Spectrograms of Representative Anandamide Analyses

SUPPLEMENTARY DATA

Figure 1

Figure 1.

Spectrograms of representative anandamide analyses.

(A) UHPLC-ESI-MS/MS spectrograms of superfusates obtained from cultures incubated in the presence of 100μM 20:4-NAPE. Spectrograms are presented for the detection of propranolol internal standard (1.45 min, m/z 260.420>116.200, represented in green colour) and for anandamide (2.3 min, m/z348.5>62.0 andm/z348.5>91.052).

(B) UHPLC-ESI-MS/MS spectrograms of superfusate obtained from control (a, b) cultures and those to which 100μM 20:4-NAPE was applied for 5 minutes (c, d). Spectrograms are presented for the detection of anandamide-d8 internal standard (a, c m/z, 356.4>63) and for anandamide (b, d,m/z 348.25>61.85).

(C) LC-APCI-MS chromatogram and mass spectra of superfusates obtained from cultures incubated in the presence of 100μM 20:4-NAPE. Analyses are carried out in the selected ion-monitoring mode using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated anandamide, respectively), as well m/z values of 347 and 349, to ensure the identification of the correct isotopic ratio for endogenous anandamide.

Figure 2

Figure 2

mRNA expression of the five known Ca2+-insensitive enzymes, which have been implicated in anandamide synthesis, in cultured rat PSN.

SYBR Green chemistry-based quantitative real-time polymerase chain reaction (PCR) was performed using cDNA which was reverse transcribed from RNA isolated from cultured rat PSN.The reaction was performed on an ABI 7900HT real-time PCR machine (Applied Biosystems). The reactions consisted of 50 cycles with 15 seconds of denaturation at 95 °C, 1 min annealing at 60 °C. Primers were designed and validated by Primer Design Ltd (UK). The expression of mRNA for each enzyme is normalised to that of GAPDH. Hence low CT values indicate high level of expression, while high CT values indicate low level of expression. Similarly to that seen with RT-PCR (Figure 2), the expression of enzymes transcripts varies: while GDE1 exhibits the highest level of expression, sPLA2G1b exhibits the lowest level of expression. The CT values (mean±SEM; n=3 cultures) for the enzymes and GAPDH are as follows: ABHd4: 23.01±0.05; GDE1: 19.39±0.04; Inpp5: 25.80±0.03; PTPn22: 25.06±0.06; sPLA2G1b: 27.30±0.05; GAPDH: 17.69±0.07.

Figure 3

Figure 3

Cultured mouse (C57BL/6 x129SvJ) primary sensory neurons were fixed in 4% paraformaldehyde for 10 minutes than washed with 0.01 M phosphate buffer saline. Combined immunostaining with anti-TRPV1-, anti-ABHd4-, anti-GDE1-, anti-Inpp5- and anti-PTPN22-antibodies were done as described in Material and Methods and Table 2 Sub-populations of cultured mouse primary sensory neurons, similarly to sub-populations of cultured rat primary sensory neurons express enzymes implicated in Ca2+-insensitive anandamide synthesis (red) and TRPV1 (green). Sub-populations of cultured mouse primary sensory neurons exhibit co-immunostaining for TRPV1 and putative anandamide-synthesising enzymes (arrows). Scale bar=20 µm.

Figure 4

Figure 4.

While application of capsaicin does, application of 20:4-NAPE does not result in increased [Ca2+]i at room temperature

Cultured rat PSN attached to glass coverslips were loaded with Fura-2 AM (5 μM) and superfused with an extracellular solution in a perfusion chamber. Capsaicin (1µM), 20:4-NAPE (50µM) or KCl (50mM) was superfused to the cells at 37 ºC (data shown in main text) or room temperature. Images were captured with a Peltier element-cooled slow scan charge-coupled camera system. Following subtraction of the background fluorescence, the ratio of fluorescence intensity at the two wavelengths as a function of time (rate 1 Hz) was calculated automatically (R = F340/F380). For more details see Materials and Methods.

(A) A representative recording of changes in intracellular Ca2+ concentration evoked by 50µM 20:4-NAPE (a), 1µM capsaicin (b) or 50mM KCl (c) at room temperature. Note that at room temperature, 60 seconds application of 50µM 20:4-NAPE (a) does not produce any change in [Ca2+]i. However, 30 seconds application of 1µM capsaicin (b) or 50mM KCl (c) increases [Ca2+]i.

(B) Relative number of neurons responding to application of 20:4-NAPEor capsaicin at room temperature, or capsaicin at 37ºC. Note that while capsaicin application results in responses in about 70% of the neurons at either temperature, 20:4-NAPE application does not induce responses at room temperature (indicated by the lack of purple and/or green proportions), hence there are no dual-responsive or 20:4-NAPE-only-responsive neurons.

Figure 5

Figure 5

Thin layer chromatography of products derived from 20:4-NAPE, the common substrate of all the putative anandamide synthesising enzymatic pathways, in the superfusate of cultured rat primary sensory neurons.

Cultured rat PSN (obtained from 2 rats) were treated with 100 μM 20:4-NAPE for 5 minutes. Lipids in the superfusate were extracted with ethylacetate. Following drying under a stream of nitrogen, the samples were reconstituted with acetonitrile and loaded onto TLC silica gel. The mobile phase was composed of chloroform:ethanol:water in 7:2.7:0.3 ratio. Lipids were visualised by potassium permanganate.Controls included 10µM anandamide and 10µM 18:1 LPA. While the samples contain anandamide, they do not contain 18:1LPA, which is another putative product derived from 20:4-NAPE, above the detection threshold. The lipid at the solvent front is 20:4-NAPE.