Expression and function of P-glycoprotein and absence of Multidrug Resistance-related Protein in rat and beige mouse peritoneal mast cells

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multid rug Resistance-related Protein (MRP), another membrane transport protein ex pressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprot ein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from th ese cells was rapid and also inhibited by verapamil and vinblastine. In con trast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were ab le to lower the concentrations of Bodipy-verapamil. Microscopical observati ons, conducted in both rat and beige mouse mast cells, demonstrated that th e fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR pe rformed on rat peritoneal mast cells revealed the presence of MDR1a and MDR 1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were furt her treated with the fluorescent probe LysoSensor Blue, a weak base that be comes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either b y treatment with P-glycoprotein modulators or with agents that disrupt pH g radients. In conclusion, these data further confirm the presence of an acti ve P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These find ings, coupled with previous ultrastructural data, lend further support to t he assumption that this protein is located on the mast cell perigranular me mbrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein mi ght be indirectly implicated in changes of pH values inside secretory granu les.