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Mu opioid receptor mRNA expression, binding, and functional coupling to G-proteins in human epileptic hippocampus

Mu opioid receptor mRNA expression, binding, and functional coupling to G-proteins in human epileptic hippocampus
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  RAPID COMMUNICATION Mu Opioid Receptor mRNA Expression, Binding, and FunctionalCoupling to G-Proteins in Human Epileptic Hippocampus Manola Cuellar-Herrera, 1 Ana Luisa Velasco, 1 Francisco Velasco, 1 Laura Chavez, 2 Sandra Orozco-Suarez, 3 Guliz Armagan, 4 Ezgi Turunc, 4 Engin Bojnik, 5 Ayfer Yalcin, 4 Sandor Benyhe, 5 Anna Borsodi, 5 Mario Alonso-Vanegas, 6 and Luisa Rocha 7 * ABSTRACT: Mu opioid receptors (MOR) are known to be involved inseizure activity. The main goal of the present study was to characterizethe MOR mRNA expression, binding, as well as G protein activationmediated by these receptors in epileptic hippocampus of patients withpharmacoresistant mesial temporal lobe epilepsy (TLE). In contrast withautopsy samples, hippocampus obtained from patients with mesial TLEdemonstrated enhanced MOR mRNA expression (116%). Saturationbinding experiments revealed significantly higher (60%)  B  max  values forthe mesial TLE group, whereas the  K  d  values were not statistically differ-ent. Although mesial TLE group demonstrated high levels of basal bind-ing for the G proteins (136%), DAMGO-stimulated [ 35 S]GTP c S bindingdid not demonstrate significant alterations. In conclusion, our presentdata provide strong evidence that the epileptic hippocampus of patientswith pharmacoresistant mesial TLE presents significant alterations inMOR. Such changes may represent adaptive mechanisms to compensatefor other as yet unknown alterations.  V V C 2010 Wiley Periodicals, Inc. KEY WORDS: mu opiod receptor; mRNA; G-protein; binding;hippocampus; mesial temporal lobe epilepsy Opioid receptors are thought to be involved in epileptic activity. Theactivation of mu opioid receptors (MOR), which interact with Go, Gi(Chan et al., 1995), and Gs proteins (Chakrabarti et al., 2005), has been associated with anticonvulsant (Albertson et al., 1984) or procon-vulsant effects (Tortella et al., 1987). Further studies support dual effectsof MOR, i.e., they facilitate the epileptogenesis process, but increaserefractoriness to subsequent seizures during the postictal period (Rocha et al., 1991). Patients with temporal lobe epilepsy (TLE), the mostcommon form of epilepsy among adults characterizedby chronic seizures srcinated in the hippocampal for-mation (Sloviter, 1994), are often resistant to medicaltherapy and become candidates to surgical resection of the epiletogenic zone. In these patients, positron emis-sion tomography (PET) studies reveal an enhancementof the opioid receptor binding availability in the tem-poral neocortex ipsilateral to the epileptic focus during the postictal and interictal periods (Frost et al., 1988;Hammers et al., 2007). Although these observationslead to suggest an enhanced activity of the opioid sys-tem in the epileptic brain, a recent study from ourgroup provides strong evidence that the signal trans-duction mechanisms downstream of MOR aredecreased in the temporal neocortex ipsilateral to theepileptic focus of patients with mesial TLE (Rocha et al., 2009). The hippocampal formation is a brainregion sensitive to the effects of opioid peptides. Acti-vation of MOR in this brain area induces excitatory effects on the activity of pyramidal neurons via disin-hibitory mechanisms involving GABAergic interneur-ons (Siggins and Zieglgansberger, 1981; Madison andNicoll, 1988). In experimental models of epilepsy, theMOR changes in hippocampus depend on the type of seizure evaluated and the rate of neuronal excitability (Bausch and Chavkin, 1997, Skyers et al., 2003,Rocha et al., 1993). Concerning human brain, PETstudies demonstrate decreased or no significantchanges in opiod receptor binding availability in theepileptic hippocampus (Frost et al., 1988). However,there is no further data concerning signal transductionmechanisms downstream of the MOR in the humanepileptic hippocampus of patients with mesialTLE. The main goal of the present study was toprovide more information concerning MOR changesassociated with epilepsy in human hippocampus. Wecharacterized MOR mRNA expression, binding, andG protein activation in hippocampus of patients withpharmacoresistant mesial TLE who underwent epi-lepsy surgery. The evaluation of this tissue is an excel-lent opportunity to determine MOR receptor changesin the brain area that represents the epileptic focus inthe mesial TLE (Sloviter, 1994). The protocoldesign was previously approved by the scientific com-mittees of the institutions involved in the present 1 Epilepsy Clinic of Hospital General de Mexico, Mexico City, Mexico; 2 Department of Pathology of Hospital General de Mexico, Mexico City,Mexico;  3 Unit for Medical Research in Neurological Diseases, NationalMedical Center, Me´xico City, Mexico;  4 Department of Biochemistry,Faculty of Pharmacy, Ege University Bornova Izmir, Turkey;  5 BiologicalResearch Center of the Hungarian Academy of Sciences, Szeged, Hun-gary;  6 National Institute of Neurology and Neurosurgery ‘‘ManuelVelasco Suarez,’’ Mexico City, Mexico;  7 Department of Pharmacobiol-ogy, Center of Research and Advanced Studies, Mexico City, Mexico Grant sponsor: National Council for Sciences and Technology of Mexico;Grant numbers: J110.0130/2009, 98386; Grant sponsor: National Instituteof Science and Technology of Distrito Federal, Academy of Sciences of Hungary; Grant numbers: ETT577/2006, RET67/2005; Grant sponsor:Technological and Scientific Council of Turkey (TUBITAK); Grant num-ber: 106S249; Grant sponsor: Hungarian-Turkish project 11–06.*Correspondence to: Dr. Luisa Rocha, Department of Pharmacobiology,Center of Research and Advanced Studies, Mexico City, Mexico.E-mail: lrocha@cinvestav.mxAccepted for publication 25 August 2010DOI 10.1002/hipo.20891Published online 3 November 2010 in Wiley Online Library( HIPPOCAMPUS 22:122–127 (2012) V V C  2010 WILEY PERIODICALS, INC.  research. Epileptic hippocampal tissue was obtained frompatients with intractable mesial TLE recruited from the Epi-lepsy Clinic Program of Hospital General de Mexico (Table 1).Each patient signed an informed consent. All the patients wereon medication with one or two of the following antiepilepticdrugs: carbamazepine, valproic acid, phenobarbital, and phenyt-oin. None of the patients involved in the present study demon-strated gross structural lesions other than hippocampal sclerosis.In 12 patients noninvasive studies with concordant results wereenough to program a temporal lobectomy. In two patients a Phase II invasive protocol was mandatory. It includes eitherbilateral hippocampal electrodes or basolateral grid implanta-tion with continuous video-EEG monitoring (Velasco et al.,2000). Epileptic patients had ‘‘en block’’ anterior lobectomy, ip-silateral to the epileptic focus at least 48 h after the last seizure.During the surgical procedure, hippocampal biopsies were col-lected immediately upon resection, quickly frozen in pulverizeddry ice and stored at  2 70 8 C. The whole hippocampusobtained at autopsy from subjects with no evidence of neuro-logical disease was used as control tissue since previous reportsindicate that MOR mRNA, binding and agonist-stimulated[ 35 S]GTP g S binding are preserved for several hours after death(Platzer et al., 2000; Gonza ´lez-Maeso et al., 2002; Escriba ´et al., 2004). Hippocampus was dissected at the time of au-topsy, with a postmortem interval of 3–10 h, immediately stored at  2 70 8 C and then manipulated as described below nolater than 1 week after they were obtained (Table 2). Forevaluation of MOR and GAPDH mRNA expression, totalRNA isolation and reverse transcription polymerase chain reac-tion for human MOR and GAPDH mRNAs were carried outaccording to Yalcin (2004). Human MOR (gene bank:NM_001145283) primers were used as described previously (Tripathi et al., 2008). Glyceraldehyde-3-phosphate dehydro-genase (GAPDH) (gene bank: NM_002046) primers werenewly designed using Primer3 software (Rozen and Scaletsky,2000). The forward primers for MOR and GAPDH geneswere 5 0 -TCTGGCTCCAAAGAAAAGGA-3 0 and 5 0 -GAGT-CAACGGATTTGGTCGT-3 0 , respectively. The reverse primersfor MOR and GAPDH were 5 0 -CAATGCAGAAGTGCCAA-GAA-3 0 , 5 0 -TTGATTTTGGAGGGATCTCG-3 0 , respective-ly. Conditions for PCRs were optimized in a gradient cycler(Techne 512, UK) with regard to primers and various anneal-ing temperatures. Optimized settings were transferred to real-time PCR protocols on a Stratagene Mx3000P real-time detec-tion system (Stratagene, USA). Amplification of 1  l l RT mix-ture (cDNA diluted 1:5) was carried out using 1  l l (10 pmol)forward and reverse primers, 12.5  l l Brilliant SYBR  1 Green Q PCR 2 3  Master Mix (Stratagene, USA) and 9.5  l l nuclease-free water in a total volume of 25  l l. Cycling parameters wereas follows: 5 min at 94 8 C, 45 s at 94 8 C followed by 40 cyclesof 30 s at 60 8 C and 50 s at 72 8 C. An additional cycle formelting curve analyses was 1 min at 95 8 C, 30 s at 55 8 C. BothcDNA synthesis and PCR amplifications included negative con-trol reactions, which were set up by excluding RNA and DNA templates, respectively. The melting temperatures ( T   m ) forMOR and GAPDH were 80.7 and 73.6 8 C, respectively.GAPDH gene was used as an endogenous control for normal-ization. The relative expressions of target genes were quantifiedaccording to ABI Prism 7700 Sequence Detection System UserBulletin No. 2 (Applied Biosystems, Foster City, CA) andLivak and Schmittengen (2001). The PCR products were ana-lyzed using 2% agarose gel which contained ethidium bromi-de. For saturation binding and [ 35 S]GTP g S functional assays,crude membrane fraction from human hippocampus was pre-pared according to the method previously described (Benyheet al., 1997). Protein levels were determined by the method of Lowry et al. (1951). Saturation binding assay was performedaccording with the procedure described previously (Gabilondo TABLE 1.Clinical Data of Patients With Pharmacoresistant Mesial Temporal Lobe Epilepsy Patient Age (years) GenderAge of seizureonset (years)Duration of epilepsy (years)Frequency of seizures (per month)Side of focusHUM-114 29 M 2 27 4 LeftHUM-115 14 F 6 8 150 LeftHUM-116 29 F 6 23 8 RightHUM-117 28 F 27 1 170 RightHUM-118 24 M 1 23 20 BilateralHUM-119 24 M 5 19 6 LeftHUM-120 30 F 8 22 10 RightHUM-121 36 F 2 34 252 BilateralHUM-122 52 F 13 39 3 RightHUM-123 24 F 9 15 10 RightHUM-124 33 F 3 30 5 LeftHUM-125 20 M 1 20 36 LeftHUM-129 38 M 33 5 15 LeftHUM-130 49 M 17 32 8 Right M, male; F, female.  Mu RECEPTORS IN HUMAN EPILEPTIC HIPPOCAMPUS  123  Hippocampus  et al., 1995). The data obtained were first fit to the rectangularhyperbolic function followed by linear transformation(Scatchard, bound/free vs. bound) to determine equilibriumdissociation constant ( K   D ) and receptor density ( B  max  ) using the program Prism (GraphPad Software). [ 35 S]GTP g S func-tional assay was used to evaluate receptor-mediated G-proteinactivation as described previously  (Rocha et al., 2009). G-pro-tein activation was given as percent of the specific [ 35 S]GTP g Sbinding observed in absence of receptor ligands (basal activity).[ 35 S]GTP g S binding experiments were performed in triplicates.Data were subjected to nonlinear regression analysis of concen-tration effect curves performed by Prism (GraphPad Software)to determine Log EC 50  and  E  max   values. Results from allexperiments were examined statistically by Student’s  t   test and a value of   P   <  0.05 was considered statistically significant. Epi-leptic hippocampi were obtained from 14 patients (six maleand eight female) ranged in age from 14 to 52 yrs (30.7  6  2.7yrs old) with intractable mesial TLE history. Their clinical data were as follows: 21.2  6  2.9 yrs of epilepsy duration; age of sei-zure onset at 9.5  6  2.6 yrs old; and 49.7  6  21.3 seizures permonth (Table 1). Autopsy samples were acquired from 8 sub- jects (4 male and 4 female) ranging in age from 20 to 77 yrs(44.5  6  7.3 yrs), who died by different causes and without his-tory of neurological disease (Table 2). Total RNA contentsfrom mesial TLE group (1.61 6 0.59) were similar to thosefound for autopsy samples (1.47 6 0.68) (Fig. 1A). Theexpected PCR products for MOR and GAPDH from autopsiesand epileptic patients were detected at 172 and 238 bp, respec-tively (Fig. 1B). We found relative gene expression levels of MOR significantly higher in hippocampus of patients withmesial TLE (2.71  6  0.38, min 0.28, max 4.78,  P   <  0.05) incontrast with autopsy samples (1.25  6  0.50, min 0.37, max 2.54) (Fig. 1C). The specific MOR binding obtained fromthe equilibrium binding experiments was found to be saturablein hippocampus of both, autopsy and mesial TLE groups.[ 3 H]DAMGO binding from autopsy samples correlated posi-tively with age at death ( r   5  0.899,  P   <  0.05) and supportsdata obtained from previous studies (Gabilondo et al., 1995).Even though autopsy samples were obtained from older sub- jects, the  B  max   value from the mesial TLE group was signifi- TABLE 2.Characteristics of Control Subjects Subject Gender Age (years) Cause of death Postmortem delay (h)C1 M 66 Gastrointestinal bleeding 4C2 M 50 Hepatitis 6C3 F 71 Diabetes 5C4 F 77 Hypovolemic shock 3C5 F 39 Ovarian cancer 3C6 F 20 Chronic renal failure 4C7 M 29 Hypovolemic shock 5C8 M 37 Hypovolemic shock 10 M, male; F, female. FIGURE 1. RT-PCR analysis showing the presence of MOR inhuman hippocampus. mRNA from patients with mesial temporallobe epilepsy (MTLE) was compared with mRNA isolated from au-topsy (control) samples. (A) Total RNA integrity analysis. Lane 1:Control RNA sample; Lane 2: total RNA isolated from a sample of a patient with MTLE; Lane 3: total RNA isolated from a controlsample. (B) Agarose gel analysis of MOR and GAPDH genes inone control and one MTLE samples. The expected PCR productsfor MOR and GAPDH were 172 and 238 bp, respectively. Lane 1:Marker DNA 100bp; Lane 2: MOR expression in the hippocampusof a patient with MTLE; Lane 3: MOR expression in a control tis-sue; Lane 4: GAPDH expression in the hippocampus of a patient  with MTLE; Lane 5: GAPDH expression in a control sample. (C)Relative gene expression levels of MOR in control ( n  5  4) and MTLE ( n  5  7) groups. Data represent mean  6  S.D of three inde-pendent real-time polymerase chain reaction experiments. * P   < 0.05 vs. autopsy samples. [Color figure can be viewed in the onlineissue, which is available at] 124  CUELLAR-HERRERA ET AL.  Hippocampus  cantly higher (60%,  P   <  0.05), whereas the  K   d  value fromboth groups (0.75  6  0.08 nM, autopsy; 1.4  6  0.61 nM, epi-lepsy) was not statistically different (Fig. 2). These results sug-gest that the hippocampus of patients with mesial TLE presentan increased density of MOR, whereas their equilibrium disso-ciation constant is not modified. MOR-mediated G-proteinactivation was assayed by the radiolabel [ 35 S]GTP g S bound tothe G protein. Basal activity from mesial TLE group was signif-icantly higher (136%,  P   <  0.05) when compared with autopsy samples (Fig. 3A). DAMGO-stimulated [ 35 S]GTP g S binding was concentration-dependent and saturable. In autopsy sam-ples, [ 35 S]GTP g S binding stimulation by DAMGO showed a positive correlation for net stimulation values in relation to ageat death ( r   5  0.870,  P   <  0.01) (data not shown), supporting the influence of aging on functional [ 35 S]GTP g S binding (Gonza ´lez-Maeso et al., 2002). [ 35 S]GTP g S binding assaysincluding the analysis of all autopsy samples revealed a maximalstimulation ( E  max  ) of 42.8% and a potency (EC 50 ) value withinthe micromolar range ( 2 6.48  6  0.16). To obtain a clear inter-pretation of the specific role of transduction mechanisms inepilepsy, values obtained from mesial TLE group were com-pared with those obtained from autopsies of subjects in a rangeof age from 20 to 50 yrs old. According to this comparison,both groups demonstrated similar Emax (27.8 and 20.8%, au-topsy and mesial TLE groups, respectively) and EC 50  ( 2 7.0 6 0.2 and  2 6.82  6  0.2, autopsy and mesial TLE groups, respec-tively) values (Fig. 3B). These results indicated that, althoughthe epileptic hippocampus presents higher density of MOR (seesaturation binding results), it does not show significant changesin the efficacy ( E  max  ) and potency (EC 50 ) of DAMGO for thestimulation of the G protein. To our knowledge, this is thefirst time that alterations in MOR mRNA expression, binding and induced G-protein activation in hippocampus of patientswith pharmacoresistance mesial TLE are described. We foundan enhancement in MOR gene expression that was consistentwith significant increases in receptor density. Surprisingly, therewere not significant alterations in DAMGO stimulated[ 35 S]GTP g S binding. According to previous studies, it is im-portant to consider that the autopsy samples were collected atpostmortem intervals that allow the preservation of their physi-cal conditions (Gonza ´lez-Maeso et al., 2002; Escriba ´ et al.,2004). Then, values obtained from autopsy samples under ourexperimental conditions can be considered as control situation.Other important factor to be considered is that the density aswell as MOR-mediated G-protein stimulation are directly cor-related with age (Gabilondo et al., 1995; Gonza ´lez-Maesoet al., 2002), a notion that was supported by our resultsobtained from autopsy samples. Gene expression is modulatedby proteins acting at DNA regulatory sequence. Regulation by transcription activators and repressors could be involved in theenhanced MOR mRNA expression found in the epileptic hip-pocampus. In this context, it has been shown that the overex-pression of poly(ADPribose) polymerase-1 (PARP-1), a 116-kDa nuclear protein known to have DNA binding activity andenzymatic activity of ADP-ribosylation (Soldatenkov et al.,2002), upregulates MOR gene transcription (Ono et al., 2009).Similarly, interleukin-6 strongly induces MOR mRNA in thehuman neuroblastoma cell line SH SY5Y, an effect dependenton the transcription factors 1 (STAT1) and STAT3 (Bo¨rneret al., 2004). PARP-1 activation has been proposed to occurfollowing status epilepticus (Fujikawa, 2005), whereas interleu-kin-6 blood concentrations are chronically increased in patientswith TLE (Liimatainen et al., 2009). In the future, moredetailed studies are needed to assess the mechanisms involvedin the augmentation of MOR mRNA expression in the humanepileptic hippocampus. In spite of their known neuronallocalization, MOR are also expressed in glia (Murphy andPearce, 1987). It is possible to suggest that the enhanced gliosisfound in hippocampus of patients with mesial TLE may account for the high [ 3 H]DAMGO binding detected in thepresent study. Although no direct evidence exists in the humanbrain, experiments carried out in rats support a reduced opioidpeptides release in hippocampus during the interictal period(Rocha et al., 1997). The enhanced density in MOR found inthe epileptic hippocampus could be a homeostatic reactioncaused by a decreased release of endogenous opioid peptides inthis brain area. Despite of the high MOR mRNA and density  FIGURE 2. Saturation analysis for [ 3 H]-DAMGO binding inhippocampus membranes of autopsy samples ( n  5  8,  n ) and epi-leptic patients ( n  5  14,  h ). Membranes were incubated withincreasing concentrations of [ 3 H]-DAMGO in the absence (totalbinding) or presence of Naloxone (10  l M, nonspecific binding).Points represent mean  6  S.E.M of the experiments performed intriplicate. The corresponding Scatchard plot is given as inset. Notethat the receptor density ( B  max  ) value for the epilepsy group washigher with respect to the autopsy samples. * P   <  0.05 vs. autopsy group.  Mu RECEPTORS IN HUMAN EPILEPTIC HIPPOCAMPUS  125  Hippocampus  levels detected in the hippocampus of patients with mesialTLE, [ 35 S]GTP g S binding experiments indicated that MOR signaling is not altered. Receptor activation of G proteins is a vital step in the signal transduction pathways determining ago-nist efficacy  (Kenakin, 1993), so the fact that the activity of theMOR signaling is not modified in the epileptic hippocampuscan be considered as an intracellular counteradaptation thatoccur in the epileptic focus preventing increased MOR binding to result in altered intracellular signaling. An explanation forthis situation is that high levels of receptor density may tend toproduce saturation of stimulus-response mechanisms, leading toa limit in the maximal ordinate response measured. Other ex-planation could be that the increased [ 3 H]-DAMGO binding could reflect an increase in the density of receptors that com-prises some with different intrinsic ability to activate G pro-teins. Indeed, since MOR couple to several different types of G a  subunits (Chakrabarti et al., 2005), epilepsy may have dif-ferent effects on specific subtypes of G a  subunits with differentaffinities for GTP. An interesting result from our functionalexperiments was that the basal binding was increased in the ep-ileptic hippocampus. It is possible that a substantial portion of the basal activity found in the present study represents preacti-vated G proteins, i.e., an spontaneous interaction establishedbetween receptors and G protein in the cell membrane, situa-tion that results in a tonic level of GTPase stimulation (Scheerand Cotecchia, 1997). Previous studies indicate that part of theapparent preactivation of basal activity may result from anopioid receptor-dependent mechanism, in as much as small butsignificant reduction of basal activity is also observed following opioid-mediated downregulation (Costa et al., 1988). In addi-tion, high levels of receptor density, as the one we found in thepresent study, are anticipated to increase basal binding as wellas the precoupled or constitutively active forms of the receptors(Costa et al., 1990). The nonsignificant increase in DAMGO-induced G protein activation in proportion to the high MOR density in the epileptic hippocampus can be explained becausethe occupation of the constitutively active receptors by agonistsmay not increase [ 35 S]GTP g S binding much above basal levels(Costa et al., 1990). Since the increase of the constitutive activ-ity of G protein-coupled receptors can be induced by muta-tions or they occur spontaneously in human diseases (Scheerand Cotecchia, 1997), we cannot exclude that it plays a signifi-cant role in the pathophysiology of the pharmacoresistant epi-lepsy. Finally, the present findings provide direct informationon the functional status of MOR in the hippocampus of patients with pharmacoresistant mesial TLE that will probably aid in the design of better antiepileptic medication for this dis-order. Further studies are necessary to elucidate the role of in-tracellular changes associated with MOR in the epileptic hippo-campus during the epilepsy process. Acknowledgments The authors thank Ms. Leticia Neri Bazan and Mr. HectorVazquez Espinosa for their excellent technical assistance. REFERENCES  Albertson TE, Joy RM, Stark LG. 1984. Modification of kindledamygdaloid seizures by opiate agonists and antagonists. J Pharma-col Exp Ther 228:620–627.Bausch SB, Chavkin C. 1997. Changes in hippocampal circuitry afterpilocarpine-induced seizures as revealed by opioid receptor distribu-tion and activation. J Neurosci 17:477–492.Benyhe S, Farkas J, To´th G, Wollemann M. 1997. Met5-enkephalin- Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioidand nonopioid sites in rat brain. J Neurosci Res 48:249–258.Bo¨rner C, Kraus J, Schro¨der H, Ammer H, Ho¨llt V. 2004. Transcrip-tional regulation of the human mu-opioid receptor gene by inter-leukin-6. Mol Pharmacol 66:1719–1726.Chakrabarti S, Regec A, Gintzler AR. 2005. Biochemical demonstra-tion of mu-opioid receptor association with Gsalpha: Enhancementfollowing morphine exposure. Mol Brain Res 135:217–224.Chan JS, Chiu TT, Wong YH. 1995. Activation of type II adenylylcyclase by the cloned mu-opioid receptor: Coupling to multiple Gproteins. J Neurochem 65:2682–2689.Costa T, Klinz FJ, Vachon L, Herz A. 1988. Opioid receptors arecoupled tightly to G proteins but loosely to adenylate cyclase inNG108–15 cell membranes. Mol Pharmacol 34:744–754.Costa T, Lang J, Gless C, Herz A. 1990. Spontaneous associationbetween opioid receptors and GTP-binding regulatory proteins innative membranes: Specific regulation by antagonists and sodiumions. Mol Pharmacol 37:383–394.Escriba ´ PV, Ozaita A, Garcı´a-Sevilla JA. 2004. Increased mRNA expression of alpha2A-adrenoceptors, serotonin receptors and FIGURE 3. (A) Representation of absolute basal valuesobtained from autopsies ( n  5  8) and epileptic patients ( n  5  14). Values are represented as mean  6  S.E.M. of fmol/mg of protein.(B) Specific [ 35 S]GTP c S binding to membranes of samples fromall autopsies (- n -), from autopsies of subjects of less than 51 yrsold ( n  5  5,   l  ) and epileptic patients (- h -) as a function of increasing concentration of the mu receptor agonist DAMGO.Each point represents the mean 6 S.E.M of the individual percent-age stimulation over basal values. Note that in epileptic patients,the [ 35 S]-GTP c S binding percentage stimulation by DAMGO wasno significantly different from autopsy samples of subjects of lessthan 51 yrs old. * P   <  0.05 vs. autopsy group. 126  CUELLAR-HERRERA ET AL.  Hippocampus
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