Cytoplasmic Mg2+ concentration in platelets: implications for determination of Ca2+ with aequorin

The concentration of cytoplasmic ionized Mg2+ ([Mg2+]i) varies considerably among different cell types. It has not been measured in platelets. Incorrect estimates of this value could markedly affect many intracellular investigations, including calibration of measurements of platelet cytoplasmic ionized Ca2+ concentration ([Ca2+]i) with the photoprotein aequorin and other Ca2+-sensitive probes. [Mg2+]i was measured in washed, gel-filtered human platelets suspended in modified Tyrode buffer by two methods: 31P-nuclear magnetic resonance (NMR) spectroscopy of intact platelets and null-point titration in platelets selectively permeabilized with digitonin. The 31P-NMR spectra demonstrated that the [Mg2+]i, as calculated from the chemical shift values of ATP resonances, was 0.23 +/- 0.02 (SD) mM in unstimulated platelets. The mean [Mg2+]i as determined by null-point titration was 0.3 +/- 0.1 mM (range: 0.1-0.6 mM). When this [Mg2+]i value was used to construct a Ca2+-calibration curve for aequorin, the indicated [Ca2+]i values in resting and stimulated platelets were lower than those obtained from curves based on previously assumed values for [Mg2+]i (1.0-1.25 mM). This finding largely resolves the discrepancy between resting [Ca2+]i as determined by aequorin or by quin2, fura-2, and indo-1.