IN VIVO NMR STUDIES OF CELLULAR MAGNESIUM

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It is generally recognized that cytosolic ions play a significant role in the regulation of cellular metabolism, and that disturbances in ionic homeostases resulting from exposure to environmental chemicals or heavy metals may be a significant factor in explaining observed toxicity. Although interest in the regulatory role of metals ions has chiefly centered around cytosolic calcium, there is an increasing appreciation of the role which cytosolic magnesium ions play in physiological and pathophysiological processes. Magnesium ions are reported to modulate many cell activities such as adenylate cyclase, calcium release from the sarcoplasmic reticulum in heart, and the plasma membrane a channel. An evaluation of the role of cytosolic magnesium as an acute or chronic regulator of cell function has been hampered by the lack of suitable methods for monitoring it. We have developed a series of intracellular indicators for magnesium which have dissociation constants in the physiological range, and dissociation constants for other cellular ions which are well above the physiological concentrations. One series of intracellular indicators contains the element fluorine and can be detected using fluorine-19 nuclear magnetic resonance. The resulting series of chelators: 4-fluoro, 5-fluoro, and 4-methyl- 5-fluoro o-aminophenol-N,N,O-triacetate (APTRA) have been characterized. A second approach combines the fluorophores developed by Tsien with the APTRA structure to yield fluorescent indicators. The indicator 2-(2-(5-carboxy)oxazole)-5-hydroxy-6- aminobenzofuran-N,N,O-triacetate (FURAPTRA) has been evaluated and the dissociation constants for Ca and Mg ions were determined to be 53 MuM (well above the physiological level) and 1.5 mM (near the physiological level), respectively.