; 10 μM). B: I-V curve summarizes experiments in which the effect of PMA (5 μM) on the whole cell Na currents was examined in the presence of DPI.
Fig. 6.
Fig. 6.
A: whole cell recording shows the effect of pyrogallol (100 μM) on amiloride-sensitive Na currents at −100 mV. B: I-V plot summarizes the effect of pyrogallol (100 μM) on the whole cell Na currents in the PC. C: bar graph summarizes the effect of pyrogallol and xanthine (0.3 mM)/xanthine oxidase (7 μU/ml) on the amiloride-sensitive whole cell Na currents at −100 mV measured with perforated patches.
Fig. 7.
Fig. 7.
H2O2 diminishes the arachidonic acid (AA)-induced inhibition of ENaC in the rat CCD. A: channel recording in a cell-attached patch shows that AA (10 μM) inhibited ENaC but the effect of AA was attenuated in the presence of 100 μM H2O2 (B). C: bar graph summarizes the effect of AA on the ENaC activity in the presence or absence of H2O2. *Significant difference from the rest of groups and #significant difference compared with its control. The experiments were performed in the CCD of rats on a high-K (2.5% K) diet for 7 days.
Fig. 8.
Fig. 8.
ANG II diminishes the AA-induced inhibition of ENaC. A: channel recording demonstrates that AA-induced inhibition of ENaC (middle trace) is reversed by adding 50 nM ANG II (bottom panel). The experiments were performed in a cell-attached patch of the rat CCD. B: bar graph summarizes the effect of AA on the ENaC activity in the presence or absence of 50 nM ANG II. *Significant difference compared with the control. The experiments were performed in the CCD of rats on a high-K (2.5% K) diet for 7 days.
Fig. 9.
Fig. 9.
Scheme illustrating the mechanism by which ANG II stimulates ENaC in PCs of the CCD. Arrows mean stimulation. 11,12-EET, epoxyeicosatrienoic acid; NOX, NADPH oxidase.
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