In all types of asthma, fragility and activation of the airway epithelium are characteristic of the disease. Indeed, the degree of epithelial damage and the number of epithelial cells in bronchoalveolar lavage fluids is associated with the degree of airway hyperresponsiveness (AHR). These hallmarks of asthma together with the following form the basis for this proposal. First, the airway epithelium, via production of nitric oxide (NO), is an important regulator of both the tone and responsiveness of airway smooth muscle. Second, the NO that is important in regulating airway responsiveness is, in part, the product of Ca2+-dependent constitutive NO synthases (cNOS). Third, several recent genetic studies have reported evidence of linkage of the diagnosis of asthma with the region harboring the neuronal NOS gene in humans. Collectively, these observations have led to the hypothesis that since epithelial Ca2+ channels play key roles in regulating airway responsiveness by controlling intracellular Ca2+ levels and thus the activity of cNOS, dysfunction of such Ca2+ channels can lead to AHR by disrupting this pathway and may underlie certain aspects of asthma. It is our contention that the relevant channels are store-operated Ca2+ channels (SOC). Thus one main objective is to investigate the basic properties of airway epithelial cell (AEC) SOC in normal and AHR animals using patch clamp techniques. If the hypothesis is correct, then agents which block SOC should cause AHR while those with an opposing action should reverse AHR. The latter might well represent the prototype for new therapeutics for asthma. Thus as a second main objective, we have developed novel cell permeant SOC regulators which will be used to explore the role of SOC in NO production and AHR in intact animals, as well as explanted tracheas. These two objectives will be met via the following specific aims: 1. Can AHR be mimicked and reversed by agents that close and open SOC respectively? 2. What are the electrophysiologic and biophysical characteristics of SOC in AEC? 3. Are SOCs in AEC from airway hyperresponsive animals dysfunctional? 4. Can the level of NO production be modulated by agents that open or close AEC SOC? 5. Which form of NOS is involved in regulation of airway responsiveness by agents that modify AEC SOC?
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