Journal: JCI Insight

Article Title: Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

doi: 10.1172/jci.insight.123467

Figure Lengend Snippet: Mice were infected with 4,000 PFU of PR8ΔGFP. NPD of control and infected mice were measured as described in Methods. (A) Recording traces depicting NPD in noninfected control mice, immediately after the probe was inserted in one the mouse nares; a negative voltage deflection was recorded and is indicative of the active Na+ ion absorption. Addition of 200 μM amiloride in the perfusate inhibited the voltage difference, indicating the role of ENaC in maintaining the initially recorded NPD. Subsequent perfusion with 10–20 μM forskolin activated a second potential difference due to the movement of Cl– and HCO3–, which was inhibited by 50 μM GlyH-101, showing the role of CFTR in this process. Representative NPD recordings from uninfected (Control) mice, 5 and 15 days p.i. are shown, with the electrical potential scale noted for each time point (bottom left). (B) ENaC activity, as measured by NPD across time course of IAV. Scatter plots with NPD value ± SEM at 2, 5, 10, and 15 days p.i. (C) Scatter plots of GlyH-101–sensitive (CFTR) NPD at days 2, 5, 10, and 15 p.i. Each data point corresponds to a single mouse, N = 9–13 per group; mean ± SEM. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons. *P < 0.0001 compared with noninfected controls.

Article Snippet: Protein was loaded into an SDS-PAGE gel in equal quantities, and bands were detected with the follow antibodies: α-ENaC (sc-20966), β-ENaC (sc-25354), and Na,K-ATPase β1 (sc-21713) (all from Santa Cruz Biotechnology) and γ-ENaC (ab3468), CFTR (ab2478), and Na,K-ATPase α (ab76020) (all from Abcam).

Techniques: Infection, Control, Activity Assay

Journal: JCI Insight

Article Title: Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

doi: 10.1172/jci.insight.123467

Figure Lengend Snippet: Mice were infected with 4,000 PFU of PR8ΔGFP, lungs were harvested 5 days p.i., and fresh 250 μM slices were prepared using a live tissue slicer from uninfected controls and infected animals as previously described (14). Slices were incubated in culture medium for 3–4 hours before ion channel activity was recorded from GFP+ and GFP- cells. Lung cryosections display widespread infection. GFP- cells exhibited single channels with conductance of 4 pS and 18 pS. Recordings were obtained at Vpatch + 100 mV when Vm was 0 mV (slices incubated in 135 mM KCl Ringer). Original magnification, ×100 (A) and ×200 (B). (C) Tracings of ENaC open probability conductance in GFP- cells. Histogram shown in D shows number of events for the 4 Ps channel only. (E) A representative recording of channel activities recorded from GFP+-infected AT2 cells. (F) GFP+ AT2 cells exhibited substantial reduction of 4 pS and 18 pS activity, without affecting their unitary conductances. Please see Figure 3 for quantification of these findings.

Article Snippet: Protein was loaded into an SDS-PAGE gel in equal quantities, and bands were detected with the follow antibodies: α-ENaC (sc-20966), β-ENaC (sc-25354), and Na,K-ATPase β1 (sc-21713) (all from Santa Cruz Biotechnology) and γ-ENaC (ab3468), CFTR (ab2478), and Na,K-ATPase α (ab76020) (all from Abcam).

Techniques: Infection, Incubation, Activity Assay

Journal: JCI Insight

Article Title: Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

doi: 10.1172/jci.insight.123467

Figure Lengend Snippet: Mice were infected with 4,000 PFU of PR8ΔGFP; lungs were harvested at days 1, 2, 4, 5, 7, 10, 13, 14, and 15 p.i.; and live slices were cut using a live tissue slicer. ENaC activities in infected AT2 cells (GFP+) were recorded using cell-attached mode of the patch-clamp technique. (A and B) Reduction of ENaC open probabilities for various times after infection. N = 5 mice and n = 29 cells per group. The maximum inhibition was seen at day 7, after which a slow recovery of both conductances was observed. (C and D) ENaC activity, measured as open probability (Po), was not affected in noninfected cells. Data are plotted as mean open probabilities, top and bottom quartiles ± SEM. Data were analyzed by 1-way ANOVA and post hoc Bonferroni correction for multiple comparisons. *P < 0.0001 for each time point compared with noninfected control values.

Article Snippet: Protein was loaded into an SDS-PAGE gel in equal quantities, and bands were detected with the follow antibodies: α-ENaC (sc-20966), β-ENaC (sc-25354), and Na,K-ATPase β1 (sc-21713) (all from Santa Cruz Biotechnology) and γ-ENaC (ab3468), CFTR (ab2478), and Na,K-ATPase α (ab76020) (all from Abcam).

Techniques: Infection, Patch Clamp, Inhibition, Activity Assay, Control

Journal: JCI Insight

Article Title: Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

doi: 10.1172/jci.insight.123467

Figure Lengend Snippet: NHBEs were grown at an air-liquid interface and infected with A/California/07/2009 H1N1 IAV at a MOI of 3 for (A) 24, (B) 48, and (C) 72 hours, at which point they were mounted in Ussing chambers for the measurement of short-circuit currents (Isc) and transepithelial resistance. Amiloride (ENaC) and forskolin-sensitive currents inhibited by the specific CFTR inhibitor CFTRinh172 were significantly reduced by IAV infection. Amilo., amiloride trough; Forsk Pk., forskolin peak; Forsk. Plt., forskolin plateau. Characteristic records (A–C) and scatter plots (n = 6 filters per groups; mean ± 1SEM) (D–F). Data were analyzed by 1-way ANOVA and post hoc Tukey test for multiple comparisons. *P < 0.0001 compared with time-matched noninfected controls.

Article Snippet: Protein was loaded into an SDS-PAGE gel in equal quantities, and bands were detected with the follow antibodies: α-ENaC (sc-20966), β-ENaC (sc-25354), and Na,K-ATPase β1 (sc-21713) (all from Santa Cruz Biotechnology) and γ-ENaC (ab3468), CFTR (ab2478), and Na,K-ATPase α (ab76020) (all from Abcam).

Techniques: Infection

Journal: JCI Insight

Article Title: Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

doi: 10.1172/jci.insight.123467

Figure Lengend Snippet: NHBEs were grown at an air-liquid interface and infected with A/California/07/2009 at a MOI of 3. (A) ENaC, CFTR, and Na/K-ATPase protein abundance was measured by Western blotting. For ENaC and CFTR measurements, biotinylated apical membrane proteins were immunoblotted with specific antibodies against ENaC subunits and CFTR, respectively. Blots are representative of 3 separate experiments. (B) Densitometry demonstrates reductions in ENaC subunits, CFTR, and α-Na/K-ATPase subunit, but no difference was observed in the expression of the regulatory β subunit Na/K-ATPase levels. Data were analyzed by 1-way ANOVA and post hoc Tukey test for multiple comparisons. N = 3 per group. *P < 0.001 compared with mock-infected controls.

Article Snippet: Protein was loaded into an SDS-PAGE gel in equal quantities, and bands were detected with the follow antibodies: α-ENaC (sc-20966), β-ENaC (sc-25354), and Na,K-ATPase β1 (sc-21713) (all from Santa Cruz Biotechnology) and γ-ENaC (ab3468), CFTR (ab2478), and Na,K-ATPase α (ab76020) (all from Abcam).

Techniques: Infection, Quantitative Proteomics, Western Blot, Membrane, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Cholinergic receptors play a role in the cardioprotective effects of anesthetic preconditioning: Roles of nitric oxide and the CaMKKβ/AMPK pathway

doi: 10.3892/etm.2020.9569

Figure Lengend Snippet: Western blot analysis of eNOS, nNOS, p-eNOS and p-nNOS levels in the homogenates of myocardial tissue from rat hearts. (A) Western blot bands of eNOS, p-eNOS, nNOS and p-nNOS. The ratio of (B) p-eNOS/total eNOS and (C) p-nNOS/total nNOS. APC-induced increases of the phosphorylation of eNOS and nNOS were reduced by the muscarinic acetylcholine receptor antagonist ATR (100 nM) and nicotinic acetylcholine receptor antagonist HEM (50 µM). The data are presented as the mean ± SD. n=5 hearts/group. *P<0.05 vs. Sham group; &P<0.05 vs. IR group; #P<0.05 vs. APC group. eNOS, endogenous nitric oxide synthase; nNOS, neuronal NOS; p, phosphorylated; IR, ischemia-reperfusion; APC, anesthetic preconditioning; ATR, atropine; HEM, hexamethonium; CTL, control.

Article Snippet: The primary antibodies were specific for eNOS (cat. no. sc-376751; 1:1,000; Santa Cruz Biotechnology, Inc.), nNOS (cat. no. sc-5302; 1:1,000; Santa Cruz Biotechnology, Inc.); AMPK (cat. no. 2352; 1:1,000; Cell Signaling Technology, Inc.); phosphorylated (p)-AMPK (Thr172; cat. no. 5884; 1:1,000; Cell signaling Technology, Inc.); p-CaMKK2 (Ser511; cat. no. AF4487; 1:1,000; Affinity Biosciences) and CAMKK2 (polyclonal antibody; cat. no. DF4793; 1:1,000; Affinity Biosciences); as well as the housekeeping protein GAPDH (cat. no. AG019; 1:1,000; Shanghai Biyuntian Biotechnology Co., Ltd.).

Techniques: Western Blot