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Human mast cells secreting leukotriene C4 express the MRP1 gene-encoded conjugate export pump
G Bartosz1, J König, D Keppler
1Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Abstract:
Mast cells are known to secrete endogenously synthesized leukotriene C4 (LTC4), but the identity of the responsible export pump in human mast cells was unknown. The multidrug resistance proteins MRP1 and MRP2 have been identified as primary-active ATP-dependent export pumps for various amphiphilic anions including the glutathione conjugate LTC4. We therefore studied the expression at the RNAand protein levels of both MRP1 and MRP2 as well as the ATP-dependent LTC4 transport in the human mast cell line HMC-1. Upon stimulation by 1 microM ionomycin, intact HMC-1 cells generated 26 pmol LTC4/10(8) cells within 20 min. Transport experiments using inside-out HMC-1 membrane vesicles demonstrated an ATP-dependent LTC4 transport amounting to 1.4 pmol x (mg protein)(-1) x min(-1). Reverse transcription PCR indicated that HMC-1 cells express mRNA of MRP1, but not of MRP2 or MRP3. Cloning and sequencing of the amplified PCR fragment confirmed its identity with the human MRP1 sequence. Immunoblots using antibodies against MRP1 and MRP2 demonstrated that HMC-1 cells contain the MRP1 but not the MRP2 protein. Our results indicate that the 190 kDa integral membrane glycoprotein MRP1 mediates the ATP-dependent export of LTC4 from human mast cells to the extracellular space.
Insights
Multidrug resistance protein 1 (MRP1) exports leukotriene C4 (LTC4) from human mast cells. This study identifies MRP1 as the key ATP-dependent pump responsible for LTC4 secretion, clarifying a crucial mechanism in mast cell biology.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Mast cells synthesize and secrete leukotriene C4 (LTC4).
- The specific export pump for LTC4 in human mast cells remained unidentified.
- Multidrug resistance-associated protein 1 (MRP1) and MRP2 are known ATP-dependent transporters for amphiphilic anions like LTC4.
Purpose of the Study:
- To identify the ATP-dependent export pump responsible for LTC4 secretion in human mast cells.
- To investigate the expression of MRP1 and MRP2 at the RNA and protein levels in HMC-1 cells.
- To characterize ATP-dependent LTC4 transport in HMC-1 membrane vesicles.
Main Methods:
- Human mast cell line HMC-1 stimulation with ionomycin.
- Measurement of LTC4 generation.
- Transport assays using inside-out HMC-1 membrane vesicles.
- Reverse transcription PCR for MRP1, MRP2, and MRP3 mRNA expression.
- Immunoblotting using antibodies against MRP1 and MRP2.
Main Results:
- HMC-1 cells generated LTC4 upon ionomycin stimulation.
- ATP-dependent LTC4 transport was observed in HMC-1 membrane vesicles.
- HMC-1 cells express MRP1 mRNA and protein, but not MRP2 or MRP3 mRNA/protein.
- MRP1 was confirmed as the transporter for LTC4 via sequencing and immunoblotting.
Conclusions:
- The integral membrane glycoprotein MRP1 mediates ATP-dependent LTC4 export from human mast cells.
- This finding elucidates the mechanism of LTC4 secretion in human mast cells.
- MRP1 is identified as the primary export pump for LTC4 in this cell type.