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'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism.

ABSTRACT: 1. The abilities of a series of saturated N-acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) has been investigated. 2. The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+-influx, nor the influx in response to a submaximal concentration of AEA (1 microM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. 3. Lauroylethanolamide (C12:0) produced a leftward shift in the dose-response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1-HEK293 cells were 1.8, 1.5, 1.1 and 0.22 microM in the presence of 0, 1, 3 and 10 microM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose - response curves to capsaicin. 4. Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (K(i(slope)) 4.1 microM, K(i(intercept)) 66 microM) of [3H]-AEA metabolism by rat brain membranes. 5. The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 microM AEA. 6. There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 microM AEA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. 7. It is concluded that although palmitoylethanolamide has entourage-like effects at VR1 receptors expressed on hVR1-HEK293 cells, other N-acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

SUBMITTER: Smart D

PROVIDER: S-EPMC1573364 | biostudies-other | 2002 Jun

REPOSITORIES: biostudies-other

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Project description:Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results have demonstrated that capsaicinoids contained in pepper spray products produce airway inflammation and cause respiratory epithelial cell death. The mechanisms of these cellular responses to capsaicinoids appear to proceed via distinct cellular pathways, but both pathways are initiated by TRPV1.

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'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism.

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