Induction of granule release by intracellular application of calcium and guanosine-5′-O-(3-thiotriphosphate) in human eosinophils

Abstract

The roles of Ca and G proteins in granule release from human eosinophils were examined by use of a patch-clamp technique in single cells. The morphologic changes and the release of eosinophil peroxidase (EPO) from single cells were simultaneously observed. In addition, the expression of small molecular weight guanine nucleotide binding protein (small G protein) mRNA ( smg p25A [ rab 3] and smg p21 [ rab 1]) was investigated. The intracellular application of Ca, 10 μmol/L, and guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S), 100 μmol/L, induced fusion of EPO containing granules with the surface membrane, which was associated with a marked increase in membrane capacitance. Ca alone caused a rapid granule release at an early stage of cell dialysis, but most granules still remained in a cluster. GTP-γ-S alone caused a gradual degranulation. Northern blot analysis revealed the definite expression of smg p21 mRNA with no appreciable expression of smg p25A. These results provide direct evidence of granule fusion by intracellular application of Ca and GTP-γ-S. In addition, Ca dependent proteins and G proteins act cooperatively in granule release, and these proteins likely regulate the different processes of degranulation. Furthermore, a protein, encoded by smg p21, may be involved in the granule release process in human eosinophils.