| Popis: |
Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes β-hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N-α-benzoyl-dl-Arg-p-nitroanilide (BAPNA). An extensively used in vitro mast cell model for studying toxicant mechanisms is the RBL-2H3 cell line. However, instead of tryptase, granule contents such as β-hexosaminidase have usually been employed as RBL-2H3 degranulation markers. To align RBL-2H3 cell toxicological studies to in vivo mast cell studies using zebrafish, we aimed to develop an RBL-2H3 tryptase assay. Unexpectedly, we discovered that tryptase release from RBL-2H3 cells is not detectable, using BAPNA substrate, despite optimized assay that can detect as little as 1 ng tryptase. Additional studies performed with another substrate, tosyl-Gly-Pro-Lys-pNA, and with an enzyme-linked immunosorbent assay, revealed a lack of tryptase protein released from stimulated RBL-2H3 cells. Furthermore, none of the eight rat tryptase genes (Tpsb2, Tpsab1, Tpsg1, Prss34, Gzmk, Gzma, Prss29, Prss41) is expressed in RBL-2H3 cells, even though all are found in RBL-2H3 genomic DNA and even though β-hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize β-hexosaminidase or another reliable marker for RBL-2H3 degranulation studies, not tryptase. Comparative toxicity testing in RBL-2H3 cells in vitro and in zebrafish mast cells in vivo will require use of a degranulation reporter different from tryptase. |
