Endocannabinoids are conserved inhibitors of the Hedgehog pathway

Significance

Hedgehog proteins regulate development and tissue homeostasis. They signal through activation of the transmembrane protein Smoothened. Smoothened hyperactivation underlies development of many tumors. Smoothened activity can be modulated by several synthetic small molecules, which have shown promise in the clinic. However, occurrence of resistance-inducing mutations limits their effectiveness. Little is known about endogenous small molecules that may inhibit Smoothened in vivo. Previous work suggested that lipids present in lipoproteins are required for Smoothened inhibition in vivo. Here, we use biochemical fractionation and lipidomics to identify these lipids as endocannabinoids and show that their activity as Smoothened inhibitors has been conserved from flies to mammals. Endocannabinoids may provide useful templates for the design of new therapeutic Smoothened antagonists.

Abstract

Hedgehog ligands control tissue development and homeostasis by alleviating repression of Smoothened, a seven-pass transmembrane protein. The Hedgehog receptor, Patched, is thought to regulate the availability of small lipophilic Smoothened repressors whose identity is unknown. Lipoproteins contain lipids required to repress Smoothened signaling in vivo. Here, using biochemical fractionation and lipid mass spectrometry, we identify these repressors as endocannabinoids. Endocannabinoids circulate in human and Drosophila lipoproteins and act directly on Smoothened at physiological concentrations to repress signaling in Drosophila and mammalian assays. Phytocannabinoids are also potent Smo inhibitors. These findings link organismal metabolism to local Hedgehog signaling and suggest previously unsuspected mechanisms for the physiological activities of cannabinoids.

Hedgehog (Hh) signaling regulates growth and differentiation during embryonic development and adult tissue homeostasis (1). It is inappropriately activated in many tumors (2) and also influences physiological functions including lipid and sugar metabolism (3, 4), nocioception (5), the response to ischemia (6), and immune activation (7, 8). Different classes of compounds with activity toward Hh signaling could have broad therapeutic applications.

Hh proteins are lipid-modified secreted ligands that can associate with lipoprotein particles (9–12). Hh signals by binding the 12-transmembrane domain protein Patched (Ptc), which prevents Ptc from repressing the seven-pass transmembrane protein Smoothened (SMO) (13). Ptc-dependent SMO repression involves alterations to SMO trafficking. In vertebrates, Ptc activity prevents SMO accumulation in the primary cilium (14, 15), whereas in Drosophila, it blocks accumulation on the basolateral membrane (16). However, Ptc must exert additional effects on SMO that are independent of SMO trafficking; localization to primary cilia, or to the basolateral membrane, is insufficient for full activation of SMO signaling (11, 17, 18). Once it is active, SMO signaling influences transcription by regulating the processing of Gli family proteins and exerts additional effects on cell migration and metabolism by Gli-independent mechanisms (1, 19–21).

Hh signaling can be targeted at different levels (2). Antibodies to mammalian Sonic Hedgehog (Shh) block ligand activity, and chemical inhibitors of Gli function block its transcriptional output. SMO is especially sensitive to chemical inhibition, and its activity can be modulated by a variety of exogenous and endogenous small molecules that interact with SMO at different binding sites. Ptc is similar to resistance-nodulation-division (RND) family transporters, which use proton gradients to transport lipophilic molecules across membranes, and exogenous SMO inhibitors are thought to mimic the activity of endogenous molecules whose availability is regulated by Ptc. However, the identity of these endogenous SMO modulators is not clear.

SMO activity can be regulated by different small molecules that bind to distinct sites. Many of these molecules target a pocket formed by the SMO transmembrane domains (22). The steroidal alkaloid cyclopamine (derived from Veratrum californicum) was the first such inhibitor to be identified (23). Chemical library screens (24, 25) subsequently found many other molecules with positive and negative effects. Many of these compete with cyclopamine for binding to SMO, suggesting they target the same site, or influence the cyclopamine binding site allosterically. Interestingly, both the SMO agonist (SAG) and the SMO antagonist 1 (SANT-1) compete with cyclopamine for SMO binding but compete less well with each other (26). Thus, the influence of these small molecules on the conformation of the SMO transmembrane domains is likely to be complex. The endogenous ligands for this site are unclear, although vitamin D is a candidate (27). Another binding site in the SMO extracellular domain is targeted by oxysterols, which activate SMO (28, 29). Competition studies and genetic analysis suggest there may be additional regulatory binding sites on SMO (2, 29). A better understanding of the endogenous compounds that regulate SMO signaling would provide insights into the logic of the pathway and provide important clues for drug design.

Genetic studies in Drosophila have played a major role in identifying Hh pathway components and elucidating their mechanism of action: The wing disk has been a particularly powerful system for understanding the Hh pathway (30). Recently, we discovered that one or more lipids present in Drosophila lipoprotein particles are required in vivo to keep Hh signaling off in wing discs in the absence of Hh ligand. These lipids destabilize Drosophila Smoothened (Smo) and promote processing of the Drosophila Gli homolog cubitus interruptus (Ci) (17). Hh associates with lipoproteins via its lipid anchors, and this association blocks their repressive function. Mammalian lipoproteins have a similar repressive activity toward Shh signaling, and association of Shh with these particles reverses their inhibitory activity in the ShhLIGHT2 reporter assay (11). In this study, we use biochemical fractionation and lipid mass spectrometry to identify these inhibitory lipids from extracts of human very low-density lipoprotein (VLDL).

Results and Discussion

Initial experiments showed that VLDLs carry lipids that repress signaling in both Drosophila discs and ShhLIGHT2 cells (Fig. S1 A–C). Therefore, we used VLDL as a starting material to identify these inhibitory lipids. To reduce the complexity of the lipid pool, we first saponified VLDL extracts to deplete glycerolipids and removed the resulting free fatty acids. Residual nonsaponifiable lipids retain inhibitory activity in Hh signaling assays (Fig. S1 D′–I). We fractionated saponified extracts by reversed phase high-performance liquid chromatography (HPLC), assaying elution fractions for inhibitory activity in ShhLIGHT2 cells. This fractionation revealed several clusters of elution fractions that inhibited Shh signaling and, surprisingly, one region with stimulatory activity (Fig. 1A).

Fig. 1.

Endocannabinoids in whole serum and active VLDL lipid fractions. (A) Ratio of reporter activity in ShhLIGHT2 cells treated as indicated. HPLC elution fractions with stimulatory/inhibitory activities are shaded in green/red. Error bars indicate SDs of three independent experiments. n = 3. (B) Distribution of endocannabinoid species, detected by MS/MS in the HPLC elution fractions tested in A. Structures of each class are shown in boxes next to distribution profiles. R, fatty acid/alcohol moiety. (C) Concentration (in nanomolars) of endocannabinoids and related molecules in human serum quantified by LC-MS/MS using the method of multiple reaction monitoring (MRM). AU, arbitrary units.

We used shotgun analyses by Fourier transform mass spectrometry (FTMS) and tandem mass spectrometry (MS/MS) to search for signaling lipids whose concentration peaked in active HPLC elution fractions (Table S1). Active fractions did not coincide with peaks of known pathway regulators, such as vitamin D3, 7-dehydrocholesterol, or hydroxysterols (Fig. S2). However, MS/MS analysis identified endocannabinoids and endocannabinoid-related molecules that cofractionated with each region of activity (Fig. 1B and Table S1). Endocannabinoids consist of fatty acids or alcohols linked to various polar head groups. Arachidonoyl derivatives of ethanolamine, dopamine, and glycerol are potent ligands for the cannabinoid receptors CB₁ and CB₂ (31). Related molecules with different fatty acid moieties and head groups have biological activities exerted through a variety of other receptors (32). We identified peaks of different N-acylethanolamide, N-acyldopamine, and 2-alkylglycerol species in repressive elution fractions and detected peaks of N-acylserines in fractions with stimulatory activity (Fig. 1 A and B).

To explore effects of different endocannabinoid classes and species on Shh signaling, we assayed synthetically produced cannabinoids in ShhLIGHT2 cells. N-acylserine 16:0 (present in stimulatory fractions) increased signaling (Fig. 2A). Surprisingly, N-acylserine 20:4 inhibited signaling (Fig. 2A). We observed similar dependence on fatty acid chain length/unsaturation for several other classes of endocannabinoids. N-acyl ethanolamides 18:2 and 20:4 inhibited Shh signaling, whereas those with shorter saturated fatty acids (16:0 or 18:0) were much less effective (Fig. 2B). Shh signaling was repressed by 2-alkylglycerol 20:4 at concentrations similar to N-acylethanolamide 20:4 (Fig. 2C). Shh signaling was also inhibited by 2-acylglycerol 20:4 (2-arachidonoylglycerol, 2-AG), but not by the 2-acylglycerol species containing the 18:2 fatty acid (Fig. 2E). Thus, N-acylserines, N-acylethanolamides, 2-acylglycerols, and 2-alkylglycerol inhibit Shh signaling with a potency that depends on the structure of their fatty acid moieties. Species with shorter fatty acids are less active, or even stimulatory in the case of N-acylserine. N-acyldopamines also repress signaling in the ShhLIGHT2 cell assay. Interestingly, however, their activities show the opposite correlation with fatty acid chain length and unsaturation; 16:0 and 18:0 species are potent inhibitors, but N-acyldopamine 20:4 is much less active (Fig. 2D). Cannabis-derived phytocannabinoids mimic the effects of endocannabinoids on a variety of receptors. These compounds also robustly suppress Shh signaling at concentrations at least 10-fold lower than endogenous cannabinoids (Fig. 2F). Taken together, these data indicate that specific endocannabinoids account for the inhibitory activity in VLDL. The dependence of inhibitory activity on fatty acid chain length suggests they influence Shh signaling through specific receptor ligand interactions, and that N-acyldopamines may act by a different mechanism than other endocannabinoids.

Fig. 2.

Synthetic endocannabinoids and phytocannabinoids repress Shh signaling. Effects of different cannabinoids on signaling by nonlipoprotein-associated Shh in ShhLIGHT2 cells: N-acylserines (A), N-acylethanolamides (B), 2-alkylglycerol 20:4 (C), N-acyldopamines (D), 2-acylglycerols (E), and phytocannabinoids (F). Different endocannabinoid species are depicted in the indicated colors. The pink line in B and blue line in E show activities of N-acylethanolamide 18:2 and N-acyldopamine 18:0 in the presence of FAAH inhibitor PF-3845. The maximum ratio of reporter activity is normalized to 100 and indicated by the orange line. In each case, this maximum ratio (i.e., fold stimulation by ShhN* over control) ranged between 8- and 10-fold. Renilla luciferase activity (reflecting cell number and viability) was unaffected at all endocannabinoid concentrations shown. Error bars indicate SDs of five independent experiments. n > 5 for each experiment.

The most potent endocannabinoids were active at low micromolar concentrations (Fig. 2 A–E). To assess whether endogenous circulating endocannabinoids are sufficiently abundant to suppress Hh signaling in vivo, we quantified them in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As reported, anandamide was present at nanomolar concentrations in human serum (33); however, other endocannabinoid species were more abundant. Total concentrations of N-acylethanolamides, 2-acylglycerols, and N-acylserines were each in the micromolar range (Fig. 1C). Thus, endocannabinoids circulate at levels sufficient to regulate Hh signaling in vivo.

Endocannabinoids are rapidly metabolized by intracellular enzymes, including fatty acid amide hydrolase (FAAH) (34). We wondered whether endocannabinoids repress the Hh pathway at lower concentrations if their rate of metabolism were reduced. Therefore, we assayed endocannabinoids in the presence of selective inhibitor of FAAH licensed by Pfizer (PF-3845), a selective inhibitor of mammalian FAAH. PF-3845 potentiated pathway inhibition by N-acylethanolamide 18:2 and N-acyldopamine 18:0, allowing them to act at 10-fold lower concentrations (Fig. 2 B and D). Thus, rapid FAAH-dependent endocannabinoid metabolism normally limits their effects on Shh signaling.

Endocannabinoids and related molecules modulate the activity of many receptors including CB₁, CB₂, transient receptor potential vanilloid channel (TRPV) channels, GPR55, peroxisome proliferator-activated receptor (PPAR)γ, and PPARα. Το ask whether endocannabinoids influenced Shh signaling indirectly through these pathways, we assayed the activity of specific agonists and antagonists of these receptors in ShhLIGHT2 cells (Fig. 3A). None repressed signaling by Shh. Furthermore, comparing our results with published values for the activities of different cannabinoids toward these pathways indicates different patterns of potency. For example, CB₁ and TRPV1 are activated by N-acyldopamine 20:4 and not by N-acyldopamine 18:0 or 16:0, whereas the reverse is true for inhibition of Shh signaling (Fig. S3). Moreover, N-acylethanolamide 18:1 and 16:0 activate PPARα better than N-acylethanolamide 20:4, but these compounds act in reverse order on Shh signaling. Thus, there are specific structural features of endocannabinoids that optimize their repressive activity toward Shh signaling compared with other pathways. These observations suggest that endocannabinoids exert their effects directly on one or more components of the Shh pathway.

Fig. 3.

Some endocannabinoids and phytocannabinoids are direct agonists/allosteric modulators of SMO. (A) Signaling by nonlipoprotein-associated Shh in ShhLIGHT2 cells in the presence of specific agonists (green-shaded regions) or antagonists (red-shaded regions) of CB₁ and CB₂ receptors, GPR55, PPAR-alpha and PPAR-gamma receptors, and TRPV channels. Error bars are SDs of three independent experiments. n = 3. (B) Hh pathway stimulation by varying concentrations of different cannabinoids in the presence of 100 nM SAG. Error bars are SDs of three independent experiments. Dotted line indicates the firefly:Renilla luciferase ratio in unstimulated cells; green line indicates the ratio observed in cells treated with 100 nM SAG alone. n > 3. (C) Hh pathway stimulation by varying concentrations of SAG in the presence of indicated endocannabinoids. Error bars are SDs of three independent experiments. Dotted line indicates the firefly:Renilla luciferase ratio in unstimulated cells, and the orange line shows the ratio observed in a parallel experiment by using Shh alone. n > 3. (D) Fluorescence of BODIPY-cyclopamine (B-C) bound to tetracycline-induced SMO in 293S cells, as monitored by FACS. Cells were treated with 5 nM B-C alone (black tracing) or with 5 nM B-C and 1 µM Cannabinol (violet), 1 µM Cannabidiol (dark red), 10 µM N-acylethanolamide 20:4 (dark green), 100 nM SANT-1 (yellow), 100 nM SAG (turquoise), 20 µM N-acyethanolamide 18:2 (pink), 20 µM N-acyldopamine 16:0 (orange), 20 µM N-acylglycerol (light green), 5 µM N-acylserine 16:0 (red), or 20 µM N-acyldopamine 18:0 (blue). As negative control, the light gray tracing shows fluorescence of cells not treated with B-C. n = 2.

To investigate the step at which endocannabinoids act, we asked whether they inhibited pathway activation by SMO agonist (SAG). SAG binds to SMO, promotes its ciliary translocation, and increases SMO signaling (18, 24, 35). Activation of SMO signaling peaks at SAG concentrations of approximately 100 nM and decreases thereafter—no pathway activation is observed at 10 µM SAG. We assayed pathway activation by 100 nM SAG in the presence of increasing concentrations of N-acyldopamine 16:0, N-acyldopamine 18:0, N-acylethanolamide 18:2, N-acylethanolamide 20:4, Cannabinol, and Cannabidiol (Fig. 3B). All reduced pathway activation by SAG at concentrations similar to those that were effective against Shh (Fig. 2). To further examine the interaction between endocannabinoids and SAG, we assayed the effectiveness of different SAG concentrations in the presence of a constant amount of different endocannabinoids. Interestingly, endocannabinoids both reduce maximal pathway activation by SAG and shift peak pathway activation to lower SAG concentrations (Fig. 3C). These findings suggest that endocannabinoids act at the level of SMO or at a subsequent step to inhibit the Shh pathway.

To investigate whether cannabinoids or endocannabinoids might bind SMO, we asked whether they could compete with BODIPY-cyclopamine for binding to fixed SMO-overexpressing cells. Cannabinol, Cannabidiol, and N-acylethanolamide 20:4 strongly reduce binding of BODIPY-cyclopamine, and N-acylethanolamide 18:2 has a weaker effect. In contrast, N-acyldopamines 16:0 and 18:0, N-acylserine 16:0, and 2-AG do not (Fig. 3D). This suggests that phytocannabinoids and N-acylethanolamides either bind SMO in the transmembrane pocket or influence the structure of this site allosterically. Thus, these molecules are direct inverse agonists or negative allosteric modulators of mammalian SMO. 2-AG and N-acyldopamines may repress SMO activity indirectly through “entourage” effects—i.e., by competing with the actual endocannabinoid ligand for endocannabinoid transporters and metabolic enzymes (36).

Activation of SMO signaling is regulated both by ciliary accumulation and also by a subsequent step that takes place in primary cilia. For example, some SMO inhibitors block ciliary accumulation, whereas cyclopamine promotes ciliary accumulation but represses the activity of SMO in the cilium (14, 35). We therefore asked whether phytocannabinoids or N-acylethanolamide 18:2 might block SAG-induced ciliary accumulation of SMO. Even at concentrations that inhibit SAG-mediated pathway activation, none of these compounds prevented ciliary SMO accumulation (Fig. S4). Thus, cannabinoid binding represses SMO activity at a step subsequent to SAG-dependent ciliary translocation.

To explore whether endocannabinoids might account for the repressive activity of lipoproteins toward the Hh pathway in Drosophila, we quantified endocannabinoid levels in larval hemolymph by LC-MS/MS. Indeed, hemolymph contains micromolar amounts of ethanolamides and acylglycerols (Table S2). Furthermore, resupplying endocannabinoids to wing imaginal discs reverses the ectopic Hh pathway activation caused by loss of lipoproteins. Knockdown of the Drosophila lipoprotein Lipophorin (Lpp) elevates accumulation of Smo on the basolateral membrane and increases the amount of full-length Ci₁₅₅ (the single Drosophila Gli homolog) (Fig. 4 A′, A′′, B′, and B′′; ref. 17). Both effects are reversed by addition of either 2-AG or N-acylethanolamide 16:0 to explanted discs (Fig. 4 A–B** and Fig. S5 A–B*). Interestingly, phytocannabinoids mimic the repressive activity of endocannabinoids in wing discs, just as they do in mammalian ShhLIGHT2 cells (Fig. S5E). These data suggest that loss of endocannabinoid delivery is responsible for the ectopic stabilization of Smo and full length Ci₁₅₅ that occurs upon lipoprotein knockdown, and that endocannabinoids therefore regulate these processes in vivo in Drosophila.

Fig. 4.

FAAH-dependent endocannabinoid homeostasis regulates Smo and Ci₁₅₅ levels in Drosophila wing imaginal discs. (A–B**) depicts wing discs from either wild-type animals (A’, A*, B’, and B*) or animals depleted of Lpp (A’’, A**, B””, and B**) that were mock-treated (A’, A”, B’, and B’’) or treated with 50 µM N-acylethanolamide 16:0 (A’’, A**, B’’, and B**), stained for Smo (A’–A**) and Ci₁₅₅ (B’–B**). A and B show the average values of Smo (A) and Ci₁₅₅ (B) staining intensities calculated from 10 wing imaginal discs for each condition. (C–D**) Wild-type wing discs either mock treated (C’ and D’) or treated with 10 µM FAAH inhibitor PF-3845 (C* and D*), stained for Smo (C’ and C*) and Ci₁₅₅ (D’ and D*). Corresponding average staining intensities calculated from 10 wing imaginal discs are shown in C and D. To estimate the significance of the difference between staining intensities in mock-treated and N-acylethanolamide 16:0- or PF-3845-treated discs, we used the mean intensity and SD values at each point along the curves in the anterior compartment. P values were calculated by applying the Student’s t test. The double-headed arrows indicate the curves that were compared. In A, **P = 1.3 × 10⁻³⁷; in B, *P = 3.4 × 10⁻². In C, **P = 2.56 × 10⁻¹⁴; in D, **P = 4.82 × 10⁻²⁶. Anterior is to the right. AP, anteroposterior. (Scale bars: 10 µm.) n > 10 discs for each quantification.

To ask whether endocannabinoid catabolism was important to maintain Hh pathway activity in wing discs, we sought to inhibit Drosophila FAAH activity. The Drosophila genome encodes six proteins similar to mammalian FAAH (Flybase). Preparing hemolymph in the presence of PF-3845 increases the yield of both ethanolamides and 2-acylglycerols (Table S2), suggesting that this compound is active against Drosophila enzymes. We therefore treated explanted wild-type discs with PF-3845 and monitored effects on Smo and Ci₁₅₅ 2 hours later. In the anterior compartment, PF-3845 treatment depletes Smo from the basolateral membrane and reduces Ci₁₅₅ accumulation (Fig. 4 C–D*). Interestingly, PF-3845, like 2-AG, also lowered Smo levels in the posterior compartment where Ptc is not present (Fig. 4 C–C* and Fig. S5 A–A**). Thus, inhibiting endocannabinoid degradation appears to circumvent the requirement for Ptc in Smo repression. The importance of FAAH-like enzymes for normal Smo trafficking and Ci₁₅₅ processing supports the idea that endocannabinoids regulate Hh signaling in the wing disk.

These experiments demonstrate that lipoprotein-derived endocannabinoids are endogenous SMO inhibitors that are conserved across phyla. Whereas oxysterols and vitamin D have clear effects on mammalian SMO signaling (Fig. S6 A–D and refs. 27 and 37), cannabinoids are the first compounds to show such conserved activity. This suggests that cannabinoids inhibit an important basic step in SMO activation that is difficult to alter during evolution, making these compounds a particularly interesting starting point for drug development. Our results indicate that phytocannabinoids and N-acylethanolamides bind mammalian SMO directly. They act at a step subsequent to SMO ciliary translocation. Whereas cannabinoids do not obviously influence trafficking of mammalian SMO—at least into the primary cilium—they influence trafficking of Drosophila Smo, preventing its accumulation on the basolateral membrane. It is possible that cannabinoids act by different mechanisms in flies and mammals. Alternatively, ciliary and basolateral membrane localization may not be functionally analogous. However, in both cases, full SMO activation appears to be a two-step process, with only one step that is influenced by endocannabinoids. Endocannabinoids regulate the ability of Drosophila Smo to stabilize the full-length form of Ci, but lipoprotein knockdown experiments show that additional steps are required for target gene activation (11, 17).

Endocannabinoids and phytocannabinoids have many physiological activities that are not completely understood. Some of these may reflect their activity toward the Hh pathway. Cannabinoids and Hh signaling regulate many of the same processes—e.g., angiogenesis (6, 38), hair follicle development (39, 40), nocioception (5, 41), bone formation (42, 43), and energy metabolism (3, 4, 44). Indeed, cannabinoids block growth of tumors known to depend on Hh signaling (45–50). Interestingly, Cannabis exposure in utero inhibits fetal growth and alters brain development (51); whether impaired Shh signaling contributes to these problems is worth investigating.

We have shown that many different species of endocannabinoids are present in circulation. Their ability to repress Hh signaling suggests a previously unidentified mechanism by which systemic metabolism could influence development, tissue homeostasis, and cancer. The fact that lipoprotein-derived endocannabinoids repress such an important tumor-promoting pathway might help explain the link between disturbed lipoprotein metabolism and cancer risk (52). Our findings forge a link between cannabinoids and Hh signaling, opening new research avenues for both important classes of signaling molecules.

Materials and Methods

Hh Signaling Assays.

Drosophila wing disk assay was performed as described (17). ShhLIGHT2 signaling assay, SAG competition assay, ciliary translocation assay, and BODIPY-cyclopamine binding assay were described (23, 24).

Column Chromatography.

Extracts containing saponification-resistant lipids from VLDL were loaded onto a C18 column (25 mm × 4.5 mm, 5 µM particles). Five hundred microliters of extract was loaded in 60% aqueous methanol and eluted with a step gradient of 80% methanol in H₂O (60 min) and a linear gradient of 80–100% methanol in water (40 min) at the flow rate of 1 mL/min. The eluate was collected into 40 fractions that were dried down and subjected to the mass spectrometric analysis and ShhLIGHT2 signaling assay.

Screening of Endocannabinoid Fractions by Shotgun Lipidomics.

Fifty microliters (1/20) of each HPLC elution fraction was mixed with sixty-five microliters of 13 mM ammonium acetate in isopropanol. Twenty microliters of sample were loaded onto 96-well plate and centrifuged for 5 min at 4,000 rpm. Mass spectrometric analyses were performed on a Q Exactive instrument with robotic nanoflow ion source TriVersa NanoMate controlled by the Chipsoft 6.4 software (nanoflow chips: 4-µm spraying nozzle diameter; ionization voltage: ±1.25kV; gas back pressure: 0.95 psi). Mass spectra were acquired in positive and negative ion mode with the target mass resolution of R_{m/z 200} = 140,000 within m/z range of 100–1,000. Precursors within m/z range of 200–500 were fragmented in a data-dependent acquisition mode with the target resolution of R_{m/z 200} = 17,000.

Extraction of Endocannabinoids from Human Blood Serum and Drosophila Larval Hemolymph.

At 4 °C, 500 µL of human serum or 500 µL of diluted larval hemolymph (equivalent to 13.5 larvae) were supplemented with 10 µM PF-3845. Then, 600 µL of ethyl acetate/n-hexane 9:1 and 17.5 µL of the internal standard mixture (14 pmol/mL d4-N-acylethanolamide 16:0, 7.39 pmol/mL d8-N-acylethanolamide 20:4, 100 pmol/mL N-acylserine 20:4, 100 pmol/mL 2-alkylglycerol 20:4, 90.3 pmol/mL d8-2-acylglycerol 20:4, and 100 pmol/mL d5-1-acylglycerol 20:4) were added. After 15 min of centrifugation at 15,000 × g and 10 min of incubation on dry ice, the organic phases were dried and reconstituted in 35 µL of water/acetonitrile/iso-propanol/formic acid (5:4:0.5:0.1), centrifuged for 5 min at 10,000 × g and transferred for LC-MS/MS analysis.

LC-MS/MS Quantification of Endocannabinioids.

The analysis was performed on Agilent LC1100 system with a C18 column (5 µm, 0.5 × 150 mm); flow rate of 20 µL/min; injection volume of 3 µL interfaced on-line to a triple quadrupole mass spectrometer TSQ Vantage. The elution gradient comprised solvent A: 0.1% formic acid in water and solvent B: acetonitrile/isopropanol/formic acid (9:1:0.1) run with the profile: 0 min, 50% B; 0–2 min, 50–66.4% B; 2–8 min, 66.4–73% B; 8–10 min, 73–95% B; 10–14 min, 95% B; 14–15 min, 95–50% B; 15–21 min, 50% B. Endocannabinoids were detected as [M+H]⁺ ions by MRM transitions in the positive mode; S-lens voltages and collision energies were optimized individually for each standard compound in direct infusion mode. The transfer capillary temperature was 275 °C and the ions isolation width was 0.7 amu.