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Guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint.

ABSTRACT: Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[beta-thio]diphosphate. ATP, adenosine 5'-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

SUBMITTER: Camps M

PROVIDER: S-EPMC1149625 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Project description:We have characterized the regulation of phospholipase D (PLD) in electropermeabilized HL-60 granulocytes in which endogenous phospholipids were pre-labelled with [3H]oleic acid. Treatment of these permeabilized cells with the non-hydrolysable GTP analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate induced a sustained (near-linear for up to 60 min) accumulation of phosphatidic acid (PA). In the presence of ethanol a sustained production of phosphatidylethanol (PEt) was also observed. With increasing concentrations of ethanol, PEt formation increased, whereas PA formation declined; this indicated involvement of a PLD-type effector enzyme. The ability of GTP[S] to stimulate this PLD activity was Mg(2+)-dependent and was inhibited by GDP and its non-hydrolysable beta-thio analogue. Ca2+, at concentrations less than or equal to nM, had no effect on the GTP[S]-dependent PLD activity. However, higher concentrations of Ca2+ produced a significant potentiation of this activity. Inclusion of MgATP (greater than or equal to 0.1 mM), but not other nucleoside triphosphates, also induced a large potentiation of GTP[S]-dependent PLD activation. In the absence of guanine nucleotides, MgATP elicited no significant activation of PLD. Significantly, this effect of ATP was not mimicked by adenosine 5'-[beta gamma-methylene]triphosphate, a non-hydrolysable ATP analogue. Rather, this analogue inhibited both basal and ATP-potentiated GTP[S]-dependent PLD activity. This suggests that the ability of ATP to potentiate GTP[S]-dependent PLD activity involves phosphotransferase action rather than simple allosteric effects induced by adenine nucleotide binding. The absolute magnitude of the GTP[S]-dependent PLD activity which could be potentiated by MgATP was decreased by 90% when the permeabilized cells were preincubated for various times before addition of these stimulatory agents. This time-dependent loss of MgATP-induced potentiation was prevented when the permeabilized cells were preincubated in the presence of GTP[S]. These results demonstrate that electropermeabilized HL-60 granulocytes can be used to discriminate synergistic roles for a GTP-binding protein(s) and an ATP-dependent process (kinase?) in the regulation of phospholipase D activity.

Project description:AimsNeutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized.MethodsGlobal gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).ResultsOur results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition.ConclusionsIDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

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Guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint.

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