Brain Arachidonic Acid Cascade Enzymes are Upregulated in a Rat Model of Unilateral Parkinson Disease

Arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. AA signaling can be coupled to D₂-like receptor initiated AA hydrolysis from phospholipids by cytosolic phospholipase A₂ (cPLA₂) and subsequent metabolism by cyclooxygenase (COX)-2. In unilaterally 6-OHDA- and sham-lesioned rats, we measured brain expression of cPLA₂, other PLA₂ enzymes, and COX-2. Activity and protein levels of cPLA₂ were significantly higher as was COX-2-protein in caudate-putamen, frontal cortex and remaining brain on the lesioned compared to intact side of the 6-OHDA lesioned rats, and compared to sham brain. Secretory sPLA₂ and Ca²⁺-independent iPLA₂ expression did not differ between sides or groups. Thus, the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA₂ and COX-2 expression within the AA metabolic cascade, which may contribute to symptoms and pathology in Parkinson disease.     

Cardiac Post-Junctional Alpha1- and Beta-Adrenoceptors: Effects of Chronic Chemical Sympathectomy with 6-Hydroxydopamine

Abstract

α₁- and β-adrenoceptor responsiveness and binding have been examined in cardiac tissues removed from guinea-pigs pretreated with 6-hydroxydopamine (6-OHDA) for 3 weeks. Results were compared with control tissues from sham-injected animals. Chemical sympathectomy with 6-OHDA resulted in an increase in the sensitivity of post junctional β-adrenoceptor-mediated responses to isoprenaline. No such increase was observed for the α₁-adrenoceptor-mediated responses to methoxamine. The increase in β-adrenergic responsiveness was accompanied by a significant (P<0.05) 52% increase in the number of [³H]-dihydroalprenolol binding sites with no change in binding affinity. [³H]-Prazosin binding was not affected by pretreatment with 6-OHDA. The results suggest that cardiac β-but not α₁-adrenergic responsiveness is regulated by the sympathetic innervation.     

Increased melatonin may play dual roles in the striata of a 6-hydroxydopamine model of Parkinson's disease

AimsTo investigate dynamic changes and roles of melatonin (MLT) in the striata of 6-hydroxydopamine (6-OHDA)-treated rats.Main methodsA Parkinson's disease (PD) rat was established by a unilateral injection of 6-OHDA into the right substantia nigra pars compacta (SNc) and the right medial forebrain bundle (MFB) to achieve a complete lesion of the ipsilateral nigrostriatal DA system. Dialysates were collected in the lesioned striatum at different time intervals by in vivo microdialysis. In addition, both contralateral and ipsilateral striatum tissues were collected at two time intervals (10:00 and 22:00 h) at 3 and 6 weeks after lesioning. The levels of DA, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the dialysates, as well as MLT in the dialysates and tissues were determined using HPLC.Key findingsThe dialysate contents of DA, DOPAC and HVA in the lesioned striatum were significantly decreased (P < 0.001) in comparison with those in the controls or in the unlesioned side 3 weeks after lesioning while the extracellular level of MLT in the lesioned striatum in these corresponding time intervals distinctly increased when compared with those in the controls (P < 0.05). The tissue MLT contents increased in the bilateral striata in different degrees at 6 weeks post-lesion (P < 0.05). Moreover, increased MLT levels correlate well with rotations or DA changes in the lesioned striatum.SignificanceThese data suggest that 6-OHDA lesion manipulates the MLT secretion pattern. Increased striatal MLT level by a unilateral intracerebral injection of 6-OHDA may play dual roles in the progression of PD in rats.     

Unilateral lesion of the nigrostriatal pathway decreases the response of fast-spiking interneurons in the medial prefrontal cortex to 5-HT1A receptor agonist and expression of the receptor in parvalbumin-positive neurons in the rat

5-Hydroxytryptamine_1A (5-HT_1A) receptors are expressed in the prefrontal cortical interneurons. Among these interneurons, calcium-binding protein parvalbumin (PV)-positive fast spiking (FS) interneurons play an important role in regulatory function of the prefrontal cortex. In the present study, the response of medial prefrontal cortex (mPFC) FS interneurons to the selective 5-HT_1A receptor agonist 8-OH-DPAT and change in expression of 5-HT_1A receptor on PV-positive neurons were examined in rats with 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNc) by using extracellular recording and double-labeling immunofluorescence histochemistry. Systemic administration of 8-OH-DPAT (1-243 μg/kg, i.v.) dose-dependently inhibited the mean firing rate of the FS interneurons in sham-operated and the lesioned rats, respectively. The cumulative doses producing inhibition in the lesioned rats (243 μg/kg) was significantly higher than that of sham-operated rats (27 μg/kg). Furthermore, the local application of 8-OH-DPAT (0.01 μg) in the mPFC inhibited the FS interneurons in sham-operated rats, while having no effect on firing rate of the FS interneurons in the lesioned rats. In contrast to sham-operated rats, the lesion of the SNc in rats did not cause the change of PV-positive neurons in the prelimbic prefrontal cortex, a subregion of the mPFC, whereas the lesion of the SNc markedly reduced in percentage of PV-positive neurons expressing 5-HT_1A receptors. Our results indicate that degeneration of the nigrostriatal pathway results in the decreased response of FS interneurons in the mPFC to 5-HT_1A receptor stimulation, which attributes to down-regulation of 5-HT_1A receptor expression in these interneurons.     

Simvastatin prevents dopaminergic neurodegeneration in experimental parkinsonian models: the association with anti-inflammatory responses

Background

In addition to their original applications to lowering cholesterol, statins display multiple neuroprotective effects. N-methyl-D-aspartate (NMDA) receptors interact closely with the dopaminergic system and are strongly implicated in therapeutic paradigms of Parkinson''s disease (PD). This study aims to investigate how simvastatin impacts on experimental parkinsonian models via regulating NMDA receptors.

Methodology/Principal Findings

Regional changes in NMDA receptors in the rat brain and anxiolytic-like activity were examined after unilateral medial forebrain bundle lesion by 6-hydroxydopamine via a 3-week administration of simvastatin. NMDA receptor alterations in the post-mortem rat brain were detected by [³H]MK-801(Dizocilpine) binding autoradiography. 6-hydroxydopamine treated PC12 was applied to investigate the neuroprotection of simvastatin, the association with NMDA receptors, and the anti-inflammation. 6-hydroxydopamine induced anxiety and the downregulation of NMDA receptors in the hippocampus, CA1(Cornu Ammonis 1 Area), amygdala and caudate putamen was observed in 6-OHDA(6-hydroxydopamine) lesioned rats whereas simvastatin significantly ameliorated the anxiety-like activity and restored the expression of NMDA receptors in examined brain regions. Significant positive correlations were identified between anxiolytic-like activity and the restoration of expression of NMDA receptors in the hippocampus, amygdala and CA1 following simvastatin administration. Simvastatin exerted neuroprotection in 6-hydroxydopamine-lesioned rat brain and 6-hydroxydopamine treated PC12, partially by regulating NMDA receptors, MMP9 (matrix metalloproteinase-9), and TNF-a (tumour necrosis factor-alpha).

Conclusions/Significance

Our results provide strong evidence that NMDA receptor modulation after simvastatin treatment could partially explain its anxiolytic-like activity and anti-inflammatory mechanisms in experimental parkinsonian models. These findings contribute to a better understanding of the critical roles of simvastatin in treating PD via NMDA receptors.     

MIF-1 fails to modify agonist-induced oral activity in neonatal 6-OHDA-treated rats

-Prolyl- -leucyl-glycinamide (MIF-1) is known to attenuate apomorphine-induced stereotypies in adult rats that are lesioned as neonates with 6-hydroxydopamine (6-OHDA). To test whether MIF-1 would affect dopamine (DA) agonist-induced and serotonin (5-HT) agonist-induced oral activity, both intact and neonatal 6-OHDA-treated rats were studied. Rats at 3 days from birth were injected with desipramine (20 mg/kg, IP), 1 h before 6-OHDA HBr (100 μg, salt form, in each lateral ventricle) or its vehicle, saline-ascorbic acid (0.1%). At approximately 6 months rats were treated with MIF-1 (0.1, 1.0, or 10.0 mg/kg, IP), 10 min before SKF 39393 HCl (1.0 mg/kg, IP) or m-chlorophenylpiperazine 2HCl (m-CPP 2HCl; 0.5 mg/kg, IP), DA D₁ and 5-HT_1C,2 receptor agonists, respectively. Although both agonists increased oral activity in control and neonatal 6-OHDA-treated rats, MIF-1 did not modify the response. In rats that received either of the three doses of MIF-1 for 21 consecutive days, there was still no observed effect of MIF-1 on the oral response of control and 6-OHDA-lesioned rats to SKF 38393 and m-CPP. These findings indicate that MIF-1 does not modify the oral activity response of supersensitized D₁ and 5-HT_1C receptors in adult rats that are lesioned neonatally with 6-OHDA.     

Effects of chronic, systemic treatment with the dopamine receptor agonist R-apomorphine in partially lesioned rat model of Parkinson's disease: an electrophysiological study of substantia nigra dopamine neurons

Previous studies have suggested that R-apomorphine (R-APO), a non-selective dopamine (DA) receptor agonist, has neuroprotective effects in the experimental models of Parkinson's disease (PD). In this study, we investigated the effects of chronic, systemic treatment with R-APO in the firing activity of substantia nigra pars compacta (SNc) DA neurons in 6-hydroxydopamine (6-OHDA) partially lesioned rats. In the 6-OHDA-lesioned rats treated with vehicle, injection of 6-OHDA (20.1 microg) into the striatum produced a partial lesion causing 41% loss of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SNc. In the partially lesioned rats, chronic, systemic treatment of R-APO (10 mg/kg/day, s.c., 11 days) attenuated loss of TH-ir neurons in the SNc. The partial lesion of the nigrostriatal pathway and R-APO treatment did not change the firing rate and firing pattern of DA neurons in the SNc of rats. In contrast, the R-APO treatment increased the number of spontaneously active DA neurons of the SNc in the partially lesioned rats, while the lesion decreased the number of spontaneously active DA neurons. In addition, the chronic R-APO treatment decreased the responsiveness of the DA neurons to intravenously administrated R-APO in the partially lesioned rats. These results indicate that chronic, systemic R-APO treatment has the neuroprotective effect, and reverses the decrease in the number of spontaneously active DA neurons in the SNc whereas the treatment induces a reduction in the sensitivity of DA receptors in the SNc to R-APO stimulation in this model.     

Turning in MFB-lesioned rats and antagonism of neuroleptic-induced catalepsy after lisuride and LSD.

The effects of lisuride, d-lysergic acid diethyl amide (LSD) and apomorphine were studied in rats with unilateral destruction of nigro-striatal nerve terminals either with 6-hydroxydopamine (6-OHDA) or 5, 6-dihydroxytryptamine (5,6-DHT). Lisuride at the dose of 50 μg kg^?1 i.p. induced contralateral turning for more than 4 hours while the circling induced by LSD (200 μg kg^?1) and apomorphine (1 mg kg^?1) persisted for only one hour. Lisuride, a compound stimulating both dopamine (DA) and 5-hydroxytryptamine (5-HT) receptors induced a more intense turning in 6-OHDA than in 5,6-DHT lesioned rats. This might indicate a modulation of 5-HT on rotational behavior. Haloperidol (1 mg kg^?1 i.p.) antagonized both lisuride- and LSD-induced turning. LSD, and much more persistently lisuride, counteracted the prochlorperazine-induced catalepsy. These findings correlate with the biochemical data indicating that lisuride is a very potent agonist at central dopaminergic receptors.     

The possible role of estrogen and selective estrogen receptor modulators in a rat model of Parkinson's disease

AimThe aim of the present study was to assess and compare the effect of 17β-estradiol and two different selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, as well as a selective estrogen receptor alpha agonist, propyl-pyrazole-triol (PPT) and a selective estrogen receptor beta agonist, diarylpropionitrile (DPN), on behavioral and biochemical alterations in 6-hydroxydopamine (6-OHDA)-induced nigral dopaminergic cell death in rats.Main methods80 female Wister rats were used. Animals were divided into eight equal groups: Group I; Sham operated, Group II; subjected to ovariectomy (OVX), Group III; OVX rats received striatal injection of 6-OHDA, Groups IV–VIII; OVX rats received striatal injection of 6-OHDA and were injected daily with 17β-estradiol, tamoxifen, raloxifene, PPT and DPN respectively for 5 days before 6-OHDA and continued for further 2 weeks.Key findingsResults showed that striatal injection of 6-OHDA produced significant behavioral alteration suggestive of PD, together with significant decrease in striatal dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) concentrations. 6-OHDA-induced nigral dopaminergic cell death was characterized by oxidative stress, evidenced by significant decrease in striatal glutathione peroxidase activity, as well as apoptosis, evidenced by significant increase in nigral caspase-3 activity. Treatment with 17β-estradiol, raloxifene, PPT, but neither tamoxifen nor DPN, resulted in significant amelioration of the behavioral and biochemical alterations induced by 6-OHDA.SignificanceThese findings suggest that estrogen and some SERMs having estrogenic agonist activity in the brain, like raloxifene, might exert beneficial effect in PD.     

Neurochemical effects of the R form of α-lipoic acid and its neuroprotective mechanism in cellular models of Parkinson’s disease

ObjectiveParkinson’s disease (PD) is a common neurodegenerative disease. This study aimed to investigate the effects of the R form of α-lipoic acid (RLA) in cellular models of PD induced by 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).MethodsCell viability and apoptosis were detected using CCK8 and Annexin V-FITC assays, respectively. Intracellular reactive oxygen species (ROS) were detected by fluorescence staining. ELISA assays were performed to detect the levels of dopamine and α-synuclein. To evaluate the effects of RLA on mitochondrial function, cytotoxicity, ATP levels, and mitochondrial gene expression were assayed. Additionally, the expression levels of autophagy-related proteins, including Parkin, PINK1, p62, ATG12, and LC3, were analyzed by western blot, and cell autophagy was visualized by immunofluorescence.ResultsRLA increased cell viability and decreased apoptosis, intracellular ROS, and cytotoxicity, and induced cell autophagy in PD models induced by 6-OHDA and MPTP. RLA also reversed the decreased dopamine and increased α-synuclein expression induced by 6-OHDA and MPTP. The mitochondrial regulatory protein PGC-1α was significantly up-regulated by RLA. The expression levels of autophagy-related proteins, including Parkin, PINK1, p62, and ATG12, were down-regulated after RLA treatment, while LC3 expression was up-regulated.ConclusionsRLA has a protective effect against cellular damage induced by 6-OHDA and MPTP. The neuroprotective mechanism of RLA may be associated with improvement of mitochondrial function and autophagy. Therefore, RLA may serve as a promising potential adjuvant for PD treatment.     

miR-506与PI3K/AKT通路在自发性高血压大鼠心脏重构中的作用

目的:探讨mi R-506和PI3K/AKT信号通路在自发性高血压大鼠心脏重构中的作用。方法:将12只雄性自发性高血压大鼠(Spontaneous Hypertension Rat, SHR)随机分为2组,每组6只。分别为SHR模型组和治疗组(卡托普利,30 mg·kg~(-1)),6只健康WKY大鼠作为空白对照组。SHR模型组和空白对照组灌胃等体积生理盐水,连续给药8周,采用尾动脉测压法测定给药前后各组大鼠血压,采用qRT-PCR法检测各组大鼠心肌miR-506表达量,并检测大鼠心肌组织中SOD和GPx mRNA表达水平,免疫印迹检测大鼠心肌中p-PI3K和p-AKT的蛋白表达量。结果:SHR模型组血压为(184.79±3.35)mmHg,与空白对照组比较显著升高(P0.05),治疗组血压为(133.57±1.43)mm Hg,与SHR模型组相比均显著降低(P0.05)。SHR模型组大鼠心肌中mi R-506、SOD、GPx的RNA相对表达量分别为(0.36±0.05)、(0.27±0.04)和(0.32±0.02),与空白对照组比较显著降低(P0.05),而p-PI3K、p-AKT蛋白水平显著降低(P0.05),与SHR模型组比较,治疗组大鼠心肌中mi R-506以及SOD、GPx的RNA水平显著升高(P0.05),p-PI3K、p-AKT蛋白水平显著升高(P0.05)。结论:在卡托普利治疗高血压的过程中,mi R-506可能通过抑制PI3K/AKT信号通路提高机体的抗氧化能力促进SHR心脏重塑。     

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin II-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR. (Mol Cell Biochem 269: 95–101, 2005)     

Development of dopamine receptor denervation supersensitivity in the neostriatum of the senescent rat

Dopamine (DA) stimulated adenylate cyclase activity and [³H]-spiroperidol specific binding were assessed in the striata from mature and old rats lesioned in the left substantia nigra with 6-hydroxydopamine (6-OHDA). Rotational behavior following the DA releasing agent, amphetamine, and the DA receptor agonist, lergotril, was also examined at 7 and 30 days, respectively, after lesioning. Results indicated that while there were rotational behavioral deficits following amphetamine in the senescent animal, none were seen with respect to lergotril. Both old and young animals showed similar degrees of contralateral rotation (with respect to the lesion) following lergotril administration. This suggested that both old and young animals showed similar development of denervation supersensitivity in the DA receptors of the lesioned striatum. Subsequent biochemical confirmation of this hypothesis was provided by findings which showed comparable relative increases in DA stimulated adenylate cyclase activity and [³H]-spiroperidol specific binding in the striata from the lesioned hemispheres of young and old rats. Additionally, high positive correlations were found between rotation and [³H]-spiroperidol specific binding, while those between DA stimulated adenylate cyclase activity and rotation were lower and dependent upon the concentration of DA used to stimulate adenylate cyclase activity (1, 5 and 100 uM). Results are discussed in terms of the specificity of the age-related deficits seen in the striatum of the senescent animal.     

Neonatal 6-Hydroxydopamine and Adult SKF 38393 Treatments Alter Dopamine D1 Receptor mRNA Levels: Absence of Other Neurochemical Associations with the Enhanced Behavioral Responses of Lesioned Rats

Abstract: To study potential biochemical correlates of dopamine (DA) and serotonin receptor supersensitivity, rats were lesioned at 3 days after birth with 6-hydroxydopamine (6-OHDA; 67 µg in each lateral ventricle; desipramine pretreatment, 20 mg/kg i.p., 1 h) and then sensitized with the DA D₁ agonist, SKF 38393 HCl (3.0 mg/kg i.p. per day) either ontogenetically (daily, for 28 consecutive days from birth) and/or in adulthood (four weekly injections, 6–9 weeks from birth). Controls received vehicle in place of 6-OHDA or SKF 38393. Enhanced locomotor responses were observed after SKF 38393 at 6 weeks, only in rats that received SKF 38393 + 6-OHDA in ontogeny. Locomotor responses were further enhanced in this group after the last of four weekly SKF 38393 injections at the 9th week. These weekly SKF 38393 treatments also produced enhanced responses in 6-OHDA rats that did not receive SKF 38393 in ontogeny. When striata were studied at 11 weeks, the percentages of high and low affinity DA D₁ binding sites were not altered. Basal as well as DA-, NaF-, and forskolin-stimulated adenylyl cyclase activities also were not changed. Dot blot analysis showed that there was a reduction of mRNA levels for DA D₁, but not serotonin_1C, receptors in the 6-OHDA groups. However, SKF 38393 at 6–9 weeks eliminated this alteration. Based on these findings it can be proposed that supersensitization may be a consequence of altered neuronal cross talk rather than an imbalance of receptor elements per se.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Kinin B1 and B2 receptor mRNA expression in the hypothalamus of spontaneously hypertensive rats

The central hypertensive effects induced by bradykinin are known to be mediated via B2 receptors, which are present constitutively in the brain. B, receptors are rapidly upregulated during inflammation, hyperalgesia, and experimental diabetes. The hypothalamus plays an important role in the regulation of cardiovascular homeostasis, and all components of kallikrein-kinin system have been identified in this area. Therefore, we analyzed the mRNA expression of B1 and B2 receptors in the hypothalamus of spontaneously hypertensive rats (SHR) by RT-PCR. Male SHR were studied at three different ages corresponding to the three phases in the development of hypertension: (i) 3-4 (prehypertensive), (ii) 7-8 (onset of hypertension), and (iii) 12-13 weeks (established hypertension) after birth, and compared with age-matched Wistar-Kyoto (WKY) rats. At all ages tested, B2 receptor mRNA levels in the hypothalamus of SHR were higher than age-matched WKY rats (p < 0.001). However, the B1 receptor mRNA levels were higher at the established phase of hypertension only. We conclude that B1 and B2 receptor mRNA are differentially expressed in the hypothalamus of SHR and may play different roles in the pathogenesis of hypertension: upregulation of B2 receptor mRNA from early age may participate in the pathogenesis of hypertension, whereas an upregulation of B1 receptor mRNA in the established phase of hypertension may reflect an epiphenomenon in essential hypertension.     

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals,birds, and amphibians

Abstract

Purpose: Previous studies have found non-CB₁ non-CB₂ G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.

Materials and methods: Antagonism of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.

Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 was not blocked by the CB₁ antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.

Conclusions: The portion of WIN55212-2-stimulated [³⁵S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB₁ receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.     

Proteomic characterization of the striatum and midbrain treated with 6-hydroxydopamine: Alteration of 58-kDa glucose-regulated protein and C/EBP homologous protein

Abstract

The present study performed proteomic analysis of the midbrain and striatum of 6-hydroxydopamine (6-OHDA)-treated neonatal rats—a model of attention-deficit hyperactivity disorder (ADHD). Proteomic analysis revealed that a 58-kDa glucose-regulated protein (Grp58) was temporarily phosphorylated and its level was elevated by 6-OHDA. Furthermore, 6-OHDA increased the expression level of C/EBP homologous protein (CHOP), a mediator of endoplasmic reticulum (ER) stress response, in the midbrain and striatum. In vitro experiments using PC12 cells revealed that 6-OHDA or hydrogen peroxide could induce the elevation of Grp58 and CHOP. 6-OHDA could induce the elevation of Grp58 and CHOP in the presence of catalase, a hydrogen peroxide-removing enzyme, suggesting that the elevation of Grp58 and CHOP are induced by both hydrogen peroxide and p-quinone generated by 6-OHDA. Collectively, these findings suggest that ER stress involving the alteration of Grp58 and CHOP play a significant role in the induction of insults by 6-OHDA in vivo.