Ding data points at each concentration of the two curves. *P
Ding data points at each concentration of the two curves. *P < 0.05, compared with control.presented as mean ?S.E.M. Statistical analyses were used with unpaired Students' t-test with Welch's correction. P < 0.05 was considered to be significant.ResultsUp-regulation of 5-HT-induced contraction Tracheal segments were cultured in the absence and presence of IL-1 (10 ng/ml) for 1, 2, 4 or 8 days. A significant increase of the maximal contractile response to 5-HT was seen at 1 day, and this up-regulation became further enhanced when the culture periods were extended. The pEC50 values remained unaltered over time (Fig. 1A ). When tracheal segments were cultured for 4 days in the presence of different concentrations of IL-1 (0.1, 1, 10 or 100 ng/ml), the 5-HT response increased in a concentration-dependent way. IL-1 concentrations higher than 10 ng/ml did not further increase the contractions. The pEC50 values remained the same in all groups (Fig. 2).IL-1 (10 ng/ml) did not affect carbachol-induced maximal contraction in the segments cultured for 1 day (con-Page 5 of(page PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25681438 number not for citation purposes)Respiratory Research 2007, 8:http://respiratory-research.com/content/8/1/Figure 3 Receptor characterization Receptor characterization. Effect of the selective 5-HT2A receptor antagonist, ketanserin (1?0 nM) on 5-HT induced contraction in the tracheal segments cultured for 4 days with IL-1 (10 ng/ml) (A). Ketanserin was added to the tissue bath 30 min before the first concentration of 5-HT. Each data point is derived from 5?3 experiments and represented as the mean ?S.E.M. Statistical analysis was performed with two-way ANOVA with Bonferroni post-test to compare the two corresponding data points at each concentration of the two curves (Fig. 3A), and unpaired Students’ t-test with Welch’s correction applied for the comparison of pEC50 values (curve shift) and the Emax (see results). *P < 0.05, compared with control. Schild analysis of data derived from panel A (B).Effects of epithelial removal In order to ascertain if epithelium is involved in the upregulation of 5-HT-induced contraction, the epithelium was removed after the trachea had been cultured for 4 days in the absence and presence of IL-1 (10 ng/ml). Removal of epithelium did not affect the contractile response to 5-HT in the fresh segments (P > 0.05, Fig. 7A) and after treatment with IL-1 (P > 0.05, Fig. 7C), whereas a not statistical significant increase was seen after organ culture (P > 0.05, Fig. 7B). Dactinomycin supplier Neither was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28914615 the mRNA level of 5-HT2A receptors nor the amount of ChAT affected by this procedure (Fig. 4). This excludes a role for epithelial factors in the IL-1 induced 5-HT hyperresponsiveness. Mitogen-activated protein kinase inhibition In order to investigate whether intracellular JNK, p38 and ERK1/2 were involved in the IL-1 induced up-regulation of the 5-HT response, a series of experiments were performed by use of the MAPK inhibitors SP600125 (10 M), SB203580 (10 M) and PD98059 (100 M), and vehicle (DMSO) as control. The inhibitors were applied 30 min prior to the first 5-HT concentration in either fresh segments or segments cultured for 4 days in the absence and presence of IL-1 (10 ng/ml). In the fresh segments, 5-HT-induced contraction was almost completely abolished by SP600125, SB203580 and PD98059, with a reduction by 98 , 68 and 82 , respectively (Fig. 8A). Segments cultured in the absence of IL-1 had a similar inhibitory pattern (85 by SP600125, 21 by SB203580 and 60.