Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi

Abstract

Background: Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP) from cigarette smoke, to nicotine and to DMSO, respectively.

Methods: Isolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ETB receptors) and endothelin-1 (an ETA and ETB receptor agonist) were studied by use of a sensitive myograph. Before ET-1 was introduced, the ETB receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ETA receptors. ETA and ETB receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ETA and ETB receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies.

Results: After being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ETA and ETB receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ETA and ETB receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ETA and ETB receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism.

Conclusion: The increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism.

Figure 1

ET-1 and S6c contraction after 24 H of DSP or nicotine incubation. Bronchial smooth muscle cell responses to the concentration-dependent application of sarafotoxin 6c (S6c) or endothelin-1 (ET-1) following incubation for 24 h together with DSP (A, C) or nicotine (B,D) as compared with incubation control (DMEM). Mean values with S.E.M. are given. Unpaired t-tests were used to compare the groups in terms of maximum induced contraction; * P < 0.05.

Figure 2

Translational inhibitor (cycloheximide) effect on DSP led to changes in ET-1 and S6c contractility. Rat bronchial segments were incubated for 24 hours in DSP in two groups. In the one group a translational inhibitor (cycloheximide, 10 μg mL^-1) was added. Two-way Anova with Bonferroni post test was used to compare contraction at different concentrations of agonist. * P < 0.05.

Figure 3

mRNA expression of ET_A receptors (A), ET_B receptors (B) and ET-1 in the bronchi. The mRNA expression of ET_A and ET_B receptors and ET-1 in the bronchi was quantified by means of real-time PCR. Each data point was derived from results for at least 3 segments. No differences in the expression level after DSP or nicotine treatment were found as compared with controls (DMSO), (p > 0.05).

Figure 4

A and B; Quantitative image analysis of the bronchi for receptor protein expression under differing experimental conditions. ET_A and ET_B receptor protein showed a significant increase (p < 0.05) in DSP treated as compared with fresh bronchi, 24 h and 24 h+DMSO treated bronchi.

Figure 5

Immunostaining of ET_A and ET_B receptor protein in bronchi. Figure C. Example of immunostaining of ET_A and ET_B receptor protein after 24 h of culture together with DMSO as compared with 24 h of culture together with DSP.