Characterisation of anandamide uptake in resting and activated murine cells
Marcus Fredriksson Sundbom
Marcus Fredriksson Sundbom Degree Thesis in Biomedicine, 30 ECTS Master’s Level
Report passed 16 Jan 2015
Supervisor Christopher Fowler and Jessica Karlsson, Dept of Pharmacology and Clinical Neuroscience
Kan man i framtiden förstärka kroppens eget smärtstillande system?
Alla drabbas vi någon gång av smärta, kanske behöver vi till och med ta till smärtstillande läkemedel som vi antingen köpt receptfritt eller som en läkare skrivit ut. Men faktum är att kroppen har sitt eget sätt att ge smärtlindring. En grupp av de kroppsegna ämnen som ger smärtlindring, verkar på liknande sätt som det aktiva ämnet i cannabis och kallas därför endocannabinoider. De bildas till exempel i samband med inflammation som uppstår efter en skada, och har sin verkan på receptorer på nervcellsytan. Receptorer är mottagarmolekyler dit en annan molekyl binder. De receptorer som endocannabinoider binder till kallas cannabinoidreceptorer. De aktiveras när endocannabinoider binder och resultatet blir att receptorerna hämmar ledning av smärtimpulser. Endocannabinoidernas effekt tas bort när de tas upp av cellen. Det som styr hur mycket som tas upp av cellen, är hur mycket som redan finns i cellen. Den endocannabinoid som vi fokuserat på i vår studie kallas anandamid, AEA. Hur mycket som finns inuti cellen regleras av enzymer som bryter ned AEA. Det främsta enzymet av dessa kallas fatty acid amide hydrolyase, FAAH, men även andra enzymer som cyclooxygenase-2, COX-2, kan också bryta ned AEA. COX-2 är ett enzym som uttrycks i samband med inflammation, och det är även på detta enzym många receptfria smärtstillande läkemedel har sin verkan. Smärtstillande tabletter på apoteket innehåller ofta acetylsalicylsyra, ibuprofen eller diklofenak och hämmar både COX-1 och COX-2, medan andra läkemedel som celecoxib och nimesulid är mer selektiva hämmare av endast COX-2. Tidigare studier har visat att om man hämmar FAAH och därmed nedbrytningen av AEA, minskar upptaget av AEA in i cellen. Detta möjliggör att AEA kan fortsätta interagera med receptorn och verka smärtstillande. Trots att man vet att COX-2 också kan bryta ned AEA, så har man inte undersökt om hämning av COX-2 leder till minskat upptag av AEA.
I vår studie har vi försökt att ta reda på vilken roll COX-2 har för upptag av AEA i en artificiell inflammationsmiljö som vi skapat i laboratoriet. För att åstadkomma detta så använde vi celler från mus och råtta, som vi utsatte för inflammationsämnen så att de skulle börja uttrycka COX-2 som vid en inflammation. Med specifika antikroppar kunde vi detekera COX-2 i cellerna.För att sedan kunna studera upptag så använde vi en metod där cellerna utsätts för radioaktivt AEA. Trots att AEA sitter ihop med en radioisotop, tas det upp av cellerna som vanligt. Vi använde hämmare av COX-2 respektive FAAH för att kunna studera hur de enzymerna var för sig bidrog till upptag av AEA. Låg mängd radioaktiv isotop inuti cellerna betydde litet upptag av AEA. Det vi fann var att FAAH är det enzym som styr upptag av AEA genom att bryta ned det inuti cellerna. COX-2 verkade inte ha någon effekt på upptaget av AEA trots att vi kunde se att det var uttryckt i muscellerna. Anledningen att vi inte kunde se COX-2 uttryckt i råttcellerna beror troligtvis på att de celler man använder i laboratoriet är cancerceller som lätt muterar, och tappar uttryck av olika proteiner ju äldre de blir. Vidare fann vi också att uttrycket av FAAH var lägre i de celler som utsattes för en inflammationsmiljö, och att de också hade ett minskat upptag av AEA. Vår tolkning är således att lägre nivåer av FAAH ger minskad nedbrytning, som i sin tur leder till minskat upptag. Detta kunde inte kompenseras av ökat uttryck av COX-2. Våra fynd bidrar till en ökad förståelse i hur man kan manipulera kroppens egna smärtstillande system. Genom att använda en hämmare för FAAH så kan det kanske leda till god smärtlindring där kroppen själv styr produktionen av smärtstillande. På detta sätt kanske överdoser och de psykiska effekterna av konventionella smärtstillande kan undvikas.
SUMMARY
Modifying the metabolism of the body’s own endocannabinoids is a novel approach for analgesia. Two key catabolic enzymes are fatty acid amide hydrolase (FAAH) and inflammation-inducible cyclooxygenase 2 (COX-2). The cellular uptake of the key endocannabinoid anandamide (AEA) has been found to be regulated by its FAAH- catalysed intracellular degradation, but COX-2 has not been investigated in this respect. We aimed to find out whether or not COX-2 in an in vitro inflammation setting would be able to gate AEA uptake. To achieve this, C6 cells and Raw 264.7 cells were stimulated with LPS/INF-γ and lysates then analyzed by immunoblot in order to verify COX-2 expression. AEA cellular uptake was quantified using a radioassay with [3H]-AEA. It was found that COX-2 was not inducible in C6 cells using the LPS/INF-γ conditions studied, while it was inducible in Raw 264.7 cells. AEA uptake in the COX-2-induced Raw 264.7 cells was not reduced by inhibitors of this enzyme.
FAAH appeared to be down-regulated in the stimulated Raw 264.7 cells, and this was reflected in an overall lower AEA uptake. Our interpretation of the data points to FAAH as gating AEA uptake. Additional experiments are required to validate our findings by verifying significance.
INTRODUCTION
Chronic pain is a major challenge for Western healthcare, and a recent study by Breivik et al., 2006 suggested that up to 19%
of Europeans suffer chronic pain. With 61% of sufferers having an impaired ability to work, it is clear the cost for society is very great. While some (but not all) patients are treatable with for example opioids, the drugs themselves carry
unwanted side-effects such dizziness, somnolence and risk for overdoses which has lately been on the increase (Debono et al., 2013).
Another class of drugs commonly used in pain management are non-steroidal anti- inflammatory drugs (NSAID). The NSAIDs inhibit both isoforms of cyclooxygenase (COX- 1, COX-2) leading to decreased prostaglandin production in an inflammatory setting, thus exhibiting its analgesic and antipyretic effects (Vane, 1971). However, this mechanism of action also produces unwanted effects, the most important being gastrointestinal toxicity and cerebrovascular events. There is thus a need for novel drugs for the treatment of pain.
The endocannabinoid system is composed by the G-protein cannabinoid receptors CB1
and CB2, with their bioactive lipid ligands (Pertwee et al., 2010) as well as the enzymes that produce and degrade them. The CB1
receptor is primarily expressed in the central nervous system (Glass et al., 1997), whereas the CB2 receptor is primarily present in peripheral tissue such as prostate gland (Ruiz- Llorente et al., 2003) and gastrointestinal tract(Pertwee, 2001). Activation of the CB1 receptor by ligands such as endocannabinoids alters memory, cognition, motor function as well as mediating analgesia (Pertwee et al., 2010).
The two most well studied endocannabinoids are anandamide (AEA) and 2- arachidonoylglycerol. Although usually produced at a very low rate, a rise in intracellular calcium concentration triggers endocannabinoid production. The production can be triggered as a result of for example mGluR1/5 activation(Ohno-Shosaku et al., 2002), lipopolysaccharide (LPS) stimulation(Hu
et al., 2012) or nerve injury (Hansen et al., 2001). The production of AEA is mediated through N-acyltransferase to yield N-acyl- phosphatidylethanolamine (NAPE) from phosphatidylethanolamine, which is then cleaved by NAPE-phospholipase D to produce anandamide (Okamoto et al., 2004). An alternative route of production is NAPE cleavage by phospholipase C (Liu et al., 2006).
In the central nervous system, the endocannabinoids produced diffuse into the synaptic cleft where they inhibit the presynaptic neuron by binding surface CB1-receptors. The mechanisms of presynaptic inhibition includes adenylylcyclase inhibition (and resulting decrease in intracellular cAMP levels), activation of mitogen-activated protein kinase as well as blockage of calcium channels and activation of potassium channels (Pertwee et al., 2010).
The presynaptic inhibition is terminated upon cellular AEA uptake and subsequent degradation. The major enzyme for AEA degradation is fatty acid amide hydrolase (FAAH) (Deutsch and Chin, 1993;Cravatt et al., 1996), but other enzymes such as COX-2 (Yu et al., 1997), lipoxygenase (Hampson et al., 1995) and P450 (Bornheim et al., 1995) also degrade AEA. Recent findings suggest that NSAIDs also mediate analgesia through the endocannabinoid system where CB1
antagonist have been shown to abolish the anti-nociceptive effects of NSAIDs, and where the profen class of compounds inhibit the COX-2 catalysed oxygenation of endocannabinoids more potently than either COX-1 or -2 catalysed oxygenation of arachidonic acid. These compounds are also modestly potent inhibitors of FAAH (review, see Fowler, 2012).
The cellular uptake of AEA has been a topic of controversy in that there has been no consensus in weather or not a designated plasma membrane transporter exists.
Substrate analogue inhibited AEA uptake without inhibiting AEA hydrolysis. The upake of AEA is saturable, but neither the influx nor the efflux of anandamide were left unaffected even when subjected to ATP and sodium alterations (Hillard et al., 1997) which would point to a mechanism of facilitated diffusion. Conversely the bulk of AEA membrane transport has been found to occur within seconds (Bojesen and Hansen, 2005), which challenges findings from previous studies that have used incubation times of minutes. Furthermore suggested AEA transporter inhibitors such as N-(4- hydroxyphenyl) arachidonylamide (AM404, Beltramo et al., 1997) did not have an effect on AEA uptake at 25 seconds, but instead showed an effect at 5 minutes (Kaczocha et al., 2006) likely mirroring inhibition of AEA hydrolysis or some other intracellular event such as inhibition of intracellular transporters.
Intracellular AEA has been shown to bind fatty- acid binding protein (FABP)-5/-7(Kaczocha et al., 2009, 2012), heat shock protein 70, albumin (Oddi et al., 2009) and the FAAH-like anandamide transporter protein (FLAT) (Fu et al., 2012, but see Leung et al. reference vid:
http://www.ncbi.nlm.nih.gov/pubmed/24223930 ).
The simplest model of AEA uptake is one of passive diffusion across the plasma membrane followed by either sequestration (or binding to FABP5 etx) or metabolism (review see Fowler, 2013). Consistent with this, inhibition of FAAH has been described to result in a reduced AEA uptake rate, where the extra-intracellular gradient is reduced (Day et al., 2001;Deutsch
et al., 2001). In theory, other metabolic enzymes for AEA such as COX-2 should also be able to gate AEA uptake, provided that their expression is sufficient. However, this has not been investigated.
In this current study we aim to examine the role of COX-2 in AEA cellular uptake by using FAAH and COX inhibitors. COX-2 has been found to be expressed primarily at inflammation, and subsequently LPS &
Interferon-γ (IFN-γ) cellular stimulation has been associated with an increase of COX-2 expression. This has been shown in both C6 glioma cells (Esposito et al., 2008) and Raw 264.7 cells(von Knethen et al., 1999). These findings enables assessment of AEA uptake when COX-2 is expressed both to a high degree under inflammatory conditions, as well as to a low degree as expected under normal conditions.
First the growth conditions must be established in regards to serum concentration in media, duration of growth and LPS concentration. Having established the growth conditions, the presence of COX-2 in murine C6 and Raw 264.7 cells will be confirmed. This will be accomplished by using a LPS/INF-γ stimulation protocol by Esposito et al (2010) to simulate inflammation in vitro, since COX-2 is expressed at low levels under normal conditions(Crofford, 1997). Upon stimulation the COX-2 presence will be confirmed by means of Western blot.
To assess the cellular uptake of AEA in the presence of FAAH or COX-2 inhibitors, a radioassay according to Sandberg and Fowler (2005) will be used. In the assay [3H]-AEA uptake will be quantified in the presence or absence of FAAH and COX inhibitors.
Figure 1.
COX-2 expression and [3H]-AEA uptake in C6 glioma cells.
(A) C6 cells were treated with INF-γ 100 U/ml and LPS 1.0 µg/ml or 0.1 µ/ml (“1:” and “0:1” respectively) and collected at either 24h or 48h (“:24” and “:48” respectively). Cell lysates were analyzed by immunoblot (n=2) using COX-2- antibodies. rCOX-2 indicates recombinant COX-2.
(B) Cell lysates from C6 cells were treated as above on then analyzed by immunoblot with β-actin antibodies (n=2)
(C) C6 cells were incubated with inhibitors or vehicle controls (DMSO and EtOH). [3H]-AEA were quantified from cell lysates (n=4) and data is presented as mean ± S.E.M.
RESULTS
COX-2 expression using LPS/INF-γ is not possible in C6 glioma cells
C6 glioma cells were stimulated with LPS/INF- γ to verify that COX-2 protein expression could be established. It was found that COX-2 was absent in vehicle control after 24h and 48h respectively, both when loading 30 µg (not shown) and 90 µg (Fig 1A). This was also found to be the case for the LPS/INF-γ treated samples, which did not produce any visible COX-2 bands following either 24 h or 48 h of treatment (Fig 1A). The recombinant COX-2 positive control was clearly visible on the immunoblot (Fig. 1A), as was β-actin in all samples (Fig. 2B).
[3H]-AEA uptake in C6 glioma cells
On the day of the experiment the cells were subjected to either vehicle control inhibitors of FAAH and/or COX-2. The FAAH inhibitor URB597, at a concentration known to inhibit FAAH completely, produced a large reduction in AEA uptake, and this was also seen with R- Flu-AM1, which inhibits both FAAH and COX-2 at the concentration used (Fig. 1C).
Flurbiprofen which inhibits COX but is a weaker inhibitor of FAAH than R-Flu-AM1, and nimesulide, which inhibits COX but noes not inhibit FAAH, produced smaller effects on AEA uptake by C6 cells (Fig 1C).
Figure 2. Immunoblot and relative COX-2 expression in Raw 264.7 cells
(A) Expression of COX-2 in Raw cells with the same conditions as for C6 cells (n=3) using immunoblotting.
(B) COX-2 expression relative to recombinant COX-2 as estimated by ImageJ. Data presented as mean with standard error, n=3.
Expression of COX-2 can be induced in Raw 264.7 cells using LPS/INF-γ
As expected, no COX-2 bands could be detected in unstimulated Raw 264.7 cells after 24h and 48h (Fig. 2A). Stimulation of Raw 264.7 using LPS/INF-γ induced COX-2 expression both at 24h and 48h (Fig. 2A).
Furthermore, a variation of COX-2 expression levels could be observed when different LPS concentrations was used. More intense COX-2 bands were visible at 48h for 0.1 µg/ml LPS compared to 48h, whereas the opposite was true for the higher 1.0 µg/ml concentration (Fig.
2B).
FAAH expression in unstimulated and LPS/INF-γ -stimulated Raw 264.7 cells
Immunoblot using an FAAH-specific antibody was performed to verify the presence of FAAH in the Raw 264.7 cell lysates. FAAH expression could be seen in all samples except negative control recombinant COX-2 (Fig. 3A).
Furthermore, FAAH expression was greater in the samples treated with vehicle compared to the LPS/INF-γ treated samples (Fig 3B).
Figure 3. FAAH expression in Raw 264.7 cells.
(A) Immunoblot displaying FAAH antibody bound protein bands from Raw 264.7 cell lysates (n=3).
(B) Relative expression of FAAH relative to 48h untreated cell lysates, as estimated by Image J.
Data presented as mean with standard error, n=3.
[3H]-AEA uptake in unstimulated and LPS/INF-γ -stimulated Raw 264.7 cells
Raw 264.7 cells were treated with LPS/INF-γ or vehicle control and were then left to grow for 48 h. On the day of the experiments, the cells were subjected to the same inhibitors as mentioned above as well as vehicle controls. It was found that in the unstimulated Raw 264.7 cells the pattern was similar to the unstimulated C6 cells, with the strongest reductions being seen with URB597 and R-Flu-
AM1 (Fig. 4A). In the stimulated population URB597 and R-Flu-AM1 appeared to have a smaller effect upon uptake compared to the unstimulated cells (Fig. 4B) and none of the other inhibitors had any substantial effect. It is worth noting that the stimulated cells showed lower levels of AEA uptake compared to the unstimulated cells. This is consistent with the lower expression of FAAH shown in the Western blots.
DISCUSSION
In this study we have reported that it is not possible to induce and detect COX-2 expression using LPS/INF-γ in C6 glioma cells (Fig. 1A). This finding is in sharp contrast to previous reports which have used almost
identical conditions and still been able to identify COX-2 on immunoblots following treatment (Esposito et al., 2008, 2010). It is possible to hypothesize that since other conditions such as blocking buffer, antibodies etc are very similar, the cancerous nature of Figure 4. [3H]-AEA uptake in Raw 264.7 cells
(A) Unstimulated cells treated with inhibitors or vehicle controls for 48h (n=3). Presented as mean ± S.E.M.
(B) Cell lysates from cells stimulated for 48h with LPS 0.1µg/ml and INF-γ 100 U/ml (n=3).
Presented as mean ± S.E.M.
C6 cells and their propensity to mutate might be the reason for COX-2 being absent (at least on a protein level). Further, since negative results are often not reported in the literature, it may be that our finding is the norm, and not the exception. The fact that COX-2 could not be identified using Western blot, is consistent with the lack of effect from any of the COX-2 inhibitors on [3H]-AEA uptake, with the exception of R-Flu-AM1, which can additionally block FAAH. The only reduction in AEA uptake was observed in samples treated with this compound and with the selective FAAH inhibitor URB597, pointing to FAAH as being the gatekeeper in AEA uptake in the C6 cells.
It would be possible to argue that since the analgesic effects of the endocannabinoid system is known to be mediated through nervous tissue, Raw 264.7 cells might not be the most suitable model to study AEA since it is a mouse macrophage derived cell line. The rat glioma C6 cell line on the other hand, has been an established model for CNS cells even since first established by Benda et al in 1968.
However, Raw 264.7 could potentially serve as an analogue for microglia, which is a type of macrophage which resides in CNS tissue (Ginhoux et al., 2013). The endocannabinoid system has been implicated in microglial regulation (for review see Stella, 2009) and it is thus relevant to be considered in respect to AEA uptake.
COX-2 was inducible in Raw 264.7 using the same LPS/INF-γ cocktail as for C6 cells (Fig.
2A) while it was absent in unstimulated cells.
This finding is consistent with previous reports (Whan Han et al., 2001) which also were able to identify COX-2 on immunoblots using similar conditions. As expected, COX-2 inhibitors did not have any effect on AEA uptake in
unstimulated cells (Fig. 4A). However, although known to degrade AEA (Yu et al., 1997), COX-2 inhibition was not found to have any effect on AEA uptake in stimulated cells.
The only substantial reductions in AEA uptake were observed in samples treated with URB597 and FluAM1 (Fig. 4B), again pointing to FAAH as being the key player even in in vitro inflammation. Our interpretation of the data is that the upregulated COX-2 expression is not sufficient to gate AEA uptake despite its ability to degrade AEA and hence reduce intracellular concentrations of this endocannabinoid.
Furthermore we also described a lower FAAH expression in the LPS/INF-γ treated samples compared to vehicle control (Fig. 3B).
This is consistent with the overall lower AEA uptake observed in the stimulated cells compared to unstimulated (Fig. 4A,B). This is further evidence indicating that FAAH is in fact the major player in AEA uptake. It is also consistent with previous findings such as with Deutsch et al. (2001), who described a twofold higher AEA uptake in Hep2 cells transfected with FAAH compared to wild-type Hep2 cells lacking FAAH. Alternatively the reduced uptake can reflect some other cellular event. In theory this could be inhibition of some other degrading enzyme, or that carrier proteins such as FABP5 is in some way inhibited by the LPS/INF-γ stimulation, and consequently AEA is not accessible to be degraded.
Unfortunately, the spread in the data from the uptake experiments was quite substantial.
As a result it was not possible to assume normal distribution of the data, and consequently no statistics were performed on the uptake experiments and thus significance could not be determined. To determine
significance it would be necessary to repeat the experiments more times, but due to time constraints this was not possible to do during this current study. It is also unfortunate that since the C6 cells lacked detectable COX-2 expression even in stimulated cells, since it restricts our conclusions the conclusions drawn about the role of COX-2 in cellular AEA uptake to a single cell line. Further cell lines should be investigated.
Future prospects include quantifying the amount of intracellular endocannabinoids in stimulated vs unstimulated cells. This would enable us to gain insight as to how endocannabinoid levels are coupled to FAAH/COX-2 expression and inhibition.
Furthermore, the expression of FAAH & COX-2 expression could also be studied using qRT- PCR and/or ELISA for a more accurate quantification. This might however be of little value for COX-2 since it not constitutively expressed, and instead has a more on/off expression pattern.
In order to have a more suitable CNS analogue a new C6 cell line could be acquired and tested, since our results are somewhat contradictory considering previous reports. It would also be interesting to investigate the contribution of carrier proteins such as FABP5 in stimulated/unstimulated cells, to elucidate if the reduction in AEA uptake in stimulated cells is a result of reduced carrier protein expression.
EXPERIMENTAL PROCEDURES
Materials
URB597 (cyclohexylcarbamic acid 3’-
carbamoylbiphenyl-3-yl ester), non-radioactive AEA and nimesulide (N-(4-Nitro-2-
phenoxyphenyl)methanesulfonamide) were purchased from Cayman Chemicals (Ann Harbour, MI USA). Flurbiprofen ((±)-2-fluoro-α- methyl(1,1′-biphenyl)-4-acetic acid) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). FluAM1 was synthesized by Cipriano et al (2013). Flurbiprofen and FluAM1 was dissolved in ethanol whereas nimesulide and URB597 was dissolved in DMSO.
Radioactive AEA [arachidonoyl
5,6,8,9,11,12,14,15-3H] (specific activity 200 Ci/mmol) was acquired from American Radiolabeled Chemicals Inc. (St. Lous, MO, USA). Mouse recombinant INF-γ was
purchased from Merck Millipore (Billerica MA, USA, lot 2453142) and LPS from E.coli Sigma- Aldrich (isotype 0111:B4). INF-γ was dissolved in a 10 mM phosphate buffer pH 8.0 and LPS was dissolved in PBS according to
manufacturers’ protocols.
Cell Lines and Growth conditions
All cells were cultured at 37°C and 5% CO2 in 75 cm2 culture flask in a humidified chamber.
C6 rat glioma cells were acquired from ECACC (Porton Down, United Kingdom) and used over a passage range of 28-45. C6 cells were grown in F10-Ham (Gibco, Grand Island NY USA) supplemented with 2mM glutamine, 100µg/ml streptomycin and 100 U/ml penicillin.
On day to day culturing, the media was supplemented with 10% Fetal Bovine Serum (FBS) purchased from Invitrogen life
technologies (Stockholm Sweden). In the experiments the C6 cells grew under serum- free conditions. The mouse macrophage
derived cell line Raw 264.7 was obtained from ECACC and grown in Dulbecco's modified Eagle's medium (DMEM) obtained from Sigma-Aldrich (St. Louis, MO USA)
supplemented with 2mM glutamine, 10% FBS, 100 U/ml penicillin and 100µg/ml of
streptomycin. Passages 5-20 were used for Raw 264.7. Fatty acid-free Bovine serum albumin (BSA) and standard BSA were acquired from Sigma Aldrich (St Louis, MO, USA).
Western blot & antibodies
Cells were plated in 6 well plates at a concentration of 2.5 x 105 cells/well and allowed to grow overnight. The following day, the wells were aspirated and fresh media added, along with 100 U/ml of INF-γ plus LPS (0.1 or 1.0 µg/ml) as well as vehicle controls.
Cells were collected at 24h and 48h. The cells were first washed with cold PBS and then lysed with NP40 lysis buffer (150 mM NaCl, 50 mM Tris, 0.1% Triton-X 100 pH 8.0) containing protease inhibitor cocktail III. Following lysis, the cellular contents were scraped off the bottom of the wells and collected into tubes.
The tubes were then shaken for 30 min at 4 °C before being sonicated with 5 pulses output 2, duty 50% using a Branson sonifier 185.
Following centrifugation (16 000 g x 5 min) at 4°C, the supernatants were collected and frozen at -80°C. Total protein concentration was determined using a BCA kit
(Thermoscientific Waltham, MA, USA) by reading absorbance at 562nm using a FLUOstar Omega (BMG Labtech) microplate reader. Aliquots (90 µg of C6 total protein or 30µg of Raw 264.7 total protein) along with recombinant COX-2 (cat. No. 60122, Cayman chemicals, Ann Arbor MI, USA) was loaded in respective wells and subsequently separated
on a SDS-PAGE gel. The separated proteins were then transferred to PVDF membranes where they were blocked in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% dry milk for 1h at room temperature on a tilting table. Following washing, primary antibodies acquired from Cayman chemicals (Ann Arbor MI, USA) were added and
incubated with the membranes overnight. The primary antibodies were polyclonal antibodies against COX-2 (1:1000 lot no 160106) and FAAH (1:1000 lot no 101600) obtained from Cayman Chemicals, and β-actin (1:1000 lot no 8827) obtained from Abcam (Cambridge, UK).
After primary antibody incubation, the membranes were again washed with TBST and then incubated with secondary antibody acquired from Dako (Copenhagen, Denmark, lot P0448) HRP goat anti-rabbit (1:2000) for one hour at RT. The antibody bound protein bands were revealed by chemiluminesence using a Bio-rad ChemiDoc XRS+ analyser after addition of Clarity Western ECL substrate (Bio-rad, Hercules CA USA). Initially, actin controls were used. However, the Bio-rad ChemiDoc XRS+ analyser can measure total protein and we used this measure instead to ensure equal well loading. The antibody bound protein bands were digitized and quantified using the Image J programme. In the case of the COX-2 experiments, the results (AUCs of the intensities) were expressed relative to the wells containing the recombinant COX-2. For the FAAH experiments, the 48 h control bands were set to unity and the other conditions expressed relative to this.
[3H]-Anadamide uptake assay
The assay used is that of Sandberg & Fowler (2005). The C6 cells were grown serum-free medium according to Esposito et al (2008) whereas the Raw 264.7 were grown with 10%
FBS culture medium according to Whan Han et al. (2001). The cells were plated in 24 well plates to an initial density of 2x105 cells/well and cultured for 48 h in the presence of 0.1µg/ml LPS + 100U/ml INF-γ or vehicle controls. Half of the wells contained the same cocktails, but contained no cells. After growth, the wells were washed with KRH buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10mM HEPES, 0.12 mM KH2PO4 and 0.12 mM MgSO4) with 1% BSA, and then washed with serum free KRH. KRH with 0.1% fatty-acid free BSA was added, and the cells were then incubated with respective inhibitor or vehicle control (solvent concentration 0.2% EtOH or 0.2% DMSO) for 10 min at 37°C. [3H]-AEA was added to a final concentration of 100 nM and then incubated in a water bath for 4 min at 37
°C. After incubation, the wells were washed three times with ice-cold KRH buffer containing 1% BSA, and after the last wash 0.2M NaOH was added. The cells were then subjected to heating at 75 °C for 15 min, and then allowed to adjust to room temperature. Aliquots (300 µL) from each well were collected and tritium uptake quantified by means of quench corrected scintillation spectroscopy.
Data presentation
Each experiment was repeated at least three times, with three observations each time per treatment. Results are presented as mean ± standard error of mean.
ACKNOWLEDGEMENTS
The author would like to especially thank Christopher Fowler, Jessica Karlsson and Mona Svensson for their kind support during the project. The author would also like to thank the rest of the staff at for the Department of Pharmacology and Clinical Neuroscience, Umeå University, Sweden for their kind support and their contribution to a fun and friendly atmosphere.
ETHICAL CONSIDERATIONS
The cell lines used in this study are well established murine immortalized cell lines which have limited ethical impact.
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