It is now known that the ectoenzyme neutral endopeptidase (NEP) catabolizes various spasmogens and mitogen peptides, including endothelin and gastrin- releasing peptide (GRP), thereby limiting their biological effects. In certain airway disorders, including those due to oxidants such as ozone, endogenous airway NEP activity is decreased. The investigators' studies concerning ozone's effect in vivo, including their most recent experiments described in this application, have suggested that increased airway epithelial cell (AEC) proliferation occurs after repeated exposure to this oxidant. This hyperproliferation may derive, at least in part, from a persistent decrease in AEC NEP expression, thereby subjecting the surviving and growing cells to unrestrained growth factor stimulation. Consequently, the broad, long-term research objectives are to: 1) evaluate the role of NEP inactivation as a mechanism of ozone- induced AEC hyperproliferation (Specific Aims 1-3); and 2) to examine in vivo whether long-term, low level ozone exposure leads to bronchial hyperactivity and AEC hyperproliferation which is linked to increased airway bombesin-like peptide (BLP) secretion and/or AEC NEP inactivation (Specific Aim 4). The investigators' first specific aim is to test in vitro the hypothesis that during acute GRP stimulation, NEP levels modulate growth of normal AEC. To address this subject in part, the investigators will make use of a NEP preparation that the investigators have partially purified in their laboratory and have shown has substantial biological activity in vivo. For this work, the growth of cells isolated from adult, specific pathogen-free miniature swine will be routinely monitored by serial measurements of cell counts and tritiated thymidine incorporation. (As an Addendum to this proposal, culture of human AEC will also be evaluated similarly.) The second specific aim is to test in vitro the hypothesis that during prolonged, GRP stimulation of normal AEC, there is compensatory down- regulated of GRP receptors and/or up-regulation of cell NEP expression (enzyme activity and mRNA synthesis) which decrease (i.e., control) continued AEC growth responsiveness. To evaluate AEC under prolonged GRP stimulation, (125) GRP radioligand-receptor binding assays of cell membrane homogenates, assessment of new NEP-specific mRNA synthesis by Northern blot analyses, and high performance liquid chromatography measurements of NEP activity will be done. The investigators' third specific aim will be to test in vitro the hypothesis that ozone-damaged AEC (under prolonged GRP stimulation) manifest abnormalities in regulation of their GRP receptors, NEP activity, and/or mRNA synthesis which thereby augment their growth responsiveness. To pursue this hypothesis, AEC will be exposed repeatedly in vitro to low level ozone and then evaluated accordingly (as described above). The investigator's forth specific aim is to test in vivo the hypotheses that: (a) long-term low level ozone leads to chronic airway hyperactivity and AEC hyperproliferation which is linked to increased BLP secretion and/or AEC NEP inactivation; and that (b) this airway NEP inactivation persists after a protracted recovery period. To pursue this hypothesis, specific airway resistance and reactivity will be repeatedly measured in guinea pigs (throughout the chronic exposure period) as indices of the degree to which biological function may have been compromised by ozone airway damage. In addition, evaluation of these animals will include post-mortem assessments of broncho-alveolar lavage (BAL) fluid, BLP activity (by ELISA), airway tissue NEP activity, airway histopathology, and AEC growth (as reflected by their of 5-bromo-deoxyuridine incorporation). These parameters will also be assessed in a subgroup of chronically exposed animals after a protracted recovery period to determine if chronic ozone-induced changes in the airways persist. It is hoped this information will increase their understanding concerning the importance of cell NEP expression in modulating airway epithelial mitogenesis, especially as it may relate to airway disease due to repeated ozone exposure.
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