Role of fatty acid amide hydrolase in the transport of the endogenous cannabinoid anandamide.

A facilitated transport process that removes the endogenous cannabinoid anandamide from extracellular spaces has been identified. Once transported into the cytoplasm, fatty acid amide hydrolase (FAAH) is responsible for metabolizing the accumulated anandamide. We propose that FAAH contributes to anandamide uptake by creating and maintaining an inward concentration gradient for anandamide. To explore the role of FAAH in anandamide transport, we examined anandamide metabolism and uptake in RBL-2H3 cells, which natively express FAAH, as well as wild-type HeLa cells that lack FAAH. RBL-2H3 and FAAH-transfected HeLa cells demonstrated a robust ability to metabolize anandamide compared with vector-transfected HeLa cells. This activity was reduced to that observed in wild-type HeLa cells upon the addition of the FAAH inhibitor methyl arachidonyl fluorophosphonate. Anandamide uptake was reduced in a dose-dependent manner by various FAAH inhibitors in both RBL-2H3 cells and wild-type HeLa cells. Anandamide uptake studies in wild-type HeLa cells showed that only FAAH inhibitors structurally similar to anandamide decreased anandamide uptake. Because there is no detectable FAAH activity in wild-type HeLa cells, these FAAH inhibitors are probably blocking uptake via actions on a plasma membrane transport protein. Phenylmethylsulfonyl fluoride, a FAAH inhibitor that is structurally unrelated to anandamide, inhibited anandamide uptake in RBL-2H3 cells and FAAH-transfected HeLa cells, but not in wild-type HeLa cells. Furthermore, expression of FAAH in HeLa cells increased maximal anandamide transport 2-fold compared with wild-type HeLa cells. These results suggest that FAAH facilitates anandamide uptake but is not solely required for transport to occur.

Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells.

Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of Delta(9)-tetrahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inactivated by mechanisms similar to those for other neurotransmitters. Of interest to the present studies are reports that anandamide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that express both a peripheral cannabinoid receptor and amidohydrolase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [(3)H]anandamide that was both time- and temperature-sensitive. This transport activity was not dependent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as Delta(9)-tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in peripheral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes.

Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.

Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.