Role of FAAH-Like Anandamide Transporter in Anandamide Inactivation

The endocannabinoid system modulates numerous physiological processes including nociception and reproduction. Anandamide (AEA) is an endocannabinoid that is inactivated by cellular uptake followed by intracellular hydrolysis by fatty acid amide hydrolase (FAAH). Recently, FAAH-like anandamide transporter (FLAT), a truncated and catalytically-inactive variant of FAAH, was proposed to function as an intracellular AEA carrier and mediate its delivery to FAAH for hydrolysis. Pharmacological inhibition of FLAT potentiated AEA signaling and produced antinociceptive effects. Given that endocannabinoids produce analgesia through central and peripheral mechanisms, the goal of the current work was to examine the expression of FLAT in the central and peripheral nervous systems. In contrast to the original report characterizing FLAT, expression of FLAT was not observed in any of the tissues examined. To investigate the role of FLAT as a putative AEA binding protein, FLAT was generated from FAAH using polymerase chain reaction and further analyzed. Despite its low cellular expression, FLAT displayed residual catalytic activity that was sensitive to FAAH inhibitors and abolished following mutation of its catalytic serine. Overexpression of FLAT potentiated AEA cellular uptake and this appeared to be dependent upon its catalytic activity. Immunofluorescence revealed that FLAT localizes primarily to intracellular membranes and does not contact the plasma membrane, suggesting that its capability to potentiate AEA uptake may stem from its enzymatic rather than transport activity. Collectively, our data demonstrate that FLAT does not serve as a global intracellular AEA carrier, although a role in mediating localized AEA inactivation in mammalian tissues cannot be ruled out

Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics

Ethical values and norms in the management of attractive natural areas

Because of their natural and anthropogenic values, attractive natural areas are protected by law. The protective activities are connected with the proper management of these areas. The paper proposes the thesis that ethical values and norms referred to ecology are not taken into consideration in the management of attractive natural areas, both in theoretical and practical terms. The study presents attractive natural areas as a valuable common good, and discusses a place of values and norms in ethics. The project of ethical concept of management in these areas containing the adopted values and moral norms that refer to ecology was also formulated. The paper was written based on the scientific literature and official documents

Etyka ekologiczna. Wartości i normy w odniesieniu do społeczności lokalnych na obszarach wiejskich

An important role in sustainable rural development, involving economy, local communities and nature, should be played by ethics. This paper presents a theoretical and empirical characterization of basic problems of ecological ethics. First and foremost, the study characterizes the philosophical fundamentals of this ethics, with emphasis on ontological and anthropological views of selected thinkers. A universal concept of ecological ethics was proposed, containing values and moral norms that point to realization of this concept. Furthermore, the paper emphasizes empirical aspects of ecological ethics which manifest themselves in the awareness of inhabitants of rural areas. With respect to this topic, we presented selected results of the empirical survey carried out in rural areas in Poland by the Ministry of Agriculture and Rural Development. The presentation of these results was preceded by a short discussion about relationships between ecological ethics and market economy ethics. The paper is based on the analysis of the related literature, legal documents and results of empirical surveys.W zrównoważonym rozwoju obszarów wiejskich, obejmującym gospodarkę, społeczności lokalne i przyrodę, istotną rolę powinna spełniać etyka. W artykule przedstawiamy teoretyczną i empiryczną charakterystykę podstawowych zagadnień etyki ekologicznej. W pierwszej kolejności scharakteryzowano jej filozoficzne podstawy, zwracając uwagę na poglądy ontologiczne i antropologiczne wybranych myślicieli. Zaproponowano uniwersalną koncepcję etyki ekologicznej, w której zawarte zostały odpowiednie wartości oraz normy moralne, wskazujące na ich realizację. W dalszej kolejności zwrócono uwagę na empiryczną stronę etyki ekologicznej, która przejawia się w świadomości mieszkańców wsi. Przedstawione zostały wybrane wyniki badań empirycznych na ten temat, przeprowadzonych na polskiej wsi przez Ministerstwo Rolnictwa i Rozwoju Wsi. Wyniki te poprzedza krótka dyskusja poświęcona relacjom etyki ekologicznej z etyką gospodarki rynkowej. Artykuł powstał na bazie analizy odpowiedniej literatury przedmiotu, dokumentów urzędowych oraz rezultatów badań empirycznych

Expression of FAAH and FLAT in mouse tissues.

<p>(A) RT-PCR analysis of FAAH and FLAT expression in mouse brain, spinal cord, and L3-L5 DRG. Transcripts representing full-length FAAH (~1740 base pair fragments) were found in all tissues examined. In contrast, FLAT (~1540 base pair fragment) was not detected in any of the tissues. (B) RT-PCR analysis of FAAH and FLAT expression in mouse tissues. (C) Western blot demonstrating the expression of FAAH (~63 kDa) in mouse brain. Increasing the amount of brain tissue from 5 to 25 µg did not reveal the presence of FLAT, which has an expected molecular weight of ~56 kDa. FAAH-transfected HeLa cells are also shown. (D) Western blot of FAAH expression in a panel of mouse tissues. </p

Localization of FLAT in HeLa cells.

<p>Subcellular distribution of FAAH, FLAT, and FABP5 in cells. FAAH and FLAT do not co-localize with the plasma membrane marker PM-GFP while FABP5 does. In contrast, FAAH and FLAT are distributed on intracellular membranes.</p

Effect of FLAT upon AEA hydrolysis and uptake.

<p>(A) AEA hydrolysis by HeLa cell homogenates transfected with FAAH or FLAT. Inset: Close up of AEA hydrolysis by FLAT-transfected and vector-transfected HeLa homogenates (n = 3). (B) Inhibition of FAAH and FLAT by URB597, PF-3845, and MAFP. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3-5). (C) AEA (100 nM) hydrolysis by FLAT normalized to its expression level relative to FAAH (n = 3). (D) FLAT hydrolyzes AEA (0.1-100 µM) with a K_m of 25.3 ± 14.2 µM and V_max of 0.29 ± 0.13 nmol/mg/min (n = 3-5). (E) AEA uptake in HeLa cells transfected with FAAH or FLAT in the absence or presence of URB597. *, p < 0.05 (n = 3). (F) Western blot showing equal expression of WT and S241A FLAT in HeLa cells. (G) Enzymatic activity of WT and S241A FLAT. (H) AEA uptake in HeLa cells expressing WT or S241A FLAT (n = 3).</p

Expression of FAAH and FLAT in cell-lines.

<p>(A) Western blot of FAAH and FLAT expression in brain, HeLa cells transfected with FAAH (0.15 µg) or FLAT (1.5 µg), and in N18TG2 neuroblastoma and CCF-STTG1 astrocytoma cells. (B) Expression of FAAH and FLAT following transfection into HEK-293 cells. (C) AEA hydrolysis in homogenates of N18TG2, CCF-STTG1, and HeLa cells (n = 3).</p