Research Project
3365033
Beta-Adrenoceptors modilates several aspects of airway function and may function abnormally in inflammatory diseases such as asthma. Previous investigations of beta-eceptors have included direct receptor binding and biochemical and functional responses to beta-agonists, but recently the availability of cDNA probes for beta-receptors has opened up new options. It is proposed to study whether inflammation or anti-inflammatory treatments, such corticosteroids, modulate beta-receptors in relevant human airway cells (cultured epithelial and smooth muscle cells) or inflammatory cells (mast cells, alveolar macrophages, monocytes, neutrophils and eosinophils). beta-2 Receptor mRNA in these cells will be measured by Northern blot analysis using a labelled cRNA probe and effects selected inflammatory mediators (e.g. histamine, leukotrienes, platelet activating factor, endothelin), protein kinase C activators and corticosteroids will be investigated. Probes for beta-1 and beta-3 receptors will also be used in parallel. If changes in mRNA are found, whether these operate at the level of gene transcription or post-transcriptional processing will be determined. The effect of steroids on alveolar macrophage and leukocyte beta-receptor mRNA in normal and asthmatic patients ex vivo, the effects of allergen challenge on leukocytes of asthmatic patients, and the effects of virus infection on leukocytes from normal subjects will also be investigated. These studies of beta-receptor mRNA will be complemented by binding studies using [125I]pindolol and measurement of cyclic AMP and protein kinase A in response to beta-agonists. In addition, in situ hybridization will be employed to study the distribution of beta-2 receptor mRNA in human and animal lung sections, and in post-mortem lung and bronchial biopsies from asthmatic patients. To investigate modulation in further detail airway beta-receptors in animals will be studied after allergic inflammation, virus infection and endotoxin exposure, all of which may reduce beta-receptor function. These studies will shed light on whether inflammation, infection and anti-inflammatory treatments may influence beta-receptors at a transcriptional level and provide further insights into abnormal beta-adrenergic control in asthma. In view of likely differences between species and in regulation between cell types, this project concentrates on the study of beta-receptor regulation from the level of gene transcription to functional effects in relevant cells, and exploits the availability of cultured and isolated human cells and of lung, airways and cells from asthmatic patients.
