EVIDENCE FOR A MODEL OF INTEGRATED INOSITOL PHOSPHOLIPID POOLS IMPLIES AN ESSENTIAL ROLE FOR LIPID TRANSPORT IN THE MAINTENANCE OF RECEPTOR-MEDIATED PHOSPHOLIPASE-C ACTIVITY IN 1321N1 CELLS

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A 'chase' protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady sta te with [H-3]inositol and the cellular [3H]inositol pool was then dilu ted selectively with nonradioactive inositol. In these cells muscarini c-receptor-stimulated phospholipase C (PLC) hydrolysed [H-3]PI at appr ox. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [H-3]Ins(1,3,4)P-3, a rapidly metabolized and sensit ive marker of PLC activity, decreased only after more than 5 min and o ver a time course similar to that during which the labelling of each [ H-3]PtdIns, [H-3]PtdInsP and [H-3]PtdInsP(2) declined by at least 50%. These results demonstrate a large receptor-responsive [H-3]PI pool th at is accessed by stimulated PLC without apparent metabolic compartmen tation, despite its probable distribution between different membrane f ractions. Support for this was obtained in intact cells by using an ac ute [H-3]inositol labelling method in which increases in the specific radioactivity of [H-3]inositol phosphates stimulated by carbachol occu rred only in parallel with similar increases in the labelling of the b ulk of cellular [H-3]]PI. In [(3)Hlinositol-prelabelled cells permeabi lized to deplete cytosolic proteins, carbachol and guanosine 5'-[gamma -thio] triphosphate stimulated the endogenous PLC to degrade only appr ox. 5% of [H-3]PI. This was increased to approx. 30% in the presence o f exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC a ctivity comparable with that observed in response to carbachol in whol e cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechan isms, this model has important implications, particularly for the hypo thesis that the ability of Li+ ions to influence these selectively mig ht account for its therapeutic actions.