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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 TRPV1-[Ca2+]i pathway on impaired airway epithelial barrier function induced by acidic stress(A) Fluorescence intensity of Ca2+ in 16HBE cells after treatment of hydrochloric acid; data were normalized to the negative controls. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group. (B) Relative 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 negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group.
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Figure 5: Effect of TRPV1-[Ca2+]i pathway on impaired airway epithelial barrier function induced by acidic stress(A) Fluorescence intensity of Ca2+ in 16HBE cells after treatment of hydrochloric acid; data were normalized to the negative controls. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group. (B) Relative 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 negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group.

Mentions: To demonstrate the effect of TRPV1 non-selective iron channel on the decline of epithelial function of human airway induced by acidification, 16HBE cells were pretreated with TRPV1 antagonist capsazepine. In consideration of the pH values of EBC and the significant decline of epithelial TJs, the culture medium with the pH of 5.0 was selected for the ideal acidic stress. As previous studies described, the normal level of intracellular calcium ([Ca2+]i) maintained the stability of cell–cell TJs [24]. Selective depletion of endoplasmic reticulum calcium stores disrupts the initiation of TJs [25,26]. However, a rapid increase of [Ca2+]i was established to be related with the loss of TJs [27,28]. To investigate the role of TRPV1 induced Ca2+ influx in the disruption of claudin-3 and claudin-4, 16HBE cells were pretreated with capsazepine (10 μM) and BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10 μM, a cell permeable Ca2+ chelator). To understand the relationship between the activation of TRPV1 and the drop of epithelial TER, 16HBE cells pretreated with TRPV1 agonist capsaicin (1 μM) were selected as a positive control. Acidification of pH 5.0 induced an approximate 2-fold increase of [Ca2+]i, which can be partly blocked by TRPV1 specific inhibitor capsazepine or BAPTA/AM (Figure 5A). Pretreatment with casazepine (10 μM) or BAPTA/AM (10 μM) exhibited a degradation of impaired TER induced by acidification of pH 5.0 culture medium (Figure 5B).


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 TRPV1-[Ca2+]i pathway on impaired airway epithelial barrier function induced by acidic stress(A) Fluorescence intensity of Ca2+ in 16HBE cells after treatment of hydrochloric acid; data were normalized to the negative controls. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group. (B) Relative 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 negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group.
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Figure 5: Effect of TRPV1-[Ca2+]i pathway on impaired airway epithelial barrier function induced by acidic stress(A) Fluorescence intensity of Ca2+ in 16HBE cells after treatment of hydrochloric acid; data were normalized to the negative controls. 16HBE cells after exposure to pH 7.4 culture medium for 8 h were set as negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group. (B) Relative 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 negative controls. Values are mean±S.D.; n=6. *P<0.05 compared with controls; #P>0.05 compared with controls; ▲P<0.05 compared with pH 5.0 exposure group.
Mentions: To demonstrate the effect of TRPV1 non-selective iron channel on the decline of epithelial function of human airway induced by acidification, 16HBE cells were pretreated with TRPV1 antagonist capsazepine. In consideration of the pH values of EBC and the significant decline of epithelial TJs, the culture medium with the pH of 5.0 was selected for the ideal acidic stress. As previous studies described, the normal level of intracellular calcium ([Ca2+]i) maintained the stability of cell–cell TJs [24]. Selective depletion of endoplasmic reticulum calcium stores disrupts the initiation of TJs [25,26]. However, a rapid increase of [Ca2+]i was established to be related with the loss of TJs [27,28]. To investigate the role of TRPV1 induced Ca2+ influx in the disruption of claudin-3 and claudin-4, 16HBE cells were pretreated with capsazepine (10 μM) and BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10 μM, a cell permeable Ca2+ chelator). To understand the relationship between the activation of TRPV1 and the drop of epithelial TER, 16HBE cells pretreated with TRPV1 agonist capsaicin (1 μM) were selected as a positive control. Acidification of pH 5.0 induced an approximate 2-fold increase of [Ca2+]i, which can be partly blocked by TRPV1 specific inhibitor capsazepine or BAPTA/AM (Figure 5A). Pretreatment with casazepine (10 μM) or BAPTA/AM (10 μM) exhibited a degradation of impaired TER induced by acidification of pH 5.0 culture medium (Figure 5B).

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