Identification of a New Class of Prostaglandin Transporter Inhibitors and Characterization of Their Biological Effects on Prostaglandin E<sub>2</sub> Transport

Chi, Yuling; Khersonsky, Sonya M.; Chang, Young‐Tae; Schuster, Victor L.

doi:10.1124/jpet.105.091975

Cited by 38 publications

(44 citation statements)

References 36 publications

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“…This transporter displays time and concentration dependent uptake kinetics for prostaglandins which are inhibited by specific transport inhibitors. However, prostaglandin efflux from cells occurs by simple diffusion (59).…”

Section: Discussionmentioning

confidence: 99%

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Kaczocha

Hermann

Glaser

et al. 2006

Journal of Biological Chemistry

113

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The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2H3) has been proposed to occur via a saturable transporter that is blocked by specific inhibitors. Measuring uptake at 25 s, when fatty acid amide hydrolase (FAAH) does not appreciably affect uptake, AEA accumulated via a nonsaturable mechanism at 37°C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37°C. Such apparent saturation can be explained by rate-limited delivery of AEA through an unstirred water layer surrounding the cells (1). In support of this, we observed kinetics consistent with rate-limited diffusion at 0°C. Novel transport inhibitors have been synthesized that are either weak FAAH inhibitors or do not inhibit FAAH in vitro (e.g. UCM707, OMDM2, and AM1172). In the current study, none of these purported AEA transporter inhibitors affected uptake at 25 s. Longer incubation times illuminate downstream events that drive AEA uptake. Unlike the situation at 25 s, the efficacy of these inhibitors was unmasked at 5 min with appreciable inhibition of AEA accumulation correlating with partial inhibition of AEA hydrolysis. The uptake and hydrolysis profiles observed with UCM707, VDM11, OMDM2, and AM1172 mirrored two selective and potent FAAH inhibitors CAY10400 and URB597 (at low concentrations), indicating that weak inhibition of FAAH can have a pronounced effect upon AEA uptake. At 5 min, the putative transport inhibitors did not reduce AEA uptake in FAAH chemical knock-out cells. This strongly suggests that the target of UCM707, VDM11, OMDM2, and AM1172 is not a transporter at the plasma membrane but rather FAAH, or an uncharacterized intracellular component that delivers AEA to FAAH. This system is therefore unique among neuro/immune modulators because AEA, an uncharged hydrophobic molecule, diffuses into cells and partial inhibition of FAAH has a pronounced effect upon its uptake. Arachidonoyl ethanolamide (AEA)2 is a neuromodulatory lipid that belongs to a family of molecules collectively termed the endocannabinoids. Like ⌬ 9 -tetrahydracannabinol, many of the actions of AEA are mediated through the G-protein-coupled cannabinoid (CB 1 and CB 2 ) and vanilloid (TRPV1) receptors (2-7). AEA signaling is terminated by a rapid reuptake mechanism followed by its hydrolysis into arachidonic acid and ethanolamine primarily by the intracellular enzyme FAAH (8 -10). By metabolizing AEA, FAAH maintains an inward gradient that drives the continued cellular accumulation of AEA (11-13).There is some controversy regarding the processes mediating AEA uptake. It was widely accepted that AEA transport is a selective, saturable process of facilitated diffusion for which there are selective transport inhibitors (For review, see Refs. 14 -16). However, in platelets, neuroblastoma, astrocytoma, primary cortical neurons, and erythrocyte ghost membranes it has been suggested that uptake occurs by passive diffusion (13,(17)(18)(19)(20). Experimental variables such as incubation times, inclusion ...

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Section: Discussionmentioning

confidence: 99%

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Kaczocha

Hermann

Glaser

et al. 2006

Journal of Biological Chemistry

113

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“…These procedures were as described previously (Chi et al, 2006). In brief, PGT-MDCK cells were seeded on 24-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we reported a new class of PGT inhibitors obtained by screening a small molecular library (Chi et al, 2006). The best hit of that screening was T34 ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, PGE 2 has to be translocated from the outside to the inside of cells for it to be metabolized to PGE 2 metabolites (PGE 2 -Ms), including 15-keto PGE 2 . The prostaglandin transporter (PGT), a member of the organic anion transporter (OATP) family (Kanai et al, 1995), mediates the energetically active influx of PGE 2 (Chi et al, 2006), accounting for almost 100% of PGE 2 internalization (Kanai et al, 1995;Chi and Schuster, 2010). In cell culture, we have demonstrated that PGT-mediated PGE 2 influx is required and is rate-limiting for PGE 2 metabolism (Nomura et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Development of a High-Affinity Inhibitor of the Prostaglandin Transporter

Chi

Min

Jasmin

et al. 2011

J Pharmacol Exp Ther

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Prostaglandin E 2 (PGE 2 ) triggers a vast array of biological signals and physiological events. The prostaglandin transporter (PGT) controls PGE 2 influx and is rate-limiting for PGE 2 metabolism and signaling termination. PGT global knockout mice die on postnatal day 1 from patent ductus arteriosus. A high-affinity PGT inhibitor would thus be a powerful tool for studying PGT function in adult animals. Moreover, such an inhibitor could be potentially developed into a therapeutic drug targeting PGT. Based on structure-activity relationship studies that built on recently identified inhibitors of PGT, we obtained N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-((4-((2-(2-(2-benzamidoethoxy) ethoxy)ethyl)amino)-6-((4-hydroxyphenyl)amino)-1,3,5-triazin-2-yl)amino)benzamide (T26A), a competitive inhibitor of PGT, with a K i of 378 nM. T26A seems to be highly selective for PGT, because it neither interacts with a PGT homolog in the organic anion transporter family nor affects PGE 2 synthesis. In Madin-Darby canine kidney cells stably transfected with PGT, T26A blocked PGE 2 metabolism, resulting in retention of PGE 2 in the extracellular compartment and the negligible appearance of PGE 2 metabolites in the intracellular compartment. Compared with vehicle, T26A injected intravenously into rats effectively doubled the amount of endogenous PGE 2 in the circulation and reduced the level of circulating endogenous PGE 2 metabolites to 50%. Intravenous T26A was also able to slow the metabolism of exogenously injected PGE 2 . These results confirm that PGT directly regulates PGE 2 metabolism and demonstrate that a high-affinity inhibitor of PGT can effectively prevent PGE 2 metabolism and prolong the half-life of circulating PGE 2 .

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“…An example of the latter is the prostaglandins, whose transporter has been cloned, and whose uptake displays time and concentration dependence and is blocked by specifi c inhibitors. 30 AEA and 2-AG may be in a special class of agonists as they are lipophilic and in terms of transport may behave like some anesthetics that freely diffuse through the membrane. To date, the identifi cation and cloning of an endocannabinoid transporter has yielded negative results.…”

Section: Resultsmentioning

confidence: 99%

2-Arachidonoylglycerol (2-AG) membrane transport: History and outlook

Hermann

Kaczocha

Deutsch

2006

AAPS J

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Identification of a New Class of Prostaglandin Transporter Inhibitors and Characterization of Their Biological Effects on Prostaglandin E₂ Transport

Cited by 38 publications

References 36 publications

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Development of a High-Affinity Inhibitor of the Prostaglandin Transporter

2-Arachidonoylglycerol (2-AG) membrane transport: History and outlook

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Identification of a New Class of Prostaglandin Transporter Inhibitors and Characterization of Their Biological Effects on Prostaglandin E2 Transport

Cited by 38 publications

References 36 publications

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Development of a High-Affinity Inhibitor of the Prostaglandin Transporter

2-Arachidonoylglycerol (2-AG) membrane transport: History and outlook

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Identification of a New Class of Prostaglandin Transporter Inhibitors and Characterization of Their Biological Effects on Prostaglandin E₂ Transport