Measurement of myo-inositol turnover in phosphatidylinositol: Description of a model and mass spectrometric method for cultured cortical neurons

Rates of myo-inositol (Ins) incorporation and turnover in phosphatidylinositol (PtdIns) were determined in cultured mouse cortical neurons. Cells were incubated with deuterium-labeled myo-inositol (Ins*) in culture medium free of unlabeled Ins. The time-dependent changes in the specific activity of cytosolic Ins* and membrane PtdIns* were measured by mass spectrometry. PtdIns turnover was modeled incorporating values for Ins* flux, cytosolic dilution, PtdIns concentration, and rate of incorporation into PtdIns. Recycled Ins diluted the labeled precursor pool, and a time course was obtained for this cytosolic process. The specific activity of the precursor pool at the plateau of the time-course curve was 0.43 ± 0.04 (mean ± SD). The incorporation of the tracer into PtdIns was linear between 4 and 10 h incubation of the neurons. After factoring in the extent of dilution of the tracer in the precursor pool, the rate of Ins incorporation into PtdIns was found to be 315 ± 51 nmol (g of protein)^-1 h^-1 The half-life of Ins in PtdIns was calculated for each point on the linear incorporation curve and then corrected for the tracer reincorporation. The half-life of Ins in PtdIns was 6.7 ± 0.2 h, which translates into a basal turnover rate of 10.3%/h in this in vitro system. The mathematical model and the stable isotope method described here should allow assessment of the dynamics of PtdIns signaling altered in certain diseases or by agents.