Ontogenesis of myosin light chain phosphorylation in guinea pig tracheal smooth muscle

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Abstract
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 (MLC20) 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 MLC20 phosphorylation achieved either in TSM crude protein homogenates exposed to Mg2+ · ATP · CaCl2 or in tracheal strips during electrical field stimulation (EFS). Phosphorylated and unphosphorylated MLC20 were separated on nondenaturing 10% polyacrylamide gels, and the ratio of phosphorylation was obtained by densitometric analysis of chemiluminescent Western immunoblots. Maximum MLC20 phosphorylation (% of total MLC20) 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 MLC20 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 MLC20 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 MLC20 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.