With the use of experimentally reconstructed steady-state cell-free systems, we propose to evaluate in a series of model systems (a) the nature of the apparent effects of Ca2+ and its segregation on e.g. fluxes through central branch-points in mitochondrial disposition of pyruvate (carboxylation and decarboxylation, vectorial movement of citrate); (b) the signals mediating persisting changes in mitochondria which result from pretreatment with glucagon or Alpha-adrenergic agonists; the extent to which these responses are correlated with altered CA2+ movements; (c) the degree to which changes in membrane-associated and bioenergetic parameters (compartmented ATP/ADP and redox potentials) and correlated under steady-state metabolizing conditions with effects of Ca2+ and by in situ endocrine treatments on mitochondria subsequently processed; (d) the quantitative role of citrate in control of glycolytic flux in muscle when other putative effectors are held constant in an 'open' system; and the influence of dietary-endocrine state of animals on citrate movements from liver mitochondria; and (e) possibilities for detecting and identifying extrinsically imposed signals generated by glucagon and Alpha-adrenergic agonists in there cell-free steady-state systems containing endocrine-responsive plasma membranes on mitochondrial function. These studies potentially have far-reaching implications in our understanding of normal- and abnormal manifestations of intrinsic (intracellular) and extrinsic (imposed from without) controls. The model systems are experimentally unique since vectorially poised multicomponent and multicompartment processes are integrated into steady-state 'open' systems resembling the intact cell. With a rat muscle preparation perfused with a synthetic medium we propose to extend studies of the quantitative flux of carbon and nitrogen under conditions in which these compounds serve as glucogenic precursors. Endocrine effects on this balance will also be evaluated. This, and the cell-free systems described, provide a framework, at several levels of organization, to evaluate intracellular, intraorgan, and interorgan metabolic fluxes under simulated physiological and pathological conditions, and to evaluate intrinsic and extrinsic influences on these processes.
