and Specific Aims.) Small-granule cells/neuroepithelial bodies (NEBs) are peptide-producing paracrine receptor-effectors dispersed throughout airways of mammalian lungs from fetal life into old age. A biphasic function is postulated: 1) during development they exert a local """"""""mitogenic effect"""""""" on the epithelium, thereby influencing the lengthening of individual airway branches; 2) later they provide local regulation of bronchial or vascular muscle tone in response to airway hypoxia and other stimuli. The proposed work addresses both phases but emphasizes the developmental role. In prenatal hamster lungs, 3H-thymidine and bromodeoxyuridine (BrdU) labeling have shown that cell division in the epithelium diminishes as a linear function of distance from differentiated NEBs. Hamsters will be the main subjects; major points will be verified in rats.
In Specific Aim 1, the onset of the mitogenic effect will be studied in two """"""""development windows"""""""" at mid-bronchial and terminal bronchiolar levels of hamster lungs staged between 13 days prenatal and adulthood. BrdU pulse labeling will be used to determine when NEB- associated epithelium shows increased labeling over NEB-unassociated epithelium. The same windows will be examined using in situ hybridization and immunocytochemistry for the first appearance of calcitonin gene-related peptide (CGRP) mRNA and peptide, the leading candidate-mitogen for hamsters and rats; coincidence of mitogenic effect and CGRP (or colocalizing substances such as serotonin) in both windows will be sought. Correlation will also be made between up- or down- regulation (change) of CGRP receptors and onset of mitogenic effect in affected airway regions. Regarding Aim 2, receptor-effector function of NEBs in mature lungs appears to correlate with acquisition of sensory innervation and appearance of a widened range of peptides in NEBs. Effects of these events on the mitogenic capacity of NEBs will be assessed: mitogenesis by BrdU labeling, peptides by immunocytochemistry, degree and character of innervation by light and transmission electron microscopy, including neurotransmitters and transmitter- associated enzyme localizations.
In Aim 3, organ cultured fetal lungs of rats and hamsters will serve as a pharmacological test system. Direct effects of exogenously administered candidate mitogens as well as indirect effects of agents affecting secretion by NEBGs will be appraised from a) changes in the growth pattern of airways, b) expression of early-immediate genes, c-fos and c-jun, assessed by in situ hybridization and confocal scanning laser microscopy, and c) quantitative analysis of BrdU or 3H-thymidine pulse labeling. Secretory responses of NEBs will be evaluated using supraoptimal dilution immunocytochemistry and electron microscopic morphometry.
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