Figure 5: Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in Figure 4. Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using β-actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student’s test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * relative to control group; NS, not significant.