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In vitro morphine metabolism by rat microglia
Neuropharmacology 75 (2013) 391e398
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
In vitro morphine metabolism by rat microglia Anna Rita Togna a, Letizia Antonilli a, Melania Dovizio b, c, Adele Salemme a, Lorenza De Carolis a, Giuseppina I. Togna a, Paola Patrignani b, c, Paolo Nencini a, * a
Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Department of Neuroscience and Imaging, “G. d’Annunzio” University, Via dei Vestini 31, 66100 Chieti, Italy c Center of Excellence on Aging (CeSI), “Gabriele d’Annunzio” University Foundation, Via dei Vestini 31, 66100 Chieti, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2013 Received in revised form 7 August 2013 Accepted 15 August 2013
Morphine is mainly transformed to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in the liver. Glucuronidation is also performed by rat brain homogenates and UDPglucuronosyltransferases (UGTs) are present in the brain. Here we investigated the possibility that microglia transforms morphine into its metabolites M3G and M6G. Primary cultures of neonatal rat microglia were incubated for different intervals of time in basal conditions or with different concentrations of morphine. The following measures were performed on these cultures and/or in the medium: (i) morphine as well as M3G and M6G concentrations; (ii) levels of mRNA coding for UGT1A1, UGT1A6, UGT1A7, and UGT2B1 as well as their protein levels; (iii) released prostaglandin (PG)E2 and nitrite concentrations. Results show that in basal conditions morphine and M3G are produced by microglia; accordingly, these cells expressed UGT1A1, UGT1A6 and UGT1A7, but not UGT2B1. When cultures were exposed to different concentrations of exogenous morphine, M6G was also synthesized. This shift in the glucuronidation was associated with variations in the expression of UGT isozymes. In particular, UGT1A7 expression was rapidly upregulated and this event was translated into enhanced protein levels of UGT1A7; lesser effects were exerted on UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Morphine exposure did not affect the synthesis of both PGE2 and nitrites, ruling out a generalized priming of microglia by morphine. In conclusion, this study suggests that morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration may at least in part be brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the bloodebrain barrier. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Morphine-3-glucuronide Morphine-6-glucuronide UGT Microglia Rat
1. Introduction The main pathway of morphine clearance in the body consists in its glucuronidation to M3G and M6G, both of which, being more polar than the parent compound, are easily excreted by kidneys (Lötsch, 2005). Interestingly enough, both M3G and M6G own pharmacological properties, the first activating excitatory responses (Yaksh and Harty, 1988; Skarke et al., 2005), the second being a full MOR agonist (Pasternak, 2001; van Dorp et al., 2006). The clinical importance of the pharmacological activity of morphine glucuronides is outlined by evidence that M6G is involved in the increased toxicity of morphine observed in patients with renal failure (Lötsch, 2005). As a matter of fact, both M3G and
* Corresponding author. Tel.: þ39 06 4991 2497; fax: þ39 06 4450618. E-mail address: [email protected] (P. Nencini). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.08.019
M6G are found in the cerebrospinal liquor upon morphine administration and the current view is that morphine glucuronides, after being formed in the liver, eventually reach the brain, notwithstanding their scarce permeability to the blood brain barrier (Bickel et al., 1996). However, there is another possibility that deserves to be taken into consideration, i.e. that the brain produces morphine glucuronides making these metabolites immediately available for inducing their central effects. This possibility is supported by evidence that morphine is metabolized by homogenates of human (Wahlström et al., 1988; King et al., 1999) and rat (Suleman et al., 1998; Nagano et al., 2000; Antonilli et al., 2003) brain. UDP-glucuronosyltransferases (UGTs) are indeed present in the brain. In particular, UGT1A1 and UGT1A6 are expressed in the rat brain (Leclerc et al., 2002), whereas the human brain expresses UGT2B7 and UGT1A6 (King et al., 1999). Using an immunohistochemical technique, Martinasevic et al. (1998) found that UGT1A6 is localized in
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neuronal cells, but not in glial cells of the human brain. However, in the rat brain the same isozyme is mainly expressed by astrocytes (Suleman et al., 1998) where it is activated by inflammatory conditions (Heurtaux et al., 2006). Notably, although in a small extent, UGT1A1 participates to morphine glucuronidation in both humans (Ohno et al., 2008) and rats (King et al., 1997). The physiological role in the central nervous system of this enzymatic pathway is not clear, but we may speculate that among the many possible substrates of UGTs in the brain there is the endogenous morphine. Indeed, evidence has been repeatedly provided that brain synthesizes its own morphine and more recent findings show that endogenous morphine and codeine, along with M3G are present in both GABA-ergic neurons and astrocytes of the spinal cord, as well as in different cerebral areas of the mouse (Laux et al., 2011, 2012). Although little is known about the physiological functions of endogenous morphine, recently it has been firmly established that both morphine and M3G activate toll-like receptor 4 (TLR4) expressed by microglia, inducing the release of a wide array of inflammatory substances (Cui et al., 2008; Hutchinson et al., 2007). It is then possible that endogenous morphine belongs to the large family of agents that modulate the inflammatory activity of microglia. In this perspective, here we formulated the hypothesis that microglia has its own system of glucuronide formation. This would not be surprising because macrophages, the peripheral analogs of microglia, have been found to express UGT isozymes (Tochigi et al., 2005). Moreover, this hypothesis may explain why during traumatic brain inflammation, liquoral concentrations of M6G and M3G, but not of morphine, directly correlate with those of IL-6, a marker of glia activation (Roberts et al., 2009). In order to test the hypothesis that microglia contributes to the synthesis of morphine glucuronides by the brain, we exposed primary cultures of rat microglia to different concentrations of morphine. The expression of different glucuronyltransferase (UGT) isozymes potentially involved in the formation of M3G and M6G (i.e. UGT1A1, UGT1A6, UGT1A7, and UGT2B1) and the consequent synthesis of morphine glucuronides in these experimental conditions were then measured. 2. Materials and methods 2.1. Chemicals Dulbecco’s MEM, and D-MEM/F12 media, trypsin, penicillin and streptomycin, FCS (fetal calf serum) were purchased from Invitrogen (Paisley, Scotland). Lipopolysaccharide (LPS), DNAse I, antibody against b-actin were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Polyclonal anti-UGT1A1, UGT1A6 and UGT1A7 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody anti-Iba-1 was from Wako (Wako Pure Chemical Industries Ltd., Osaka, Japan). Western blot enhanced chemiluminescence detection system was from Bio-Rad Laboratory (Hercules, CA, USA). Morphine hydrochloridre and morphine-3-glucuronide (M3G) were purchased from S.A.L.A.R.S. (Como, Italy), morphine-6-glucuronide (M6G) was obtained from Sigma Aldrich (Milan, Italy). Acetonitrile, methanol, both gradient grades were purchased from Merck (Darmstadt, Germany) and ammonium formate from Sigma Aldrich (Milan, Italy). Ultrapure water was prepared using a Milli-Q system (Millipore, MA USA). 2.2. Cell cultures All the animal-related procedures were conducted in accordance with European Communities Council Directive n 86/609/EEC. Microglial cells were obtained from the cerebral cortex of 1- or 2-day old decapitated rats as previously described (Togna et al., 2013). Briefly the cortices were dissected and digested for 20 min at 37 C in 0.125% trypsin and for further 5 min in presence of 50 KU/ml of Dnase I. Cells were plated at a density of 4.5 104 cells/cm2 in T75 flasks in 10 ml D-MEM supplementing with 10% FCS and antibiotics (100 IU/ml of penicillin and 100 mg/ml of streptomycin). The medium was changed within 24 h, and then twice a week. After 10e14 days from dissection, microglia was detached from the astrocyte monolayer by shaking, and the cells re-suspended in D-MEM/F12 (10% FCS and antibiotics). Thereafter the
cells were placed in 24-well plastic plates at a density of 0.5 106 cells/ml, incubated at 37 C in a humidified atmosphere containing 5% CO2. Purity of microglial cell populations (>98%) was verified by staining with IBA-1 (1:1000) antibody. After 2 h the medium was replaced with 1 ml of fresh medium. 2.2.1. Evaluation of morphine, M3G and M6G in microglia cell cultures Microglial cells were cultured for 6 and 24 h with fresh medium containing saline or different morphine concentrations (2.5, 5.0 and 10 mM). Supernatants were then collected and 0.2 ml of ice-cold acetontrile was added to 0.3 ml of sample. The samples were then centrifuged for 5 min at 600 g and the resulting supernatants underwent solid phase extraction on reversed-phase/strong cation-exchange sorbent Strata-X-C (96-well plates, 30 mg) (Phenomenex, Torrance, CA). Cartridges were conditioned with methanol (0.6 ml) followed by water (0.6 ml) and phosphate buffer (0.01 M pH 3.0, 0.6 ml). The sample (0.2 ml) was applied to the column and absorbed by gravity; then the column was washed with phosphate buffer (0.01 M pH 3.0, 0.6 ml) and dried for 30 s. The analytes were eluted with 0.6 ml of NH4OH 1% in methanol. The eluate was evaporated to dryness at 37 C under a nitrogen stream. The residue was dissolved in 0.2 ml of 5 mM ammonium formate buffer (pH 4.0) and stored at 4 C until assayed for morphine and its metabolites M3G and M6G by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 2.2.2. Kinetics of morphine glucuronidation by microglia Microglia cells, treated for 6 h with medium containing saline, were lysed for 5 min with phosphate buffer 1M pH 7.4 and homogenate in the same buffer. Homogenates were incubated for 10 min with 0.05% deoxycholic acid at 5 C and used as the source of morphine glucuronidation. Enzymatic reactions were conducted at 37 C for 30 and 120 min in 0.1 M phosphate buffer pH 7.4. The incubation mixture consisted of morphine (0e2.5 mM), 100 mM phosphate buffer (pH 7.4) and cells homogenates (50 mg protein/ml) to a final volume of 0.3 ml. The reactions were terminated by addition of ice-cold acetonitrile and centrifuged for 5 min at 600 g. The resulting supernatants were extracted on reversed-phase/strong cation-exchange sorbent as described above. The extracts were stored at 20 C until assayed for M3G and M6G by LC/ MS/MS. 2.3. Liquid chromatography-tandem mass spectrometry The HPLC system consisted of a PerkinElmer 200 Series binary pump and autosampler (PerkinElmer, Norwalk, CT, USA) and an SCIEX API2000 MS/MS triple quadrupole mass spectrometer (Applied Biosystem-MDS SCIEX, Thornhill, Ontario, Canada). Chromatographic separation was performed on a Synergi Polar RP column (150 2.0 mm, 4 mm), protected by a guard column with identical packing material (4 2.0 mm; Phenomenex, Torrance, CA, USA) The mobile phase consisted of a linear gradient (3e80% with respect to acetonitrile) formed by combination of 5 mM ammonium formate buffer in water (pH 4.0, eluent A) and acetonitrile (eluent B). Flow rate of the mobile phase was set at 0.2 ml/min. Morphine, M3G, and M6G were detected using multiple reaction monitoring (MRM) in positive ionization mode. Selected ion masses of the protonated precursors and fragmented ions (m/z) were 286.3/201.0 for morphine and 462.2/286.0 for M3G and M6G. Chromatographic peaks were integrated using AnalystÔ software (version 1.4.1, SCIEX). The detection limits (LOD) and quantification limits (LOQ) for all analytes were 1.5 and 3.4 pg/ml. 2.4. mRNA expression of UGTs by quantitative (q)RT-PCR Microglial cells were cultured for 1, 3 and 6 h in the absence and presence of morphine 5 mM. Total RNA was extracted from 2 106 cells using the total RNA purification kit (Norgen Biotek Corp., Ontario, Canada), according to the manufacturer’s protocols. Two mg of total RNA were treated with DNAse kit (Fermentas, St. Leon-Rot, Germany) and subsequently reverse transcribed into cDNA using IscriptcDNA Synthesis Kit (Bio-rad Laboratories, CA, USA), according to the manufacturer’s protocols. One hundred ng of cDNA was used for the reaction mixture and the amplification of UGT1A1, UGT1A6, UGT1A7 and UGT2B1 was performed using TaqMan gene expression assay’s (Rn00754947_m1; Rn00756113_mH, Rn02749780_s1; Rn00756519_m1, respectively) (Applied Biosystem, CA, USA), as per the manufacturer’s instructions using a 7900HT Real-Time PCR system (Applied Biosystems). Expression levels of UGT mRNAs were normalized with those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Rn99999916_s1)(Applied Biosystem). Gene expression assays were performed by relative quantification with comparative Ct using SDS 2.4 software (Applied Biosystem). 2.5. Protein expression of UGTs by Western blot analysis Microglial cells were cultured for 6 and 24 h in the absence and presence of morphine 5 mM. Then, the cells were washed with PBS three times, and lysed, by adding icecold lysis buffer (10 mmol/L TriseHCl, pH 7.5; 150 mmol/L NaCl; 1 mmol/L EDTA; 50 mmol/L sodium fluoride; 1% Triton X-100; 10% glycerol; 1 mmol/L sodium orthovanadate; 1 mmol/L phenylmethanesulfonyl fluoride; 25 mmol/L glycerol-
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Fig. 1. A. Generation of morphine and M3G by microglia cells under basal conditions. Cells (0.5 106) were cultured for 6 or 24 h and the culture medium was assayed for morphine and M3G concentration. B. Morphine levels were detected in the extracellular medium (extracellular) and intracellular compartment (intracellular) of microglial cells (0.5 106 cells) cultured for 6 and 24 h. All data are expressed as mean values S.E.M. of three different experiments. In panel A, **P < 0.01 vs 6 h; xP < 0.01 vs morphine; in panel B, **P < 0.01 vs extracellular levels at the same time; xP < 0.01 vs extracellular (6 h) and #P < 0.01 vs extracellular (24 h).
2-phosphate; 10 mg/mL aprotinin and 1 mg/mL leupeptin) for 10 min. Lysates were then centrifuged at 1000 g for 10 min a 4 C. Total protein content of cell lysates was assessed using the Bradford method. Equal amount of proteins (30 mg) were electrophoresed on a 10% SDS-polyacrylamide gel (Mini-PROTEAN II apparatus, Bio-Rad Laboratories) before being transferred onto nitrocellulose membranes with the Mini-Trans blot system (Bio-Rad Laboratories). Membrane was blocked with 5% non-fat milk and incubated overnight at 4 C with polyclonal primary antibodies against UGT1A1 (diluted 1:200),UGT1A6 (1:200) or UGT1A7 (1:200). Membranes were then washed and incubated with a peroxidase-coniugated antirabbit secondary antibody (1:1000) for 1 h at room temperature using chemiluminescence (ECL) reagents for detection. To ensure equal protein loading, the membranes were separately probed for b-actin protein. Scanning densitometry was performed using the ImageJ 1.47 program, and signal density was normalized to b-actin density.
2.6. Biosynthesis of PGE2 and nitric oxide (NO) Microglial cells (0.5 106 cells), were incubated with LPS 10 ng/ml or morphine 5 mM, alone or together, for 6 h. PGE2 biosynthesis was assessed in culture medium by previously described, validated, and specific radioimmunoassay technique (Patrignani et al., 1994). The production of NO was measured as accumulated nitrite (NO 2 ), one of the stable end products of NO, by a Griess reaction. Briefly, a volume of conditioned culture medium from each sample was mixed with the same volume of the Griess reagent [50 mM sulfanylamide, 5% H3PO4 and 3 mM N-(1-naphthyl) ethylenediamine dihydrocloride]. The amount of NO-2 was measured through the determination of A540 nm in a microplate spectrophotometer.
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Fig. 2. Kinetic of M3G and M6G formations by microglia cells incubated for 30 min (A) or 120 min (B) with increasing concentration of morphine (0e2.5 mM). Each data points represent an average of triplicate determinations S.E.M.
2.7. Statistics All values were reported as mean SEM. Statistical analysis was performed using GraphPad Prism Software (version 5.00 for Windows, GraphPad, San Diego, California). One-way ANOVA followed by Newman-Keuls post-test was used to compare the means of more than two independent groups. Values of P < 0.05 were considered statistically significant.
3. Results Microglial cells cultured for 6 h released detectable levels of morphine (129 12 pmol/0.5 106 cells, n ¼ 12) and M3G (13.4 1.8 pmol/0.5 106 cells, n ¼ 12) while M6G was undetectable (<3.2 pmol/0.5 106 cells) (Fig. 1A). In contrast, neither morphine nor M3G were detected in medium incubated without cells (data not shown). Hence, in our experimental conditions, primary microglia cultures of the rat both synthesized morphine and transformed it in its glucuronide M3G (Fig. 1A). At 24 h of culture, the levels of morphine detected in the medium of microglial cells were profoundly reduced while M3G levels remained stable and M6G was undetectable (Fig. 1A). In a further experiment, we measured the intra- and extracellular morphine content at both 6 and 24 h (Fig. 1B). At 6 h, morphine was mostly present in the medium and accounted for 132.3 7.5 pmol/0.5 106 cells, whereas the intracellular levels were significantly lower (2.0 0.5 pmol/0.5 106 cells; P < 0.01); at 24 h, extracellular morphine levels (86.8 2.8 pmol/0.5 106 cells) were significantly (P < 0.01) lower than those detected at 6 h. At the
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Fig. 4. Effect of morphine on UGT1A1 mRNA (A) and protein (B, C) expression in rat microglial cells. Primary rat microglia cultures were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A1 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A1 (B), protein expression by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of bactin (C). Values are expressed as mean SEM of four experiments. In panel A, **P < 0.01 vs vehicle (1 h); *P < 0.05 vs morphine (1 h); xP < 0.01 vs vehicle (1 h). Fig. 3. M3G (A) and M6G (B) formation by microglial cells exposed to different concentrations of morphine for 6 and 24 h. Data are expressed as mean S.E.M. of three different experiments. In panel A, **P < 0.01 vs vehicle; xP < 0.05 vs 5 mM; *P < 0.05 vs vehicle; #P < 0.05 vs 2.5 mM and 5 mM; f P<0.01 vs 10 mM (6 h). In panel B, @P < 0.01 vs 2.5 mM (6 h); f P<0.05 vs 2.5 mM (6 h). (C) Ratio between M3G and M6G concentrations. *P < 0.05 vs 6 h. Data are expressed as mean S.E.M.
same time point, the levels of intracellular morphine (47.7 3.9 pmol/0.5 106 cells) were significantly higher that those detected at 6 h (P < 0.01). Summing up, on the long run morphine synthesis appears to be stopped and its content redistributed across the cell membrane. In order to exclude that astrocytes were the real source of endogenous morphine and that microglia takes morphine up before being isolated, we measured intra- and extra-cellular levels of morphine up to 2 h following microglia isolation. In these conditions, we found that intracellular morphine concentrations were below assay detection limit whereas in the medium low levels were detected (5.54 0.62 pmol/0.5 106 cells, n ¼ 6). Altogether our results show that microglial cells can synthesize, release and re-
uptake morphine; moreover they can transform morphine in its major metabolite M3G. Then, we aimed to study the capacity of microglial cells to glucuronide exogenously added morphine (Fig. 2A and B). We used morphine concentrations that are in the range of plasmalevels associated with analgesic effects (Smith et al., 2000). In these conditions, glucuronidation was extended to the formation of both M3G and M6G occurring with saturable kinetics (Fig. 2A and B). It is worth noting that at 120 min both kinetics did not fit MichaelisMenten equation showing sigmoid shape curves with values of the Hill coefficient of 5.32 and 2.13 for M3G and M6G, respectively. These values suggest positive cooperation between different enzyme molecules. Altogether our results suggest that microglial cells express UGTs that metabolize morphine to M3G and M6G. M3G is the major product generated at a fast rate but at longer incubation times with the opioid, a substantial concentration of M6G is produced which is 40% of M3G.
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Fig. 5. Effect of morphine on UGT1A6 mRNA (A) and protein (B, C) expression in rat microglial cells. Cells were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A6 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A6 (B), protein expressions by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of b-actin (C). Values are expressed as mean S.E.M. of four experiments. In panel A, **P < 0.01 vs vehicle (1 h); xP < 0.01 vs morphine (1 h); @P < 0.02 vs vehicle (6 h). In panel C, *P < 0.05 vs vehicle (24 h); xP < 0.05 vs vehicle (6 h).
Then, we aimed to study whether the capacity of microglial cells to metabolize exogenously added morphine to M3G and M6G may increase in a time-dependent fashion in association with changes of UGTs expression. As shown in Fig. 3A, when cultures were exposed for 6 h to micromolar concentrations (2.5, 5 and 10 mM) of morphine, M3G levels were significantly increased by roughly 2fold, at each concentration, versus baseline levels. Moreover, microglial cells exposed to morphine released M6G that increased in a concentration-dependent fashion (Fig. 3B). At 24 h of microglial cultures, baseline M3G levels were similar to those detected at 6 h (Fig. 3A). Differently, after exposure to exogenous morphine for 24 h, M3G levels were significantly decreased versus those detected at 6 h (Fig. 3A). The levels of M3G resulted marginally increased versus baseline values. In contrast, M6G levels were not significantly different from those measured at 6 h of incubation with morphine 5 and 10 mM (Fig. 3B).
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Fig. 6. Effect of morphine on UGT1A7 mRNA (A) and protein (B, C) expression in rat microglial cells. Cells were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A7 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A7 (B), protein expressions by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of b-actin (C). Values are expressed as mean SEM of four experiments. In panel A, *P < 0.05 vs vehicle (1 h); **P < 0.01 vs vehicle (1 h); xP < 0.01 vs morphine (1 h); @P < 0.05 vs vehicle (1 h); f P ¼ 0.056 vs vehicle (3 h); #P < 0.01 vs vehicle (6 h). In panel C, **P < 0.01 vs vehicle (6 h); *P < 0.05 vs morphine (6 h).
As shown in Fig. 3C, in microglial cells incubated for 6 h with morphine 2.5, 5 and 10 mM, M3G/M6G ratio values were approximately equal to three. After 24 h of incubation with morphine, M3G/M6G ratios decreased in a concentration-dependent fashion. In the presence of morphine 10 mM, M3G/M6G ratio was 1.4 0.05 (n ¼ 10), showing that M6G concentrations were not so far distant from those of M3G (Fig. 3C). Next we aimed to verify whether the exposure of microglial cells to morphine (5 mM) led to changes of mRNA and protein expression and of UGT1A1, UGT1A6, UGT1A7 and UGT2B1, which are involved in morphine metabolism (De Gregori et al., 2012). At 1 h of culture of rat microglial cells without morphine, baseline mRNAs for UGT1A1, UGT1A6, and UGT1A7 were detected whereas mRNA for UGT2B1 was undetectable. The levels of these three mRNAs increased in a time-dependent fashion up to 6 h (Fig. 4 A, 5 A and 6 A, respectively). This was associated with a constitutive protein expression of UGT1A1, 1A6 and 1A7 (Fig. 4B,C, 5B,C and 6B,C). At 6
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M3G may induce a pro-inflammatory response through the release of PGE2 as well as of nitrites. The effects of morphine were compared to those of a known inflammation inducer, i.e. LPS. As shown in Fig. 7 A and B, microglial cells exposed for 24 h to LPS were associated with enhanced release of both PGE2 and nitrites while morphine did not affect their synthesis neither in basal conditions neither under LPS stimulation. Moreover, exposing microglia to two different concentrations of LPS (10 and 50 ng/ml) did not affect basal levels of endogenous morphine or change morphine glucuronidation in presence of the drug (data not shown). 4. Discussion
Fig. 7. Effect of LPS (10 ng/ml) and morphine (5 mM)(MORF) on inducible PGE2 generation (A) and nitrite production (B) in rat microglial cells. Microglial cells were incubated with LPS or morphine alone or together for 6 h. PGE2 release was assessed in the medium by immunoassay and nitrite production by a Griess reaction. A and B: **P < 0.01 vs vehicle or morphine alone.
and 24 h of cultures, microglial cells expressed comparable protein levels of UGT1A1 and UGT1A7 (Fig. 4B,C and Fig. 6B, C). UGT1A6 protein levels were significantly, slightly increased at 24 h versus 6 h (Fig. 5B, C). The exposure of cells to morphine was associated with a rapid (at 1 h) increase of mRNA levels of UGT1A1 and UGT1A7 versus baseline values that declined at 3 h (Figs. 4A and 6A, respectively). At 6 h of incubation without morphine, mRNAs of UGT1A1 and UGT1A7 were maximally increased by 70- and 5.5-fold, respectively (versus 1 h). The presence of morphine constrained the enhanced expression of these mRNAs (Figs. 4A and 6A). UGT1A6 mRNA levels were not significantly affected by morphine exposure up to 3 h while at 6 h they were significantly reduced versus the levels detected in the absence of morphine, at the same time-point (Fig. 5A). Protein expression of the three isozymes was assessed after 6 and 24 h of exposure to morphine (Fig. 4B and C, 5B and C, 6B and C). UGT1A7 was the only UGT significantly increased at 6 h (Fig. 6B and C). At 24 h of exposure with morphine, all UGT protein levels tended to be lower than those detected at the same time-point without morphine; however, only UGT1A6 levels were significantly reduced (Fig. 5B and C). No significant change in the content of UGT1A1 was detected upon exposure to morphine (Fig. 4B and C). Altogether these results suggest that mRNAs of UGT1A1, 1A6 and 1A7 were constitutively expressed in primary rat microglial cells and that morphine modulates the rate of their expression. Interaction of morphine with the MOR and/or TLRs has been associated with the induction of pro-inflammatory response and oxidative stress (Dave and Khalili, 2010). Thus, we addressed the hypothesis that morphine by itself and/or through the formation of
Our study provides three major findings: i) morphine and its metabolite M3G are detectable in primary cultures of rat microglia; ii) the presence of M3G is consistent with the expression of different UGT isozymes, namely UGT1A1, UGT1A6, UGT1A7; iii) glucuronidation is extended to M6G formation when microglia is exposed to micromolar concentrations of exogenous morphine; iv) this was associated with substantial changes in the expression of these three UGT genes and enhanced protein expression of UGT1A7. At the best of our knowledge, this is the first report showing the presence of morphine and M3G in cultures of primary microglia of rats, extending to microglia the capability of forming endogenous morphine, already demonstrated in both GABAergic neurons and astrocytes (Laux et al., 2011, 2012). In our experimental conditions, endogenous morphine showed a time-dependent redistribution between extra and intra cellular space suggesting the existence of an active transport of the molecule, consistently with previous findings that morphine is taken up by different cell types in a timedependent manner (Déchelotte et al., 1993). The co-presence of M3G suggests the existence in the microglia of a clearance system capable of disposing endogenous morphine and here we give evidence that this system consists in the presence of constitutive UGT isozymes. That microglia can express UGT1A1, UGT1A6, as well as UGT1A7 is not surprising since the same UGT isozymes have been detected in macrophages that share with microglia mesodermic origin and inflammatory functions (Tochigi et al., 2005). Hence, it is not a chance that both microglia and macrophages do not express UGT2B1 (this study and Tochigi et al., 2005). Although our results are apparently in contrast with the common opinion that in the rat UGT2B1 is the only catalyser of M3G formation and that M6G is not formed at all (Milne et al., 1996), several findings have already weakened this notion. First, it has been demonstrated that UGT1A1, although with a limited activity, also contributes to M3G formation (King et al., 1997; Ishii et al., 1997; Ohno et al., 2008). Moreover, recombinant human UGT1A1 has been found to catalyse M6G formation at therapeutic morphine concentrations (10e100 mM), whereas at higher concentrations M6G formation exhibited substrate inhibition kinetics (Ohno et al., 2008). Finally, both M3G and M6G are formed when morphine is added to homogenates of rat brain (Nagano et al., 2000). Consistently with these findings, when we exposed microglia cultures to concentrations of exogenous morphine that corresponded to the upper analgesic range in both humans (Tiseo et al., 1995; Klepstad et al., 2003, 2004) and rats (Mas et al., 2000; Smith et al., 2000), we found that M3G synthesis increased by 2-fold over control levels and that glucuronidation was extended to the formation of M6G, otherwise undetectable in basal conditions. Prolonging the exposure to morphine up to 24 h, M3G concentrations even declined whereas M6G formation remained stable. As a consequence M3G/ M6G ratio was progressively reduced, approximating the unit when the culture were exposed for 24 h to the higher morphine concentration (10 mM). A different timing in the formation of the two
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metabolites is also suggested by the results of the experiment wherein microglia homogenates were incubated with increasing concentrations of morphine for 30 and 120 min M3G formation developed much faster than that of M6G: from 30 to 120 min Vmax of M3G formation just doubled whereas that of M6G showed a 6 time increase. In microglia homogenates, Vmax of both M3G and M6G formation was in the picomolar order, that were quite lower with respect to the Vmax of M3G formation by liver microsomes (i.e. nanomolar range) (Antonilli et al., 2003, 2005). Yet, it was close to the physiological concentrations of endogenous morphine and sufficient to permit an incremental formation of morphine glucuronides upon the exposure to the drug. As already mentioned, during traumatic brain inflammation, liquoral concentrations of M6G and M3G, but not of morphine, directly correlate with those of IL-6, a marker of glia activation (Roberts et al., 2009). Our results point to microglia as a possible source of morphine glucuronides when the brain is exposed to therapeutic concentrations of the parent compound. Morphine glucuronidation is consistent with the expression of different UGT isozymes by microglia. Although we did not attempt to assess what was the contribution that the three UGT isoforms gave to morphine glucuronidation, some cues may be drawn from the shape of saturation curves of M3G and M6G formation (see Fig. 2). Both of them showed a point of flex and then deviated from the exponential reaction curve representing the result of monomeric enzymatic activity, fully described by the MichaeliseMenten equation. Sigmoid saturation curves are considered to reflect homo- or ethero-cooperation between enzymatic molecules, as it is the case of estradiol-3-glucuronide formation due to homotropic UGT1A1 activation, which in turn it depends on the multimeric structure of this enzyme. The binding of a molecule of substrate to a subunit produces an increase in the affinity of a neighbouring subunit for the substrate (Senafi et al., 1994; Williams et al., 2002; Ghosh et al., 2001; Antonilli et al., 2008). It is then possible that a likewise homotropic UGT1A1 activation is involved in the formation of both M3G and M6G by microglia. More studies are request to test this possibility. An important finding of the present study is that the exposure to morphine led to a rapid upregulation of the expression of genes coding for UG1A1 and UGT1A7, which at 6 h translated into enhanced protein levels of UGT1A7, but not of UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Incidentally, although changes of UGT expression were temporally coincident with the enhanced release of M3G and M6G, we should be cautious in considering the two events causally related since UGT1A7 has been found to be almost inactive in catalysing morphine glucuronidation (Ohno et al., 2008). We have already found relevant changes in the induction of genes coding for the UGT isozymes here examined, as well as for UGT2B1, by hepatocytes exposed to either heroin or codeine (Antonilli et al., 2012). Mechanisms responsible for these modulations of UGT expression are not clear but it is well known that prolonged exposure to morphine causes a host of changes in the cellular signalling. For instance, interesting results have shown that morphine has an important role in the regulation of different microRNAs (miRNA) involved in the expression of morphine receptor (MOR), such as miR-let-7, which works as a mediator of the movement of the MOR mRNA into P-bodies, leading to translational repression, miR-23b, which is involved in linking MOR expression and morphine treatment at the post-transcriptional level (reviewed in Rodríguez, 2012). Further studies are necessary to verify whether morphine-induced changes in these miRNAs play a role in the regulation of UGT levels in microglial cells.
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Importantly, morphine-induced modulation of UGT expression does not seem to belong to a generalized priming of microglia since in the present experimental conditions the drug failed to affect the synthesis of both PGE2 and nitrites either under basal circumstances or upon LPS stimulation. Moreover, exposing microglia cultures to LPS did not alter the rate of morphine glucuronidation. Altogether these results suggest that at least in microglial cells the generation of M3G is independent from the signalling that triggers the upregulation of cyclooxygenase-2 involved in delayed generation of PGE2 occurring in inflammatory responses (Ricciotti and FitzGerald, 2011). 5. Conclusions In conclusion, here we provide evidence that glucuronidation belongs to the wide array of functions exerted by microglia; whether this function is only addressed to endogenous morphine or is also aimed to clear other brain-born compounds, such as neurosteroids, is presently unknown. It is most interesting that primary microglia cultures respond to pharmacological concentrations of morphine changing the expression of different UGT isozymes and expanding glucuronidation to the 6-OH moiety, i.e. forming M6G. Although in our experimental conditions microglia transformed morphine into M6G at a rather low rate, the amount of the metabolite may be pharmacologically significant, taking into account the finding that in the rat brain M6G, unlike morphine, is mainly distributed in the extracellular space and very little into neurons; hence, the actual concentration of the metabolite available for receptor binding is close to that of morphine upon systemic administration of the drug (Stain-Texier et al., 1999). It is then possible that at least in part morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration are brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the bloodebrain barrier. Moreover, evidence that microglia can form highly active morphine metabolites incentives more study on the role of these metabolites in the overall effects produced by chronic exposure to phenanthrenic compounds, such as heroin and codeine, which converge into the common process of morphine formation and metabolism. Acknowledgements This work was supported by the Sapienza University of Rome intramural grant C26A11ATBL to PN and by MIUR(ex 60%) to PP. The authors wish to thank Sabina Moschini for technical assistance in cells culturing and Francesca Filipponi for her skilful technical assistance in the analytical procedure for glucuronide determinations. References Antonilli, L., Suriano, C., Paolone, G., Badiani, A., Nencini, P., 2003. Repeated exposures to heroin and/or cadmium alter the rate of formation of morphine glucuronides in the rat. J. Pharmacol. Exp. Ther. 307, 651e660. Antonilli, L., Petecchia, E., Caprioli, D., Badiani, A., Nencini, P., 2005. Effect of repeated administrations of heroin, naltrexone, methadone, and alcohol on morphine glucuronidation in the rat. Psychopharmacology (Berl) 182, 52e64. Antonilli, L., Brusadin, V., Milella, M.S., Sobrero, F., Badiani, A., Nencini, P., 2008. In vivo chronic exposure to heroin or naltrexone selectively inhibits liver microsome formation of estradiol-3-glucuronide in the rat. Biochem. Pharmacol. 76, 672e679. Antonilli, L., De Carolis, L., Brusadin, V., Togna, A.R., Dovizio, M., Togna, G.I., Patrignani, P., Nencini, P., 2012. Repeated exposure to codeine alters morphine glucuronidation by affecting UGT gene expression in the rat. Eur. J. Pharmacol. 693, 7e14. Bickel, U., Schumacher, O.P., Kang, Y.S., Voigt, K., 1996. Poor permeability of morphine 3-glucuronide and morphine 6-glucuronide through the blood-brain barrier in the rat. J. Pharmacol. Exp. Ther. 278, 107e113.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
In vitro morphine metabolism by rat microglia Anna Rita Togna a, Letizia Antonilli a, Melania Dovizio b, c, Adele Salemme a, Lorenza De Carolis a, Giuseppina I. Togna a, Paola Patrignani b, c, Paolo Nencini a, * a
Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Department of Neuroscience and Imaging, “G. d’Annunzio” University, Via dei Vestini 31, 66100 Chieti, Italy c Center of Excellence on Aging (CeSI), “Gabriele d’Annunzio” University Foundation, Via dei Vestini 31, 66100 Chieti, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2013 Received in revised form 7 August 2013 Accepted 15 August 2013
Morphine is mainly transformed to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in the liver. Glucuronidation is also performed by rat brain homogenates and UDPglucuronosyltransferases (UGTs) are present in the brain. Here we investigated the possibility that microglia transforms morphine into its metabolites M3G and M6G. Primary cultures of neonatal rat microglia were incubated for different intervals of time in basal conditions or with different concentrations of morphine. The following measures were performed on these cultures and/or in the medium: (i) morphine as well as M3G and M6G concentrations; (ii) levels of mRNA coding for UGT1A1, UGT1A6, UGT1A7, and UGT2B1 as well as their protein levels; (iii) released prostaglandin (PG)E2 and nitrite concentrations. Results show that in basal conditions morphine and M3G are produced by microglia; accordingly, these cells expressed UGT1A1, UGT1A6 and UGT1A7, but not UGT2B1. When cultures were exposed to different concentrations of exogenous morphine, M6G was also synthesized. This shift in the glucuronidation was associated with variations in the expression of UGT isozymes. In particular, UGT1A7 expression was rapidly upregulated and this event was translated into enhanced protein levels of UGT1A7; lesser effects were exerted on UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Morphine exposure did not affect the synthesis of both PGE2 and nitrites, ruling out a generalized priming of microglia by morphine. In conclusion, this study suggests that morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration may at least in part be brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the bloodebrain barrier. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Morphine-3-glucuronide Morphine-6-glucuronide UGT Microglia Rat
1. Introduction The main pathway of morphine clearance in the body consists in its glucuronidation to M3G and M6G, both of which, being more polar than the parent compound, are easily excreted by kidneys (Lötsch, 2005). Interestingly enough, both M3G and M6G own pharmacological properties, the first activating excitatory responses (Yaksh and Harty, 1988; Skarke et al., 2005), the second being a full MOR agonist (Pasternak, 2001; van Dorp et al., 2006). The clinical importance of the pharmacological activity of morphine glucuronides is outlined by evidence that M6G is involved in the increased toxicity of morphine observed in patients with renal failure (Lötsch, 2005). As a matter of fact, both M3G and
* Corresponding author. Tel.: þ39 06 4991 2497; fax: þ39 06 4450618. E-mail address: [email protected] (P. Nencini). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.08.019
M6G are found in the cerebrospinal liquor upon morphine administration and the current view is that morphine glucuronides, after being formed in the liver, eventually reach the brain, notwithstanding their scarce permeability to the blood brain barrier (Bickel et al., 1996). However, there is another possibility that deserves to be taken into consideration, i.e. that the brain produces morphine glucuronides making these metabolites immediately available for inducing their central effects. This possibility is supported by evidence that morphine is metabolized by homogenates of human (Wahlström et al., 1988; King et al., 1999) and rat (Suleman et al., 1998; Nagano et al., 2000; Antonilli et al., 2003) brain. UDP-glucuronosyltransferases (UGTs) are indeed present in the brain. In particular, UGT1A1 and UGT1A6 are expressed in the rat brain (Leclerc et al., 2002), whereas the human brain expresses UGT2B7 and UGT1A6 (King et al., 1999). Using an immunohistochemical technique, Martinasevic et al. (1998) found that UGT1A6 is localized in
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neuronal cells, but not in glial cells of the human brain. However, in the rat brain the same isozyme is mainly expressed by astrocytes (Suleman et al., 1998) where it is activated by inflammatory conditions (Heurtaux et al., 2006). Notably, although in a small extent, UGT1A1 participates to morphine glucuronidation in both humans (Ohno et al., 2008) and rats (King et al., 1997). The physiological role in the central nervous system of this enzymatic pathway is not clear, but we may speculate that among the many possible substrates of UGTs in the brain there is the endogenous morphine. Indeed, evidence has been repeatedly provided that brain synthesizes its own morphine and more recent findings show that endogenous morphine and codeine, along with M3G are present in both GABA-ergic neurons and astrocytes of the spinal cord, as well as in different cerebral areas of the mouse (Laux et al., 2011, 2012). Although little is known about the physiological functions of endogenous morphine, recently it has been firmly established that both morphine and M3G activate toll-like receptor 4 (TLR4) expressed by microglia, inducing the release of a wide array of inflammatory substances (Cui et al., 2008; Hutchinson et al., 2007). It is then possible that endogenous morphine belongs to the large family of agents that modulate the inflammatory activity of microglia. In this perspective, here we formulated the hypothesis that microglia has its own system of glucuronide formation. This would not be surprising because macrophages, the peripheral analogs of microglia, have been found to express UGT isozymes (Tochigi et al., 2005). Moreover, this hypothesis may explain why during traumatic brain inflammation, liquoral concentrations of M6G and M3G, but not of morphine, directly correlate with those of IL-6, a marker of glia activation (Roberts et al., 2009). In order to test the hypothesis that microglia contributes to the synthesis of morphine glucuronides by the brain, we exposed primary cultures of rat microglia to different concentrations of morphine. The expression of different glucuronyltransferase (UGT) isozymes potentially involved in the formation of M3G and M6G (i.e. UGT1A1, UGT1A6, UGT1A7, and UGT2B1) and the consequent synthesis of morphine glucuronides in these experimental conditions were then measured. 2. Materials and methods 2.1. Chemicals Dulbecco’s MEM, and D-MEM/F12 media, trypsin, penicillin and streptomycin, FCS (fetal calf serum) were purchased from Invitrogen (Paisley, Scotland). Lipopolysaccharide (LPS), DNAse I, antibody against b-actin were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Polyclonal anti-UGT1A1, UGT1A6 and UGT1A7 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody anti-Iba-1 was from Wako (Wako Pure Chemical Industries Ltd., Osaka, Japan). Western blot enhanced chemiluminescence detection system was from Bio-Rad Laboratory (Hercules, CA, USA). Morphine hydrochloridre and morphine-3-glucuronide (M3G) were purchased from S.A.L.A.R.S. (Como, Italy), morphine-6-glucuronide (M6G) was obtained from Sigma Aldrich (Milan, Italy). Acetonitrile, methanol, both gradient grades were purchased from Merck (Darmstadt, Germany) and ammonium formate from Sigma Aldrich (Milan, Italy). Ultrapure water was prepared using a Milli-Q system (Millipore, MA USA). 2.2. Cell cultures All the animal-related procedures were conducted in accordance with European Communities Council Directive n 86/609/EEC. Microglial cells were obtained from the cerebral cortex of 1- or 2-day old decapitated rats as previously described (Togna et al., 2013). Briefly the cortices were dissected and digested for 20 min at 37 C in 0.125% trypsin and for further 5 min in presence of 50 KU/ml of Dnase I. Cells were plated at a density of 4.5 104 cells/cm2 in T75 flasks in 10 ml D-MEM supplementing with 10% FCS and antibiotics (100 IU/ml of penicillin and 100 mg/ml of streptomycin). The medium was changed within 24 h, and then twice a week. After 10e14 days from dissection, microglia was detached from the astrocyte monolayer by shaking, and the cells re-suspended in D-MEM/F12 (10% FCS and antibiotics). Thereafter the
cells were placed in 24-well plastic plates at a density of 0.5 106 cells/ml, incubated at 37 C in a humidified atmosphere containing 5% CO2. Purity of microglial cell populations (>98%) was verified by staining with IBA-1 (1:1000) antibody. After 2 h the medium was replaced with 1 ml of fresh medium. 2.2.1. Evaluation of morphine, M3G and M6G in microglia cell cultures Microglial cells were cultured for 6 and 24 h with fresh medium containing saline or different morphine concentrations (2.5, 5.0 and 10 mM). Supernatants were then collected and 0.2 ml of ice-cold acetontrile was added to 0.3 ml of sample. The samples were then centrifuged for 5 min at 600 g and the resulting supernatants underwent solid phase extraction on reversed-phase/strong cation-exchange sorbent Strata-X-C (96-well plates, 30 mg) (Phenomenex, Torrance, CA). Cartridges were conditioned with methanol (0.6 ml) followed by water (0.6 ml) and phosphate buffer (0.01 M pH 3.0, 0.6 ml). The sample (0.2 ml) was applied to the column and absorbed by gravity; then the column was washed with phosphate buffer (0.01 M pH 3.0, 0.6 ml) and dried for 30 s. The analytes were eluted with 0.6 ml of NH4OH 1% in methanol. The eluate was evaporated to dryness at 37 C under a nitrogen stream. The residue was dissolved in 0.2 ml of 5 mM ammonium formate buffer (pH 4.0) and stored at 4 C until assayed for morphine and its metabolites M3G and M6G by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 2.2.2. Kinetics of morphine glucuronidation by microglia Microglia cells, treated for 6 h with medium containing saline, were lysed for 5 min with phosphate buffer 1M pH 7.4 and homogenate in the same buffer. Homogenates were incubated for 10 min with 0.05% deoxycholic acid at 5 C and used as the source of morphine glucuronidation. Enzymatic reactions were conducted at 37 C for 30 and 120 min in 0.1 M phosphate buffer pH 7.4. The incubation mixture consisted of morphine (0e2.5 mM), 100 mM phosphate buffer (pH 7.4) and cells homogenates (50 mg protein/ml) to a final volume of 0.3 ml. The reactions were terminated by addition of ice-cold acetonitrile and centrifuged for 5 min at 600 g. The resulting supernatants were extracted on reversed-phase/strong cation-exchange sorbent as described above. The extracts were stored at 20 C until assayed for M3G and M6G by LC/ MS/MS. 2.3. Liquid chromatography-tandem mass spectrometry The HPLC system consisted of a PerkinElmer 200 Series binary pump and autosampler (PerkinElmer, Norwalk, CT, USA) and an SCIEX API2000 MS/MS triple quadrupole mass spectrometer (Applied Biosystem-MDS SCIEX, Thornhill, Ontario, Canada). Chromatographic separation was performed on a Synergi Polar RP column (150 2.0 mm, 4 mm), protected by a guard column with identical packing material (4 2.0 mm; Phenomenex, Torrance, CA, USA) The mobile phase consisted of a linear gradient (3e80% with respect to acetonitrile) formed by combination of 5 mM ammonium formate buffer in water (pH 4.0, eluent A) and acetonitrile (eluent B). Flow rate of the mobile phase was set at 0.2 ml/min. Morphine, M3G, and M6G were detected using multiple reaction monitoring (MRM) in positive ionization mode. Selected ion masses of the protonated precursors and fragmented ions (m/z) were 286.3/201.0 for morphine and 462.2/286.0 for M3G and M6G. Chromatographic peaks were integrated using AnalystÔ software (version 1.4.1, SCIEX). The detection limits (LOD) and quantification limits (LOQ) for all analytes were 1.5 and 3.4 pg/ml. 2.4. mRNA expression of UGTs by quantitative (q)RT-PCR Microglial cells were cultured for 1, 3 and 6 h in the absence and presence of morphine 5 mM. Total RNA was extracted from 2 106 cells using the total RNA purification kit (Norgen Biotek Corp., Ontario, Canada), according to the manufacturer’s protocols. Two mg of total RNA were treated with DNAse kit (Fermentas, St. Leon-Rot, Germany) and subsequently reverse transcribed into cDNA using IscriptcDNA Synthesis Kit (Bio-rad Laboratories, CA, USA), according to the manufacturer’s protocols. One hundred ng of cDNA was used for the reaction mixture and the amplification of UGT1A1, UGT1A6, UGT1A7 and UGT2B1 was performed using TaqMan gene expression assay’s (Rn00754947_m1; Rn00756113_mH, Rn02749780_s1; Rn00756519_m1, respectively) (Applied Biosystem, CA, USA), as per the manufacturer’s instructions using a 7900HT Real-Time PCR system (Applied Biosystems). Expression levels of UGT mRNAs were normalized with those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Rn99999916_s1)(Applied Biosystem). Gene expression assays were performed by relative quantification with comparative Ct using SDS 2.4 software (Applied Biosystem). 2.5. Protein expression of UGTs by Western blot analysis Microglial cells were cultured for 6 and 24 h in the absence and presence of morphine 5 mM. Then, the cells were washed with PBS three times, and lysed, by adding icecold lysis buffer (10 mmol/L TriseHCl, pH 7.5; 150 mmol/L NaCl; 1 mmol/L EDTA; 50 mmol/L sodium fluoride; 1% Triton X-100; 10% glycerol; 1 mmol/L sodium orthovanadate; 1 mmol/L phenylmethanesulfonyl fluoride; 25 mmol/L glycerol-
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Fig. 1. A. Generation of morphine and M3G by microglia cells under basal conditions. Cells (0.5 106) were cultured for 6 or 24 h and the culture medium was assayed for morphine and M3G concentration. B. Morphine levels were detected in the extracellular medium (extracellular) and intracellular compartment (intracellular) of microglial cells (0.5 106 cells) cultured for 6 and 24 h. All data are expressed as mean values S.E.M. of three different experiments. In panel A, **P < 0.01 vs 6 h; xP < 0.01 vs morphine; in panel B, **P < 0.01 vs extracellular levels at the same time; xP < 0.01 vs extracellular (6 h) and #P < 0.01 vs extracellular (24 h).
2-phosphate; 10 mg/mL aprotinin and 1 mg/mL leupeptin) for 10 min. Lysates were then centrifuged at 1000 g for 10 min a 4 C. Total protein content of cell lysates was assessed using the Bradford method. Equal amount of proteins (30 mg) were electrophoresed on a 10% SDS-polyacrylamide gel (Mini-PROTEAN II apparatus, Bio-Rad Laboratories) before being transferred onto nitrocellulose membranes with the Mini-Trans blot system (Bio-Rad Laboratories). Membrane was blocked with 5% non-fat milk and incubated overnight at 4 C with polyclonal primary antibodies against UGT1A1 (diluted 1:200),UGT1A6 (1:200) or UGT1A7 (1:200). Membranes were then washed and incubated with a peroxidase-coniugated antirabbit secondary antibody (1:1000) for 1 h at room temperature using chemiluminescence (ECL) reagents for detection. To ensure equal protein loading, the membranes were separately probed for b-actin protein. Scanning densitometry was performed using the ImageJ 1.47 program, and signal density was normalized to b-actin density.
2.6. Biosynthesis of PGE2 and nitric oxide (NO) Microglial cells (0.5 106 cells), were incubated with LPS 10 ng/ml or morphine 5 mM, alone or together, for 6 h. PGE2 biosynthesis was assessed in culture medium by previously described, validated, and specific radioimmunoassay technique (Patrignani et al., 1994). The production of NO was measured as accumulated nitrite (NO 2 ), one of the stable end products of NO, by a Griess reaction. Briefly, a volume of conditioned culture medium from each sample was mixed with the same volume of the Griess reagent [50 mM sulfanylamide, 5% H3PO4 and 3 mM N-(1-naphthyl) ethylenediamine dihydrocloride]. The amount of NO-2 was measured through the determination of A540 nm in a microplate spectrophotometer.
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Fig. 2. Kinetic of M3G and M6G formations by microglia cells incubated for 30 min (A) or 120 min (B) with increasing concentration of morphine (0e2.5 mM). Each data points represent an average of triplicate determinations S.E.M.
2.7. Statistics All values were reported as mean SEM. Statistical analysis was performed using GraphPad Prism Software (version 5.00 for Windows, GraphPad, San Diego, California). One-way ANOVA followed by Newman-Keuls post-test was used to compare the means of more than two independent groups. Values of P < 0.05 were considered statistically significant.
3. Results Microglial cells cultured for 6 h released detectable levels of morphine (129 12 pmol/0.5 106 cells, n ¼ 12) and M3G (13.4 1.8 pmol/0.5 106 cells, n ¼ 12) while M6G was undetectable (<3.2 pmol/0.5 106 cells) (Fig. 1A). In contrast, neither morphine nor M3G were detected in medium incubated without cells (data not shown). Hence, in our experimental conditions, primary microglia cultures of the rat both synthesized morphine and transformed it in its glucuronide M3G (Fig. 1A). At 24 h of culture, the levels of morphine detected in the medium of microglial cells were profoundly reduced while M3G levels remained stable and M6G was undetectable (Fig. 1A). In a further experiment, we measured the intra- and extracellular morphine content at both 6 and 24 h (Fig. 1B). At 6 h, morphine was mostly present in the medium and accounted for 132.3 7.5 pmol/0.5 106 cells, whereas the intracellular levels were significantly lower (2.0 0.5 pmol/0.5 106 cells; P < 0.01); at 24 h, extracellular morphine levels (86.8 2.8 pmol/0.5 106 cells) were significantly (P < 0.01) lower than those detected at 6 h. At the
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Fig. 4. Effect of morphine on UGT1A1 mRNA (A) and protein (B, C) expression in rat microglial cells. Primary rat microglia cultures were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A1 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A1 (B), protein expression by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of bactin (C). Values are expressed as mean SEM of four experiments. In panel A, **P < 0.01 vs vehicle (1 h); *P < 0.05 vs morphine (1 h); xP < 0.01 vs vehicle (1 h). Fig. 3. M3G (A) and M6G (B) formation by microglial cells exposed to different concentrations of morphine for 6 and 24 h. Data are expressed as mean S.E.M. of three different experiments. In panel A, **P < 0.01 vs vehicle; xP < 0.05 vs 5 mM; *P < 0.05 vs vehicle; #P < 0.05 vs 2.5 mM and 5 mM; f P<0.01 vs 10 mM (6 h). In panel B, @P < 0.01 vs 2.5 mM (6 h); f P<0.05 vs 2.5 mM (6 h). (C) Ratio between M3G and M6G concentrations. *P < 0.05 vs 6 h. Data are expressed as mean S.E.M.
same time point, the levels of intracellular morphine (47.7 3.9 pmol/0.5 106 cells) were significantly higher that those detected at 6 h (P < 0.01). Summing up, on the long run morphine synthesis appears to be stopped and its content redistributed across the cell membrane. In order to exclude that astrocytes were the real source of endogenous morphine and that microglia takes morphine up before being isolated, we measured intra- and extra-cellular levels of morphine up to 2 h following microglia isolation. In these conditions, we found that intracellular morphine concentrations were below assay detection limit whereas in the medium low levels were detected (5.54 0.62 pmol/0.5 106 cells, n ¼ 6). Altogether our results show that microglial cells can synthesize, release and re-
uptake morphine; moreover they can transform morphine in its major metabolite M3G. Then, we aimed to study the capacity of microglial cells to glucuronide exogenously added morphine (Fig. 2A and B). We used morphine concentrations that are in the range of plasmalevels associated with analgesic effects (Smith et al., 2000). In these conditions, glucuronidation was extended to the formation of both M3G and M6G occurring with saturable kinetics (Fig. 2A and B). It is worth noting that at 120 min both kinetics did not fit MichaelisMenten equation showing sigmoid shape curves with values of the Hill coefficient of 5.32 and 2.13 for M3G and M6G, respectively. These values suggest positive cooperation between different enzyme molecules. Altogether our results suggest that microglial cells express UGTs that metabolize morphine to M3G and M6G. M3G is the major product generated at a fast rate but at longer incubation times with the opioid, a substantial concentration of M6G is produced which is 40% of M3G.
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Fig. 5. Effect of morphine on UGT1A6 mRNA (A) and protein (B, C) expression in rat microglial cells. Cells were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A6 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A6 (B), protein expressions by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of b-actin (C). Values are expressed as mean S.E.M. of four experiments. In panel A, **P < 0.01 vs vehicle (1 h); xP < 0.01 vs morphine (1 h); @P < 0.02 vs vehicle (6 h). In panel C, *P < 0.05 vs vehicle (24 h); xP < 0.05 vs vehicle (6 h).
Then, we aimed to study whether the capacity of microglial cells to metabolize exogenously added morphine to M3G and M6G may increase in a time-dependent fashion in association with changes of UGTs expression. As shown in Fig. 3A, when cultures were exposed for 6 h to micromolar concentrations (2.5, 5 and 10 mM) of morphine, M3G levels were significantly increased by roughly 2fold, at each concentration, versus baseline levels. Moreover, microglial cells exposed to morphine released M6G that increased in a concentration-dependent fashion (Fig. 3B). At 24 h of microglial cultures, baseline M3G levels were similar to those detected at 6 h (Fig. 3A). Differently, after exposure to exogenous morphine for 24 h, M3G levels were significantly decreased versus those detected at 6 h (Fig. 3A). The levels of M3G resulted marginally increased versus baseline values. In contrast, M6G levels were not significantly different from those measured at 6 h of incubation with morphine 5 and 10 mM (Fig. 3B).
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Fig. 6. Effect of morphine on UGT1A7 mRNA (A) and protein (B, C) expression in rat microglial cells. Cells were incubated with vehicle or morphine (5 mM) up to 24 h. Total RNA extracted was assayed for UGT1A7 mRNA levels by qPCR (A) and its levels were normalized to GAPDH mRNA levels. Cell lysates were assayed for UGT1A7 (B), protein expressions by Western Blot analysis and quantification of optical density (OD) of specific bands was calculated and normalized to the OD of b-actin (C). Values are expressed as mean SEM of four experiments. In panel A, *P < 0.05 vs vehicle (1 h); **P < 0.01 vs vehicle (1 h); xP < 0.01 vs morphine (1 h); @P < 0.05 vs vehicle (1 h); f P ¼ 0.056 vs vehicle (3 h); #P < 0.01 vs vehicle (6 h). In panel C, **P < 0.01 vs vehicle (6 h); *P < 0.05 vs morphine (6 h).
As shown in Fig. 3C, in microglial cells incubated for 6 h with morphine 2.5, 5 and 10 mM, M3G/M6G ratio values were approximately equal to three. After 24 h of incubation with morphine, M3G/M6G ratios decreased in a concentration-dependent fashion. In the presence of morphine 10 mM, M3G/M6G ratio was 1.4 0.05 (n ¼ 10), showing that M6G concentrations were not so far distant from those of M3G (Fig. 3C). Next we aimed to verify whether the exposure of microglial cells to morphine (5 mM) led to changes of mRNA and protein expression and of UGT1A1, UGT1A6, UGT1A7 and UGT2B1, which are involved in morphine metabolism (De Gregori et al., 2012). At 1 h of culture of rat microglial cells without morphine, baseline mRNAs for UGT1A1, UGT1A6, and UGT1A7 were detected whereas mRNA for UGT2B1 was undetectable. The levels of these three mRNAs increased in a time-dependent fashion up to 6 h (Fig. 4 A, 5 A and 6 A, respectively). This was associated with a constitutive protein expression of UGT1A1, 1A6 and 1A7 (Fig. 4B,C, 5B,C and 6B,C). At 6
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M3G may induce a pro-inflammatory response through the release of PGE2 as well as of nitrites. The effects of morphine were compared to those of a known inflammation inducer, i.e. LPS. As shown in Fig. 7 A and B, microglial cells exposed for 24 h to LPS were associated with enhanced release of both PGE2 and nitrites while morphine did not affect their synthesis neither in basal conditions neither under LPS stimulation. Moreover, exposing microglia to two different concentrations of LPS (10 and 50 ng/ml) did not affect basal levels of endogenous morphine or change morphine glucuronidation in presence of the drug (data not shown). 4. Discussion
Fig. 7. Effect of LPS (10 ng/ml) and morphine (5 mM)(MORF) on inducible PGE2 generation (A) and nitrite production (B) in rat microglial cells. Microglial cells were incubated with LPS or morphine alone or together for 6 h. PGE2 release was assessed in the medium by immunoassay and nitrite production by a Griess reaction. A and B: **P < 0.01 vs vehicle or morphine alone.
and 24 h of cultures, microglial cells expressed comparable protein levels of UGT1A1 and UGT1A7 (Fig. 4B,C and Fig. 6B, C). UGT1A6 protein levels were significantly, slightly increased at 24 h versus 6 h (Fig. 5B, C). The exposure of cells to morphine was associated with a rapid (at 1 h) increase of mRNA levels of UGT1A1 and UGT1A7 versus baseline values that declined at 3 h (Figs. 4A and 6A, respectively). At 6 h of incubation without morphine, mRNAs of UGT1A1 and UGT1A7 were maximally increased by 70- and 5.5-fold, respectively (versus 1 h). The presence of morphine constrained the enhanced expression of these mRNAs (Figs. 4A and 6A). UGT1A6 mRNA levels were not significantly affected by morphine exposure up to 3 h while at 6 h they were significantly reduced versus the levels detected in the absence of morphine, at the same time-point (Fig. 5A). Protein expression of the three isozymes was assessed after 6 and 24 h of exposure to morphine (Fig. 4B and C, 5B and C, 6B and C). UGT1A7 was the only UGT significantly increased at 6 h (Fig. 6B and C). At 24 h of exposure with morphine, all UGT protein levels tended to be lower than those detected at the same time-point without morphine; however, only UGT1A6 levels were significantly reduced (Fig. 5B and C). No significant change in the content of UGT1A1 was detected upon exposure to morphine (Fig. 4B and C). Altogether these results suggest that mRNAs of UGT1A1, 1A6 and 1A7 were constitutively expressed in primary rat microglial cells and that morphine modulates the rate of their expression. Interaction of morphine with the MOR and/or TLRs has been associated with the induction of pro-inflammatory response and oxidative stress (Dave and Khalili, 2010). Thus, we addressed the hypothesis that morphine by itself and/or through the formation of
Our study provides three major findings: i) morphine and its metabolite M3G are detectable in primary cultures of rat microglia; ii) the presence of M3G is consistent with the expression of different UGT isozymes, namely UGT1A1, UGT1A6, UGT1A7; iii) glucuronidation is extended to M6G formation when microglia is exposed to micromolar concentrations of exogenous morphine; iv) this was associated with substantial changes in the expression of these three UGT genes and enhanced protein expression of UGT1A7. At the best of our knowledge, this is the first report showing the presence of morphine and M3G in cultures of primary microglia of rats, extending to microglia the capability of forming endogenous morphine, already demonstrated in both GABAergic neurons and astrocytes (Laux et al., 2011, 2012). In our experimental conditions, endogenous morphine showed a time-dependent redistribution between extra and intra cellular space suggesting the existence of an active transport of the molecule, consistently with previous findings that morphine is taken up by different cell types in a timedependent manner (Déchelotte et al., 1993). The co-presence of M3G suggests the existence in the microglia of a clearance system capable of disposing endogenous morphine and here we give evidence that this system consists in the presence of constitutive UGT isozymes. That microglia can express UGT1A1, UGT1A6, as well as UGT1A7 is not surprising since the same UGT isozymes have been detected in macrophages that share with microglia mesodermic origin and inflammatory functions (Tochigi et al., 2005). Hence, it is not a chance that both microglia and macrophages do not express UGT2B1 (this study and Tochigi et al., 2005). Although our results are apparently in contrast with the common opinion that in the rat UGT2B1 is the only catalyser of M3G formation and that M6G is not formed at all (Milne et al., 1996), several findings have already weakened this notion. First, it has been demonstrated that UGT1A1, although with a limited activity, also contributes to M3G formation (King et al., 1997; Ishii et al., 1997; Ohno et al., 2008). Moreover, recombinant human UGT1A1 has been found to catalyse M6G formation at therapeutic morphine concentrations (10e100 mM), whereas at higher concentrations M6G formation exhibited substrate inhibition kinetics (Ohno et al., 2008). Finally, both M3G and M6G are formed when morphine is added to homogenates of rat brain (Nagano et al., 2000). Consistently with these findings, when we exposed microglia cultures to concentrations of exogenous morphine that corresponded to the upper analgesic range in both humans (Tiseo et al., 1995; Klepstad et al., 2003, 2004) and rats (Mas et al., 2000; Smith et al., 2000), we found that M3G synthesis increased by 2-fold over control levels and that glucuronidation was extended to the formation of M6G, otherwise undetectable in basal conditions. Prolonging the exposure to morphine up to 24 h, M3G concentrations even declined whereas M6G formation remained stable. As a consequence M3G/ M6G ratio was progressively reduced, approximating the unit when the culture were exposed for 24 h to the higher morphine concentration (10 mM). A different timing in the formation of the two
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metabolites is also suggested by the results of the experiment wherein microglia homogenates were incubated with increasing concentrations of morphine for 30 and 120 min M3G formation developed much faster than that of M6G: from 30 to 120 min Vmax of M3G formation just doubled whereas that of M6G showed a 6 time increase. In microglia homogenates, Vmax of both M3G and M6G formation was in the picomolar order, that were quite lower with respect to the Vmax of M3G formation by liver microsomes (i.e. nanomolar range) (Antonilli et al., 2003, 2005). Yet, it was close to the physiological concentrations of endogenous morphine and sufficient to permit an incremental formation of morphine glucuronides upon the exposure to the drug. As already mentioned, during traumatic brain inflammation, liquoral concentrations of M6G and M3G, but not of morphine, directly correlate with those of IL-6, a marker of glia activation (Roberts et al., 2009). Our results point to microglia as a possible source of morphine glucuronides when the brain is exposed to therapeutic concentrations of the parent compound. Morphine glucuronidation is consistent with the expression of different UGT isozymes by microglia. Although we did not attempt to assess what was the contribution that the three UGT isoforms gave to morphine glucuronidation, some cues may be drawn from the shape of saturation curves of M3G and M6G formation (see Fig. 2). Both of them showed a point of flex and then deviated from the exponential reaction curve representing the result of monomeric enzymatic activity, fully described by the MichaeliseMenten equation. Sigmoid saturation curves are considered to reflect homo- or ethero-cooperation between enzymatic molecules, as it is the case of estradiol-3-glucuronide formation due to homotropic UGT1A1 activation, which in turn it depends on the multimeric structure of this enzyme. The binding of a molecule of substrate to a subunit produces an increase in the affinity of a neighbouring subunit for the substrate (Senafi et al., 1994; Williams et al., 2002; Ghosh et al., 2001; Antonilli et al., 2008). It is then possible that a likewise homotropic UGT1A1 activation is involved in the formation of both M3G and M6G by microglia. More studies are request to test this possibility. An important finding of the present study is that the exposure to morphine led to a rapid upregulation of the expression of genes coding for UG1A1 and UGT1A7, which at 6 h translated into enhanced protein levels of UGT1A7, but not of UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Incidentally, although changes of UGT expression were temporally coincident with the enhanced release of M3G and M6G, we should be cautious in considering the two events causally related since UGT1A7 has been found to be almost inactive in catalysing morphine glucuronidation (Ohno et al., 2008). We have already found relevant changes in the induction of genes coding for the UGT isozymes here examined, as well as for UGT2B1, by hepatocytes exposed to either heroin or codeine (Antonilli et al., 2012). Mechanisms responsible for these modulations of UGT expression are not clear but it is well known that prolonged exposure to morphine causes a host of changes in the cellular signalling. For instance, interesting results have shown that morphine has an important role in the regulation of different microRNAs (miRNA) involved in the expression of morphine receptor (MOR), such as miR-let-7, which works as a mediator of the movement of the MOR mRNA into P-bodies, leading to translational repression, miR-23b, which is involved in linking MOR expression and morphine treatment at the post-transcriptional level (reviewed in Rodríguez, 2012). Further studies are necessary to verify whether morphine-induced changes in these miRNAs play a role in the regulation of UGT levels in microglial cells.
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Importantly, morphine-induced modulation of UGT expression does not seem to belong to a generalized priming of microglia since in the present experimental conditions the drug failed to affect the synthesis of both PGE2 and nitrites either under basal circumstances or upon LPS stimulation. Moreover, exposing microglia cultures to LPS did not alter the rate of morphine glucuronidation. Altogether these results suggest that at least in microglial cells the generation of M3G is independent from the signalling that triggers the upregulation of cyclooxygenase-2 involved in delayed generation of PGE2 occurring in inflammatory responses (Ricciotti and FitzGerald, 2011). 5. Conclusions In conclusion, here we provide evidence that glucuronidation belongs to the wide array of functions exerted by microglia; whether this function is only addressed to endogenous morphine or is also aimed to clear other brain-born compounds, such as neurosteroids, is presently unknown. It is most interesting that primary microglia cultures respond to pharmacological concentrations of morphine changing the expression of different UGT isozymes and expanding glucuronidation to the 6-OH moiety, i.e. forming M6G. Although in our experimental conditions microglia transformed morphine into M6G at a rather low rate, the amount of the metabolite may be pharmacologically significant, taking into account the finding that in the rat brain M6G, unlike morphine, is mainly distributed in the extracellular space and very little into neurons; hence, the actual concentration of the metabolite available for receptor binding is close to that of morphine upon systemic administration of the drug (Stain-Texier et al., 1999). It is then possible that at least in part morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration are brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the bloodebrain barrier. Moreover, evidence that microglia can form highly active morphine metabolites incentives more study on the role of these metabolites in the overall effects produced by chronic exposure to phenanthrenic compounds, such as heroin and codeine, which converge into the common process of morphine formation and metabolism. Acknowledgements This work was supported by the Sapienza University of Rome intramural grant C26A11ATBL to PN and by MIUR(ex 60%) to PP. The authors wish to thank Sabina Moschini for technical assistance in cells culturing and Francesca Filipponi for her skilful technical assistance in the analytical procedure for glucuronide determinations. References Antonilli, L., Suriano, C., Paolone, G., Badiani, A., Nencini, P., 2003. Repeated exposures to heroin and/or cadmium alter the rate of formation of morphine glucuronides in the rat. J. Pharmacol. Exp. Ther. 307, 651e660. Antonilli, L., Petecchia, E., Caprioli, D., Badiani, A., Nencini, P., 2005. Effect of repeated administrations of heroin, naltrexone, methadone, and alcohol on morphine glucuronidation in the rat. Psychopharmacology (Berl) 182, 52e64. Antonilli, L., Brusadin, V., Milella, M.S., Sobrero, F., Badiani, A., Nencini, P., 2008. In vivo chronic exposure to heroin or naltrexone selectively inhibits liver microsome formation of estradiol-3-glucuronide in the rat. Biochem. Pharmacol. 76, 672e679. Antonilli, L., De Carolis, L., Brusadin, V., Togna, A.R., Dovizio, M., Togna, G.I., Patrignani, P., Nencini, P., 2012. Repeated exposure to codeine alters morphine glucuronidation by affecting UGT gene expression in the rat. Eur. J. Pharmacol. 693, 7e14. Bickel, U., Schumacher, O.P., Kang, Y.S., Voigt, K., 1996. Poor permeability of morphine 3-glucuronide and morphine 6-glucuronide through the blood-brain barrier in the rat. J. Pharmacol. Exp. Ther. 278, 107e113.
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