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Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations.

ABSTRACT: Rabbit neutrophils labelled with [3H]inositol and permeabilized with saponin produced [3H]inositol trisphosphate (InsP3) when incubated with stable analogues of GTP or millimolar concentrations of Ca2+. [3H]InsP3 production elicited by guanosine 5'-[gamma-thio]triphosphate was enhanced by the chemoattractant formylmethionyl-leucyl-phenylalanine and inhibited by pertussis-toxin pretreatment. A pertussis-toxin-sensitive stimulation of [3H]InsP3 concentration was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with guanosine 5'-[beta-thio]diphosphate or GTP. Millimolar Ca2+ alone was sufficient to stimulate [3H]InsP3 production; however, in the presence of guanosine 5'-[gamma-thio]triphosphate, the Ca2+ dose-response curve was shifted to submicromolar concentrations. These findings directly confirm the role of a pertussis-toxin-sensitive guanine nucleotide regulatory protein (G protein) in chemoattractant-stimulated phospholipase C activity in rabbit neutrophils. Moreover, the ability of guanine nucleotides to sensitize phospholipase C to physiologically relevant Ca2+ concentrations suggests that the role of the activated G protein may be to enhance the apparent affinity of phospholipase C for Ca2+ and thus to activate the enzyme without an increase in the Ca2+ concentration.

SUBMITTER: Bradford PG

PROVIDER: S-EPMC1147244 | biostudies-other | 1986 Oct

REPOSITORIES: biostudies-other

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Project description:The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.

Project description:We have measured the time course of secretion of hexosaminidase from rat mast cells permeabilized (in simple buffered NaCl solutions) in response to guanine nucleotides [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])] and Ca2+. In these experiments, ATP was excluded from the system (and the cells were pretreated with metabolic inhibitors). For cells permeabilized in the absence of Mg2+ but in the presence of Ca2+, secretion commences promptly in response to addition of GTP; when Mg2+ (2 mM) is provided, secretion commences after an extended delay, much higher concentrations of GTP are required, and the final extent of secretion is decreased. Ongoing secretion due to GTP and Ca2+ is abruptly terminated by addition of Mg2+ to cells initially stimulated in its absence. In contrast, although Mg2+ has no effect on the sensitivity to the non-hydrolysable analogue GTP[S], its absence does nevertheless cause delays in the onset of secretion triggered by the addition of GTP[S] to cells initially permeabilized in the presence of Ca2+ (micromolar range, again in the absence of ATP). However, exocytosis from cells triggered with Ca2+ after permeabilization in the presence of high concentrations of GTP[S] is instantaneous. The delays due to triggering by GTP[S] have GTP[S]-concentration-dependent and -independent components. The guanine-nucleotide-concentration-dependent component is expressed as an extended duration of delay as the concentration of GTP[S] is decreased, and may reflect the binding of GTP[S] to GE. The concentration-independent component is manifested as a limiting delay which cannot be further diminished by increasing the guanine nucleotide concentration. The duration of the limiting delay is sensitive to the identity of the stimulating nucleotide (GTP < GTP[S] < p[NH]ppG) and may reflect the time taken for an activating conformational change to occur after binding. Since both components of the delays are abolished by the presence of Mg2+, both the binding of guanine nucleotide and the activation of GE appear to be Mg(2+)-dependent. We therefore conclude that nucleotide binding, activation and the GTPase activity of GE are strongly dependent on Mg2+, in common with the same three processes in Gs and Gi.

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Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations.

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