8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties

Abstract

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

Fig. 1.

Activation of human αβγ-epithelial Na⁺ channels (ENaC) by 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) in Xenopus oocytes. 8-pCPT-cGMP Na salts and amiloride (Amil) were continuously perfused on oocytes. Whole cell currents were monitored at 10-s intervals. A: representative inward and outward current traces in the presence of 8-pCPT-cGMP (CPT-cGMP) and amiloride. Currents were digitized at both −100 and +70 mV. Application of drugs is indicated by solid horizontal lines. B: average currents at −100 mV (means ± SE). *P < 0.05 compared with basal level. n = 6. C: representative current trace showing no effect of cell-impermeable standard cGMP (Std cGMP). Amiloride was applied at the end of experiments. D: corresponding average currents at −100 mV (means ± SE). E: application of 8-pCPT-cGMP ranged from 0.1 to 1,000 μM. Current trace was recorded at −100 mV. F: dose-response curve. The dashed line was created by fitting the raw data with the Hill equation. n = 10.

Fig. 2.

8-pCPT-cGMP activates mouse and rat αβγ-ENaC heterologously expressed in oocytes. A and B: representative current traces showing the effects of 8-pCPT-cGMP (CPT-cGMP) on mouse and rat ENaC channels, respectively. Membrane potential was −100 mV. C: summary of current levels. The numbers in parentheses are the number of oocytes examined for each group. *P < 0.05, **P <0.01. D: the ratio of activated and basal currents (I_CPT-cGMP/I_basal) in the presence and absence of 8-pCPT-cGMP. *P < 0.05, **P <0.01.

Fig. 3.

8-pCPT-cGMP-activated currents associated with human αβγ-ENaC in oocytes perfused with alkali ions. To ascertain that the 8-pCPT-cGMP-activated current was associated with αβγ-ENaC, the effects of 8-pCPT-cGMP (CPT-cGMP) were examined when oocytes were bathed in LiCl and KCl solutions. A and B: representative whole cell current traces elicited in LiCl and KCl media, respectively. Bath solution was switched to LiCl or KCl solution from regular ND-96 medium containing 96 mM NaCl. 8-pCPT-cGMP was perfused to the bath followed by the perfusate with amiloride. C and D: summary of corresponding inward currents at −100 mV. **P <0.01. E and F: summary of corresponding outward currents at +70 mV. *P < 0.05. n = 3.

Fig. 4.

Regulation of αβγ-ENaC activity by PKG isoform-specific modulators. A: effects of PKGI and PKGII activators on ENaC activity. 8-pCPT-cGMP (CPT-cGMP) was a cell-permeable PKG activator, predominantly activating PKGII isoform. Sp isomer of β-phenyl-1,N²-etheno-8-bromo-cGMP (Sp-8-Br-PET-cGMP) was a specific PKGI (both I-α and I-β) activator. Oocytes were perfused with 8-pCPT-cGMP, and then it was washed out, followed by application of Sp-8-Br-PET-cGMP. *P < 0.05. n = 6. B: blockade of the stimulating effect in oocytes pretreated with PKG inhibitor. Cells were pretreated with 250 μM Rp-8-Br-cGMP to block both PKGI and PKGII for 1 h at room temperature. n = 6. C: competition of standard cGMP (Std cGMP) and 8-pCPT-cGMP. The activation efficacy of 8-pCPT-cGMP (I_CPT-cGMP/I_basal) was compared in the absence and presence of standard cGMP. P > 0.05. D: PKG enzymatic activity. Normalized PKG activity in oocytes was incubated with vehicle (Control), 8-pCPT-cGMP (0.2 mM), Rp-8-Br-cGMP (0.5 mM), or both for 1 h. **P < 0.01 compared with control. Numbers in parentheses, number of oocytes examined for each group.

Fig. 5.

Acute and chronic effects of kinase blocker cocktail on the 8-pCPT-cGMP-mediated stimulation of αβγ-ENaC activity. A: current trace from an oocyte perfused with 8-pCPT-cGMP (CPT-cGMP) and followed by the kinase inhibitor cocktail (PKA inhibitor, Rp-cAMP 100 μM; PKC inhibitor, acryriarubin A 2 μM; MEK inhibitor, PD98059 20 μM; and p38 MAP inhibitor, SB-202190 30 μM). 8-pCPT-cGMP (CPT-cGMP) and the kinase inhibitor cocktail were washed out with ND-96 medium before addition of amiloride. B: pretreatment of oocytes with kinase blocker cocktail. Cells were incubated with the kinase blocker cocktail for 1 h at room temperature, and then the responses of ENaC channels to 8-pCPT-cGMP and amiloride were recorded. Data are represented as means ± SE; n = 4–6. *P < 0.05, **P <0.01.

Fig. 6.

Response of potential PKG phosphorylation mutants and cytosolic N- and C-tail truncation mutants to 8-pCPT-cGMP. A–C: amiloride-sensitive (AS) currents associated with single (A) and COOH- (B) and NH₂-terminal tail truncation mutants (C). *P < 0.05 and **P < 0.01 compared with wild-type channels. Holding potential, −100 mV. D: single-point mutation of intrinsic PKG phosphorylation sites. The current ratios of activated and basal current levels were shown. E: sensitivity of intracellular COOH- and NH₂-terminal tail truncation mutants to 8-pCPT-cGMP. Numbers in parentheses, number of oocytes examined for each group.

Fig. 7.

Knockdown of PKG expression in oocytes. A: expression of PKG isoforms at the mRNA level. Typical mRNA blot shows PKGI (300 bp) and PKGII (108 bp) expression in oocytes. B and C: PKG isoform I (PKGI) and II (PKGII) expressed at the protein levels. Mouse brain extract (mBE) and purified PKGII protein from bovine lung served as positive controls for PKGI and PKGII, respectively. n = 4. D: knockdown of PKGII. The knockdown effects of three small interfering RNAs (siRNAs; 30 ng/cell) against PKGII were tested using Western blot. GAPDH was used as loading control. E: activation of 8-pCPT-cGMP on ENaC in PKGI and PGKII knockdown oocytes. n = 5.

Fig. 8.

Domain-dependence of CPT-cGMP and CPT-cAMP analogs. A: modified positions are shown for 8-pCPT-cGMP and 8-CPT-cAMP. The responses of human αβγ-ENaC to these compounds (0.2 mM) were compared: 8-pCPT-cGMP (CPT-cGMP), 8-CPT-cAMP (CPT-cAMP), 2-aminophenylthio (APT)-cGMP, 8-pCPT-methylated ribose 2′-hydroxy (2′-O-Me)-cGMP (CPT-2′OMe-cGMP), Sp-8-Br-cGMP (Br-cGMP), 8-CPT-2′-O-Me-cAMP (CPT-2′OMe-cAMP), 4-chlorothiolphenol (CPT), and the mixture of CPT and KOH (CPT+OH). The current ratios after and before application of these compounds were compared with that of the standard cGMP molecule (Std cGMP). **P < 0.01, *P < 0.05. Numbers in parentheses are oocytes examined for each group.

Fig. 9.

Stimulatory effects of 8-pCPT-cGMP and 8-CPT-cAMP on human αβγ-ENaC single-channel activity. A: representative outside-out recording. The patch potential was −60 mV. Closed (0) and open (1–3) levels were marked. Perfusion of 8-CPT-cAMP (CPT-cAMP, 0.2 mM) and subsequently 8-pCPT-cGMP (CPT-cGMP, 0.2 mM) and amiloride (Amil, 10 μM) was indicated. B: expanded segments for basal (a), 8-CPT-cAMP activated (b), and 8-pCPT-cGMP activated (c) current traces. C: single-channel activity (NP_o). *P < 0.05 compared with basal or washout level using paired t-test.