One aim of this proposal is to study the mechanisms of relaxin action in several rat target sites - the interpubic ligament, uterine cervix, myometrium and mammary gland. Rat relaxin will be purified and radiolabeled. Relaxin-binding sites will be characterized with respect to their affinity and concentration, subcellular location and specificity. Changes in the concentration of relaxin receptors will be measured in gestation, labor and in the postpartum period. The effects of estrogen and progesterone treatment on relaxin receptors will be studied. We plan to determine if altered sensitivity of target cells to relaxin can be explained by alterations in relaxin-receptor interaction. We will study relaxin-stimulated adenylate cyclases (AC) in rat target organs. Optimum assay conditions and the effects of regulatory nucleotides will be investigated. Relaxin activation will be compared to that of beta-agonists. The relationship between the occupancy of receptor sites by relaxin and the stimulation of AC will be studied. We will compare the dose response curves, half times for relaxin binding and AC activation and the relative ability of relaxin analogues to inhibit the binding of 125I relaxin and stimulate AC. The amount of relaxin bound will be compared to the degree of AC activation in pregnancy and lactation. The second aim is to initiate studies on the biosynthesis of relaxin. PolyA mRNA will be isolated from ovaries of rats in late gestation by oligo(dT)cellulose chromatography. Changes in relaxin mRNA activity during pregnancy will be assessed by in vitro translation in the wheat germ system. The labeled protein will be immunoprecipitated with antibody to rat relaxin. The identity of the immunoprecipitated protein(s) will be verified by SDS-gel electrophoresis. With these methods we hope to determine the molecular weight(s) of putative prorelaxin(s). The ability of microsomal enzymes and trypsin to convert immunoprecipitable, higher molecular weight forms of relaxin will be investigated. Intermediates in relaxin synthesis will be studied by pulse labeling of relaxin-related proteins in isolated corpora lutea.