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Syed Benazir Alam, Marianna Kulka, Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms, The Journal of Immunology, Volume 206, Issue 1_Supplement, May 2021, Page 23.14, https://doi.org/10.4049/jimmunol.206.Supp.23.14
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Abstract
Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules of myeloid-derived mast cells, but, the mechanism of berberine’s internalization is poorly understood. Our data showed that 84±3.3% of highly granulated bone marrow-derived mast cells (BMMC) internalized BBR (10 ug/mL) after 45 hr. Methanol fixation completely blocked this process, suggesting an energy-dependent active transport mechanism, and 3-mtheyladenine (600 uM) inhibited internalization by ~43%, indicating that lysosomal self-degradation via the autophagy pathway was involved. BBR incorporation into lipoplexes did not improve BBR internalization, but, rather decreased it by ~30% (1 ug/mL Lipo/BBR; n=3, p<0.05), suggesting that BBR internalization may utilize clathrin-dependent endosomal endocytosis. Interleukin-3 (IL-3)-mediated activation of BMMC augmented BBR internalization (~54% by 40 ng/mL IL-3) in a concentration-dependent manner, further suggesting that clathrin-mediated endocytosis as well as active cell division, have a profound role in BBR internalization. Altogether, our data suggest that internalization of BBR in resting and IL-3 activated BMMC, utilizes clathrin-dependent pathway. Our findings provide important new mechanistic insights into the internalization of benzylsoquinoline compounds by resting and activated mast cells and open new avenues for designing organelle-targeted nanotherapeutics.