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1.
Figure 8

Figure 8. PKGII inhibitor eliminates cGMP-activated whole-cell Na+ currents in voltage clamped H441 cells. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, amiloride-sensitive step IV traces before (control) and after addition of the mixture of 8-pCPT-cGMP (1 mm) and PKGII inhibitor, Rp-8-pCPT-cGMP (10 μm, +Rp-8-pCPT-cGMP). B, normalized whole-cell current densities (I/Icontrol). See Fig. 5 legend for additional details.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
2.
Figure 4

Figure 4. Activation of epithelial Na+ channels in confluent H441 monolayers. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, representative current trace showing elevated Na+ current (Isc) in the presence of various concentrations of 8-pCPT-cGMP (cGMP). B, dose–response curve. The cGMP-activated Isc fraction (ΔI) was normalized to the maximal cGMP-activated Isc level (ΔImax). This plot was fitted with the Hill equation to compute the EC50 value. n= 5.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
3.
Figure 10

Figure 10. Effects of cGMP on ENaC activity in H441 cells pre-exposed to CFTR and CNG channel inhibitors. From: Regulation of epithelial sodium channels by cGMP/PKGII.

H441 cells were first perfused with CFTRinh-172 (20 μm) and diltiazem (50 μm) to inhibit CFTR and CNG channels. Addition of either 0.5 mm 8-pCPT-cGMP (A) or 8-Br-cGMP (B) to the bath is indicated by arrows. Holding potential was −100 mV. C and D, average amiloride-sensitive whole-cell current (ASI) density.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
4.
Figure 7

Figure 7. Inhibition of cGMP-activated epithelial Na+ channel activity in H441 monolayers by isoform specific protein kinase G inhibitors. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, representative Isc trace showing the inhibitory effects of Rp-8-Br-PET-cGMP (PKGI Inh) and Rp-8-pCPT-cGMP (PKGII Inh) on the 8-pCPT-cGMP-elevated Isc. B, amiloride-sensitive (AS) Isc levels before (control) and after addition of 8-pCPT-cGMP (cGMP), Rp-8-Br-PET-cGMP (PKGI Inh), and Rp-8-pCPT-cGMP (PKGII Inh). n= 10.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
5.
Figure 1

Figure 1. Up-regulation of mouse alveolar fluid clearance (AFC) in vivo by 8-pCPT-cGMP. From: Regulation of epithelial sodium channels by cGMP/PKGII.

Anaesthetized, ventilated mice were intratracheally instilled with 5% BSA and the instillate was collected after 15 min. Reabsorption rate of instillate was computed as the percentage of instilled volume (% AFC15). Data are presented as means ±s.e.m. Numbers within the brackets are total mice examined for control, amiloride (1 mm), 8-pCPT-cGMP (cGMP, 1 mm), and cGMP+amiloride (+amiloride). P values were computed with one-way ANOVA.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
6.
Figure 6

Figure 6. Activation of epithelial Na+ channels by cGMP in basolaterally permeabilized H441 monolayers. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, representative short-circuit current (Isc) trace. Amphotericin B (am-B, 100 μm) was added to the basolateral bath as indicated by an arrow. 8-pCPT-cGMP (0.2 mm cGMP) was applied to the apical bath followed by amiloride (100 μm). B, summary of Isc levels. Mean amiloride-sensitive (AS) Isc values before and after addition 8-pCPT-cGMP (cGMP) were averaged. n= 8. C and D, representative trace and average Isc levels in amiloride-pretreated H441 monolayers. n= 7.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
7.
Figure 3

Figure 3. Stimulation of heterologously expressed human ENaCs in Xenopus oocytes by cGMP. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, representative current traces recorded at +40 mV and −100 mV. Oocytes were perfused with 8-pCPT-cGMP ranging from 1 μm to 1 mm as indicated by arrows. Amiloride was added to the bath before the recordings were terminated. B, dose–response curve. Average cGMP-activated current fraction in the presence of cGMP (ΔI) was normalized to the maximal cGMP-elevated current (ΔImax) and plotted as a function of cGMP contents. Smooth lines and the EC50 value was created by fitting the raw data with the Hill equation. n= 9. C, average amiloride-sensitive (AS) αβγδ ENaC activities before (control) and after cGMP perfusion. Holding potential, −100 mV. n= 10.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
8.
Figure 5

Figure 5. Activation of amiloride-sensitive Na+ currents by cGMP in patch clamped H441 cells. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, representative time course showing 8-pCPT-cGMP (cGMP)-activated whole cell current trace at −40 mV. B, amiloride-sensitive current traces recorded in a cell before (control) and after delivery of 8-pCPT-cGMP (cGMP) to the bath. Dashed lines indicate the zero current levels. C, IV curves for average control currents (control), in the presence of 8-pCPT-cGMP (cGMP), and amiloride plus 8-pCPT-cGMP (amil+cGMP). Amiloride-sensitive currents (ASIs) were normalized to the current density at −120 mV of control. D, normalized whole-cell current densities (I/Icontrol). Whole-cell currents in the presence of 8-pCPT-cGMP (cGMP), diltiazem (50 μm) and cGMP (diltia + cGMP), amiloride (10 μm) + cGMP, and amiloride alone (amil) were normalized to the control. Data were pooled from five independent experiments performed by three patch clampers. Numbers in brackets are patched cells.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
9.
Figure 9

Figure 9. Expression of protein kinase G isoforms in H441 cells and human lung tissues (HLT). From: Regulation of epithelial sodium channels by cGMP/PKGII.

A and C, reverse transcription-PCR of protein kinase G mRNA expression. The expression of three protein kinase G isoforms, PKGIα (Iα), PKGIβ (Iβ) and PKGII (II), was examined in H441 cells (A) and human lung tissues (C). M and N denote molecular mass markers and the running buffer only without RNA. Arrows indicate the 500 bp marker. n= 3. B and D, Western blots of protein kinase G isoform I and II. H441 cell and human lung tissue lysates were probed with isoform specific anti-PKG antibodies. Mouse brain extract (mBE) was used as a positive control for PKGI and purified cGKII protein for PKGII. These experiments were repeated 3–5 times.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.
10.
Figure 2

Figure 2. Expression of ENaC δ subunit in lung epithelial tissues. From: Regulation of epithelial sodium channels by cGMP/PKGII.

A, reverse transcription-PCR analysis of δ ENaC mRNA expression in human lung. This RT-PCR represents 3 independent experiments with similar results. H441 cells were used as a positive control (Ji et al. 2006). Lanes from left to right represent human alveolar type II cells (hATII), human lung tissue (HLT), H441 cells, rabbit lung tissue (RLT), and mouse lung tissue (MLT). B, indirect immunofluorescence detection of δ ENaC in human lung tissue. Human lung sections were incubated with specific antibody against δ ENaC. ENaC δ protein was detected (green channel) with FITC conjugated secondary antibody. Left panel is a negative control incubated with non-immune IgG substituted for the anti-δ ENaC antibody (control). Cell nuclei were stained with DAPI (blue channel). Bar equals 20 μm; n= 5. C, immunoblotting assay of δ ENaC protein. A polypeptide of approximately 85 kDa was detected in the lysate of human lung tissue (HLT). H441 cell lysate serves as a positive control (Ji et al. 2006). n= 3.

Hong-Guang Nie, et al. J Physiol. 2009 June 1;587(Pt 11):2663-2676.

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