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The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor.

Xu R, Li Q, Zhou J, Zhou XD, Perelman JM, Kolosov VP - Biosci. Rep. (2013)

Bottom Line: Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly.However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells.The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)].

View Article: PubMed Central - PubMed

Affiliation: *Department of Respiratory Medicine, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.

ABSTRACT
Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca2+]i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)].

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Effect of acidification on airway epithelial barrier function(A) 16HBE cells exposed to pH 7.4 culture medium for 8 h were set as negative control. TER values of 16HBE cells were recorded before and after exposure to acidic stress. Values are mean±S.D.; n=6. *P<0.05. #P>0.05. (B) Fold changes of TER values in 16HBE cells after acidic stress. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as base line. Values are mean±S.D.; n=6. *P<0.05 compared with the base line. #P>0.05 compared with the base line.
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Figure 2: Effect of acidification on airway epithelial barrier function(A) 16HBE cells exposed to pH 7.4 culture medium for 8 h were set as negative control. TER values of 16HBE cells were recorded before and after exposure to acidic stress. Values are mean±S.D.; n=6. *P<0.05. #P>0.05. (B) Fold changes of TER values in 16HBE cells after acidic stress. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as base line. Values are mean±S.D.; n=6. *P<0.05 compared with the base line. #P>0.05 compared with the base line.

Mentions: Airway epithelial function was estimated by TER as described in the experimental section. 16HBE cells exposed in DMEM with the pH of 7.4 was set as the negative control. For statistic analysis, TER values of 16HBE cells after exposure to different acidification culture mediums were normalized by TER of 16HBE cells after exposure to pH 7.4 culture medium. No significant changes were found in TER values of 16HBE cells in pH=7.4 or 7.0 culture medium 8 h later. Interestingly, TER values of 16HBE cells exhibited a slightly increase after exposure to the culture medium with pH=6.0. However, there was no statistical significance. Stimulated with higher acidification (pH=5.0, 4.0) increased the permeability of 16HBE cells. (Figure 2A) Normalized by negative control, TER of 16HBE cells exhibited an insignificant increase after slight acidification (pH=6.0). However, TER values of 16HBE cells were significantly decreased after pH=5.0 or 4.0 acidic stimulation compared with basal level, which indicated a loss of airway epithelial barrier function followed by higher dose of acidic stimulation. Exposed to pH 5.0 culture medium, the TER value of 16HBE cells approximately decreased more than 20% compared with the basal level. Acidification of pH 4.0 induced a nearly 40% reduction of TER values in 16HBE cells (Figure 2B).


The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor.

Xu R, Li Q, Zhou J, Zhou XD, Perelman JM, Kolosov VP - Biosci. Rep. (2013)

Effect of acidification on airway epithelial barrier function(A) 16HBE cells exposed to pH 7.4 culture medium for 8 h were set as negative control. TER values of 16HBE cells were recorded before and after exposure to acidic stress. Values are mean±S.D.; n=6. *P<0.05. #P>0.05. (B) Fold changes of TER values in 16HBE cells after acidic stress. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as base line. Values are mean±S.D.; n=6. *P<0.05 compared with the base line. #P>0.05 compared with the base line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814059&req=5

Figure 2: Effect of acidification on airway epithelial barrier function(A) 16HBE cells exposed to pH 7.4 culture medium for 8 h were set as negative control. TER values of 16HBE cells were recorded before and after exposure to acidic stress. Values are mean±S.D.; n=6. *P<0.05. #P>0.05. (B) Fold changes of TER values in 16HBE cells after acidic stress. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as base line. Values are mean±S.D.; n=6. *P<0.05 compared with the base line. #P>0.05 compared with the base line.
Mentions: Airway epithelial function was estimated by TER as described in the experimental section. 16HBE cells exposed in DMEM with the pH of 7.4 was set as the negative control. For statistic analysis, TER values of 16HBE cells after exposure to different acidification culture mediums were normalized by TER of 16HBE cells after exposure to pH 7.4 culture medium. No significant changes were found in TER values of 16HBE cells in pH=7.4 or 7.0 culture medium 8 h later. Interestingly, TER values of 16HBE cells exhibited a slightly increase after exposure to the culture medium with pH=6.0. However, there was no statistical significance. Stimulated with higher acidification (pH=5.0, 4.0) increased the permeability of 16HBE cells. (Figure 2A) Normalized by negative control, TER of 16HBE cells exhibited an insignificant increase after slight acidification (pH=6.0). However, TER values of 16HBE cells were significantly decreased after pH=5.0 or 4.0 acidic stimulation compared with basal level, which indicated a loss of airway epithelial barrier function followed by higher dose of acidic stimulation. Exposed to pH 5.0 culture medium, the TER value of 16HBE cells approximately decreased more than 20% compared with the basal level. Acidification of pH 4.0 induced a nearly 40% reduction of TER values in 16HBE cells (Figure 2B).

Bottom Line: Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly.However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells.The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)].

View Article: PubMed Central - PubMed

Affiliation: *Department of Respiratory Medicine, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.

ABSTRACT
Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca2+]i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)].

Show MeSH
Related in: MedlinePlus