RESUMO
Airway smooth muscle (ASM) displays a hyperresponsive phenotype at young age and becomes less responsive in adulthood. We hypothesized that allergic sensitization, which causes ASM hyperresponsiveness and typically occurs early in life, prevents the ontogenetic loss of the ASM hyperresponsive phenotype. We therefore studied whether neonatal allergic sensitization, not followed by later allergen challenges, alters the ontogenesis of ASM properties. We neonatally sensitized guinea pigs to ovalbumin and studied them at 1 week, 3 weeks, and 3 months (adult). A Schultz-Dale response in isolated tracheal rings confirmed sensitization. The occurrence of inflammation was evaluated in the blood and in the submucosa of large airways. We assessed ASM function in tracheal strips as ability to produce force and shortening. ASM content of vimentin was also studied. A Schultz-Dale response was observed in all 3-week or older sensitized animals. A mild inflammatory process was characterized by eosinophilia in the blood and in the airway submucosa. Early life sensitization had no effect on ASM force generation, but prevented the ontogenetic decline of shortening velocity and the increase in resistance to shortening. Vimentin increased with age in control but not in sensitized animals. Allergic sensitization at birth without subsequent allergen exposures is sufficient to prevent normal ASM ontogenesis, inducing persistence to adulthood of an ASM hyperresponsive phenotype.
RESUMO
Increased airway responsiveness occurs in normal young individuals compared to adults. A maturation of airway smooth muscle (ASM) contractility is likely a mechanism of this juvenile airway hyperresponsiveness. Indeed, we showed in guinea pig tracheal smooth muscle (TSM) that maximum shortening velocity decreases dramatically after the first 3 weeks of life. Because the phosphorylation of the 20-kDa myosin light chain (MLC(20)) was shown to be a key event in ASM contractility, in the present work we sought to investigate it during ontogenesis. In three age groups (1-week-old, 3-week-old, and adult guinea pigs), we assessed the amount of MLC(20) phosphorylation achieved either in TSM crude protein homogenates exposed to Mg(2+) . ATP . CaCl(2) or in tracheal strips during electrical field stimulation (EFS). Phosphorylated and unphosphorylated MLC(20) were separated on nondenaturing 10% polyacrylamide gels, and the ratio of phosphorylation was obtained by densitometric analysis of chemiluminescent Western immunoblots. Maximum MLC(20) phosphorylation (% of total MLC(20)) in TSM tissue homogenate was, respectively, 32.6 +/- 5.7, 32.2 +/- 5.7, and 46.8 +/- 5.8 in 1-week, 3-week, and adult guinea pigs. Interestingly, in nonstimulated intact tracheal strips, we found a substantial degree of MLC(20) phosphorylation: respectively, 42.2 +/- 5.8, 36.5 +/- 7.8, and 46.4 +/- 4.7 in 1-week, 3-week, and adult guinea pigs. Maximal EFS-induced MLC(20) phosphorylation (% increase over baseline) in the 3-week age group was attained after 3 sec of EFS, and was 161.2 +/- 17.6, while in 1-week and adult guinea pigs, it was attained at 1.5 sec of EFS and was, respectively, 133.3 +/- 9.3 and 110.2 +/- 3.9 (P < 0.05). We conclude that MLC(20) phosphorylation in guinea pig intact tracheal strips correlates with ontogenetic changes in shortening velocity and changes in myosin light chain kinase content. These results further suggest that the maturation of ASM contractile properties plays a role in the greater airway responsiveness reported in children and young animals.
