1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity.
2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [14C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes.
3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [14C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [3H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [14C]-anandamide uptake in HeLa (FAAH−/−) cells.
4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH−/−) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.
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ACKNOWLEDGMENTS
Julie-the-Mentor Remembered
As a young graduate student, I was not immune to the temptation to worship the great minds of science not as mere mortals, but as demigods blessed with special powers. The challenges associated with consistently winning grants and delivering high quality, big impact and frequently cited manuscripts seemed overwhelming and mysterious. As chair of the student committee on external speakers, I can distinctly remember our naïve selection process that began ambitiously with a list of Nobel Laureates. Surprisingly, after only one very nervous phone call, we managed to attract Julius Axelrod to speak at Georgetown University simply because, as he put it, he most enjoyed speaking directly with students. When he arrived on the big day, I was very surprised at his calm, unassuming, and friendly demeanor. We chatted easily about mutual interests as I was fumbling his 35 mm slides into a carousel and maneuvered him through the halls to the lecture room. To my complete dismay, the plush amphitheater that was reserved for his talk was occupied by a medical student class and the professor refused to relinquish the space “no matter who was waiting outside the door.” Although my own composure was melting fast, it was remarkable to watch Julie help steer a gathering crowd to a neighboring lecture hall bedecked with harsh lighting and wooden seats. His talk was both inspiring and memorable, but his personable style was the most compelling for such an accomplished individual. Several months later, I began my staff fellowship at the NIMH in Julie's laboratory. Over time, I realized that Julie's mysterious powers had encouraged most of his former students to undergo a transmutation that created more than just a legacy of neuroscience discovery, but a true revolution in neuroscience leadership. His approach to collaborations and solutions to interesting questions was always well grounded in authenticity and simplicity. I share below my own perhaps over-simplified interpretation of Julie's philosophy of a typical day in his laboratory. As his last official post doc, I feel extremely fortunate to have had the opportunity to share ten years of scientific discovery with such a truly human and caring man of science.
Julie's Philosophy on Research
•Ask simple questions–but look beyond the obvious
•Do something new, but not too new–work just left or right of mainstream questions
•Talk to people … read … … talk to even more people … read some more … … one never knows where the next idea will come from
•Science is 99% discouragement … … stay focused
•Do one good experiment a day
•Find and exploit your scientific style
•Skimming the cream is a good thing–but do enough science to know that the cream is real
•Do not dwell too much on the details of the unknown–focus on your hypothesis and do not get swayed by complexity
•Publish often to clarify your thinking and your hypothesis
•Know when to park an idea and move on
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Dickason-Chesterfield, A.K., Kidd, S.R., Moore, S.A. et al. Pharmacological Characterization of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Inhibitors. Cell Mol Neurobiol 26, 405–421 (2006). https://doi.org/10.1007/s10571-006-9072-6
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DOI: https://doi.org/10.1007/s10571-006-9072-6