Cytoplasmic free Mg2+ in rat ventricular myocytes studied with the fluorescent indicator furaptra

To estimate cytoplasmic Mg2+ concentration ([Mg2+]i), ventricular myocytes enzymatically isolated from rat hearts were loaded with the fluorescent indicator, furaptra, and the fluorescence signals of single quiescent myocytes were measured at 32 degrees C. The excitation spectrum of furaptra measured in the myocytes was well-fitted by the spectra obtained in vitro; thus it was possible to calibrate the fluorescence signal in terms of [Mg2+]i. The analysis implied that about 20% of the indicator molecules were Mg(2+)-bound. Considering that the indicator likely reacts with Mg2+ with a larger KD value in cytoplasm than in vitro (by a factor of 1.2-2 as suggested for mouse and frog skeletal muscles), the [Mg2+]i for the resting single myocytes was estimated to be within 0.8-1.3 mM. Superfusion with a high extracellular Mg2+ concentration (20 mM) caused a slow and slight elevation in [Mg2+]i over a period of a few hours. Other experimental interventions, including application of a low extracellular Na+ concentration and isoproterenol, and CO2 acidosis, did not cause a detectable change in [Mg2+]i, whereas the application of an uncoupler, a blocker of oxidative phosphorylation in mitochondria, caused a rapid and large increase in [Mg2+]i. It is suggested that the [Mg2+]i is tightly maintained at around 1 mM, unless intracellular ATP is depleted.