The cardiac glycosides (CG) have long played a central role in the treatment of heart failure and supraventricular arrhythmias. Hypotheses that an endogenous mammalian counterpart of CG exists have culminated in recent evidence that ouabain is present in mammalian plasma and adrenal cortex and is secreted from cultured adrenocortical cells. The presence of mammalian production of ouabain will be examined in this study. Using solid phase extraction, HPLC purification and a specific and sensitive radioimmunoassay for ouabain, we will determine whether ouabain can routinely be shown to be present in normal plasma from a variety of mammalian species. We will evaluate whether the source of such material is endogenous by studying adrenal secretion of this material. The ability of adrenocortical tissue to take up and release CGs which may be derived elsewhere, including the diet, will also be examined. We will evaluate the adrenal synthesis of ouabain and related CG-like material from cholesterol using radiolabelled cholesterol and analogs. Previous work in this laboratory has characterized the secretion of a material from cultured adrenocortical cells which is CG-like, is recognized by anti-digoxin antibodies, competes with ouabain for binding to sodium, potassium ATPase (NKA) and inhibits the ion translocating action of the enzyme. This material is neither ouabain, nor digoxin. We propose to purify and identify this material. Finally, we will examine the effect of high local concentrations of material with CG properties on the function of NKA in adrenocortical tissue. The pattern of alpha isoform expression will be quantitated by solution hybridization in an RNase protection assay, the translation into protein will be examined by Western blot and the ouabain sensitivity of ion translocation by NKA in adrenal tissue will be studied.
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