Involvement of guanine nucleotides in superoxide release by fluoride-treated neutrophils. Implications for a role of a guanine nucleotide regulatory protein

D. English, M. T. Rizzo, G. Tricot, R. Hoffman

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28 Scopus citations


Previous studies demonstrating hydrolysis of phosphatidylinositol bisphosphate (PIP2) and generation of inositol phosphates in neutrophils exposed to 20.0 mM NaF provide indirect evidence that activation of phospholipase-associated guanine nucleotide regulatory protein, a guanine nucleotide binding protein which regulates the activation of a membrane inositol-specific phospholipase C, is an early event in the neutrophil stimulus-response pathway triggered by fluoride. Consistent with this hypothesis, exposure of a plasma membrane rich preparation isolated from 32P labeled neutrophils to 20.0 mM NaF resulted in hydrolysis of labeled PIP2. Levels of other phospholipids were not affected. Inositol bisphosphate and inositol trisphosphate were detected in extracts of neutrophil plasma membranes exposed to fluoride. To further explore the involvement of guanine nucleotides in functional responses of intact neutrophils triggered by fluoride, we preincubated cells with 2-β-D-ribofuranosylthiazole-4-carboxamide (tiazofurin), a selective inhibitor of inosine monophosphate dehydrogenase, to diminish guanine nucleotide synthesis and then compared superoxide generation induced by FMLP, PMA, digitonin, and 20.0 mM NaF to intracellular levels of guanine nucleotides. Preincubation of neutrophils for 2.5 h at 37°C with tiazofurin resulted in dose-dependent depletion of GTP and GDP. Maximal depletion of guanine nucleotides required relatively high levels of tiazofurin (200 to 400 μM) and resulted in a 55 to 60% reduction of GTP and GDP. The effects of tiazofurin on guanine nucleotides levels were not observed when neutrophils were preincubated at 4°C. At 37°C, tiazofurin also decreased intracellular ATP and ADP levels but adenine nucleotide depletion was less pronounced than guanine nucleotide depletion for each concentration of tiazofurin used. When tiazofurin was removed by washing cells after incubation, adenine nucleotide quickly returned to preincubation values but guanine nucleotide levels remained depressed. Addition of exogenous guanosine (200 μM) prevented tiazofurin-dependent depletion of guanine nucleotides but had no influence on adenine nucleotide depletion. Superoxide released triggered by FMLP and F- was inhibited to an extent similar to that of guanine nucleotide depletion under different conditions of preincubation. Inhibition of superoxide release was not observed if cells were preincubated at 4°C, was not rapidly reversible, and was not observed when guanosine was added with tiazofurin. Tiazofurin depletion of guanine nucleotides did not result in inhibition of superoxide release triggered by either digitonin or PMA. Thus, neutrophil responses to F-, like those induced by FMLP, are regulated by availability of intracellular guanine nucleotides. Inasmuch as guanine nucleotides potentially regulate neutrophil G proteins involved in activation of both the initial and terminal events of signal transduction in neutrophils, we assayed the influence of tiazofurin on F--induced generation of inositol phosphates in intact cells. Tiazofurin preincubation decreased F- triggered inositol phosphate generation to an extent similar to that observed for inhibition of F--induced superoxide generation. These results are consistent with the hypothesis that activation of a plasma membrane PIP2-specific guanine nucleotide regulatory protein is an early event in the neutrophil stimulus response pathway triggered by 20.0 mM NaF.

Original languageEnglish
Pages (from-to)1685-1691
Number of pages7
JournalJournal of Immunology
Issue number5
StatePublished - 1989
Externally publishedYes


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