The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover

Abstract

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [14C]inositol and then supplying them with [3H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively 3H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li+). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F2 alpha, have largely failed to demonstrate the formation of relatively 3H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher 3H: 14C ratios than phosphatidylinositol, but the 3H: 14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the 3H: 14C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.

Evidence for a single pool of myo‐inositol in hormone‐responsive WRK‐1 cells

“…Since greater than 95% of this radioactivity is in the form of phosphatidylinosi-to1 (PdtIns), it is referred to as such in the text, figures, and tables. As previously demonstrated, there was no qualitative difference in the behavior of the three phosphoinositide lipids under various experimental conditions [MacCallum et al, 1989;Koreh and Monaco, 19861.…”

Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle

“…WRK1 cells make little or no inositol de novo from glucose 6-phosphate [30]. In Table 1 and Figure 3, therefore, we have calculated intracellular concentrations for each inositol derivative from: (a) the specific radioactivity of the [ 3 H]inositol used to label the cells, (b) the radioactivity found in a particular cell constituent, and (c) the average cell volume at the relevant stage in the cell cycle.…”

“…Some time ago, we defined the complex inositol phosphate complement of the rat mammary tumour cell line WRK-1, and described how activating Gq-coupled V 1 -vasopressin receptors influences the formation and metabolism of inositol phosphates in these cells [27][28][29][30][31][32][33][34]. Many of the inositol polyphosphates res-Abbreviations used: EMEM, Eagle's minimal essential medium; GroPIns, glycerophosphoinositol; PIC, phosphoinositidase C; PTEN, phosphatase and tensin homologue deleted on chromosome 10.…”

Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling

“…This approach was employed successfully in the study of inositol polyphosphates generated after vasopressin or prostaglandin stimulation of vascular tissue. Their source was found to be rapidly labelled phosphoinositides, while the bulk of the highly phosphorylated inositol polyphosphates InsP 5 and InsP 6 were not created from the rapid phosphoinositide turnover, and were therefore deemed metabolically inert [56]. Dual labels can also have more technical uses.…”