Mouse ileal Peyer's patches are covered by a mucus layer.

<p>(A) Stereo microscope image of an ileal explant containing a PP. Domes are indicated by black arrows. Charcoal particles were added to visualize the otherwise transparent mucus layer (bar = 0.5 mm). (B) Two mucus filled goblet cells (black arrows) in a dome stained by PAS (bar = 10 µm). (C) Mucus on top of the FAE was removed and remaining mucus thickness measured every 20 minutes for an hour (open circles; n = 6) or mucus thickness was measured at time 0 and 20 min and a combination of carbachol and PGE₂, 10 µM of each, was perfused after the second measurement (arrow, closed circles; n = 6). (D) Initial mucus thickness was measured on the villi of the PP, mucus was removed and remaining mucus thickness measured at time 20 min. Half the number of explants were left unstimulated (open circles; n = 10) and half of the explants were stimulated with carbachol and PGE₂ (10 µM of each; arrow), and mucus thickness was measured at time 40 and 60 min (closed circles; n = 10). (E) Mucus penetrability to beads the size of bacteria was assessed by confocal imaging of mouse ileal explants containing a PP. Tissue is visualized in blue and beads are red (0.5 µm), purple (1 µm) and green (2 µm). (F) To clarify how beads penetrate to the FAE surface, a flat section of the epithelium (blue) is shown. Note how some beads (red, purple and green) are suspended in the mucus. Bars in E and F = 50 µm.</p

Transmission electron micrographs of mouse, rat and human Peyer's patches show secreting goblet cells.

<p>(A) Secreting goblet cell in mouse FAE. (B) M cell in mouse FAE. (C) Secreting goblet cell in a rat FAE. (D) Two M cells next to each other in a rat FAE. (E) Secreting goblet cell in human FAE. (F) Mucus on top of a human FAE, mucus border indicated by black arrow and mucus marked by black star. Bars = 2 µm.</p

MUC2 positive cells on domes of mouse, rat and human Peyer's patches.

<p>Fluorescent staining of Muc2 reveals mucin containing cells in the FAE of a mouse (A), rat (B) and human (C) ileal PP. Bars = 50 µm. Inset in panel C shows the MUC2 positive cells at higher magnification (bar = 10 µm). Muc2 staining is green, nuclei are blue and FAE is indicated by dashed lines. (D) MUC2 positive cells and nuclei in FAE were counted in sections from 5 mice, 5 rats and 5 humans. Values are presented as median (25^th and 75^th percentile). The percentage of goblet cells was larger in human FAE compared to mouse FAE (<i>P</i><0.001, ***) and rat domes (<i>P</i><0.05, *).</p

The monoacylglycerol lipase inhibitor JZL184 produces TRPV1-mediated antinociception.

<p>Intraplantar injection of formalin (2.5%) into the mouse paw produced a biphasic nociceptive response. (A) Intracerebroventricular (i.c.v.) injection of the monoacylglycerol lipase inhibitor JZL184 (10 nmol) 10 min before formalin injection produced antinociception in the first phase response and this effect was prevented by i.c.v co-administration of 10 nmol capsazepine (Cz). (B) The antinociceptive effect of JZL184 on the first phase of the formalin test was lost in TRPV1 knock-out mice. (C) The second nociceptive phase of the formalin response was unaffected by JZL184 (10 nmol) given i.c.v. 10 min after the intraplantar injection of formalin. Data are presented as mean ± s.e.m (n = 8–9). Kruskal-Wallis one-way ANOVA followed by Dunn's <i>post hoc</i> test was used for comparing groups of data.*<i>P</i><0.05.</p

2-Arachidonoylglycerol and 1-arachidonoylglycerol activate heterologously expressed rat and human TRPV1.

<p>Current-voltage relationships after application of vehicle (Basal) or 2-arachidonoylglycerol (2-AG, 10 µM) to rat TRPV1-expressing CHO cells in the absence (A) and presence (B) of the protein kinase C inhibitor bisindoylmaleimide IV (BIM; 10 µM). Each current-voltage curve is representative of 3 cells. (C) Current traces showing responses to 10 µM 2-AG (left) and 10 µM 1-arachidonoyl glycerol (1-AG, right) in rat TRPV1-expressing CHO cells at a holding potential of −60 mV. Application of 10 µM of the TRPV1 antagonist capsazepine (Cz) immediately reversed the inward currents. (D) Excised inside-out patches from rat TRPV1-expressing CHO cells responded to 10 µM 2-AG with robust outward currents at a membrane potential of +60 mV. (E) Current-voltage relationships before and after application of 2-AG (10 µM) in HEK293 cells stably expressing human TRPV1. (F) Inward currents in HEK293 cells transiently expressing human TRPV1 at a holding potential of −50 mV. Traces show inward currents elicited by 2 µM capsaicin (CAP) and 10 µM 2-AG in a calcium free solution. Exposure to capsaicin and 2-AG a second time produced currents of similar magnitude. Data are presented as mean ± s.e.m. Kruskal-Wallis one-way ANOVA followed by Dunn's <i>post hoc</i> test was used for comparing groups of data. **<i>P</i><0.01, ***<i>P</i><0.001.</p

Contribution of 2-arachidonoylglycerol to TRPV1 activation evoked by stimulation of phospholipase C-coupled histamine H₁ receptors in HEK293 cells.

<p>(A) Monoacylglycerol lipase (MAGL) expression in HEK293 cells, as shown by immunocytochemistry. Upper and lower left images show staining with human MAGL antibodies from Abcam and Pierce Biotechnology, respectively. Right hand images were obtained after incubation of the primary antibodies with respective blocking peptide. (B) Histamine (His, 100 µM) increased the content of 2-arachidonoylglycerol (2-AG) in HEK293 cells transiently expressing the phospholipase C-coupled histamine H₁ receptor. Incubation (30 min) with the MAGL inhibitor JZL184 (JZL, 10 µM) further increased the 2-AG content, whereas the diacylglycerol lipase (DAGL) inhibitor tetrahydrolipstatin (THL, 10 µM) prevented the histamine-induced 2-AG increase. The 2-AG content was normalized to the average 2-AG content induced by histamine in vehicle-treated HEK293 cells in each experiment. The results are presented as mean ± s.e.m (n = 6). One-way ANOVA followed by Bonnferroni's <i>post hoc</i> test was used for comparing groups of data. (C) Current-voltage relationships in HEK293 cells transiently co-expressing TRPV1 and the rat histamine H₁ receptor before (red trace) and after (black trace) exposure to histamine 100 µM in the absence and presence of JZL184 (10 µM), THL (10 µM), JZL184 plus THL (each 10 µM) or Cz (10 µM). The control trace shows the average current with error bars obtained from 119 cells. (D) The mean current amplitude elicited by histamine at a test potential of +100 mV in non-treated (n.t.) cells and in cells treated (at least 20 minutes) with enzyme inhibitors. Data are presented as mean ± s.e.m (n = 6–8). Kruskal-Wallis one-way ANOVA followed by Dunn's <i>post hoc</i> test was used for comparing groups of data. **<i>P</i><0.01, ***<i>P</i><0.001.</p

Bradykinin and ATP selectively increase the content of 2-arachidonoylglycerol in rat dorsal root ganglia.

<p>Isolated dorsal root ganglia from newborn rats were incubated with a mixture of 10 µM bradykinin and 1 mM ATP (Stim), or vehicle (Unstim) for 2 min and the contents of 2-arachidonoylglycerol (2-AG, A), 2-oleoylglycerol (2-OG, B) and anandamide (AEA, C) determined by mass-spectrometry. Y-axis indicates normalized peak area (nPA). Data are presented as mean ± s.e.m. (n = 6). Paired Student's <i>t</i>-test on log transformed values was used for comparing groups of data. *<i>P</i><0.05</p

The monoacylglycerol lipase inhibitors MAFP and JZL184 potentiate 2-arachidonoylglycerol and 1-arachidonoylglycerol sensory nerve-mediated vasodilation.

<p>(A) Concentration-response curves for (i) 2-arachidonoylglycerol (2-AG), (ii) 1-arachidonoylglycerol (1-AG), and (iii) anandamide (AEA) in the presence of MAFP (n = 6–11), JZL184 (n = 6–8) or vehicle (n = 13–18). The area under the curve was larger in the presence than in the absence of MAFP (<i>P</i><0.05, 2-AG; <i>P</i><0.001, 1-AG) or JZL184 (<i>P</i><0.05, 2-AG; <i>P</i><0.001, 1-AG). (iv) Concentration-response curves for noladin ether (NE), the stable analog of 2-AG (n = 6–8). The vasodilation evoked by 2-AG, 1-AG, AEA and NE but not arachidonic acid were abolished by capsaicin pretreament (A–D). Traces show vasodilator responses to (B) 2-AG, (C) 1-AG and (D) arachidonic acid after pretreatment with vehicle (left) or capsaicin (right) to cause desensitization and/or neurotransmitter depletion of sensory nerve endings in rat mesenteric arteries. Subsequent application of acetylcholine (ACh) was used to confirm that arteries were able to respond with vasorelaxation (mediated by endothelium-derived hyperpolarising factor; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081618#pone.0081618-Zygmunt4" target="_blank">[83]</a>). The capsaicin pretreatment consisted of a 30 min exposure to 10 µM capsaicin followed by washout of capsaicin. The arterial segments were submaximally contracted with phenylephrine (PhE) before addition of the test drugs. The dashed line shows the basal tension level before addition of PhE. MAFP (30 nM) was present in experiments with 2-AG (B) and 1-AG (C). Control experiments were performed in the presence of vehicle (0.1% ethanol). Data are expressed as mean ± s.e.m.</p

Diagram showing the contribution of 2-arachidonoyl glycerol to phospholipase C-dependent activation of TRPV1.

<p>Ligand-Gq-receptor interaction causes activation of phospholipase C (PLC), hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and formation of diacylglycerol (DAG), which is further metabolized to 2-arachidonoylglycerol (2-AG) by diacylglycerol lipase (DAGL). 2-Arachidonoylglycerol undergoes a spontaneous acylmigration to yield 1-arachidonoylglycerol (1-AG). Both 2-AG and 1-AG directly activate TRPV1, and their effects are terminated by monoacylglycerol lipase (MAGL) and related enzymes, including the alpha/beta-hydrolases ABHD6 and ABDH12. Diacylglycerol indirectly regulates TRPV1 via protein kinase C (PKC)-dependent phosphorylation of the ion channel, whereas PIP₂ has complex and opposing effects on TRPV1 channel gating. Hydrolysis of PIP₂ also yields inositol 1,4,5-triphosphate (IP₃), which via release of intracellular calcium causes activation of calcium-calmodulin-dependent protein kinase II (CaMK II). The phosphatase calcineurin dephosphorylates TRPV1. Protein phosphorylation, protonation, PIP₂ modulation and binding of membrane-derived lipids provide an intricate system for fine-tuning of TRPV1 activity.</p

The calcineurin inhibitor ciclosporine and palmitoylethanolamide potentiate the sensory nerve-dependent vasodilator response to 2-arachidonoylglycerol.

<p>Effects of ciclosporine (A) and palmitoylethanolamide (PEA, B) on the vasorelaxation evoked by 2-arachidonoylglycerol (2-AG) in rat isolated mesenteric arterial segments. Pretreatment with 10 µM capsaicin almost abolished the 2-AG-induced relaxation in the presence of ciclosporine or PEA. The MAGL inhibitor methylarachidonylfluorophosphonate (30 nM) was present throughout. Data are presented as mean ± s.e.m (n = 6–8).</p