Maturation of Normal & Sensitized Airway Contractility

描述(申请人提供)：儿童时期哮喘患病率较高，早期呼吸道侮辱可能会影响成人哮喘的表现。几个物种的呼吸道反应性在几天到几周内从胎儿生命和出生期间的最低水平增加到相当高的水平。我们发现肌球蛋白轻链激酶(MLCK)对肌球蛋白轻链(MLC20)的磷酸化起着关键和平行的作用，在3周龄的幼年豚鼠中，对正常增强的气道平滑肌(ASM)缩短的机械阻力减少，在新生儿致敏后的成年豚鼠中发现免疫增强缩短。最大力的产生不随成熟度和敏化而变化。根据我们的结果：ASM缩短从少年到成年期下降，而对缩短RSI的内部阻力和肌肉的被动僵硬增加。MLCK水平和MLC20的磷酸化水平平行下降。在初步实验中，新生儿致敏只增加成人ASM的RSI，增加ASM的缩短和MLCK含量。在这项应用中，这些发现被整合到一个新的假设机制中，该机制反映了在正常的非新生儿青少年和以前作为新生儿敏感的成年人中增强的平滑肌缩短的新范式。这一范式表明，细胞骨架基质中促进缩短的变化与促进肌球蛋白轻链磷酸化的那些因素具有类似的重要性。这也表明，呼吸道高反应性可能有其起源(因此需要干预)在生命早期。其具体目的是：1.确定MLCK含量增加是否在正常幼年气管和新生儿致敏成人ASM缩短增加中起关键调节作用。为了确定肌球蛋白重链的快速亚型SM1B和SM2B的含量增加是否会增加ASM的缩短。2.探讨MLCK在发育和新生儿致敏后表达的遗传和蛋白调控。3.确定正常幼年气管和新生儿致敏的成年气管的缩短增加是否与被动机械性能有关，后者降低了对缩短的内部阻力。目的：探讨中间丝结蛋白和波形蛋白的含量/磷酸化在气道平滑肌收缩内阻中的作用。

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.