We will contiune studies on the molecular and cellular mechanisms of androgenic and Beta-adrenergic responses in heart myocytes. Previous studies in rodent kidney cortex and heart, using tissue slices, revealed that nanomolar concentrations of testosterone and 1-isoproterenol induce a rapid (less the 1 min) Ca2+ - and receptor-dependent stimulation of Ca2+ fluxes, endocytosis and transport of hexose and amino acid; and an early stimulation of ornithine decarboxylase (ODC) activity and polyamine synthesis. Studies with the ODC inhibitor Alpha-difluoromethylornithine (DFMO) showed that rapid stiumulation of ODC activity and polyamine synthesis is obligatory for androgenic and Beta-adrenergic stimulation of Ca2+ fluxes and membrane transport processes. On the basis of these and other findings a new model for signal transduction and stimulus-response (permeability) was proposed in which stimulaltion of ODC activity and polyamine synthesis plays a pivotal role in regulating Ca2+ fluxes. In this model, activation of cell surfar receptors induces an early, transient, Ca2+ - (or Ca2+ -calmodulin), prostaglandin- and cyclic AMP-dependent stimulation of the catalytic activity of a cryptic ODC in or near the plasma membrane by a phosphorylation-dephosphorylation sequence involving ODC (or possibly an ODC regulatory protein). The delayed increase in ODC activity results from an induction of new ODC protein. Newly synthesized polyamines serve as messengers to generate a Ca2+ signal by increasing Ca influx or mobilizing intracellular Ca, or both, via a cation exchange reaction. Polyamines are thought to be involved in the regulation of membrane transport, cytoskeletal, mitochondrial, lysosomal function, and other cell responses by: (a) generating local Ca2+ signals, and mediating Ca2+ -dependent processes; and (b) binding to cytomembranes and other components with acidic sites and inducing conformational changes by displacing bound Ca or decreasing surface charge density.
The specific aims of this project are to study in testosterone- and isoproterenol-stimulated Langendorff-perfused rat heart and acutely isolated cardiac myocytes: (1) Positive inotropic effects and their polyamine dependence. (2) Conversion of phosphorylase b to phosphorylase a and glycogenolysis. (3) Changes in Ca2+ fluxes and free cytosolic Ca2+ concentration and their polyamine dependence. (4) Changes in Na22+ transport, Rb86+ transport and intracellular pH and their polyamine dependence. (5) The molecular and cellular mechanisms underlying the rapid stimulation of ODC activity. (6) Ca-dependent effects of the cardiac glycosides digoxin and ouabain, and the antiarrythmogenic drug phenytoin and the role of polyamine synthesis of their effects.
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