Studies are proposed to continue investigation of the ontogeny of airway hyperresponsiveness. The central aim of this proposal is to examine the role of granulocytic inflammation on epithelial modulation of airway contractility during maturation. Initial studies are directed to determine the role and mechanism by which granulocytes induce airway hyperresponsiveness in neonatal and 3 month old guinea pigs. Additional studies are proposed to examine in vitro the effect of inflammation on indices of airway smooth muscle shortening during maturation. Preliminary studies suggest that inflammatory induction of airway hyperresponsiveness caused by activated eosinophils or exogenous endothelin-1 diminishes during maturation as a consequence of maturational alterations in metabolic function of epithelium. Using an in situ """"""""living explant"""""""" preparation developed in the prior grant period, studies are proposed to examine the maturational development of 1) epithelial barrier function, 2) transduction of inflammatory stimulation and 3) modulation of the response to endothelin-1 by maturing epithelium. In the prior grant period, methods were developed for immunomagnetic separation of human eosinophils and neutrophils (PMN), enabling > 98% purification of eosinophils. Further studies are proposed to examine the contribution and mechanism of inflammatory transduction by either PMN or eosinophils after in vitro activation in maturing airways. In preliminary studies in the prior grant period, a model was developed to assess transduction of hyperpnea-induced bronchoconstriction mediated by epithelial c-fibers int he guinea pig. Further studies are proposed to study the maturation of this neural response in the epithelium. A final series of experiments is proposed to examine the effect of epithelial maturation on isotonic airway smooth muscle shortening. Pilot studies have been completed using a specially developed computerized electromagnetic level system that permits measurement of indices of actomyosin cross linkage and maximal shortening capacity. Studies are proposed to examine the biophysical properties of excised airway smooth muscle after inflammatory stimulation with activated granulocytes. Preliminary studies also indicate that smooth muscle acetylcholinesterase is not expressed phenotypically in immature animals and that inflammation downregulates the activity of this enzyme. Further studies are proposed to examine the role of inflammatory stimulation of the parasympathetic response to electrical field stimulation in vitro in maturing airway smooth muscle. These studies will establish the basis for intrinsic changes in basal responsiveness that may not be related directly to cell- cell interactions. Data derived from these studies will define alterations in epithelial-smooth muscle functions during inflammatory states and determine the potential role of activated granulocytes. Further studies will elucidate the mechanism of these maturational changes and suggest potential therapeutic strategies related to the ontogenic state of the airway.
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