Cellular mechanism underlying formaldehyde-stimulated Cl- secretion in rat airway epithelium

Abstract

Background: Recent studies suggest that formaldehyde (FA) could be synthesized endogeneously and transient receptor potential (TRP) channel might be the sensor of FA. However, the physiological significance is still unclear.

Methodology/principal findings: The present study investigated the FA induced epithelial Cl(-) secretion by activation of TRPV-1 channel located in the nerve ending fiber. Exogenously applied FA induced an increase of I(SC) in intact rat trachea tissue but not in the primary cultured epithelial cells. Western blot and immunofluorescence analysis identified TRPV-1 expression in rat tracheal nerve ending. Capsazepine (CAZ), a TRPV-1 specific antagonist significantly blocked the I(SC) induced by FA. The TRPV-1 agonist capsaicin (Cap) induced an increase of I(SC), which was similar to the I(SC) induced by FA. L-703606, an NK-1 specific inhibitor and propranolol, an adrenalin β receptor inhibitor significantly abolished the I(SC) induced by FA or Cap. In the ion substitute analysis, FA could not induce I(SC) in the absence of extracelluar Cl(-). The I(SC) induced by FA could be blocked by the non-specific Cl(-) channel inhibitor DPC and the CFTR specific inhibitor CFTR(i-172), but not by the Ca(2+)-activated Cl(-) channel inhibitor DIDS. Furthermore, both forskolin, an agonist of adenylate cyclase (AC) and MDL-12330A, an antagonist of AC could block FA-induced I(SC).

Conclusion: Our results suggest that FA-induced epithelial I(SC) response is mediated by nerve, involving the activation of TRPV-1 and release of adrenalin as well as substance P.

Figure 1. Effect of FA on I _SC in rat tracheal epithelium.

(A) FA 200 µM applied to the basolateral side resulted in an increase in I _SC in intact tracheal tissue. (B) FA (0.1 µM-10 mM) stimulated a concentration-dependent I _SC response(n = 5). Each data point represents a mean ± SEM (n = 3∼6). (C) FA (200 µM) applied to the basolateral side resulted no increase in I _SC in cultured tracheal epithelial cells. Adr (5 µM) was added to demonstrate the good activity of epithelial cells. (D) Comparison of the peak magnitude of I _SC induced by FA in tracheal tissue and cultured cells. Values are mean ± SEM (n = 4, ***p<0.001).

Figure 2. Representative western blot analysis for TRPV-1.

T: tracheal tissue; D: dorsal root ganglion (DRG). β-actin served as loading control.

Figure 3. Double immunofluorescence localization of TRPV-1 and neurofilament in rat trachea.

(A) and (D) fluorescence images of tracheal sections showing FITC immunoreactivity for TRPV-1. (B) and (E) fluorescence images of tracheal sections showing TR immunoreactivity for NF-H. (C) overlapping of (A) and (B). (F) overlapping of (D) and (E). (G) (H) and (I) negative control, no first antibody was used.

Figure 4. Effects of TRPV-1 agonist and antagonist on I _SC induced by FA.

(A) I _SC response induced by FA (200 µM). (B) Representative mechanograms show the inhibitory effect of exogenously applied TRPV-1 antagonist CAZ (5 µM) on I _SC induced by FA (200 µM). (C) Representative mechanograms show the effect of TRPV-1 agonist Cap (5 µM) applied to the basolateral side on I _SC. (D) Representative mechanograms show the inhibitory effect of CAZ (5 µM) applied to the basolateral side on I _SC induced by Cap (5 µM). FSK (10 µM) applied to the basolateral side was added to demonstrate the good activity of tracheal tissue. (E) Summary results showing the effects of TRPV-1 agonist and antagonist on I _SC induced by FA (n = 5, mean ± SEM,* p<0.05, *** p<0.001).

Figure 5. Effect of β-adrenergic receptor and neurokinin-1 receptor on I _SC induced by FA or

Cap. Representative mechanograms show the inhibitory effects of exogenously applied the adrenalin β-receptor inhibitor propranolol (Prop, 10 µM) to the basolateral side on I _SC induced by (A) FA (200 µM) or (B) Cap (5 µM) and exogenously applied Prop (10 µM) as well as the neurokinin-1 (NK-1) specific inhibitor L-703606 (20 nM) to the basolateral side on I _SC induced by (C) FA (200 µM) or (D) Cap (5 µM). CCH (5 µM) was added to the basolateral side to demonstrate the good activity of tracheal tissue. (E) Summary results showing the effects of Prop (10 µM) and L-703606 (20 nM) on I _SC induced by FA (200 µM, n = 5) or Cap (5 µM, n = 4, mean ± SEM,* p<0.05, ** p<0.01, *** p<0.001).

Figure 6. Replacement of extracellular anion resulted in different I _SC induced by FA.

Representative I _SC recordings with arrows indicating FA (200 µM) were added in different K-H solutions. (A) Normal K-H (B) HCO₃ ⁻ free K-H (C) Cl^- free K-H and (D) Cl^- and HCO₃ ⁻ both free K-H. (E) Comparison of the peak magnitude of I _SC in different bathing solutions induced by FA(200 µM). Values are mean ± SEM (n = 5, ***p<0.001 vs. control).

Figure 7. Effect of different chloride channel inhibitors on I _SC induced by FA.

Representative traces showing the ISC induced by FA (200 µM) pretreated with (A) the non-specific chloride channel inhibitor DPC (1 mM) to the mucosal side (B) the CFTR specific inhibitor CFTR_i-172 (10 µM) to the mucosal side and (C) the Ca²⁺-activated chloride channel (CaCC) inhibitor DIDS (100 µM) to the mucosal side. (D) Summary results showing the effects of different chloride channel inhibitor on I _SC induced by FA (200 µM). Values are mean ± SEM (n = 6, ***p<0.001 vs. control).

Figure 8. Effect of adenylate cyclase (AC) agonist or antagonist on I _SC induced by FA.

(A) Pretreatment with the AC agonist forskolin (10 µM) to the basolateral side abolished FA-induced I _SC. (B) Pretreatment with the AC antagonist MDL-12330A (10 µM) to the basolateral side decreased FA-induced I _SC. CCH (5 µM) was added to demonstrate the good activity of tracheal tissue. (C) Comparison of the peak magnitude of I _SC induced by FA with or without MDL-12330A. Values are mean ± SEM (n = 4, ***p<0.001).

Figure 9. Cellular mechanism of formaldehyde caused Cl^- secretion in nerve-dependent epithelium.

FA activates TRPV-1 in tracheal nerve ending, causing a Ca²⁺ influx, thus induces the release of SP and Adr in the nerve. SP activates NK1R and Adr activates β-AdrR in epithelium, evoking the increase of cAMP and finally activating CFTR to induce a Cl^- secretion in tracheal epithelium.