Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: PAR-2-activated secretion by airway gland serous cells: role for CFTR and inhibition by Pseudomonas aeruginosa

doi: 10.1152/ajplung.00411.2020

Figure Lengend Snippet: Submucosal exocrine gland serous acinar cells and protease-activated receptor 2 (PAR-2). A: diagram of anatomical structure of an airway submucosal gland, showing cystic fibrosis transmembrane conductance regulator (CFTR)-expressing serous cells at the distal acini, which have been demonstrated to be the origin of the majority of secreted fluid (14). B: differential interference contrast (DIC) and immunofluorescence images of middle turbinate submucosal gland stained with anti-PAR-2 antibody SAM11 (65). Scale bar is 50 µm. C: image of isolated serous acini. Scale bar is 25 µm. D: image of isolated acinar cells loaded with Ca2+ indicator fura-2. Scale bar is 10 µm.

Article Snippet: Immunofluorescence was carried out using PAR-2 mouse monoclonal antibody SAM11 (1:50 in DPBS + 1% BSA, 2% normal donkey serum, 0.2% saponin; Abcam) using previously described methods ( 20 , 58 ).

Techniques: Expressing, Immunofluorescence, Staining, Isolation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: PAR-2-activated secretion by airway gland serous cells: role for CFTR and inhibition by Pseudomonas aeruginosa

doi: 10.1152/ajplung.00411.2020

Figure Lengend Snippet: Polarized fluid secretion in air-liquid interface (ALI) cultures of primary nasal serous cells supports synergistic cystic fibrosis transmembrane conductance regulator (CFTR)-dependent secretion elicited by low-level protease-activated receptor 2 (PAR-2) and vasoactive intestinal peptide (VIP)ergic stimulation. A: confocal micrograph of methanol-fixed primary serous ALI showing immunofluorescence of Na+/K+ ATPase (magenta; Abcam ab76020 rabbit monoclonal antibody) and PAR-2 (cyan; SAM11 mouse monoclonal antibody). Both antibodies exhibited lateral membrane staining. Results representative of independent cultures from three patients. B: Western blot for PAR-2 (Abcam ab180953 rabbit monoclonal antibody) from ALIs from two separate patients. Results representative of four ALIs from four patients. C: representative orthogonal sections from confocal stacks [×60 1.4 numerical aperture (NA) oil objective, 0.3 µm step size] showing Texas red dextran-labeled airway surface liquid (ASL) in individual cultures from non-cystic fibrosis (CF, left) and ΔF508/ΔF508 CF cultures (right) after stimulation as indicated. Note z-axis scale bar is not calibrated for refractive index mismatch, as discussed in methods. D: bar graph of ASL heights from independent experiments as in C (n = 5 per condition using ALIs from five patients). ASL heights in non-CF cultures were (in µm) 10 ± 1 (unstimulated), 12 ± 2 [500 nM 2-Furoyl-LIGRLO-NH2 (2FLI)], 36 ± 5 (5 µM 2FLI; *P < 0.05 vs. unstimulated), 11 ± 1 (5 µM 2FLI + 50 µM GB83; †P < 0.05 vs. 5 µM 2FLI alone), 11 ± 1 (100 nM VIP), 34 ± 2 (1 µM VIP; *P < 0.05 vs. unstimulated), 41 ± 4 (500 nM 2FLI + 100 nM VIP; *P < 0.05 vs unstimulated; #P < 0.05 vs. either 500 nM 2FLI or 100 nM VIP alone), 15 ± 2 (500 nM 2FLI + 100 nM VIP + 100 µM bumetanide; ‡P < 0.05 vs. 500 nM 2FLI + 100 nM VIP without bumetanide). ASL heights in CF cultures were 9 ± 1 (unstimulated), 11 ± 1 (500 nM 2FLI), 31 ± 3 (5 µM 2FLI; *P < 0.05 vs. unstimulated), 13 ± 2 (5 µM 2FLI + 50 µM GB83; †P < 0.05 vs. 5 µM 2FLI alone), 12 ± 1 (100 nM VIP), 11 ± 2 (1 µM VIP), 10 ± 1 (500 nM 2FLI + 100 nM VIP). Significance by one-way ANOVA with Bonferroni post-test with paired comparisons. E: expression of TMEM16A (Ca2+-activated Cl− channel), BEST1 (Ca2+-activated Cl− channel), and UBC1 (housekeeping control) relative to GAPDH ± scrambled, TMEM16A, or BEST1 siRNAs (n = 4 ALIs, two each from two individual patients). Expression of TMEM16A was 0.10 ± 0.02 (scrambled siRNA), 0.025 ± 0.01 (TMEM16A siRNA; *P < 0.05 vs. scrambled), and 0.10 ± 0.02 (BEST1 siRNA). Expression of BEST1 was 0.03 ± 0.007 (scramble siRNA), 0.04 ± 0.006 (TMEM16A siRNA), 0.007 ± 0.003 (BEST1 siRNA; *P < 0.05 vs. scrambled). Expression of UBC1 was 0.60 ± 0.07 (scramble siRNA), 0.56 ± 0.05 (TMEM16A siRNA), and 0.61 ± 0.08 (BEST1 siRNA). Significance by one-way ANOVA with Bonferroni post-test. F: representative orthogonal sections of unstimulated (top) or 5 µM 2FLI stimulated ALIs treated with scrambled, TMEM16A, or BEST1 siRNAs. G: bar graph showing mean ASL heights from experiments as in F (n = 6 ALIs, three each from two individual patients). Mean ASL height was 10 ± 1 (unstimulated + scrambled siRNA), 32 ± 2 (5 µM 2FLI + scrambled siRNA; **P < 0.01 vs. unstimulated + scrambled siRNA), 11 ± 1 (unstimulated + TMEM16A siRNA), 16 ± 5 (5 µM 2FLI + TMEM16A siRNA; n.s. vs. unstimulated + TMEM16A siRNA; #P < 0.05 vs. 5 µM 2FLI + either scrambled or BEST1siRNA), 11 ± 1 (unstimulated + BEST1 siRNA), and 38 ± 2 (5 µM 2FLI + BEST1 siRNA; **P < 0.01 vs. unstimulated + BEST1 siRNA). Significance by one-way ANOVA with Bonferroni posttest. Data points in all bar graphs are independent experiments and error bars are SE.

Article Snippet: Immunofluorescence was carried out using PAR-2 mouse monoclonal antibody SAM11 (1:50 in DPBS + 1% BSA, 2% normal donkey serum, 0.2% saponin; Abcam) using previously described methods ( 20 , 58 ).

Techniques: Immunofluorescence, Membrane, Staining, Western Blot, Labeling, Refractive Index, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: PAR-2-activated secretion by airway gland serous cells: role for CFTR and inhibition by Pseudomonas aeruginosa

doi: 10.1152/ajplung.00411.2020

Figure Lengend Snippet: Ca2+ responses to protease protease-activated receptor 2 (PAR-2) activation is inhibited by Pseudomonas aeruginosa secreted products in Calu-3 air-liquid interfaces (ALIs). A: representative immunofluorescence image showing punctate lateral membrane staining of PAR-2 (SAM11) in methanol-fixed Calu-3s, with GLUT1 staining (Abcam SPM498/ab40084 mouse monoclonal) for comparison. B: Western (Abcam ab180953 rabbit monoclonal antibody) showing expression of PAR-2 in submerged (left) and ALI (right) Calu-3s. C: Fluo-4 intensity measurements of Calu-3 air-liquid interface cultures were made using the entire field of view as the region of interest, which thus averaged intensity over hundreds of cells grown on the transwell. Pseudo-colored F/Fo images are shown are shown for one representative experiment showing response to 10 µM basolateral trypsin application. Images were created using the average of the first five images of the series as baseline using “F div Fo” plugin from the ImageJ Cookbook plugin set (https://imagej.net/Cookbook). Background was estimated based on imaging a non-Fluo-4-loaded ALI at identical settings and subtracted before calculation of F/Fo. D: representative Ca2+ (Fluo-4 F/Fo) responses to basolateral trypsin (10 µM) but not apical thrombin (10 µM), apical trypsin, or basolateral thrombin. E: Fluo-4 traces showing Ca2+ response to basolateral 2-Furoyl-LIGRLO-NH2 (2FLI) (10 µM) but not apical 2FLI, apical AY-NH2 (10 µM), or basolateral AY-NH2. Representative Fluo-4 traces showing loss of responses to 10 µM trypsin (F) or 10 µM tryptase (G) after 5 min pretreatment with PAO-1 conditioned media (CM) but not heat-inactivated PAO-1 CM. 2FLI response was intact after PAO-1 CM pretreatment. Representative Fluo-4 traces showing intact trypsin response after pretreatment with PAO-1 CM + phosphoramidon (10 µM; H) or PAO-JP2 CM (I). J: Fluo-4 traces showing loss of 10 µM trypsin Ca2+ response with clinical P. aeruginosa (PAO-CRS01) CM pretreatment (left) intact trypsin response after heat-inactivated PAO-CRS01 pretreatment (right). K: bar graph of apical vs. basolateral responses from experiments as in E and F. Peak ΔFluo-4 F/Fo was 0.05 ± 0.02 (apical thrombin), 0.09 ± 0.04 (apical trypsin) 0.07 ± 0.04 (apical 2FLI), 0.06 ± 0.02 (apical AY-NH2), 0.15 ± 0.06 (basolateral thrombin), 2.0 ± 0.18 (basolateral trypsin; **P < 0.01 vs. apical trypsin), 2.3 ± 0.25 (basolateral 2FLI; **P < 0.01 vs. apical 2FLI), 0.12 ± 0.06 (basolateral AY-NH2); n = 4 independent experiments each. Significance by one-way ANOVA with Bonferroni post-test with paired comparisons. L: bar graph of peak Ca2+ responses with 10 µM trypsin from experiments as shown in G–K. Peak ΔFluo-4 F/Fo with 10 µM trypsin was 2.1 ± 0.15 (control LB only pretreatment), 0.25 ± 0.1 (PAO-1 CM; **P < 0.01 vs. LB only), 1.81 ± 0.13 (heat-inactivated PAO-1 CM; ##P < 0.01 vs. PAO-1 CM), 1.70 ± 0.1 (PAO-1 CM + phosphoramidon; ##P < 0.01 vs. PAO-1 CM), 1.94 ± 0.22 (PAO-JP2 CM; ##P < 0.01 vs. PAO-1 CM), 0.19 ± 0.08 (PA-CRS01 CM; **P < 0.01 vs. LB only), 0.12 ± 003 (PA-CRS02 CM; **P < 0.01 vs. LB only), 0.11 ± 0.02 (PA-CRS03 CM; **P < 0.01 vs. LB only), 1.94 ± 0.19 (heat-inactivated PA-CRS01 CM; ††P < 0.01 vs. PA-CRS01 CM), 2.06 ± 0.19 (heat-inactivated PA-CRS02 CM; ‡‡P < 0.01 vs. PA-CRS02 CM). Significance determined by one-way ANOVA with Bonferroni post-test; n = 4–5 independent experiments. M: bar graph of peak Ca2+ responses with 10 µM lung tryptase from experiments as shown in G–K. Peak ΔFluo-4 F/Fo with 10 µM tryptase was 1.85 ± 0.2 (control LB only pretreatment), 0.09 ± 0.02 (PAO-1 CM; **P < 0.01 vs. LB only control), 1.63 ± 0.09 (heat-inactivated PAO-1 CM; ##P < 0.01 vs. PAO-1 CM), 2.0 ± 0.11 (PAO-1 CM + phosphoramidon; ##P < 0.01 vs. PAO-1 CM), 2.2 ± 0.13 (PAO-JP2 CM; ##P < 0.01 vs. PAO-1 CM), 0.083 ± 0.01 (PA-CRS01 CM; **P < 0.01 vs. LB only control), 0.080 ± 0.02 (PA-CRS02 CM; **P < 0.01 vs. LB only control), 0.09 ± 0.04 (PA-CRS03 CM; **P < 0.01 vs. LB only control), 1.55 ± 0.06 (Heat-inactivated PA-CRS03 CM; ††P < 0.01 vs. PA-CRS03 CM). Significance determined by one-way ANOVA with Bonferroni post-test; n = 4–5 independent experiments. N: bar graph of peak Ca2+ responses with 10 µM 2FLI from experiments as shown in G–K. Peak ΔFluo-4 F/Fo with 10 µM 2FLI were 1.83 ± 0.18 (control LB only pretreatment), 1.98 ± 0.15 (PAO-1 CM), 1.90 ± 0.21 (heat-inactivated PAO-1), 2.05 ± 0.13 (PAO-1 CM + phosphoramidon), 1.95 ± 0.24 (PAO-JP2 CM), 1.83 ± 0.15 PA-CRS01, 1.93 ± 0.11 PA-CRS02, 1.91 ± 0.09 (PA-CRS03), 1.90 ± 0.13 (heat-inactivated PA-CRS01). No significant differences by one-way ANOVA; n = 4 experiments per condition. For all bar graphs, data points are independent experiments and error bars are SE. Note that pretreatment of cultures with dialyzed LB media alone had no effect on the magnitude of Ca2+ responses.

Article Snippet: Immunofluorescence was carried out using PAR-2 mouse monoclonal antibody SAM11 (1:50 in DPBS + 1% BSA, 2% normal donkey serum, 0.2% saponin; Abcam) using previously described methods ( 20 , 58 ).

Techniques: Activation Assay, Immunofluorescence, Membrane, Staining, Comparison, Western Blot, Expressing, Imaging, Control