The objective of this proposal is to elaborate the mechanism by which glucocorticoids modulate the synthesis of the 35kDa surfactant associated protein (PSP-A). At first the developmental profile of PSP-A synthesis under the influence of glucocorticoids at the protein and RNA level will be studied using an in vivo system. The effects of various doses of steroid on PSP-A synthesis will be examined. The steroid effect on PSP-A synthesis as a function of gender will also be examined to determine whether the sex of the animal modulates the glucocorticoid effect on PSP-A synthesis. These studies may provide insight as to why male fetuses show a higher incidence of respiratory distress syndrome and respond poorly to glucocorticoid treatment. Towards these goals newly synthesized PSP-A from control and dexamethasone-treated animals will be analyzed by two- dimensional gel electrophoresis. PSP-A primary translation products of RNA from the same animals will be analyzed similarly. RNA levels of PSP-A from the two groups will also be examined directly by Northern blotting using a rat PSP-A cDNA probe. Subsequently, experiments will be carried out to determine whether the increased PSP-A synthesis following glucocorticoid treatment results from an increased number of Type II epithelial cells synthesizing PSP-A or whether higher amounts of PSP-A message are produced per Type II cell. Using biochemical approaches we will address the questions of whether glucocorticoids modulate PSP-A synthesis by acting directly at the transcriptional level. Newly synthesized PSP-A mRNA in isolated nuclei from whole lung tissue will be obtained from control or dexamethasone treated animals and its rate of synthesis will be examined by filter hybridization to rat PSP-A cDNA. The stability of PSP-A mRNA will also be examined by pulse-chase experiments if this appears to be a factor in the regulation of PSP-A synthesis. We will also use the morphological technique of tissue in situ hybridization, to determine directly whether the glucocorticoid enhanced PSP-A synthesis reflects a higher number of Type II cells synthesizing PSP-A or increased accumulation of PSP-A mRNA in Type II cells. The experiments outlined here taken together will contribute significantly to our understanding about how glucocorticoids modulate PSP-A synthesis and accelerate lung maturity.
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