Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells.
The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.
Kato
Naoto
N
Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Nakanishi
Mamoru
M
Hirashima
Naohide
N
D000818:Animals; D000941:Antigens / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D015550:Cell Degranulation / Q000187:drug effects; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism* / Q000502:physiology; D003505:Cyclodextrins / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D047392:beta-Cyclodextrins; D019061:src-Family Kinases / Q000378:metabolism
D000941:Antigens; D003505:Cyclodextrins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000001:Calcimycin; D019284:Thapsigargin; D002784:Cholesterol; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium