CYCLIC AMP-MEDIATED RELAXATION OF AIRWAY SMOOTH MUSCLE

呼吸道阻塞是哮喘的必要条件。虽然粘液过多 分泌物导致这种现象，主要原因是分泌物减少。 由于气道平滑肌收缩，导致气道管径增加。 细胞内环磷酸腺苷水平的增加与 气道平滑肌松弛。目前，有两个相互的 环磷酸腺苷介导的松弛的非排他性假设。一 假说认为松弛是通过磷酸化来调节的。 肌球蛋白轻链激酶(MLCK)通过环磷酸腺苷依赖的蛋白激酶表达； 另一种观点认为，松弛是细胞内减少的结果。 钙随着环磷酸腺苷水平的升高而升高。我们建议使用 采用多学科方法来确定其中一项或两项 机制有助于环磷酸腺苷介导的松弛。 第一，cAMP介导的MLCK在犬体内的磷酸化作用 将对气管平滑肌(犬TSM)松弛进行研究 确定(A)MLCK磷酸化和时间关系 松弛，(B)被磷酸化的部位和(C) 磷酸化对MLCK活性的影响。第二，MLCK之间的相互作用 皮肤松弛过程中磷酸化和胞浆钙的变化 将对完整的犬TSM进行研究。在这些实验中，细胞质 钙浓度将被操纵，抑制MLCK的抗体 磷酸化将被用来研究这些过程如何影响每个 其他的。最后，细胞内钙离子对药物反应的变化 对完整犬TSM的刺激作用将被量化。《钙离子》 然后将测量结果与循环AMP水平的变化相关联， 犬TSM的MLCK磷酸化、肌球蛋白去磷酸化和松弛。 这些实验利用生理学、药理学、生物化学和 免疫学方法。这样的方法应该提供对 两个重要的细胞信使(钙和环磷酸腺苷)和两个 重要的酶(肌球蛋白和肌球蛋白轻链激酶)在调节 气道平滑肌的收缩特性。这些研究还应该 指出对呼吸道口径进行药理学操作的途径。

Airways obstruction is the sine qua non of asthma. Although excess mucus secretions contribute to this phenomenon, the primary cause is a decrease in airway caliber due to airway smooth muscle contraction. An increase in cellular cyclic AMP levels has been associated with the relaxation of airway smooth muscle. There are, at present, two mutually non-exclusive hypotheses for cyclic AMP-mediated relaxation. One hypothesis asserts that relaxation is mediated through the phosphorylation of myosin light chain kinase (MLCK) by cyclic AMP-dependent protein kinase; the other asserts that relaxation results from a decrease in intracellular calcium following a rise in cyclic AMP levels. We propose to use a multidisciplinary approach to determine if either, or both, of these mechanisms contribute to cyclic AMP-mediated relaxation. First, the role of cyclic AMP-mediated phosphorylation of MLCK in canine tracheal smooth muscle (canine TSM) relaxation will be studied by determining (a) the temporal relationship between MLCK phosphorylation and relaxation, (b) the sites that are phosphorylated and (c) the effect of phosphorylation on MLCK activity. Second, the interaction between MLCK phosphorylation and cytoplasmic calcium during relaxation of skinned and intact canine TSM will be studied. In these experiments, the cytoplasmic calcium concentration will be manipulated and antibodies that inhibit MLCK phosphorylation will be used to study how these processes affect each other. Finally, changes in cell calcium in response to pharmacological stimulation of intact canine TSM will be quantitated. The Ca2+ measurements will then be correlated with changes in cyclic AMP levels, MLCK phosphorylation, myosin dephosphorylation and relaxation of canine TSM. These experiments utilize physiological, pharmacological, biochemical and immunological methods. Such an approach should provide insights into the roles of two important cellular messengers (calcium and cyclic AMP) and two important enzymes (myosin and myosin light chain kinase) in regulating the contractile properties of airway smooth muscle. These studies should also indicate avenues for pharmacological manipulation of airway caliber.