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Uptake of a Fluorescently Labeled Alkamide by Mammalian Cells.

Johnstone, SE;

Alkamides are fatty acid amides produced by a variety of different plants. Alkamides exert immunomodulatory and anti-nociceptive effects and most studies of their mechanism of action have focused on the effects of alkamides on receptors and ion channels that mediate pain and inflammatory signaling. Whether alkamides can enter cells and potentially exert intracellular effects is unknown and will be the subject of this dissertation. To address this question, we produced a fluorescently labeled alkamide by replacing the isobutyl head group of the natural product (2E,4E)-N-isobutyldodeca-2,4-dienamide with a fluorescein (FITC) molecule. We found that this molecule (FITC-Alk) retained the ability to inhibit production of TNF-? from LPSstimulated RAW 264.7 macrophage-like cells. Confocal microscopy was then used to characterize the entry and intracellular localization of FITC-Alk in RAW 264.7 cells. Our experiments revealed that the FITC-Alk entered cells within minutes and formed both large, bright puncta and diffuse pinpoint staining in the cytosol. Three-dimensional Z-stack depth coding revealed that puncta formation occurred throughout the cytoplasm in a uniform, non-polarized manner. The same pattern was seen also seen in Vero green monkey kidney cells and RBL-2H3 basophilic leukemia cells, suggesting a common pathway of uptake unrelated to the cell type or tissue of origin. Staining intensity was found to be both time and concentration dependent and continued linearly for at least 4 hours. Co-staining with Texas Red-dextran indicated that the large puncta produced by FITCAlk uptake were intracellular endocytic compartments. Additionally, our data suggest a role for actin filaments in FITC-Alk uptake, and that the FITC-Alk molecule likely accumulates in Rab7 positive, late-stage endosomes. Intracellular proteins should therefore be considered as targets for alkamide activity and alkamides may be useful for endocytic drug targeting.