Cloning and expression of DAGLs
A full-length clone of the human α gene (gi|20521122) was obtained from the Kazusa DNA Research Institute (KIAA0659), and the mouse β gene (gi|16359288) was obtained from I.M.A.G.E. Consortium (4921222). The coding sequence for the α gene was amplified by PCR and inserted into pcDNA3.1D/V5-His-TOPO (Invitrogen) to generate an expression construct with an in-frame 3′ V5 epitope tag. The coding sequence for the β gene was amplified by PCR and subcloned into pCMV-Tag4A (Stratagene) using the NotI and XhoI restriction sites, in-frame with a 3′ FLAG epitope tag. Single point mutations in the β gene were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). Plasmids were transfected into COS-7 cells using lipofectamine plus (Invitrogen), and stable transfected clones were selected using G418. For Western blotting, equal amounts of protein lysate were separated on SDS–polyacrylamide gels and transferred to nitrocellulose Hybond ECL (Amersham Biosciences). Primary antibodies used were mouse anti-V5 (Invitrogen) at 1:5,000 and mouse anti-Flag (Stratagene) at 1:1,000. The secondary antibody was anti–mouse HRP (Vector Laboratories) used at 1:3,000.
Synthesis of substrates
In brief, the compounds were obtained from the R (−) solketal esterified with either unlabeled or 14
C-labeled oleic acid using N
-ethylcarbodiimide hydrochloride/4-dimethylaminopyridin, and deprotecting the acetonide with hydrochloride/methanol. The primary alcoholic group was protected selectively with triisopropylsilyl chloride, whereas the free secondary alcohol was esterified with various fatty acids, either unlabeled or 14
C-labeled. Finally, the sn
-1,2-diacyl-glycerol with two different acyl groups in position sn
-1 and 2 was obtained by removing selectively the sylyl group with tetrabutylammonium fluoride/acetic acid. To prepare sn
C]oleoyl-2-arachidonyl glyceryl ether, the previously prepared 2-AG ether (noladin) was esterified with [14
C]oleic acid using N
-ethylcarbodiimide hydrochloride /4-dimethylaminopyridin. 2-[3
H]arachidonoyl-glycerol and arachidonoyl-[14
C]ethanol-amide were synthesized as described previously (Bisogno et al., 1997
) with minor modifications.
Confluent cells were harvested in Tris-HCl buffer, pH 7, and homogenized in a homogenizer (Dounce). The homogenates were centrifuged at 4°C sequentially at 800 g (5 min), 10,000 g (25 min), and 100,000 g (70 min). Each fraction or, for most of the experiments, the 10,000-g fraction was incubated at pH 7.0 (or in citrate 50 mM or Tris-HCl 50 mM buffers at different pH values) at 37°C for 15 min, with different radiolabeled substrates (i.e., for DAGL activity, with various synthetic 14C-labeled DAGs [1.0 mCi/mmol, 50 μM] or with sn-1-stearoyl-2-[14C]arachidonoyl-glycerol from Amersham Biosciences, 56.0 mCi/mmol, at different concentrations; for monoacylglycerol lipase activity, with synthetic 2-[3H]arachidonoyl-glycerol, 1.0 mCi/mmol, 50 μM; for triacylglycerol lipase activity, with 1,2,3-tri-[14C]oleoyl-glycerol from NEN, 100.0 mCi/mmol, 50 μM; for phospholipase A1/A2 activity, or sn-1-[14C]oleoyl-2-[14C]oleoyl-phosphatidylcholine from NEN, 104.0 mCi/mmol, 20 μM; and for fatty acid amide hydrolase activity, with synthetic arachidonoyl-[14C]ethanolamide, 5.0 mCi/mmol, 25 μM). After the incubation, lipids were extracted three times with 2 vol chloroform/methanol 2:1 (by vol), and the extracts were lyophilized under vacuum. Extracts were fractionated by TLC on silica on polypropylene plates using chloroform/methanol/NH4OH (85:15:0.1, by vol) as the eluting system. Under these conditions, the migration index of free fatty acids, monoacylglycerols, and diacylglycerols was 0.25, 0.65, and 0.9, respectively; the migration index of phospholipids and triacylglycerols was 0.05 and 1.0, respectively. Bands corresponding to each class of lipids were cut, and their radioactivity was counted with a β-counter. Using sn-1-stearoyl-2-[14C]arachidonoyl-glycerol as the substrate, the DAGL reaction rate was linear up to 20 min and reached a plateau after 30 min, with both DAGLα and β. Saturation of DAGLα and β activity was reached with 500 and 400 μM, respectively, when using sn-1-stearoyl-2-[14C]arachidonoyl-glycerol as the substrate.
Intact cell stimulation and 2-AG analyses
Confluent cells were stimulated for 20 min at 37°C with either vehicle or 4 μM ionomycin or 1 μM ionomycin + THL, after a 5-min preincubation, with THL in DME medium without serum. Immediately after the stimulation, cells, medium, or cells plus medium were extracted three times with 2 vol chloroform/methanol 2:1 (by vol), and the extracts were lyophilized under vacuum. Each extract was purified by open bed chromatography over silica columns, followed by 2-AG quantification by means of isotope dilution atmospheric pressure chemical ionization–liquid chromatography–mass spectrometry (Marsicano et al., 2002
Rabbit antibodies, raised and affinity purified against the GASPTKQDDLVISAR epitope in DAGLα, and the SSDSPLDSPTKYPTL epitope in DAGLβ, were used at 1.5–3.0 μg/ml for immunostaining. An affinity-purified antibody against the CB1 receptor (PA1-745; Affinity BioReagents, Inc.) was used at 20 μg/ml IgG. An antineurofilament monoclonal antibody (N5264; Sigma-Aldrich) was used at a dilution of 1:1,000. 6-μm sections of formalin-fixed, paraffin wax–embedded tissues were subjected to heat-mediated antigen retrieval to disclose antigenic sites before being incubated in primary antibody solutions overnight at 4°C. After washing, the sections were incubated with biotinylated secondary antibodies (E0432 and E0433; Dakopatts) followed by detection with an HRP-streptavidin-biotin kit (model K377; Dakopatts). To visualize the V5 epitope tag, cells were fixed with 4% PFA and processed for indirect immunofluorescence. Cells were permeabilized with 0.2% Triton X-100/PBS and stained with mouse anti-V5 primary antibody at 1:200, followed by Alexa Fluor 488 goat anti–mouse IgG secondary (Molecular Probes) at 1:2,000. Slides were viewed on a microscope (model Axiovert 135; Carl Zeiss MicroImaging, Inc.), using 10×/0.25 Achrostigmat or 63×/1.4 Oil Pan-Apochromat lens. Images were captured with an AxioCam using KS300 software (Carl Zeiss MicroImaging, Inc.). Images were processed in Adobe Photoshop, with brightness and contrast being the only adjustments made.
Neurite outgrowth studies
Neurite outgrowth studies and reagents used therein were as described previously (Williams et al., 2003
Real-time RT-PCR analysis of DAGLα expression in various tissues
TaqMan RT-PCR for DAGLα was performed essentially as described previously (Bond et al., 2002
). Primers were based on sequences encoded by exon 19; for the mouse, the forward and reverse primers were ttcgccgagttcattgacag and tctcaggcaccatcatgca. For the human, the forward and reverse primers were cctcttcaacctggacagcaa and gggccctcagcgtagtca. To normalize data and to correct for variations in RNA and/or cDNA quality and quantity, parallel TaqMan assays were run for two housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.