International Journal of Molecular Sciences

Article Selective Blockade of the Metabotropic mGluR5 Protects Mouse Livers in In Vitro and Ex Vivo Models of Ischemia Reperfusion Injury

Andrea Ferrigno 1,* ID , Clarissa Berardo 1, Laura Giuseppina Di Pasqua 1, Veronica Siciliano 1, Plinio Richelmi 1, Ferdinando Nicoletti 2,3 and Mariapia Vairetti 1 ID 1 Department of Internal Medicine and Therapeutics, Cellular and Molecular Pharmacology and Toxicology Unit, University of Pavia, 27100 Pavia, Italy; [email protected] (C.B.); [email protected] (L.G.D.P.); [email protected] (V.S.); [email protected] (P.R.); [email protected] (M.V.) 2 Department of Physiology and Pharmacology, Sapienza University, 00185 Roma, Italy; [email protected] 3 I.R.C.C.S. Neuromed, 86077 Pozzilli, Italy * Correspondence: [email protected]; Tel.: +39-0382-986451

Received: 20 November 2017; Accepted: 22 January 2018; Published: 23 January 2018 Abstract: 2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike . This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions.

Keywords: mGluR5; MPEP; MTEP; fenobam; ischemia; liver; mice; liver transplantation; mitochondria; hepatocytes

1. Introduction Glutamate is one of the major excitatory neurotransmitters in the central nervous system, signaling through ionotropic receptors, which are glutamate-gated, fast-responding ion channels, and metabotropic receptors (mGluR), which are slower responders, able to activate an intracellular cascade. Eight different mGluRs have been classified into three groups, on the basis of gene-sequence homology and transduction mechanism. Group I includes mGluR1 and mGluR5, which are coupled to the inositol phosphate/diacylglycerol signaling pathway and induce intracellular calcium mobilization. On the contrary, mGluRs belonging to Group II (mGluR2 and mGluR3 subtypes) and Group III (mGluR4, mGluR6, mGlu7, and mGluR8 subtypes) inhibit the production of cyclic AMP [1,2]. Evidence of the presence of glutamate receptors in peripheral organs has been published in recent years and numerous studies have demonstrated the presence of mGluRs in peripheral cells, many of

Int. J. Mol. Sci. 2018, 19, 314; doi:10.3390/ijms19020314 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 314 2 of 16 them not originating from neural crests [3]. mGluRs have been found in the pancreas [4], in the liver [5], in the stomach mucosa [6], in human testes [7], and in primate ovaries [8]. These peripheral mGluRs can be activated by non-synaptic glutamate, probably synthesized from α-oxo-glutarate, a byproduct of the Krebs cycle. Glutamate can be transported outside the cell, where it intervenes in cell-to-cell communication or self-regulation by activating the mGluRs [9]. Sureda et al. (1997) first demonstrated the presence of a metabotropic glutamate receptor in the liver, showing that the competitive agonists quisqualate and 1-Amino-1,3-dicarboxycyclopentane (ACPD) stimulated [3H]inositolmonophosphate ([3H]InsP) formation in primary rat hepatocytes [10]. The existence of the mGluR5 subtype in primary rat hepatocytes was demonstrated in two articles by Storto and colleagues, using Western blot and polymerase chain reaction (PCR) analysis. More interestingly, these papers showed that hypoxic hepatocytes released more glutamate, probably saturating and hyperactivating mGluR5. Furthermore, hypoxic rat hepatocytes treated with the mGluR5 negative modulator 2-methyl-6-(phenylethynyl)pyridine (MPEP) or hepatocytes from mice knock-out (KO) for mGluR5s, were more resistant to hypoxic injury, suggesting that the activation of mGluR5s by endogenous glutamate promotes the progression of hypoxic injury [5,11]. Recently, MPEP has been found to reduce ATP content in microglia cells and astrocytes; direct or indirect receptor-mediated mechanisms have been proposed by the authors [12,13]. This findings, are, apparently, in contrast with the ability of MPEP to protect against ischemic injury, since the failure of ATP formation is the fundamental stressor in ischemic injury [14]. Hence this study attempted (1) to verify these published data in rat hepatocytes; (2) to compare the ATP-depleting ability of MPEP, with other negative allosteric modulators (NAMs), such as: 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) and fenobam, or orthosteric ligands, such as the agonist (DHPG) and the antagonist (S)-4-carboxyphenylglycine (CPG); (3) to understand the mechanism of MPEP-mediated ATP depletion and its possible impact in the protection from ischemic injury in vitro. Finally, the goal of the study was to verify, for the first time, whether MPEP is able to protect whole livers in murine models of cold or warm ischemia-reperfusion injury. We found that MPEP, MTEP, but not fenobam, DHPG, or CPG, deplete ATP in cells, mitochondrial suspensions, and acellular solutions without altering mitochondrial functionality. MPEP and MTEP showed good protection from ischemic injury in vitro despite their ability to deplete ATP. Finally, we showed, for the first time, that mouse livers treated with MPEP or from mice knocked out for mGluR5, are more resistant to both cold and warm ischemia-reperfusion injury.

2. Results

2.1. mGluR5 NAMs Protect Rat Hepatocytes from Ischemic Injury Regardless of Their Ability to Deplete ATP

2.1.1. Effects of MPEP and MTEP on Cellular and Mitochondrial ATP Content ATP was measured in rat liver mitochondria in the presence of MPEP, MTEP, fenobam, CPG, and DHPG at different concentrations (0, 0.3, 3 and 30 µM). MPEP and MTEP reduced ATP content, in a dose-dependent way, to 13.0 ± 1.3% and 53.7 ± 12.5%, with respect to control mitochondria. Fenobam and CPG had no effect (Figure1a) nor did DHPG alter ATP concentration [ 15]. In primary rat liver hepatocytes, MPEP dose-dependently reduced ATP concentration, producing a near-significant effect at 3 µM and a markedly significant effect at 30 µM(p = 0.003). At 0.3 µM MPEP did not reduce ATP concentration in freshly isolated hepatocytes (Figure1b). When added to plain phosphate-buffered saline (PBS), 10 µM ATP was significantly reduced by the co-administration of MPEP (16.8 ± 0.5% of control solution at 30 µM) and MTEP (47.1 ± 1.7% of control solution, at 30 µM). Fenobam, CPG, and DHPG did not change ATP in PBS, reproducing the same trend observed in Figure1a (Figure1c). To rule out the possibility that MPEP might reduce ATP concentration in a receptor-dependent way, we evaluated ATP in hepatocyte extracts from mice KO for mGluR5, with respect to extracts from wild-type mice. No significant difference was found (20.03 ± 3.69 nmol/mL and 23.08 ± 6.63 nmol/mL in mGluR5 KO and wild type, respectively, p = 0.71). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 16

Fenobam, CPG, and DHPG did not change ATP in PBS, reproducing the same trend observed in Figure 1a (Figure 1c). To rule out the possibility that MPEP might reduce ATP concentration in a receptor-dependent way, we evaluated ATP in hepatocyte extracts from mice KO for mGluR5, with respect to extracts Int.from J. Mol. wild-type Sci. 2018 ,mice.19, 314 No significant difference was found (20.03 ± 3.69 nmol/mL and 23.08 ±3 of6.63 16 nmol/mL in mGluR5 KO and wild type, respectively, p = 0.71).

Figure 1. MPEP and MTEP deplete ATP from isolated mitochondria, primary hepatocytes, and Figure 1. MPEP and MTEP deplete ATP from isolated mitochondria, primary hepatocytes, and acellular, acellular, ATP-containing solutions. (a) MPEP and MTEP reduce ATP content in a suspension of ATP-containing solutions. (a) MPEP and MTEP reduce ATP content in a suspension of isolated hepatic isolated hepatic mitochondria, in a dose dependent way, to 13.0 ± 1.3% and 53.7 ± 12.5% with respect mitochondria, in a dose dependent way, to 13.0 ± 1.3% and 53.7 ± 12.5% with respect to control to control mitochondria. Fenobam and CPG did not show any effect. DHPG did not alter ATP content mitochondria. Fenobam and CPG did not show any effect. DHPG did not alter ATP content [15]; [15]; (b) In primary rat liver hepatocytes, MPEP dose-dependently reduced ATP concentration, (b) In primary rat liver hepatocytes, MPEP dose-dependently reduced ATP concentration, producing a near-significantproducing a near-significant effect at 3 µM effect and a at markedly 3 µM and significant a markedly effect significant at 30 µM( effectp = 0.003).at 30 µM At 0.3(p =µ 0.003).M MPEP At did0.3 µM not reduceMPEP did ATP not concentration reduce ATP in concentration freshly isolated in hepatocytes.freshly isolated DHPG hepatocy did nottes. alter DHPG ATP did content not alter [15]; (ATPc) 10 contentµM ATP [15]; was ( addedc) 10 µM to aATP plain was PBS added buffer. to ATP a plain was PBS significantly buffer. ATP reduced was significantly by the addition reduced of MPEP by (16.8the addition± 0.5% ofof controlMPEP (16.8 solution ± 0.5% at 30 ofµ controlM) and solu MTEPtion (47.1 at 30± µM)1.7% and of control MTEP solution,(47.1 ± 1.7% at 30 ofµ M)control but notsolution, fenobam at 30 or µM) CPG, but reproducing not fenobam the or sameCPG,trend reproducing observed the in same (a). The trend asterisk observed indicates in (a). aThe significant asterisk differenceindicates a (Tukey’s significant Test) difference with respect (Tukey’s to controls Test) with (0 µ M).respect to controls (0 µM).

2.1.2. MPEP (30 µM) Does Not Alter Mitochondrial Functionality 2.1.2. MPEP (30 µM) Does Not Alter Mitochondrial Functionality In order to exclude the possibility that MPEP and MTEP reduced mitochondrial functionality, In order to exclude the possibility that MPEP and MTEP reduced mitochondrial functionality, mitochondria isolated from rat livers were assayed for respiratory control index (RCI), membrane mitochondria isolated from rat livers were assayed for respiratory control index (RCI), membrane potential, FOF1 ATPase activity and ROS production. When added to a mitochondrial suspension, potential, FOF1 ATPase activity and ROS production. When added to a mitochondrial suspension, MPEP 30 µM did not change RCI (Figure 2a). MPEP 30 µM, MTEP 30 µM, fenobam 30 µM and CPG MPEP 30 µM did not change RCI (Figure2a). MPEP 30 µM, MTEP 30 µM, fenobam 30 µM and CPG 30 µM did not alter mitochondrial membrane potential and Complex V (FOF1ATPase) activity when 30 µM did not alter mitochondrial membrane potential and Complex V (FOF1ATPase) activity when tested with respect to control mitochondria (Figure 2b,c). Finally, different concentrations of MPEP tested with respect to control mitochondria (Figure2b,c). Finally, different concentrations of MPEP and and DHPG did not change mitochondrial ROS production with respect to controls. With increasing DHPG did not change mitochondrial ROS production with respect to controls. With increasing MPEP MPEP concentrations, there was a slight statistically insignificant decrease in ROS (Figure 2d). We do concentrations, there was a slight statistically insignificant decrease in ROS (Figure2d). We do not not consider this phenomenon to be associated with mitochondrial activity, since we observed no consider this phenomenon to be associated with mitochondrial activity, since we observed no related related changes in mitochondrial respiration or mitochondrial membrane potential. changes in mitochondrial respiration or mitochondrial membrane potential.

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Figure 2. MPEP did not alter mitochondrial functionality. (a) MPEP 30 µM did not change respiratory Figure 2. MPEP did not alter mitochondrial functionality. (a) MPEP 30 µM did not change respiratory control index; (b) MPEP 30 µM, MTEP 30 µM, fenobam 50 µM and CPG 100 µM did not alter control index; (b) MPEP 30 µM, MTEP 30 µM, fenobam 50 µM and CPG 100 µM did not alter mitochondrial membrane potential; (c) MPEP 30 µM, MTEP 30 µM, fenobam 30 µM and CPG 30 µM mitochondrial membrane potential; (c) MPEP 30 µM, MTEP 30 µM, fenobam 30 µM and CPG 30 µM did not alter Complex V (FOF1-ATPase) activity with respect to control mitochondria; (d) MPEP and did not alter Complex V (FOF1-ATPase) activity with respect to control mitochondria; (d) MPEP and DHPG did not change mitochondrial ROS production with respect to controls. As negative controls DHPGmitochondria did not changetreated mitochondrialwith olicomycin ROS 2 µg/mL production (Oligomycin) with respect and/or to subjecte controls.d to As three negative freeze/thaw controls µ mitochondriacycles (Uncoupled) treated were with used. olicomycin 2 g/mL (Oligomycin) and/or subjected to three freeze/thaw cycles (Uncoupled) were used. 2.1.3. MPEP, MTEP and Fenobam Protect Rat Hepatocytes from Warm Ischemic Injury 2.1.3. MPEP, MTEP and Fenobam Protect Rat Hepatocytes from Warm Ischemic Injury The mortality rate of hypoxic hepatocytes was evaluated by means of Trypan blue (TB) exclusion.The mortality MPEP, rate MTEP of hypoxic and fenobam hepatocytes reduced was mort evaluatedality rates by meanswith respect of Trypan to untreated blue (TB) hypoxic exclusion. MPEP,hepatocytes. MTEP andBy comparing fenobam reducedmortality mortality curves by rates means with of respecta linear tomixed-effects untreated hypoxic fitting analysis, hepatocytes. it Byemerged comparing that, mortality in the 0′–90 curves′ time by range, means the ofmortality a linear rate mixed-effects for hepatocytes fitting treated analysis, with itMPEP emerged 30 µM that, inwas the 0lower0–900 timethan range,in control the anoxic mortality hepatocytes rate for hepatocytes(Figure 3a, p treated= 0.003). with The MPEPp value 30 forµ Mthis was comparison lower than inincreased control anoxic in the hepatocytes 0′–75′ interval (Figure (Figure3a, 3a,p p= = 0.003). 0.00009). The Ap significantvalue for difference this comparison was also increased observed in in the 00–75this0 intervaltime range (Figure for anoxic3a, p hepatocytes= 0.00009). Atreated significant with MPEP difference 3 µM was (Figure also 3b, observed p = 0.047) in and this fenobam time range for50 anoxic µM (Figure hepatocytes 3c, p = treated0.05) with with respect MPEP to 3untreatedµM (Figure hypoxi3b, cp hepatocytes.= 0.047) and Furthermore, fenobam 50 µhepatocytesM (Figure3 c, treated with fenobam 50 µM and MPEP 3 µM also had lower a mortality rate compared with anoxic p = 0.05) with respect to untreated hypoxic hepatocytes. Furthermore, hepatocytes treated with fenobam controls in the 0′–60′ time range in which a significant difference was also observed for hepatocytes 50 µM and MPEP 3 µM also had lower a mortality rate compared with anoxic controls in the 00–600 treated with MTEP 3 µM (p = 0.047). The orthosteric antagonist CPG showed no protective effect time range in which a significant difference was also observed for hepatocytes treated with MTEP 3 µM (Figure 3d). Administration of DHPG 100 µM-DFB 10 µM did not worsen cellular injury (Figure 3d), (p = 0.047). The orthosteric antagonist CPG showed no protective effect (Figure3d). Administration of probably because, as previously demonstrated [5], mGluR5 receptors are already saturated with DHPG 100 µM-DFB 10 µM did not worsen cellular injury (Figure3d), probably because, as previously glutamate. Analyzing the 45′ time point by ANOVA, it emerged that, compared to anoxic 0controls, demonstratedthere was a statistical [5], mGluR5 difference receptors in arethe alreadyviability saturated of cells treated with glutamate. with fenobam Analyzing 1 µM and the fenobam 45 time point10 byµM, ANOVA, (Figure it emerged3c, p = 0.05 that, and compared 0.003, respectively). to anoxic controls,The addition there of was MPEP a statistical 30 µM, MTEP difference 30 µM, in thefenobam viability of50 cells µM, treated or DHPG with 100 fenobam µM to 1oxygenatedµM and fenobam hepatocytes 10 µ didM, (Figurenot produce3c, p any= 0.05 changes and 0.003, when respectively).compared Theto addition control oxygenated of MPEP 30 hepatocytes.µM, MTEP 30 µM, fenobam 50 µM, or DHPG 100 µM to oxygenated hepatocytes did notThe produce mortality any rate changes of hypoxic when comparedhepatocytes to was control also oxygenatedevaluated by hepatocytes. means of lactate dehydrogenase (LDH)The mortalityrelease. Inrate the oftime hypoxic range of hepatocytes 0′–90′, a statistical was also difference evaluated was by meansobserved of between lactate dehydrogenase anoxic cells (LDH)treated release. with MPEP In the 30 time µM range (p = 0.004, of 00 –90linear0, a mixed-effects statistical difference fitting analysis) was observed and corresponding between anoxic anoxic cells treateduntreated with controls. MPEP 30 TheµM( significancep = 0.004, fo linearr this comparison mixed-effects decreased fitting analysis) in the 0′–75 and′ time corresponding range (p = 0.009) anoxic untreatedand further controls. decreased The significancein the 0′–60′ time for this range comparison (p = 0.048) decreased(Figure 4a). in No the further 00–750 statisticaltime range difference (p = 0.009 ) andwas further observed, decreased although in the mortality 00–600 timerates rangehad a (simip = 0.048)lar trend (Figure compared4a). No with further the one statistical observed difference using wasTB observed, exclusion although(Figure 4b–d). mortality The addition rates had of a MP similarEP 30 trend µM, comparedMTEP 30 µM, with fe thenobam one observed50 µM or DHPG using TB exclusion100 µM (Figure to oxygenated4b–d). The hepatocytes addition of MPEPdid not 30 µprM,oduce MTEP any 30 µchangesM, fenobam when 50 comparedµM or DHPG to 100controlµM to oxygenatedoxygenated hepatocytes hepatocytes. did not produce any changes when compared to control oxygenated hepatocytes.

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Int. J. Mol.100 Sci.µM 2018to ,oxygenated19, 314 hepatocytes did not produce any changes when compared to control5 of 16 oxygenated hepatocytes.

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Figure 3. MPEP, MTEP and fenobam reduce mortality rate (measured by TB exclusion) of hypoxic Figure 3. MPEP, MTEP and fenobam reduce mortality rate (measured by TB exclusion) of hypoxic primary hepatocytes. (a) The mortality rate of hepatocytes treated with MPEP 30 µM was lower than Figure 3. MPEP, MTEP and fenobam reduce mortality rate (measured by TB exclusion)µ of hypoxic primaryin control hepatocytes. anoxic hepatocytes, (a) The mortality in the 0′ rate–90′ time of hepatocytes range (p = 0.003, treated linear with mixed-effects MPEP 30 fittingM was analysis). lower than primary hepatocytes. (a) The mortality0 rate0 of hepatocytes treated with MPEP 30 µM was lower than in controlThe p anoxicvalue for hepatocytes, this comparison in the increased 0 –90 timein the range 0′–75′ (rangep = 0.003, (p = 0.00009, linear mixed-effectslinear mixed-effects fitting fitting analysis). in control anoxic hepatocytes, in the 0′–90′ time range0 0(p = 0.003, linear mixed-effects fitting analysis). The panalysis);value for (b this) MTEP comparison 3 µM reduced increased the mortalit in they rate 0 –75 in thisrange time (p range= 0.00009, (p = 0.048, linear linear mixed-effects mixed-effects fitting The p value for this comparison increased in the 0′–75′ range (p = 0.00009, linear mixed-effects fitting analysis);fitting ( banalysis);) MTEP 3(c)µ fenobamM reduced improved the mortality viability rate of hypoxic in this timehepatocytes range (inp =both 0.048, the 0 linear′–60′ and mixed-effects 0′–75′ analysis); (b) MTEP 3 µM reduced the mortality rate in this time range (p = 0.048, linear mixed-effects fittingtime analysis); range (p (=c 0.03) fenobam and 0.05, improved respectively, viability linear mixed-effe of hypoxiccts hepatocytes fitting analysis). in bothFurthermore, the 00–60 fenobam0 and 0 0–750 fitting analysis); (c) fenobam improved viability of hypoxic hepatocytes in both the 0′–60′ and 0′–75′ time range1 µM and (p = 10 0.03 µM and improved 0.05, respectively, the vitality of linear hepatocytes mixed-effects at 45′ and fitting 60′ (p analysis).= 0.05 and Furthermore,0.003, respectively, fenobam time range (p = 0.03 and 0.05, respectively, linear mixed-effects fitting analysis). Furthermore, fenobam 1 µMTukey’s and 10 Test);µM improved (d) the orthosteric the vitality antagonist of hepatocytes CPG showed at 45no0 protectiveand 600 (p effect.= 0.05 The and administration 0.003, respectively, of 1 µM and 10 µM improved the vitality of hepatocytes at 45′ and 60′ (p = 0.05 and 0.003, respectively, Tukey’sDHPG Test); 100 ( dµM–DFB) the orthosteric 10 µM did antagonist not worsen CPGcellular showed injury. no protective effect. The administration of Tukey’s Test); (d) the orthosteric antagonist CPG showed no protective effect. The administration of DHPG 100 µM–DFB 10 µM did not worsen cellular injury. DHPG 100 µM–DFB 10 µM did not worsen cellular injury.

Figure 4. MPEP improves the viability (measured by LDH release) of hypoxic hepatocytes: (a) a statistical difference was observed in the 0′–90′ time range, between anoxic cells treated with MPEP Figure30 µM 4. (MPEPp = 0.004, improves linear themixed-effects viability (measuredfitting analys byis) LDH and release)the corresponding of hypoxic untreated hepatocytes: anoxic (a) a Figure 4. MPEP improves the viability (measured by LDH release) of hypoxic hepatocytes: (a) a statisticalcontrols. difference The significance was observed for this comparison in the 0′–90 decreased′ time range, in the between 0′–75′ time anoxic range cells (p = 0.009)treated and with further MPEP statistical difference was observed in the 00–900 time range, between anoxic cells treated with MPEP 30 decreasedµM (p = in0.004, the 0 ′linear–60′ time mixed-effects range (p = 0.048); fitting (b )analys no statisticalis) and difference the corresponding was observed, untreated by means anoxic of 30 µM(p = 0.004, linear mixed-effects fitting analysis) and the corresponding untreated anoxic controls. controls.linear mixed-effectsThe significance fitting for analysthis comparisonis or Tukey’s decreased Test, for in MTEP, the 0′–75 although′ time range mortality (p = 0.009)rates showed and further a The significance for this comparison decreased in the 00–750 time range (p = 0.009) and further decreased decreasedsimilar trendin the compared 0′–60′ time with range that (p observed= 0.048); (usingb) no statisticalTB exclusion; difference (c) no wasstatistical observed, difference by means was of in the 00–600 time range (p = 0.048); (b) no statistical difference was observed, by means of linear linear mixed-effects fitting analysis or Tukey’s Test, for MTEP, although mortality rates showed a mixed-effectssimilar trend fitting compared analysis with or that Tukey’s observed Test, forusing MTEP, TB exclusion; although ( mortalityc) no statistical rates showeddifference a similarwas trendobserved, compared by means with thatof linear observed mixed-effects using TB fitting exclusion; analysis (c or) no Tukey’s statistical Test, difference for fenobam, was although observed, bymortality means of rates linear were mixed-effects similar to the fitting rates analysis observed or using Tukey’s TB Test,exclusion; for fenobam, (d) no statistical although difference mortality was rates wereobserved similar using to the linear rates mixed-effects observed using fitting TB analysis exclusion; or Tukey’s (d) no statistical Test for CPG, difference an orthosteric was observed antagonist. using linear mixed-effects fitting analysis or Tukey’s Test for CPG, an orthosteric antagonist.

Int. J. Mol. Sci. 2018, 19, 314 6 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 16 2.1.4. MPEP Prevents ATP DepletionDepletion inin HypoxicHypoxic HepatocytesHepatocytes 2.1.4. MPEP Prevents ATP Depletion in Hypoxic Hepatocytes Although ATPATP is is depleted depleted by by MPEP MPEP and and MTEP MTEP in normoxic in normoxic hepatocytes, hepatocytes, this tendency this tendency is reversed is Although ATP is depleted by MPEP and MTEP in normoxic hepatocytes, this tendency is inreversed hypoxic in hepatocytes.hypoxic hepatocytes. In particular, In particular, hypoxic hepatocyteshypoxic hepatocytes treated with treated MPEP with 3 µMPEPM and 3 30µMµ Mand had 30 reversed in hypoxic hepatocytes. In particular, hypoxic0 hepatocytes0 treated with MPEP 3 µM and 30 higherµM had ATP higher content ATP thancontent hypoxic than hypoxic controls, controls, after 30 afterand 30 45′ andhypoxia. 45′ hypoxia. No statistical No statistical difference difference was µM had higher ATP content than hypoxic controls, after 30′ and 45′ hypoxia. No statistical difference observedwas observed for treatments for treatments with with other other mGluR5 mGluR5 ligands ligands (Figure (Figure5). 5). was observed for treatments with other mGluR5 ligands (Figure 5).

Figure 5. MPEP attenuates the time dependent ATP decrease induced by oxygen deprivation, 300′ and Figure 5. MPEP attenuates the time dependent ATP decrease inducedinduced byby oxygenoxygen deprivation,deprivation, 3030′ and 450′ after N2 insufflation (Tukey’s test). 45′ after N2 insufflationinsufflation (Tukey’s (Tukey’s Test).test). 2.2. MPEP Protects Mouse Livers in an Ex Vivo Model of Cold Storage/Reperfusion Injury 2.2. MPEP Protects Mouse Livers in an Ex VivoVivo ModelModel ofof ColdCold Storage/ReperfusionStorage/Reperfusion InjuryInjury Mouse livers were maintained for 18 h at 4 ◦°C in Belzer preservation solution to simulate organ Mouse livers were maintained for 18 hh at 44 °CC in Belzer preservation solution to simulate organ preservation for transplantation; livers were then reperfused with oxygenated Krebs–Henseleit preservationpreservation forfor transplantation; transplantation; livers livers were were then th reperfuseden reperfused with oxygenated with oxygenated Krebs–Henseleit Krebs–Henseleit buffer at buffer◦ at 37 °C to simulate reperfusion and induce ischemia reperfusion injury. Livers treated with 37bufferC to at simulate 37 °C to reperfusion simulate reperfusion and induce and ischemia induce reperfusion ischemia injury.reperfusion Livers injury. treated Livers with MPEP treated 300 with nM MPEP 300 nM (added to the preservation solution and the reperfusion solution) and livers from (addedMPEP 300 to the nM preservation (added to the solution preservation and the reperfusionsolution and solution) the reperfusion and livers solution) from knock-out and livers mice from for knock-out mice for mGluR5 showed a significant decrease in LDH release after cold preservation mGluR5knock-out showed mice for a significant mGluR5 showed decrease a insignificant LDH release decrease after coldin LDH preservation release after (Figure cold6 a)preservation as well as (Figure 6a)0 as well as during 60′ of oxygenated, normothermic reperfusion (Figure 6b). during(Figure 606a)of as oxygenated, well as during normothermic 60′ of oxygenated, reperfusion normothermic (Figure6b). reperfusion (Figure 6b).

Figure 6. MPEP 300 nM reduced LDH release during cold storage and LDH release rate during warm Figure 6. MPEP 300 nM reduced LDH release during cold storage and LDH release rate during warm Figurereperfusion. 6. MPEP (a) 300LDH nM was reduced measured LDH in release the solution during flushed cold storage out of and the LDH liver release after 18 rate h duringcold storage. warm reperfusion. (a) LDH was measured in the solution flushed out of the liver after 18 h cold storage. reperfusion.Control ischemic (a) LDH livers was released measured significantly in the solution more LDH flushed than out livers of the preserved liver after in 18MPEP h cold containing storage. Control ischemic livers released significantly more LDH than livers preserved in MPEP containing Controlsolution ischemicand in livers livers from released mGluR5 significantly KO mice ( morep = 0.03 LDH and than 0.05, livers respectively, preserved with in MPEPTukey’s containing test). The solution and in livers from mGluR5 KO mice (p = 0.03 and 0.05, respectively, with Tukey’s test). The solutionaddition andof DHPG in livers 100 from µM mGluR5and DFB KO 10 miceµM to (p the= 0.03 preservation and 0.05, solution respectively, produced with Tukey’sno statistical Test). addition of DHPG 100 µM and DFB 10 µM to the preservation solution produced no statistical Thedifference addition vis-à-vis of DHPG ischemic 100 µ Mcontrols; and DFB (b 10) duringµM to theoxygenated preservation warm solution reperfusion, produced control no statisticalischemic difference vis-à-vis ischemic controls; (b) during oxygenated warm reperfusion, control ischemic differencelivers hadvis- a significantlyà-vis ischemic higher controls; LDH (b) release during oxygenatedrate than livers warm treated reperfusion, with controlMPEP ischemicor livers liversfrom livers had a significantly higher LDH release rate than livers treated with MPEP or livers from hadmGluR5 a significantly KO mice ( higherp = 0.022 LDH and release 0.014, raterespectively, than livers in treated a linear with mixed-effects MPEP or livers fitting from analysis). mGluR5 When KO mGluR5 KO mice (p = 0.022 and 0.014, respectively, in a linear mixed-effects fitting analysis). When miceDHPG-DFB (p = 0.022 was and added, 0.014, the respectively, LDH release in a rate linear did mixed-effects not differ from fitting ischemic analysis). controls. When DHPG-DFB was DHPG-DFB was added, the LDH release rate did not differ from ischemic controls. added, the LDH release rate did not differ from ischemic controls. At the end of 60′ warm reperfusion, the Bax/Bcl-2 ratio in livers from mice knock-out for mGluR5 At the end of 600′ warm reperfusion, the Bax/Bcl-2 ratio in livers from mice knock-out for mGluR5 was significantlyAt the end of lower 60 warm than reperfusion, wild-type ischemic the Bax/Bcl-2 controls, ratio whereas in livers livers from micetreated knock-out with 300 for nM mGluR5 MPEP was significantly lower than wild-type ischemic controls, whereas livers treated with 300 nM MPEP wasshowed significantly no significant lower tendency than wild-type to decrease ischemic (Figure controls, 7). whereas livers treated with 300 nM MPEP showed nono significantsignificant tendencytendency toto decreasedecrease (Figure(Figure7 7).).

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Figure 7. Tissue hepatic Bax/Bcl-2 ratio was significantly reduced in livers from mGluR5 KO mice Figure 7. Tissue hepatic Bax/Bcl-2 ratio was significantly reduced in livers from mGluR5 KO mice (Tukey’s Test) after 18 h preservation by cold storage and 60′ warm reperfusion. Livers treated with (Tukey’s Test) after 18 h preservation by cold storage and 600 warm reperfusion. Livers treated with MPEP during preservation and reperfusion had a near-significant reduction of Bax/Bcl-2 ratio, at the MPEP during preservation and reperfusion had a near-significant reduction of Bax/Bcl-2 ratio, at the end of 60′ reperfusion. end ofFigure 600 reperfusion. 7. Tissue hepatic Bax/Bcl-2 ratio was significantly reduced in livers from mGluR5 KO mice (Tukey’s Test) after 18 h preservation by cold storage and 60′ warm reperfusion. Livers treated with At the end of 18 h preservation and 60′ warm reperfusion, the expression of tumor necrosis At theMPEP end during of 18 preservation h preservation and reperfusion and 60 had0 warm a near reperfusion,-significant reduction the expression of Bax/Bcl-2 ratio, of tumor at the necrosis factor-α (TNF-α) was significantly lower in livers from mGluR5 KO mice and in livers treated with α end of α60′ reperfusion. factor-MPEP (TNF-300 nM) (Figure was significantly 8a). Similarly, lower the inexpression livers from of mGluR5inducible KOnitric mice oxide and synthase in livers (iNOS) treated was with MPEPsignificantly 300At nM the lower (Figureend of in 818 liversa). h Similarly,preservation from mGluR5 the and expression KO 60′ micewarm and ofreperfusion, induciblein livers treatedthe nitric expression with oxide MPEP synthaseof tumor 300 nM (iNOS)necrosis (Figure was significantly8b).factor- On theα lower(TNF- contrary,α in) was livers no significantly statistical from mGluR5 differencelower KO in livers mice was andfrom observed in mGluR5 livers in treated KOthe miceexpression with and MPEP in liversof endothelial 300 treated nM (Figure with nitric 8b). Onoxide theMPEP contrary,synthase 300 nM no(eNOS), (Figure statistical an 8a). endothelial Similarly, difference theenzyme was expression observed involved of inducible inin thevasodilation expression (Figure of synthase endothelial 8c). (iNOS) nitric was oxide synthasesignificantlyATP (eNOS), has been lower an endothelialevaluated in livers from in enzymeextracts mGluR5 involvedfrom KO liversmice in and collected vasodilation in livers at treatedthe (Figure end with of8 c).oxygenated MPEP 300 nM reperfusion. (Figure TheATP8b). trend On has ofthe been ATP contrary, evaluatedreflected no statistical the in level extracts differenceof injury from or liverswas pr otection,observed collected within atthe higher the expression end levels of oxygenated of in endothelial livers treated reperfusion. nitric with TheMPEP/KO trendoxide ofsynthase ATPlivers reflected (eNOS),and lower an the endothelial levels level in of injurylivers enzyme treated or involved protection, with in DHPGvasodilation with 100 higher µM. (Figure levels However, 8c). in livers no significant treated with MPEP/KOdifferenceATP liverswas has found been and evaluated lower(Table levels1). in extracts in livers from treated livers collected with DHPG at the 100end µofM. oxygenated However, reperfusion. no significant The trend of ATP reflected the level of injury or protection, with higher levels in livers treated with difference was found (Table1). MPEP/KOTable 1. liversATP measuredand lower in levels liver extractsin livers at treated the end with of 1DHPG h reperfusion 100 µM. (nmol/mg However, proteins). no significant No difference was found (Table 1). Tablesignificant 1. ATP difference measured was in liver found extracts among at groups the end (ANOVA). of 1 h reperfusion (nmol/mg proteins). No significant difference was found among groups (ANOVA). Table 1. ATP measuredCTRL in liver MPEP extracts 300 atnM the end KOof 1 h reperfusion DHPG 100(nmol/mg µM proteins). No significant difference2.39 was± 0.34 found among 3.52 ± groups 1.56 (ANOVA). 2.81 ± 0.92 1.82 ± 0.87 CTRL MPEP 300 nM KO DHPG 100 µM CTRL MPEP 300 nM KO DHPG 100 µM 2.39 ± 0.34 3.52 ± 1.56 2.81 ± 0.92 1.82 ± 0.87 2.39 ± 0.34 3.52 ± 1.56 2.81 ± 0.92 1.82 ± 0.87

Figure 8. Cont.

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Figure 8. Hepatic expression of TNF-α, iNOS and eNOS in a model of cold storage/reperfusion injury. Figure 8. Hepatic expression of TNF-α, iNOS and eNOS in a model of cold storage/reperfusion injury. (a) Tissue hepatic TNF-α was significantly lower in livers from mGluR5 KO mice and MPEP treated a α ( ) Tissuelivers hepatic(Tukey’s TNF- test) afterwas 18significantly h preservation lower by cold in storage livers fromand 60 mGluR5′ warm reperfusion. KO mice and Livers MPEP treated treated livers (Tukey’s Test) after 18 h preservation by cold storage and 600 warm reperfusion. Livers treated Figurewith DHPG 8. Hepatic during expression preservation of TNF- andα, iNOSreperfusion and eNOS showed in a modelno significant of cold storage/reperfusion variation compared injury. with with DHPG during preservation and reperfusion showed no significant variation compared with (controla) Tissue ischemic hepatic livers, TNF- αat was the significantlyend of 60′ reperfusion; lower in livers (b) tissue from hepaticmGluR5 iNOS KO micewas significantlyand MPEP treated lower 0 controlliversin livers ischemic (Tukey’s from livers, mGluR5test) after at theKO 18 endmiceh pr ofeservation and 60 MPEPreperfusion; by treate coldd storage livers (b) tissue (Kruskal–Wallisand 60 hepatic′ warm iNOSreperfusion. non-parametric was significantly Livers test treated and lower in liverswithDunn’s fromDHPG Test) mGluR5 during after 18 preservation KO h preservation mice and and MPEPby reperfusion cold treated storage showed liversand 60 (Kruskal–Wallisno′ warm significant reperfusion. variation non-parametric Livers compared treated withwith test and 0 Dunn’scontrolDHPG Test) ischemicduring after 18preservation livers, h preservation at the andend ofre by perfusion60 cold′ reperfusion; storage showed ( andb) notissue 60 significant warmhepatic reperfusion. iNOSvariation was significantlyrespect Livers to treatedcontrol lower with DHPGinischemic duringlivers fromlivers, preservation mGluR5 at the endKO and miceof reperfusion 60 ′and reperfusion; MPEP showed treate (c) dno no livers significant significant (Kruskal–Wallis difference variation non-parametricwas respect observed to control intest eNOS and ischemic livers,Dunn’sexpression at the Test) end at afterthe of 60end 180 reperfusion; hof preservation 18 h cold storage (c by) no cold and significant storage 60′ warm and difference reperfusion, 60′ warm was reperfusion. when observed comparing Livers in eNOSKO treated vs. expressionCTRL with groups (p = 0.08, ANOVA). at theDHPG end ofduring 18 h coldpreservation storage and and re 60perfusion0 warm reperfusion,showed no significant when comparing variation KOrespect vs. to CTRL control groups (p = 0.08,ischemic ANOVA). livers, at the end of 60′ reperfusion; (c) no significant difference was observed in eNOS 2.3. MPEPexpression Protects at the Mouse end of Livers 18 h cold in an storage Ex Vivo and Model 60′ warm of Warm reperfusion, Ischemia when Reperfusion comparing Injury KO vs. CTRL groups (p = 0.08, ANOVA). 2.3. MPEPMouse Protects livers Mouse were Livers perfused in anwith Ex deoxygenated Vivo Model of Kreb Warms–Henseleit Ischemia buffer Reperfusion at 37 °C Injury for 30 min, then reperfused with oxygenated Krebs–Henseleit buffer at 37 °C for 120 min in order to induce warm Mouse2.3.ischemia/reperfusion MPEP livers Protects were Mouse perfusedinjury. Livers Livers in with an treated Ex deoxygenated Vivo with Model MPEP of Warm Krebs–Henseleit300 IschemianM (added Reperfusion to bufferthe perfusion Injury at 37 ◦solutionC for 30at min, ◦ thenevery reperfusedMouse stage liversof with the were oxygenatedexperiment) perfused andwith Krebs–Henseleit livers deoxygenated from knock-out Kreb buffers–Henseleit mice at 37 forC buffermGluR5 for 120 at 37showed min °C for in 30a order significant min,to then induce warmreperfuseddecrease ischemia/reperfusion in with the oxygenatedLDH release injury. Krebs–Henseleit rate Liversduring treated 120buffer′ of at withnormothermic 37 °C MPEP for 120 300 minreperfusion nM in order (added to(Figure induce to the 9). warmperfusion The solutionischemia/reperfusionadministration at every stage of DHPG ofinjury. the 100 experiment)Livers µM and treated DFB andwith10 µM liversMPEP did fromnot300 worsen nM knock-out (added ischemia/reperfusion to mice the perfusion for mGluR5 solutioninjury showed in at a a α significanteverysignificant stage decrease way. of the The in experiment) therelease LDH of TNF- releaseand livers inrate the from perfusion during knock-out 120 solution,0 ofmice normothermic atfor the mGluR5 end of 120showed reperfusion′ reperfusion, a significant (Figure was 9). lower in mGluR5 KO group and in the MPEP 300 nM group (Figure 10). The administrationdecrease in the of LDH DHPG release 100 µ Mrate and during DFB 10120µ′ Mof did normothermic not worsen ischemia/reperfusionreperfusion (Figure 9). injury The in a administration of DHPG 100 µM and DFB 10 µM did not worsen ischemia/reperfusion injury in a significant way. The release of TNF-α in the perfusion solution, at the end of 1200 reperfusion, was significant way. The release of TNF-α in the perfusion solution, at the end of 120′ reperfusion, was lowerlower in mGluR5 in mGluR5 KO KO group group and and in in the the MPEP MPEP 300300 nMnM group group (Figure (Figure 10). 10 ).

Figure 9. After 30′ perfusion with anoxic medium at 37 °C, during oxygenated warm reperfusion, the LDH release rate of control ischemic livers was significantly higher than livers treated with MPEP and livers from mGluR5 KO mice (p = 0.05 and 0.005, respectively, in a linear mixed-effects fitting Figureanalysis). 9. After The 30addition′ perfusion of DHPG-DFB with anoxic wasmedium not atassociated 37 °C, during to changes oxygenated in LDH warm release reperfusion, rate when the Figure 9. After 300 perfusion with anoxic medium at 37 ◦C, during oxygenated warm reperfusion, LDHcompared release to ischemicrate of control controls. ischemic livers was significantly higher than livers treated with MPEP the LDHand livers release from rate mGluR5 of control KO ischemicmice (p = livers0.05 and was 0.005, significantly respectively, higher in a thanlinear livers mixed-effects treated withfitting MPEP and liversanalysis). from The mGluR5 addition KO of miceDHPG-DFB (p = 0.05 was and not 0.005,associated respectively, to changes in in a LDH linear release mixed-effects rate when fitting analysis).compared The to addition ischemic controls. of DHPG-DFB was not associated to changes in LDH release rate when compared to ischemic controls.

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Figure 10. TNF-α release measured at the end of 120′ reperfusion was significantly lower in livers Figure 10. TNF-α release measured at the end of 1200 reperfusion was significantly lower in livers treated with MPEP and livers from mice KO for mGluR5 (Tukey’s test). treated with MPEP and livers from mice KO for mGluR5 (Tukey’s Test).

ATP hashas beenbeen evaluatedevaluated inin liverliver extractsextracts atat thethe endend ofof 22 hh anoxicanoxic perfusion.perfusion. NoNo significantsignificant difference waswas foundfound (Table(Table2 ).2).

TableTable 2.2. ATPATP measured measured in in liver liver extracts extracts at at the the end end of of2 h 2 anoxic h anoxic perfusion perfusion (nmol/mg (nmol/mg proteins). proteins). No Nosignificant significant difference difference was was found found among among groups groups (ANOVA). (ANOVA). CTRL MPEP 300 nM KO DHPG 100 µM CTRL MPEP 300 nM KO DHPG 100 µM 2.48 ± 0.55 2.90 ± 0.70 2.44 ± 0.82 2.13 ± 0.77 2.48 ± 0.55 2.90 ± 0.70 2.44 ± 0.82 2.13 ± 0.77 3. Discussion 3. Discussion In this work, we demonstrated for the first time that the pharmacological blockade of mGluR5 protectsIn this whole work, livers we demonstratedfrom ischemia/r foreperfusion the first timeinjury that in themurine pharmacological ex vivo models blockade of cold of and mGluR5 warm protectsischemia. whole Furthermore, livers from we ischemia/reperfusion showed that MPEP,injury MTEP in and murine fenobam ex vivo share models the ofability cold andto protect warm ischemia.primary rat Furthermore, hepatocytes we from showed ischemic that injury, MPEP, MTEPdespite and their fenobam differences share with the abilityregard toto protectATP-depleting primary ratproperties. hepatocytes from ischemic injury, despite their differences with regard to ATP-depleting properties.

3.1. MPEP Sequesters ATP fromfrom AcellularAcellular SolutionSolution andand CellularCellular SuspensionsSuspensions The blockade of mGluR5 with MPEP 100 µµMM was recently described as the cause of a reduction inin ATPATP content content in in murine murine BV-2 BV-2 microglia microglia cells. cells. Since theSince blockade the blockade of mGluR5 of mGluR5 in microglia in ismicroglia associated is 2+ withassociated an increase with an in increase Ca release, in Ca the2+ release, opening the of opening the mitochondrial of the mitochondrial membrane membrane permeability permeability transition pore,transition mitochondrial pore, mitochondrial membrane membrane depolarization depolarizat and increaseion and inincrease ROS production, in ROS production, the authors the positedauthors 2+ that,posited ATP that, depletion ATP depletion in BV-2 cellsin BV-2 might cells be might due tobe overworkeddue to overworked ATPases ATPases during during Ca reuptake Ca2+ reuptake to the ERto the [12 ].ER Furthermore, [12]. Furthermore, in astrocytes, in astrocytes, the activation the activation of a glutamate of a glutamate receptor receptor was correlatedwas correlated to ATP to release:ATP release: 100 µ M100 glutamate µM glutamate caused caused an increase an increase in ATP in up ATP to 0.3–0.4up to 0.3–0.4 pmol/min pmol/min [16]; on [16]; the on other the hand,other MPEPhand, MPEP completely completely inhibited inhibited the glutamate-evoked the glutamate-evoked increase increase in extracellular in extracellular ATP. ATP. This This effect effect was was not attributednot attributed to the to activation/blockade the activation/blockade of mGluR5, of mGlu asR5, mature as mature astrocytes astrocytes do not expressdo not thisexpress receptor; this however,receptor; however, the authors the posited authors involvement posited invo oflvement the kainate of the receptor kainate glutamate receptor receptor,glutamate ionotropic receptor, kainateionotropic 2 (GluK2) kainate [13 2]. (GluK2) We found [13]. that We addition found of that MPEP addition 3–30 µ Mof reduces MPEP ATP3–30 concentration µM reduces whenATP addedconcentration to suspended when added rat hepatocytes’ to suspended mitochondria rat hepatocyte ands’ mitochondria acellular buffers. and acellular We also buffers. found thatWe also the additionfound that of MTEP,the addition which of only MTEP, differs which from only MPEP differs through from the MPEP substitution through of the a benzene substitution ring withof a abenzene tiazole ring group, with reduces a tiazole the group, ATP content reduces in the mitochondria ATP content and in mitochondria buffered solutions, and buffered albeit tosolutions, a lower extent.albeit to Both a lower MPEP extent. and Both MTEP MPEP are aromaticand MTEP alkynes are aromatic characterized alkynes by characterized the presence by of the two presence aromatic of ringstwo aromatic joined together rings joined by a together triple bond. by a Ontriple the bond. contrary, On the fenobam contrary, and fenobam CPG, which and CPG, do not which possess do annot alkyne possess triple an alkyne bond, dotriple not bond, sequester do not ATP sequester either in ATP acellular either solutions in acellular or insolutions cellular or suspensions. in cellular Oursuspensions. findings Our suggest findings that thesuggest potential that the role potentia of glutamatel role of receptors glutamate in receptors MPEP- and in MPEP- MTEP-associated and MTEP- ATPassociated depletion, ATP shoulddepletion, be reconsidered, should be reconsidered, in view of the in factview that of the MPEP fact and that MTEP MPEP dose-dependently and MTEP dose- sequesterdependently ATP sequester in acellular ATP solutions in acellular at concentrations solutions at concentrations equal to or above equal 3 µtoM. or above 3 µM.

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3.2. NAMs Protect Hepatocytes from Ischemic Injury, Regardless of Their Ability to Induce ATP Depletion Considering the importance of ATP in the progression of ischemic injury, we assessed whether MPEP, MTEP, Fenobam and CPG had different propensities as regards protecting rat hepatocytes from warm ischemia/reperfusion injury in vitro. We found that the blockade of mGluR5 protects from ischemia, independently of any ATP-depleting ability. Surprisingly, the most significant protection was found for cells treated with MPEP 30 µM(p = 0.00009 in the time range of 00–750). CPG did not reduce the mortality rate of ischemic hepatocytes, although we think that the tested CPG concentration (100 and 200 µM) was too low. In fact, CPG is an orthosteric antagonist with very low potency compared to MPEP, MTEP and fenobam; its IC50 in HEK-293 cells was estimated to be 1970 µM[17], compared to the IC50 of 36 nM found for MPEP [18]. In these experiments, the addition of DHPG 100 µM–DFB 10 µM did not significantly change mortality rates for hypoxic hepatocytes as compared with hypoxic control cells [15]. Similar findings are found in literature and have been justified in terms of excessive release of glutamate during ischemia, causing the saturation of mGluR5 [5]. Glutamate is the primary excitatory neurotransmitter in the CNS, playing a key role in cell excitotoxicity, via activation of glutamate receptors. In ischemic tissues, sustained cell membrane depolarization causes increased glutamate release; simultaneously, the process of glutamate reuptake is suppressed. The activation of glutamate receptors is a major route for excessive Ca2+ influx, believed to trigger excitotoxicity, a process responsible for acute neuronal death [19,20]. In particular, the activation of mGluR5 is linked to the formation of inositoltrisphosphate and diacylglycerole, which in turn induce oscillatory increases in cytosolic Ca2+ [21]. These intracellular changes are known to produce mitochondrial dysfunction, DNA fragmentation and oxidative stress, finally leading to cell death, especially in cells subjected to stress stimuli such as ischemia or hypoxia [22]. High glutamate and Ca2+ release have been previously associated to ischemic injury, in primary hepatocytes. An increase in extracellular glutamate release was observed in primary hepatocytes after 2 h warm ischemia. Blocking mGluR5s with the administration of MPEP 30 µM protected ischemic rat hepatocytes, suggesting that overactivation of glutamate receptors promotes progression of ischemic injury [23]. Furthermore, hepatocytes from mice knocked out for mGluR5s were more resistant to warm ischemia compared to control hepatocytes from wild-type mice [11]. In a paper by Elimadi and colleagues (2001), rat hepatocytes showed a significant increase in cytosolic Ca2+, in an in vitro model of cold ischemia/reperfusion. Primary rat hepatocytes were preserved in Belzer solution at 4 ◦C for 0, 24 and 48 h, then reoxygenated at 37 ◦C for 1 h. Pretreatment of hepatocytes with m-iodobenzylguanidine, which stabilizes Ca2+ homeostasis, improved hepatocyte viability [24]. Here we showed for the first time that MTEP and fenobam, well known selective mGluR5 negative allosteric modulators, protect rat hepatocytes from ischemia, probably by blocking part of the cytotoxic injury triggered by the excessive glutamate release. We also demonstrated that this protection does not seem to be affected by the ATP-depleting properties that MPEP and MTEP share. Surprisingly, the administration of MPEP 3 µM and 30 µM to hypoxic hepatocytes attenuated the hypoxia-driven ATP depletion. These data, insofar as they appear to be contradictory, suggest that, among the different variables affecting cellular ATP content during ischemia, including MPEP receptor-independent ATP depletion, lack of oxygen and MPEP receptor-dependent protection from ischemia, the prevailing variable may be MPEP protection from ischemic injury, producing as a consequence the attenuation of ATP depletion.

3.3. The Blockade of mGluR5 Protects Mouse Livers from Cold Ischemia and Warm Ischemia Reperfusion Injury

After inducing cold or warm ischemia in mouse livers, we evaluated hepatic conditions in a standard model of isolated perfused liver. Isolated perfused liver represents a suitable model when monitoring liver injury in rat and mouse livers [25,26] even in absence of hepatic artery reconstruction [27]. In this study, we found for the first time that mGluR5 blockade significantly reduces Int. J. Mol. Sci. 2018, 19, 314 11 of 16 the LDH release rate, in both cold ischemia and warm ischemia reperfusion models. In livers preserved by cold storage, we found a significant decrease in LDH release rate, both at the end of 18-h preservation and during 600 warm reperfusion, in MPEP and KO groups; we also found a reduction in TNF-α and iNOS expression, in KO and MPEP treated livers. The role of TNF-α has been widely studied in hepatic ischemia. Ischemic stress enhances the expression and release of proinflammatory molecules, such as TNF-α and iNOS; in addition, TNF-α can directly trigger apoptosis [28] and, thanks to its central role in promoting an inflammatory response, suppressing the formation of TNF-α might well be effective in attenuating acute post-ischemic inflammation and injury [29,30]. The expression of TNF-α and iNOS seems to be connected: the cloning of the promoter region of the iNOS gene showed the presence of TNF responsive elements [31]. Moreover, in a model of indomethacin-induced jejunoileitis in rats, it has been shown that treatment with TNF antibody reduces iNOS expression, suggesting that TNF-α may directly modulate iNOS expression [32]. To our knowledge, no publication in the literature has demonstrated a direct link between mGluR5 blockade and TNF-α downregulation and a similar conclusion is surely not deducible from our data. More reasonably, mGluR5 blockade is probably involved in halting excitotoxic events triggered by mGluR5 hyperactivation. The pharmacological inhibition of mGluR5-mediate excitotoxicity might play a role in indirectly reducing TNF-α and iNOS expression by reducing the source of cellular stress. In conclusion, in this work, we confirmed for the first time in the whole liver, that mGluR5 plays a key role in the ischemic injury progression. Furthermore, we showed that various mGluR5 NAMs share the ability to protect primary rat hepatocytes from ischemic injury; this protective effect was more significant for MPEP 30 µM, despite its ability to deplete cellular ATP. The mechanism underlying protection against ischemic injury reasonably consists in the block of cytotoxic events triggered by the overactivation of mGluR5s, such as the increase of Ca2+ release and the consequent mitochondrial dysfunction. The role of this receptor in liver has to be fully explored, although our data suggest their possible protective deployment in liver ischemic and inflammatory injury.

4. Materials and Methods Original studies in animals have been carried out in accordance with a protocol approved by the local committee for animal welfare and MIUR, the Italian Ministry for University and Research, according to the strictest European Directives. MTEP and MPEP were purchased from Tocris Cookson Ltd. (Bristol, UK). All other chemicals used for experiments were of analytical grade and were purchased from Sigma (Milan, Italy).

4.1. In Vitro Studies

4.1.1. Mitochondrial Isolation and Assays Mitochondrial isolation and assays: whole rat livers (9–15 g) were washed with ice-cold saline and processed immediately for mitochondria isolation. Minced tissue was homogenized in ice cold medium containing 0.25 M sucrose, 1 mM EDTA, 5 mM HEPES (pH 7.2) using a Teflon/glass Potter homogenizer (B. Braun, Melsungen, Germany). The homogenate was centrifuged at 1000× g for 10 min. The supernatant was centrifuged for 10 min at 10,000× g. The resulting pellet was resuspended in 0.25 M sucrose, 5 mM HEPES and centrifuged again 10 min at 10,000× g [33,34]. Single mitochondrial preparations were obtained for each individual animal (n = 6). Protein concentration was determined using the Lowry method [35]. Mitochondrial suspensions added with mGluR5 ligands or vehicle, were assayed for: ATP, mitochondrial membrane potential, RCI and FOF1 ATPase activity. Mitochondrial membrane potential was assessed by measuring the uptake of the fluorescent dye rhodamine 123 as previously described [33,34]. Briefly: after 150 treatment with mGluR5 modulators, mitochondria were resuspended in a phosphate buffer with 0.3 µmol/L rhodamine 123. Rhodamine 123 fluorescence was monitored using a Perkin Elmer LS 50B fluorescence spectrometer at 503 (excitation wavelength) and 527 nm (emission wavelength). ∆Ψ (mV) was calculated according to Int. J. Mol. Sci. 2018, 19, 314 12 of 16 the following relationship: ∆Ψ = −59 log (rhodamine 123) in/(rhodamine 123) out. Each measurement was performed in triplicate using fresh mitochondria preparations. RCI was measured using a Clarke electrode in a sealed mitochondrial chamber. Briefly, O2 consumption was measured in a sealed chamber at 25 ◦C by a Clark-type electrode. Mitochondria (1 mg/mL) were added to 1 mL of phosphate buffer containing 5 mM Mg2+ and 0.5 mM EGTA. Mitochondrial respiration was initiated with the addition of 10 mM succinate and 1 µM rotenone and oxidative phosphorylation was initiated with the addition of 5 mM adenosine diphosphate (ADP). O2 consumption recordings allowed the calculation of RCI, defined as the ratio between State 3 (maximum respiration rate reached in presence of respiratory substrates and ADP) and State 4 (low respiratory rate, respiratory substrates are present but ADP is exhausted) [36]. FOF1 ATPase activity was assayed using the Complex V MitoCheck kit from Cayman (Cayman Chemical, Ann Arbor, MI, USA). ROS formation was monitored by preloading mitochondria with 5 µM dichlorodihydrofluorescein diacetate (DCFH-DA). After 300 mitochondria were withdrawn and centrifuged at 10,000× g for 100. Pellets were resuspended in 0.5 mL KRH buffer and transferred to a 96-well plate for fluorescence measurements. Fluorescence values were measured on a Perkin Elmer Victor II (Perkin Elmer Inc., Waltham, MA, USA), using the excitation wavelength of 485 nm and emission wavelength of 530 nm [37].

4.1.2. Hepatocyte Isolation Hepatocytes were isolated from male Wistar rats (200–250 g, fasted for 18 h) by collagenase perfusion of the liver as previously described [38,39]. Hepatocytes were layered on top of a Percoll suspension and centrifuged at 500× g for 3 min. After removing the supernatant and the Percoll layer, cells were suspended in Krebs–Ringer–HEPES buffer containing 115 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L KH2PO4, 1.2 mmol/L MgSO4, and 2.5 mmol/L HEPES at pH 7.4 and incubated at 37 ◦C (final cell density, 106/mL).

4.1.3. Warm Ischemia Experiments on Rat Hepatocytes Anoxia was induced by blowing nitrogen for 3 min into hermetically sealed vials containing the cell suspension, while control vials were exposed to an oxygen-containing atmosphere. Hepatocytes were pre-incubated with the negative allosteric modulators MPEP (3 and 30 µM), MTEP (3 and 30 µM) and fenobam (1, 10 and 50 µM); with the orthosteric antagonist CPG (100 and 200 µM) and with the orthosteric agonist DHPG (100 µM) plus the mGluR5 selective positive allosteric modulator DFB (10 µM), for 15 min. The sealed vials were maintained at 37 ◦C in a water bath for 900 and cell samples were collected at 00, 300, 450, 600, 750 and 900.

4.1.4. Measurement of Cell Viability Cell viability was monitored by TB exclusion [40] and by release of LDH into the medium, as described by Bergmeyer [41].

4.1.5. Measurement of ATP ATP variation in presence of mGluR5 modulators was evaluated in PBS added with ATP 10 µM, in mitochondria and in cell suspensions. ATP was measured by the luminescence method using the ATPlite luciferin/luciferase kit (Perkin Elmer Inc., Waltham, MA, USA). Luminescence was evaluated on a Perkin Elmer Victor II, using a white 96-well plate.

4.2. Ex Vivo Studies

4.2.1. Cold Ischemia Reperfusion Model Livers were isolated from Balb c wild-type mice and mGluR5 knock-out mice, washed with Belzer cold preservation solution and stored on ice for 18 h, then reperfused with oxygenated Krebs–Henseleit at 37 ◦C for 1 h. After 18 h preservation, livers were washed with 5 mL of Krebs–Henseleit and Int. J. Mol. Sci. 2018, 19, 314 13 of 16 the outflowing perfusate was stored at −80 ◦C for further analysis. Samples of the reperfusion solution were collected at 00, 150, 300, 450, and 600. At the end of reperfusion, liver samples were snap-frozen in liquid nitrogen for further analysis. MPEP 0.3 µM or dimethyl sulfoxide (DMSO) was added to the preservation solution during cold storage and to the Krebs–Henseleit buffer during oxygenated reperfusion.

4.2.2. Warm Ischemia Model Livers were isolated from Balb c, wild-type mice and mgluR5 knockout mice, washed with Krebs–Henseleit solution and perfused for 300 with deoxygenated Krebs–Henseleit at 37 ◦C. Deoxygenated Krebs–Henseleit was obtained by bubbling the solution with N2CO2 (95–5%). At the end of the ischemic period, livers were reperfused with oxygenated Krebs–Henseleit solution at 37 ◦C for 2 h. Samples of the reperfusion solution were collected at 00, 300, 600, 900, and 1200. At the end of reperfusion, liver samples were snap-frozen in liquid nitrogen for further analysis. MPEP 30 µM or DMSO was added to the preservation solution during warm ischemia and during oxygenated reperfusion.

4.2.3. Perfusate Analysis Liver parenchyma viability was assessed by measuring the release of LDH into the effluent perfusate, as described by Bergmeyer [41].

4.2.4. Western Blot Analysis Liver biopsies were homogenized in an ice cold sodium dodecyl sulfate (SDS) lysis buffer (pH 7.4) containing 1 mM PMSF and a mixture of protease inhibitors. Equal amounts of proteins were suspended in a SDS–bromophenol blue reducing buffer with 20 mM 1,4-dithiothreitol (DTT). Protein electrophoresis was performed on 7.5% SDS polyacrylamide gels run on a minigel apparatus (BioRad, Mini Protean II Cell); gels were electroblotted on ImmunBlot polyvinylidene difluoride (PVDF) Membrane (Biorad Laboratories Inc., Hercules, CA, USA) for 2 h using a Wet/Tank blotting system (BioRad Mini Trans-blot Cell); membranes were blocked in tris-buffered saline (TBS) buffer containing 5% bovine serum albumin or blotting grade blockers. Blots were then incubated at 4 ◦C overnight with primary antibodies all at 1:1000 dilution, with the exception of anti-actin (1:10,000). As primary antibodies we used: mouse monoclonal anti-Tubulin (Sigma Aldrich, Milan, Italy); goat polyclonal anti-TNF-α, rabbit polyclonal anti-Bax, mouse monoclonal anti-Bcl-2, rabbit polyclonal anti-eNOS and rabbit polyclonal anti-actin (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-iNOS (Cayman chemical, Ann Arbor, MI, USA). Specific peroxidase-conjugated anti-IgG antibodies were from Santa Cruz Biotechnology. Blots were incubated for 1 h with secondary antibodies (peroxidase-coupled anti-mouse and anti-rabbit, Biorad) diluted 1:2000 with PBS. Immunostaining was revealed by ECL using a Chemidoc XRS Plus (Biorad Laboratories Inc., Hercules, CA, USA) [42].

4.2.5. Statistical Analysis Statistical analysis was performed by means of R Statistical software (v. 3.3.0) and the graphical interface R Studio (v. 1.0.143). The normality and homogeneity of variances were verified using Shapiro’s test and Levene’s test, respectively. In the majority of cases, data had normal distribution curves, where analyzed with ANOVA, followed by Tukey’s HSD Test for multiple comparisons. The Kruskal–Wallis non-parametric test was used for iNOS data since they had dishomogeneous variances. Dunn’s test was used for multiple comparisons. With measurements repeated against different time points, significance was analyzed by fitting data in a linear mixed-effects model. Int. J. Mol. Sci. 2018, 19, 314 14 of 16

Acknowledgments: This work was not sponsored by public or private institutions and was carried out using the internal resources of the Department of Internal Medicine and Therapeutics, Cellular and Molecular Pharmacology and Toxicology Unit. We thank Micaela Ascoli, Nicoletta Breda, and Massimo Costa for their skillful technical assistance. We also thank Mario Comelli for helping us with statistical evaluations and Anthony Baldry for revising the English. Author Contributions: Andrea Ferrigno, Ferdinando Nicoletti, and Mariapia Vairetti conceived and designed the experiments; Andrea Ferrigno and Mariapia Vairetti performed the in vivo experiments; Laura G. Di Pasqua, Clarissa Berardo, and Veronica Siciliano performed the in vitro experiments; Andrea Ferrigno, Plinio Richelmi, and Mariapia Vairetti analyzed the data from the ex vivo experiments; Clarissa Berardo, Laura G. Di Pasqua, and Veronica Siciliano analyzed the data from the in vitro experiments; Andrea Ferrigno wrote the draft; Laura G. Di Pasqua, Clarissa Berardo, and Veronica Siciliano revised the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

[3H]InsP [3H]inositolmonophosphate ACPD (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ADP adenosine diphosphate DCFH-DA dichlorodilhydrofluorescein diacetate DFB 3,30-difluorobenzaldazine DMSO dimethyl sulfoxide DTT 1,4-dithiothreitol eNOS endothelial nitric oxide synthase ER endoplasmic reticulum Gluk2 glutamate receptor, ionotropic kainate 2 iNOS inducible nitric oxide synthase KO knock-out PCR polymerase chain reaction PVDF polyvinylidene difluoride SDS sodium dodecyl sulfate TBS tris-buffered saline TNF-α tumor necrosis factor-α

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