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 phosphatidylinosi tol (PtdIns) were determined in cultured mouse cortical neurons. Cells were incubated with deuterium-labeled myo-inositol (Ins*) in culture medium fre e of unlabeled Ins. The time-dependent changes in the specific activity of cytosolic Ins* and membrane PtdIns* were measured by mass spectrometry. Ptd Ins turnover was modeled incorporating values for Ins* flux, cytosolic dilu tion, 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 incorpor ation 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 th e 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 w as calculated for each point on the linear incorporation curve and then cor rected 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 thi s in vitro system. The mathematical model and the stable isotope method des cribed here should allow assessment of the dynamics of PtdIns signaling alt ered in certain diseases or by agents.