Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction

Abstract

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

FIGURE 1. Representative traces of the inhibitory effect of AngII (100 nM) on the apical ROMK channel in a CCD from LK-fed rats

A, the top trace shows the time course of the experiment. Three parts of the trace are expanded (traces 1–3) to show detailed channel activity at faster time resolution. The channel closed state (“C”) is indicated by a dashed line. Current levels are indicated by the short bars on the right of each trace. The experiment was performed in a cell-attached patch at a holding potential of 0 mV. B, channel recording showing that the inhibitory effect of AngII is partially reversible after washout of AngII from bathing solution.

FIGURE 2. Dose-response curves for the AngII-induced inhibition of the ROMK channel in CCDs

AngII led to a reduction in NPo of ROMK channels in CCDs from LK (open circles)- but not from NK (closed circles)-fed rats at concentrations ≥ 1 nM. *, p < 0.05; **, p < 0.01 versus 0.1 nM AngII. #, p < 0.05 versus 100 nM AngII in NK-fed rats only.

FIGURE 3. Effect of AT1 and AT2 receptor antagonists on the AngII-induced inhibition of ROMK channels in CCDs from LK-fed rats

A, representative channel recording obtained in a principal cell-attached patch at a holding potential of 0 mV. The inhibitory effect of AngII was completely blocked by pretreatment of the CCD with losartan (20 μM), an AT1 receptor antagonist. The top trace demonstrates the time course of the experiment; the recording is expanded below (traces 1 and 2) to show fast time resolution. The channel-closed state (“C”) is indicated by a dashed line. Current levels are indicated by the short bars on the right of each trace. B, bar graph summarizing the effects of antagonists of the AT1 receptor (losartan) and AT2 receptor (PD123319) on the AngII-induced reduction of ROMK channel activity. C, bar graph summarizing the effect of oral losartan administration (10 mg/kg/day added to the drinking water) in LK-fed rats. ROMK channel activity (NPo) in losartan-treated rats significantly exceeded that measured in LK-fed animals provided vehicle alone in drinking water.

FIGURE 4. Apical membrane expression of AT1 receptors in rat CCD

CCD from LK rat was co-labeled with an anti-AT1 receptor antibody and visualized with an Alexa-488-conjugated secondary antibody (green) and rhodamine-conjugated DBA (red), a marker of distal nephron principal cells. The CCD exhibits abundant apical membrane expression of AT1 receptors.

FIGURE 5

The inhibitory effect of AngII on ROMK channel activity in CCDs is mediated by activation of PLC-PKC. In CCDs from LK-fed rats, PMA (10 μM), an activator of PKC, inhibited ROMK channel activity. Application of inhibitors of PLC (U73122; 10 μM) or PKC (calphostin C; 100 nM) alone had no effect on ROMK channel activity but prevented the AngII-induced reduction in NPo. In CCDs pretreated with U73122 and exposed to AngII, subsequent PMA application inhibited channel activity due to direct stimulation of PKC.

FIGURE 6. The inhibitory effect of AngII on ROMK channel activity is mediated, in part, by activation of PTK

HA (1 μM), an inhibitor of PTK, increased ROMK channel activity in CCDs from LK-fed rats. In CCDs pretreated with HA, AngII led to a partial reduction in channel activity. In this figure, mean NPo is presented relative to that measured in untreated control CCDs.

FIGURE 7. The inhibitory effect of AngII on ROMK channel activity is mediated, in part, by NADPH oxidase

A, pretreatment of CCDs from LK rats with one of two NADPH oxidase inhibitors, IDP (10 μM) or apocynin (100 nM), blocked the effect of AngII on the ROMK channel. Neither one of these inhibitors had a significant effect on base-line channel activity. CCDs pretreated with IDP failed to respond to PMA with an inhibition of ROMK channel activity, suggesting that AngII inhibits the channel via the activation of the PKC-NADPH oxidase pathway. B, relative expression of mRNA encoding p22^phox and p47^phox in CCDs from NK- and LK-fed rats. Dietary potassium restriction for 4 – 6 days increased expression of p47^phox (but not p22^phox) when compared with NK-fed rats.

FIGURE 8

Proposed signaling pathway by which AngII inhibits the activity of ROMK channels in the CCD from LK-fed rats.