Inositol 1:2(cyclic),4,5-trisphosphate is not a major product of inositol phospholipid metabolism in vasopressin-stimulated WRK1 cells

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.

The inositol phosphates in WRK1 rat mammary tumour cells

“…Insufficient material was available from the unstimulated and 5 s samples for their enantiomeric configurations to be determined. The small amounts of iditol and glucitol found in the oxidation products do, however, suggest the presence in peak 11 of small amounts of Ins(1,2-cyclic,4,5)P3, as previously reported in these cells by Wong et al (1988): opening of the cyclic phosphate ring, presumably during periodate treatment, would give rise to Ins(1,4,5)P3 and Ins(2,4,5)P3, which would yield these two hexitols after oxidation.…”

“…Peak 2: Ins(1,2cyc1ic)P. The early elution of peaks 1 and 2 suggested that one of them might be Ins(1:2cyclic)P, whose presence was expected, since Ins(I:2cyclic,4,5)P3 (Wong et al, 1988) and Ins(l :2cyclic,4)PJ (see below) have been seen in these cells. These peaks were isolated from control cells and those stimulated for 5 s, 30 s and 10 min, by extraction with the neutral phenol/chloroform medium: they were then acid-treated to open any acid-labile five-membered cyclic phosphate ring.…”

“…Inositol 1,2-cyclic monophosphate [Ins(1 :2-cyclic)P] and inositol (1,2-cyclic,4)bisphosphate 4)PJ] are acidlabile and could only be recovered from neutral phenol extracts (Wong et al, 1988 Elution was for 32 min with water at 0.2 ml/min. Fractions (0.1 ml) were collected and their radioactivity was determined by scintillation counting.…”

“…[14C]Ins2P was produced by acid-catalysed migration of [14C]Ins3P (Pizer & Ballou, 1959 in > 90 % yield by incubation at 37°C for 10 min in 5 ml of 0.1 M-glycine buffer, pH 9.0, containing 2 mM-Mg2+, 0.5 ,ug of calf intestinal alkaline phosphatase/ml and 7.5 ,tg of bovine plasma albumin/ml, followed by h.p.l.c. [Hughes & Drummond (1987) Wong et al (1988): when LiCl was present, the cells were incubated in 10 mM-LiCl for 10 min before addition of vasopressin. Incubations were terminated either with phenol/chloroform-saturated phosphate buffer or with HClO4 (neutralization of acid extracts was with KOH), and the extracts were stored at -20°C: the methods are specified in the legends, and are described by Wong et al (1988) and Kirk et al (1990).…”

“…[Hughes & Drummond (1987) Wong et al (1988): when LiCl was present, the cells were incubated in 10 mM-LiCl for 10 min before addition of vasopressin. Incubations were terminated either with phenol/chloroform-saturated phosphate buffer or with HClO4 (neutralization of acid extracts was with KOH), and the extracts were stored at -20°C: the methods are specified in the legends, and are described by Wong et al (1988) and Kirk et al (1990).…”