Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate

1. A Herpes-based medium has been devised which allows rapid P(i) exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [³²P]P(i) in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [³²P]P(i) in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P₂) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P₂ remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate group. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4P, PtdIns4,5P₂ and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the γ-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca²⁺-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P₂ and 50% of ³²P-labelled PtdIns4,5P₂ present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P₂ are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P₂ are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.