Virtually all animal cells depend upon the activity of sodium-potassium pumps (Na pumps) in their surface membrane. Cardiac glycosides (CG) are plant-derived steroids long recognized for their utility in medicine and the basic sciences. It is widely accepted that the binding of CG to Na pumps inhibits the active transport of sodium and potassium ions. Yet while CGs all target Na pumps, the consequences of their action differ dramatically among different cell types - and profound physiological actions occur with CG concentrations below the therapeutic level. In addition, the Na pump is heterogeneous - comprised of a family of four known isoforms. Three of the isoforms have retained the ability to bind CGs with extraordinary specificity and high affinity over the eons. The impression is that the Na pump, in addition to a role in ion transport, functions as a physiological receptor for one or more endogenous CGs. Indeed, overwhelming evidence has shown that an analog of the arrow poison, ouabain, is a ubiquitous component of the circulation in animals and humans. The endogenous """"""""ouabain"""""""" (EO) together with aldosterone influences cardiovascular and electrolyte homeostasis. The inappropriate elevation of circulating EO is linked with common essential hypertension. In spite of the importance and novelty of this system, the origin of the endogenous """"""""ouabain"""""""" remains uncertain. The adrenal cortex has been repeatedly implicated as a major source of EO but the biosynthetic pathway is not described. This proposal investigates the metabolism of the adrenal gland and specifically explores the possibility of EO biosynthesis by bovine adrenocortical tissue and cells. The approach is multifaceted. It involves the investigation of classical steroid intermediates as possible fuels for EO biosynthesis and studies of the fate of labeled compounds to probe for possible similarities and differences from known steroid biosynthetic pathways. The investigation will be complemented by studies of the bioactivity and structure of EO and its precursors as well as novel binding proteins. The successful outcome of the proposed studies would be revolutionary - representing the first new physiologically significant steroid to be identified from the adrenal since the discovery of aldosterone. The implications for the life sciences and medicine are both broad and profound and will form the basis for fundamentally new therapeutic modalities for hypertension and heart failure. ? ?
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