TY - JOUR
T1 - 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
AU - Maccallum, S. H.
AU - Barker, C. J.
AU - Hunt, P. A.
AU - Wong, N. S.
AU - Kirk, C. J.
AU - Michell, R. H.
PY - 1989/1/1
Y1 - 1989/1/1
N2 - 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 relatively3H-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 F(2α), have largely failed to demonstrate the formation of relatively3H-enriched inositol phosphates. There was a tendency for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bis-phosphate to have slightly higher3H :14C ratios than phosphatidylinositol, but the3H :14C ratios than phosphatidylinositol, but the3H :14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the3H :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.
AB - 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 relatively3H-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 F(2α), have largely failed to demonstrate the formation of relatively3H-enriched inositol phosphates. There was a tendency for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bis-phosphate to have slightly higher3H :14C ratios than phosphatidylinositol, but the3H :14C ratios than phosphatidylinositol, but the3H :14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the3H :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.
UR - http://www.scopus.com/inward/record.url?scp=0024351886&partnerID=8YFLogxK
U2 - 10.1677/joe.0.1220379
DO - 10.1677/joe.0.1220379
M3 - Article
C2 - 2769159
AN - SCOPUS:0024351886
SN - 0022-0795
VL - 122
SP - 379
EP - 389
JO - Journal of Endocrinology
JF - Journal of Endocrinology
IS - 1
ER -