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Auto-oxidation Products of Epigallocatechin Gallate
A study in the Oxford Journals' Chemical Senses reports that auto-oxidation products of epigallocatechin gallate (EGCG) activate TRPA1 and TRPV1 in sensory neurons. The goal of the study was to further investigate the sensation mechanism of astringency induced by green tea polyphenols that have been shown to activate nociception receptors TRPV1 and TRPA1 in past studies, by testing how the auto-oxidized products of tea catechins affect these receptors in rodents.
Seeing as the receptors of focus are TRPV1 and TRPA1, those who designed the study chose to conduct calcium imaging analysis of various cells types either engineered to or naturally able to express the TRPV1 and TRPA1 receptors, and incubated in the calcium indicator Fluo-8. These cell subjects included HEK293 cells transfected with rat TRPV1, mouse TRPA1, chick TRPV1/TRPA1, or rattlesnake TRPV1/TRPA1 expression vector, and dorsal root ganglion (DRG) cells which inherently express the two nociceptors. An observed intracellular calcium response of the cells upon incubation in fresh EGCG, incubation-oxidized EGCG, or EGCG dimer theasinensin A (TS-A) would be indicative of the TRPV1 and TRPA1 receptors being activated by the respective substance. Their findings suggested that species-specific mammalian TRPV1 and TRPA1 were only able to sense auto-oxidized EGCG and not fresh EGCG, because auto-oxidized EGCG produced TS-A which activates both TRP channels.
The scientists were able to apply the robust Fluo-8 calcium dye broadly throughout their experiments in this study for fluorescence calcium imaging. Fluo-8 is one of the best options for monitoring calcium response because of its optimized brilliance and sensitivity in signal and ease in cell loading preparation. The Materials and Methods describes their procedure for Fluo-8 staining of HEK and DRG cells as they did in their last study, which states that cells were "incubated in HBSS containing 5 µM Fluo8-AM for 30 min at room temperature". This method is of interesting note because many calcium indicators require loading at 37 degrees Celsius (which may subject cells to more heat stress), excess reagent (which may alter intracellular calcium activity), or longer incubation time (which is inconvenient). This study strongly demonstrates that Fluo-8 heavily improves upon these common issues, making it a time-saving and user-friendly product ready to be applied to any study.
Seeing as the receptors of focus are TRPV1 and TRPA1, those who designed the study chose to conduct calcium imaging analysis of various cells types either engineered to or naturally able to express the TRPV1 and TRPA1 receptors, and incubated in the calcium indicator Fluo-8. These cell subjects included HEK293 cells transfected with rat TRPV1, mouse TRPA1, chick TRPV1/TRPA1, or rattlesnake TRPV1/TRPA1 expression vector, and dorsal root ganglion (DRG) cells which inherently express the two nociceptors. An observed intracellular calcium response of the cells upon incubation in fresh EGCG, incubation-oxidized EGCG, or EGCG dimer theasinensin A (TS-A) would be indicative of the TRPV1 and TRPA1 receptors being activated by the respective substance. Their findings suggested that species-specific mammalian TRPV1 and TRPA1 were only able to sense auto-oxidized EGCG and not fresh EGCG, because auto-oxidized EGCG produced TS-A which activates both TRP channels.
The scientists were able to apply the robust Fluo-8 calcium dye broadly throughout their experiments in this study for fluorescence calcium imaging. Fluo-8 is one of the best options for monitoring calcium response because of its optimized brilliance and sensitivity in signal and ease in cell loading preparation. The Materials and Methods describes their procedure for Fluo-8 staining of HEK and DRG cells as they did in their last study, which states that cells were "incubated in HBSS containing 5 µM Fluo8-AM for 30 min at room temperature". This method is of interesting note because many calcium indicators require loading at 37 degrees Celsius (which may subject cells to more heat stress), excess reagent (which may alter intracellular calcium activity), or longer incubation time (which is inconvenient). This study strongly demonstrates that Fluo-8 heavily improves upon these common issues, making it a time-saving and user-friendly product ready to be applied to any study.
References
- Mako Kurogi, Yasushi Kawai, Katsuhiro Nagatomo, Michihiro Tateyama, Yoshihiro Kubo, and Osamu Saitoh. Auto-oxidation Products of Epigallocatechin Gallate Activate TRPA1 and TRPV1 in Sensory Neurons. Chem Senses. 2015; 40:27-46
Original created on January 6, 2017, last updated on January 6, 2017
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