ENaC is regulated by natriuretic peptide receptor-dependent cGMP signaling

Abstract

Epithelial sodium channels (ENaCs) located at the apical membrane of polarized epithelial cells are regulated by the second messenger guanosine 3',5'-cyclic monophosphate (cGMP). The mechanism for this regulation has not been completely characterized. Guanylyl cyclases synthesize cGMP in response to various intracellular and extracellular signals. We investigated the regulation of ENaC activity by natriuretic peptide-dependent activation of guanylyl cyclases in Xenopus 2F3 cells. Confocal microscopy studies show natriuretic peptide receptors (NPRs), including those coupled to guanylyl cyclases, are expressed at the apical membrane of 2F3 cells. Single-channel patch-clamp studies using 2F3 cells revealed that atrial natriuretic peptide (ANP) or 8-(4-chlorophenylthio)-cGMP, but not C-type natriuretic peptide or cANP, decreased the open probability of ENaC. This suggests that NPR-A, but not NPR-B or NPR-C, is involved in the natriuretic peptide-mediated regulation of ENaC activity. Also, it is likely that a signaling pathway involving cGMP and nitric oxide (NO) are involved in this mechanism, since inhibitors of soluble guanylyl cyclase, protein kinase G, inducible NO synthase, or an NO scavenger blocked or reduced the effect of ANP on ENaC activity.

Fig. 1.

Western blot (WB) analysis of natriuretic peptide receptor (NPR) subtypes in 2F3 renal cells. A: NPR-A antibody detected a distinct immunoreactive band at ∼130 kDa. B: NPR-B antibody detected multiple immunoreactive bands, including one at ∼130 kDa. C: NPR-C antibody detected a distinct immunoreactive band of ∼60 kDa.

Fig. 2.

Immunofluorescence analysis of NPR expression in 2F3 and mouse kidney cells. Expression of NPR-A (A), NPR-B (B), and NPR-C (C) in 2F3 cells is shown. Immunoreactive staining for NPR-A (D), NPR-B (E), and NPR-C (F) in mouse kidney slices is shown. Zonula occludens-1 is red and a marker for tight junctions. The z-axis shows all three natriuretic peptide subtypes are mainly expressed at the apical membrane. Red scale bar is 10 μm.

Fig. 3.

Distribution of NPRs in mouse kidney. NPRs are expressed in aquaporin-2 (AQP2)-positive cells of mouse kidney. We wished to show that NPRs were in principal cells of mouse collecting ducts where they could influence epithelial sodium channel (ENaC) activity. A–C: distribution of NPR-A, -B, and -C, respectively. For reference, differential interference contrast images are included on the bottom right of each panel (A–C). The images show that all three types of NPRs are present in the cells that express AQP2. A: all of the receptors colocalize at the apical membrane with AQP2, but NPR-A almost exclusively colocalizes with AQP2 in the apical membranes of principal cells. B: NPR-B also colocalizes with AQP2, but is also present at locations different from AQP2. C: NPR-C is present in all cell types. D: the distributions of the three receptor types in the renal slices can best be appreciated in the bottom magnification image. Red scale bar in all images is 10 μm.

Fig. 4.

Amiloride-sensitive transepithelial current measurements in 2F3 cells after application of exogenous ligands. Cells were cultured on permeable supports until confluent (10 days). When confluent, transepithelial voltage and resistance was measured after vehicle (ethanol), 1 μM atrial natriuretic peptide (ANP), 1 μM β-phenyl-1,N²-etheno-8-bromo-cGMP (8-Br-PET-cGMP), or 1 μM 8-(4-para-chlorophenylthio)-cGMP (8-pCPT-cGMP) were applied to the apical side of the cell monolayers. Total transepithelial current was calculated as the ratio of voltage to resistance. A statistically significant decrease in transepithelial current was observed in response to ANP and 8-pCPT-cGMP compared with vehicle alone, but no significant difference for 8-Br-PET-cGMP-treated cells. Values are the mean current of three plates and the standard deviation. Statistical differences are marked with asterisks and were calculated using a one-way ANOVA with a Holm-Sidak posttest for comparison to the vehicle-treated cells.

Fig. 5.

Effect of ANP on the open probability (P_o) of ENaC in 2F3 cells. A: representative single-channel current traces obtained from cell-attached patches after application of 1 μM ANP to the apical side of 2F3 cells. The closed level of the channel is marked with c and an arrow. B: summary plot showing a statistically significant decrease (marked with an asterisk) in the P_o of ENaC in response to the ANP treatment. For this figure, we determined mean P_o for 5 min before addition of any agent and for 5 min after addition.

Fig. 6.

Effect of C-type natriuretic peptide (CNP) and NPR-C-specific agonist cANP on the P_o of ENaC in 2F3 cells. A: representative single-channel current traces obtained from cell-attached patches after application of 1 μM CNP to the apical side of 2F3 cells. B: summary plot showing little if any effect on the P_o of ENaC in response to the CNP treatment. C: representative single-channel current traces obtained from cell-attached patches after application of 1 μM cANP (an NPR-C agonist) to the apical side of 2F3 cells. D: summary plot showing little if any effect on the P_o of ENaC in response to the cANP treatment. For this figure, we determined mean P_o for 5 min before addition of any agent and for 5 min after addition.

Fig. 7.

Protein kinase G (PKG) II mediates the effect of ANP. A: effect of a PKG II activator on the P_o of ENaC in 2F3 cells. Representative single-channel current (C) record was obtained from cell-attached patches after application of 1 μM 8-pCPT-cGMP to the apical side of 2F3 cells. B: summary plot showing a statistically significant decrease (marked with an asterisk) in the P_o of ENaC in response to the 8-pCPT-cGMP treatment. C: effect of a PKG-1α and PKG-1β activator on the P_o of ENaC in 2F3 cells. Representative single-channel current traces were obtained from cell-attached patches after application of 10 μM 8-Br-PET-cGMP to the apical side of 2F3 cells. D: summary plot showing no change in the P_o of ENaC in response to the 8-Br-PET-cGMP treatment. For this figure, we determined mean P_o for 5 min before addition of any agent and for 5 min after addition.

Fig. 8.

Effect of ANP on the P_o of ENaC in 2F3 cells after inhibiting PKG. A: representative single-channel current record obtained from cell-attached patches after application of 1 μM KT5823 to the apical side of 2F3 cells. B: summary plot showing KT5823 treatment did not have a significant effect on the P_o of ENaC. For this figure, we determined mean P_o for 5 min before addition of any agent and for 5 min after addition.

Fig. 9.

Effect of ANP on the P_o of ENaC in 2F3 cells after inhibiting nitric oxide (NO). A: representative single-channel current record obtained from cell-attached patches after application of 200 μM (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) to the apical side of 2F3 cells. B: summary plot showing C-PTIO treatment blocked the effect of ANP on the P_o of ENaC. C: effect of ANP on the P_o of ENaC in 2F3 cells after inhibiting inducible NO synthase (iNOS). (The large upward deflections in the current record are spurious electrical noise from ventilation motors in the vicinity of the patch setup.) Representative single-channel current record was obtained from cell-attached patches after application of 50 μM N^G-nitro-l-arginine methyl ester (l-NAME) to the apical side of 2F3 cells. D: summary plot showing l-NAME treatment blocked the effect of ANP on the P_o of ENaC. For this figure, we determined mean P_o for 5 min before addition of any agent and for 5 min after addition.