CALCIUM AND ADENYLATE CYCLASE IN AIRWAY SMOOTH MUSCLE

气道平滑肌中的钙和腺苷酸环化酶

基本信息

批准号：
2224218
负责人：
JOHN Mark MADISON
金额：
$ 23.06万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-04-13 至 1995-03-31
项目状态：
已结题

项目摘要

Because airway smooth muscle contraction is an important contributor to airflow obstruction in asthma, the long term objective is to characterize the mechanisms underlying contraction by muscarinic agonists and relaxation by beta-adrenergic agonists in airway smooth muscle. Several studies have indicated that airway smooth muscle from patients with asthma have reduced relaxant responsiveness to beta-adrenergic agonists and, therefore, it is important to understand molecular mechanisms that may underlie resistance to beta-adrenergic agonists. Because contractions induced by muscarinic agonists arc relatively resistant to relaxation by beta-adrenergic agonists and because decreasing cytosolic calcium concentrations is one mechanism underlying the adenylate cyclase-dependent relaxant effects beta-adrenergic agonists, it is hypothesized that muscarinic inhibition of adenylate cyclase activity is an important, and possibly physiologically regulated, determinant of the inhibitory effects that beta-adrenergic agonists have on cytosolic calcium. Using cells isolated from bovine tracheal smooth muscle, the specific aims are: 1) Characterize the receptor subtype, location, and G-protein coupling ofthe muscarinic receptors mediating increases in cytosolic calcium and inhibition of adenylate cyclase. 2) Show that inhibiting muscarinic regulation of adenylate cyclase increases the sensitivity of cytosolic calcium concentrations to the inhibitory effects of beta-adrenergic agonists. 3) Determine whether prolonged exposure to muscarinic agonists decreases muscarinic receptor expression, muscarinic inhibition of adenylate cyclase activity, or alters calcium concentration sensitivity to beta-adrenergic agonists. The methods used to accomplish these specific aims for isolated cells will be digital imaging of fura-2 fluorescence in single cells to measure cytosolic calcium, the development of an intact cell radioligand binding assay for isolated trachealis cells, the development of new fluorescent probes for the muscarinic receptor, and the measurement of cAMP in trachealis cells. The results of these experiments will characterize the molecular mechanisms underlying adenylate cyclase regulation in airway smooth muscle cells and will, demonstrate the functional consequences that inhibiting adenylate cyclase activity has for the regulation of calcium by beta-adrenergic agonists. The results of these studies will provide new information on the relative resistance that muscarinic responses have to the inhibitory effects of beta-adrenergic agonists.

因为气道平滑肌收缩是造成的重要因素哮喘中的气流阻塞，长期目标是表征毒蕈碱激动剂和气道平滑肌中的β-肾上腺素能激动剂放松。一些研究表明，患者的气道平滑肌哮喘降低了对β-肾上腺素能激动剂的松弛反应性因此，了解分子机制很重要可能是对β-肾上腺素能激动剂的抵抗。因为宫缩由毒蕈碱激动剂诱导 β-肾上腺素能激动剂，因为胞质钙降低浓度是腺苷酸盐的一种机制依赖环酶的松弛效果β-肾上腺素能激动剂，它是假设毒蕈碱抑制腺苷酸环化酶活性是一个重要的，可能受到生理调节的，决定因素 β-肾上腺素激动剂对胞质的抑制作用钙。使用从牛气管平滑肌分离的细胞，具体目的是： 1）表征受体亚型，位置和G蛋白偶联毒蕈碱受体介导的胞质钙和抑制腺苷酸环化酶。 2）证明抑制腺苷酸环化酶的毒蕈碱调节增加了胞质钙浓度对 β-肾上腺素激动剂的抑制作用。 3）确定长时间暴露于毒蕈碱激动剂会降低毒蕈碱受体表达，毒蕈碱抑制腺苷酸盐环化酶活性，或改变钙浓度敏感性 β-肾上腺素能激动剂。用于实现这些特定目标的方法将是单个细胞中Fura-2荧光的数字成像以测量胞质钙，完整细胞放射性结合的发展分离的气管细胞的测定，新荧光的发展毒蕈碱受体的探针，以及在训练营的测量气管细胞。这些实验的结果将表征气道中腺苷酸环化酶调节的分子机制平滑肌细胞，并将证明功能后果抑制腺苷酸环化酶活性的调节 β-肾上腺素能激动剂的钙。这些研究的结果将提供有关毒蕈碱的相对抵抗力的新信息对β-肾上腺素能激动剂的抑制作用的反应。