Maturation of Normal & Sensitized Airway Contractility

描述（由申请人提供）：儿童期哮喘患病率较高，早期气道损伤可能影响成人哮喘的表达。几个物种的气道反应性从胎儿生命和出生期间的最低水平增加到几天到几周的实质性水平。我们已经发现了肌球蛋白轻链激酶（MLCK）磷酸化肌球蛋白轻链（MLC 20）的关键和平行作用，以及在3周龄幼年豚鼠的气道平滑肌（ASM）正常增强缩短和新生儿致敏后成人的免疫增强缩短中减少对缩短的机械对抗。最大力产生在成熟和致敏后不变。根据我们的结果：从青少年到成年，ASM缩短下降，而内部阻力缩短Rsi和肌肉的被动刚度增加。MLCK水平和MLC 20的磷酸化水平平行下降。在初步实验中，新生儿致敏增加ASM缩短和MLCK含量，并降低Rsi的成人ASM。在这个应用程序中，这些发现被整合到一个新的假设机制，反映了一个新的范例，增强平滑肌缩短在正常的非新生儿青少年和成人以前致敏的新生儿。这种模式表明，细胞骨架基质的变化，以促进缩短是类似的重要性，这些因素，促进肌球蛋白轻链磷酸化。这也表明气道高反应性可能起源于生命早期（因此需要干预）。具体目标是：1.确定MLCK含量增加是否在正常青少年气管和新生儿致敏成人的ASM缩短增加中起关键调节作用。确定肌球蛋白重链的快速亚型SM 1B和SM 2B的含量增加是否会增加ASM缩短。2.确定MLCK表达随发育和新生儿致敏的遗传和蛋白调节。3.确定正常青少年气管和新生儿致敏成人气管缩短的增加是否与降低缩短内阻的被动机械特性相关。确定中间丝结蛋白和波形蛋白的含量/磷酸化是否在气道平滑肌缩短的内部阻力中起作用。

DESCRIPTION (provided by applicant): Asthma prevalence is greater during childhood and early airway insults may affect the expression of asthma in adults. Airway reactivity in several species increases from minimal during fetal life and birth to a substantial level in a few days to weeks. We have discovered key and parallel roles for the phosphorylation of myosin light chain (MLC20) by myosin light chain kinase (MLCK) and the reduced mechanical opposition to shortening in the normally augmented shortening of airway smooth muscle (ASM) in 3 week old juvenile guinea pigs and immune-augmented shortening in adults following neonatal sensitization. Maximal force generation is unchanged with maturation and following sensitization. Based on our results: ASM shortening declines from juveniles to adulthood while the internal resistance to shortening Rsi and the passive stiffness of the muscle increase. MLCK levels and the phosphorylation of MLC20 decline in parallel. In preliminary experiments neonatal sensitization increases ASM shortening and MLCK content and decreases the Rsi of adult ASM only. In this application, these findings are integrated into a novel hypothetical mechanism that reflects a new paradigm for augmented smooth muscle shortening in normal non-neonatal juveniles and in adults previously sensitized as neonates. This paradigm suggests that changes in the cytoskeleton matrix to facilitate shortening are of similar importance to those factors that facilitate the phosphorylation of myosin light chain. It also suggests that airway hyperresponsiveness may have its origins (and thus the need for intervention) early in life. The specific aims are: 1. To determine whether or not increased MLCK content plays a key regulatory role in the increased ASM shortening in normal juvenile trachealis and adults sensitized as newborns. To determine whether or not the increased content of the fast isoforms SM1B and SM2B of myosin heavy chain increases ASM shortening. 2. To determine the genetic and protein regulation of MLCK expression with development and following neonatal sensitization. 3. To determine whether the increased shortening in normal juvenile trachealis and in adults sensitized as newborns is associated with passive mechanical properties that reduce the internal resistance to shortening. To determine whether the content/phosphorylation of the intermediate filaments desmin and vimentin plays a role in the internal resistance to shortening of airway smooth muscle.