Inflammation research : official journal of the European Histamine Research Society ... [et al.] · 1997
Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine.
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Edgar A J, Davies G R, Anwar M A, Bennett J P
Аннотация
OBJECTIVE: We studied changes in cell surface morphology following treatment with secretagogue or trifluoperazine in a mast cell model.
MATERIALS AND METHODS: Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 microM trifluoperazine (TFP). The release of a secretory granule enzyme, beta-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of cell surface microvilli was determined by using a computer with input from a digitising tablet from scanning electron micrographs. Cytoskeletal proteins present in microvilli were analysed by confocal immunofluorescence.
RESULTS: When RBL cells are stimulated to secrete with an antigen, the cell surface is transformed from a microvillous morphology to a ruffled one. The cell surface rearrangement preceded beta-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t1/2 of 39 s) whereas secretion can only be measured after a lag of 47 s. The calmodulin antagonist, TFP did not inhibit antigen-induced secretion or loss of microvilli, however TFP alone caused a similar loss of microvilli but was unable to stimulate or potentiate secretion. The microvilli mostly disappeared within 30 s, and a half-maximal effect occurred at approximately 8 microM TFP. Using immunofluorescence, calmodulin was localized to punctate structures on the dorsal cell surface which presumably correspond to the microvilli, and which also stained for F-actin and myosin I.
CONCLUSIONS: Loss of cell surface microvilli on RBL cells precedes secretion and could reflect a cytoskeletal rearrangement which facilitates fusion of secretory granules with the membrane. It can be mimicked using trifluoperazine and we suggest it may involve calmodulin-binding components of the microvillus cytoskeleton such as myosin I.
MATERIALS AND METHODS: Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 microM trifluoperazine (TFP). The release of a secretory granule enzyme, beta-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of cell surface microvilli was determined by using a computer with input from a digitising tablet from scanning electron micrographs. Cytoskeletal proteins present in microvilli were analysed by confocal immunofluorescence.
RESULTS: When RBL cells are stimulated to secrete with an antigen, the cell surface is transformed from a microvillous morphology to a ruffled one. The cell surface rearrangement preceded beta-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t1/2 of 39 s) whereas secretion can only be measured after a lag of 47 s. The calmodulin antagonist, TFP did not inhibit antigen-induced secretion or loss of microvilli, however TFP alone caused a similar loss of microvilli but was unable to stimulate or potentiate secretion. The microvilli mostly disappeared within 30 s, and a half-maximal effect occurred at approximately 8 microM TFP. Using immunofluorescence, calmodulin was localized to punctate structures on the dorsal cell surface which presumably correspond to the microvilli, and which also stained for F-actin and myosin I.
CONCLUSIONS: Loss of cell surface microvilli on RBL cells precedes secretion and could reflect a cytoskeletal rearrangement which facilitates fusion of secretory granules with the membrane. It can be mimicked using trifluoperazine and we suggest it may involve calmodulin-binding components of the microvillus cytoskeleton such as myosin I.