We propose to solubilize, purify and characterize the structure of oxytocin (OT) receptors from rat mammary gland and uterus. Receptors will be purified by affinity chromatography with OT-linked gels and subjected to 1- and 2-dimensional gel electrophoresis. Mice will be immunized with the purest receptor preparations and hybridomas will be screened for production of anti-receptor monoclonal antibodies. Mice also will be immunized with plasma membranes containing OT receptors, in the event that we are unable to solubilize or purify active receptors. Antibodies will be screened by inhibition of [3H]OT binding to membranes and/or immunoadsorption of solubilized [3H]OT binding proteins. The specificity of the antibody will be verified by its binding to other OT target tissues and inhibition of OT-induced contractions of mammary and uterine strips. Antibodies will be used to determine whether changes in OT binding in the uterus and mammary gland during pregnancy and lactation are due to the de novo synthesis, activation or unmasking of cryptic receptors. Receptors in explants and/or myometrial minces will be labeled biosynthetically in pulse-chase studies with [35S]-methionine. Receptors also will be surface-labeled with 125I and lactoperoxidase. The molecular mechanisms by which OT inhibits (Ca++ + MG++)-ATPase will be studied. We plan to solubilize and characterize the enzyme from rat myometrium. Linkage between the enzyme and OT receptors will be sought by cofractionation of the two activities by affinity chromatography on columns of OT-agarose, calmodulin-agarose and/or by immunoaffinity chromatography. The mechanisms of OT inhibition of (Ca++ + Mg++)-ATPase will be studied by examination of the effect of OT on phosphorylation and dephosphorylation rates of phosphoenzyme intermediate(s). We also will study the effects of OT on calcium efflux from myometrial plasma membrane vesicles. Factors regulating OT receptor concentration in the mammary gland will be studied with explants maintained in culture. These factors include prolactin, growth hormone and relaxin. Many studies have indicated that OT plays a role in learning and behavior. We plan to use autoradiographic techniques to localize OT binding sites in the rat brain. We also will use these techniques to determine if OT receptors are localized primarily in the circular or longitudinal muscle layers of rat myometrium.
