Preliminary characterization of diacylglycerol generation in human basophils: Temporal relationship to histamine release and resolution of degranulation

Alfonso Oriente, Thomas Hundley, Walter C. Hubbard, Donald W. MacGlashan

Research output: Contribution to journalArticle

Abstract

Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 μM fMLP (basal levels of 15 pmol/106 basophils vs. 45 pmol/106 basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/106 basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/106 basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.

Original languageEnglish (US)
Pages (from-to)637-642
Number of pages6
JournalJournal of Leukocyte Biology
Volume61
Issue number5
DOIs
StatePublished - May 1997

Keywords

  • lipids
  • molecular species
  • signal transduction

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Cell Biology

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