The increasing incidence of asthma in the general population results in increased numbers of patients with poorly controlled asthma presenting for surgery and to the intensive care unit. Such patients are at increased risk for perioperative bronchospasm, rapid onset of which implicates airway smooth muscle in its etiology. The primary therapy of acute bronchospasm includes agents (i.e., beta-adrenergic agonists) that increase levels in airway smooth muscle cells of cAMP, a second messenger that plays a pivotal role in airway smooth muscle tone. This pathway is down regulated in patients with asthma, but the mechanism by which this occurs is unknown. In the asthmatic lung chronic activation of irritant refleses releases acetylcholine from parasympathetic nerves which, along with inflammatory mediators, chronically activate the Gq/PKC sinalling pathway. Preliminary data suggest that this chronic Gq/PKC activation cross-regulates and down regulates adenylyl cyclase enzymes responsible for the synthesis of cAMP in response to the therapeutic agents used to treat asthma. The overall plan of this proposal is to understand the mechanisms by which chronic activation of the Gq/PKC signalling pathway in asthma down regulates the functiona and expression of adenylyl cyclases in airway smooth muscle. The time course, dose response and revesibility of chronic activation of Gq/PKC on decreased adenylyl cyclase function and protein expression will be determined. The isoforms of adenylyl cyclase expressed in airway smooth muscle will be identified by northern blot analysis of poly A+ RNA. The mechanism of the down regulation in response to chronic Gq/PKC activation will be characterized at the protein level using 3H-forskolin binding and at the mRNA level using northern blot, ribonuclease protection and competitive PCR approaches.
