Transport of doxorubicin in mast cell granules and the effect of the calcium antagonist verapamil.
P-glycoprotein has been identified in mast cells stabilized in culture as well as in rat peritoneal mast cells, and is primarily concentrated on the granular membrane. This study aimed to define the role of this protein in the transport and accumulation of doxorubicin in mast cell granules and in its histamine releasing effect. The reverting agent verapamil, that is a substrate for P-glycoprotein, inhibited doxorubicin uptake in intact mast cells in a dose and time dependent manner, but had no effect on the exocytotic action of the antineoplastic drug. Doxorubicin was also concentrated in granules with intact membranes and the uptake was dependent on temperature and showed a trend for saturation. Verapamil and vinblastine, another substrate for P-glycoprotein, significantly reduced doxorubicin concentrations in intact granules. Similar results were obtained with the metabolic inhibitors sodium metavanadate, N-ethylmaleimide, and sodium azide, whereas ouabain, an inhibitor of sodium-potassium ATPase, was without effect. Doxorubicin was taken also up in granule remnants, consisting of a proteoglycan matrix without membrane, that are extruded from mast cells upon stimulation. However, the uptake was not dependent on temperature and was not modified by P-glycoprotein substrates or metabolic inhibitors. Rat peritoneal mast cells were examined for the expression of P-glycoprotein at the protein level with C219 monoclonal antibody, using Western blot, confirming that P-glycoprotein was expressed in mast cells. These data suggest the presence of a P-glycoprotein active in the transport of doxorubicin, in mast cell granules.
Candussio
L
L
Department of Biomedical Sciences, University of Trieste, Italy. candussi@univ.trieste.it
Decorti
G
G
Crivellato
E
E
Rosati
A M
AM
Traversa
U
U
Klugmann
F B
FB
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000502:physiology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics*; D001692:Biological Transport; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004317:Doxorubicin / Q000493:pharmacokinetics*; D008407:Mast Cells / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar; D014700:Verapamil / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D002121:Calcium Channel Blockers; D004317:Doxorubicin; D014700:Verapamil