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Breakdown of phosphatidylinositol 4,5-bisphosphate in a T-cell leukaemia line stimulated by phytohaemagglutinin is not dependent on Ca2+ mobilization.

ABSTRACT: Addition of phytohaemagglutinin (PHA) to the [32P]Pi-prelabelled JURKAT cells, a human T-cell leukaemia line, resulted in a decrease of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to about 35% of the control value. The decrease was almost complete within 30s after the PHA addition. This decrease was followed by an increase in the 32P-labelling of phosphatidic acid (maximally 2.8-fold at 2 min). The stimulation of myo-[2-3H]inositol-prelabelled JURKAT cells by PHA induced an accumulation of [2-3H]inositol trisphosphate in the presence of 5 mM-LiCl. The result indicates hydrolysis of PtdIns (4,5)P2 by a phospholipase C. The PHA stimulation of JURKAT cells induced about 6-fold increase in the cytosolic free Ca2+ concentration, [Ca2+]i, which was reported by Quin-2, a fluorescent Ca2+ indicator. Studies with partially Ca2+-depleted JURKAT cells, with the Ca2+ ionophore A23187, and with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate indicate that the breakdown of PtdIns(4,5)P2 is not mediated through changes of [Ca2+]i. These results therefore indicate that the PHA-induced breakdown of PtdIns(4,5)P2 in JURKAT cells is not dependent on the Ca2+ mobilization.

SUBMITTER: Sasaki T

PROVIDER: S-EPMC1144929 | biostudies-other | 1985 May

REPOSITORIES: biostudies-other

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Project description:Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.

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Breakdown of phosphatidylinositol 4,5-bisphosphate in a T-cell leukaemia line stimulated by phytohaemagglutinin is not dependent on Ca2+ mobilization.

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