ABSTRACT: 1. A method has been devised for quenching cell incubations with an aqueous phenol/chloroform/EDTA mixture of neutral pH, to allow the analysis of acid-labile cell components. 2. Using this method, we have searched for the appearance of Ins(1:2cyclic,4,5)P3 [inositol 1:2(cyclic),4,5-trisphosphate] in WRK1 mammary tumour cells that were labelled to high specific radioactivity with [3H]inositol and then stimulated with 0.4 microM-vasopressin. 3. Vasopressin caused a very rapid accumulation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate), followed by a slower decline towards the original concentration. An acid-labile and inositol-labelled compound with the chromatographic properties of Ins(1:2cyclic,4,5)P3 was present in unstimulated cells at less than 5% of the elevated concentration of Ins(1,4,5)P3. Its concentration rose 2-3-fold during stimulation for 3 min, at which time its concentration was about 5% of the elevated concentration of Ins(1,4,5)P3. 4. We conclude that Ins(1,4,5)P3 is the major product of phosphoinositidase C-catalysed phosphatidylinositol 4,5-bisphosphate hydrolysis in vasopressin-stimulated WRK1 cells. Ins(1:2cyclic,4,5)P3 is unlikely to be an important intracellular messenger in these cells, at least during the first few minutes of stimulation.
