The major effort will be directed at the characterization of the mechanisms involved in the activation, release and metabolic degradation of the endogenous digitalis-like substances (EDLS) of the toad, Bufo Marinus. We wish to explore the hypothesis that EDLS act as hormonal regulators of Na+ Homeostasis in the toad, with the hormonal role of EDLS being of greater physiological importance than a presumed protective role. Having completed the chemical identification and biochemical characterization of the major EDLS of toad bile, we plan next to identify and characterize EDLS recently isolated from toad serum and skin. Having developed the capacity to monitor changes in serum concentrations of EDLS, we now propose to delineate the physiological and pharmacological stimuli which modulate changes in serum levels of toad EDLS. The role of EDLS in modulating active transepithelial Na+ transport will be explored in toad bladder and cultured toad kidney cells. Having established the ability of the toad to inactivate EDLS, we no wish to characterize the metabolic fate of radiolabeled EDLS, with special emphasis on the identification of inactive EDLS, particularly those with altered lactone rings, which are not detectable by currently available biochemical, immunochemical or spectrophotometric methods. A second series of studies will involved the extension of the information obtained from the toad experiments to the study of mamalian body fluids. We propose to explore the hypotheses; (i) that there is a teleological requirement for the prompt, effective inactivation of EDLS in sensitive mammalian species; and (ii) that the current inability of so many laboratories to identify authentic mamalian EDLS stems not from their nonexistence, but rather from the inability of currently available assay methods to detect inactive derivatives of EDLS, particularly derivatives with altered lactone rings. These experiments will involve two approaches to the characterization of inactive derivatives of putative mammalian EDLS: The detailed study of mammalian metabolism of radiolabeled authentic active toad EDLS; and efforts at detection of mamalian analogues of inactive amphibian EDLS derivative. Other studies will be directed at more precise delineation of environmental factors responsible for modulating clinical variability in the phenotypic expression of reductive inactivation of digoxin by the anaerobe, Eubacteriu lentum, in the intestinal flora of digoxin-treated carriers of the microbe.
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