Studies conducted during the previous funding period and by other laboratories indicate that specific hormonal stimulation or intracellular signals rapidly mobilize large amounts of Mg2+ from myocytes (or other cells) to the extracellular space, and vice versa. On the other hand, the general understanding, supported by experimental data, assumes that cytosolic free Mg2+ is held relatively constant at a millimolar- submillimolar concentration. Hence, the main hypothesis of the research of this Component Project is to demonstrate that hormonal stimulation of myocytes induces redistribution of Mg2+ between intracellular organelles and the extracellular space, leaving cytosolic Mg2+ virtually unchanged. Key to athe hypothesis is the demonstration that specific intracellular signals induce large fluxes of Mg2+ across organelles such as mitochondria, sarcoplasmic reticulum and the cytosol, and that these fluxes are coupled with the activation of powerful sarcolemma Mg2+ transporter(s). The five specific aims proposed will define the operation and regulation of various mechanisms used by the myocardial cell to translocate Mg2+ across the sarcolemma, sarcoplasmic reticulum and mitochondria. Some of the aims proposed a broad and systemic investigation, whereas others are very specific. One example of the latter is the investigation of the cAMP binding to two isozymes of the adenine nucleotide translocase and the resulting Mg-ATP transport from mitochondrial when cAMP is bound. A large variety of models (anesthetized animals, perfused hearts, myocytes, other isolated cells, permeabilized cells, isolated organelles, purified proteins, liposomes) and experimental approaches (31P NMR, Electron Probe Microanalysis, cell imaging, isotopic, potentiometric techniques) will be used to confirm or to integrate the acquired knowledge on myocyte Mg2+ homeostasis. Additional integration is proposed in Aim 1 by information derived from experiments using anesthetized animals. Using this model, the overall results and consequences of Mg2+ redistribution between tissues and plasma will be investigated. Finally, in collaboration with other investigators of this Program Project, the results of Mg2+ redistribution within the myocyte and the changes in organelle Mg2+ will be correlated with the function of the organelles or myocytes in terms of ATP synthesis and utilization, and regulation of ion gradients.
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