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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 TJ proteins(A) Cell extractions of insoluble fraction were loaded. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. (B) Relative expression of TJ proteins. The expression levels of TJ proteins in insoluble fracture were normalized to those of negative controls respectively. Values are mean±S.D.; n=6. *P<0.05 compared with controls.
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Figure 3: Effect of acidification on TJ proteins(A) Cell extractions of insoluble fraction were loaded. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. (B) Relative expression of TJ proteins. The expression levels of TJ proteins in insoluble fracture were normalized to those of negative controls respectively. Values are mean±S.D.; n=6. *P<0.05 compared with controls.

Mentions: To determine the influence of different degrees of acidification on the expression of TJ proteins, claudin-1, claudin-3, claudin-5, claudin-7 and ZO-1 were tested in membranal extracts. Acidification of the culture medium changed claudin-3 and claudin-4 levels in insoluble fraction. However, other proteins of TJs in 16HBE cells as claudin-1, claudin-5, claudin-7 and ZO-1 were stable to acidic stress (Figure 3A). Normalized with negative controls, claudin-3 and claudin-4 displayed a significant decline followed an acidic stimulation (pH 5.0, 4.0). An approximate 26% degradation of relative quantities of claudin-3 and claudin-4 were detected by Western blot of insoluble fraction by pH 5.0 acid stimulation; more than 30% degradation by pH 4.0 acid stimulation. However, no significant changes were discovered in insoluble fraction of claudin-1, claudin-5, claudin-7 and ZO-1 (Figure 3B). The TJ proteins were visualized by cell immunofluorescence staining and confocal microscopy. As shown in Figure 4, acidification of pH 5.0 culture medium for 8 h caused reduction of claudin-3 and claudin-4, but not claudin-1, claudin-5, claudin-7 and ZO-1.


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 TJ proteins(A) Cell extractions of insoluble fraction were loaded. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. (B) Relative expression of TJ proteins. The expression levels of TJ proteins in insoluble fracture were normalized to those of negative controls respectively. Values are mean±S.D.; n=6. *P<0.05 compared with controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of acidification on TJ proteins(A) Cell extractions of insoluble fraction were loaded. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. (B) Relative expression of TJ proteins. The expression levels of TJ proteins in insoluble fracture were normalized to those of negative controls respectively. Values are mean±S.D.; n=6. *P<0.05 compared with controls.
Mentions: To determine the influence of different degrees of acidification on the expression of TJ proteins, claudin-1, claudin-3, claudin-5, claudin-7 and ZO-1 were tested in membranal extracts. Acidification of the culture medium changed claudin-3 and claudin-4 levels in insoluble fraction. However, other proteins of TJs in 16HBE cells as claudin-1, claudin-5, claudin-7 and ZO-1 were stable to acidic stress (Figure 3A). Normalized with negative controls, claudin-3 and claudin-4 displayed a significant decline followed an acidic stimulation (pH 5.0, 4.0). An approximate 26% degradation of relative quantities of claudin-3 and claudin-4 were detected by Western blot of insoluble fraction by pH 5.0 acid stimulation; more than 30% degradation by pH 4.0 acid stimulation. However, no significant changes were discovered in insoluble fraction of claudin-1, claudin-5, claudin-7 and ZO-1 (Figure 3B). The TJ proteins were visualized by cell immunofluorescence staining and confocal microscopy. As shown in Figure 4, acidification of pH 5.0 culture medium for 8 h caused reduction of claudin-3 and claudin-4, but not claudin-1, claudin-5, claudin-7 and ZO-1.

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