Orexin-A up-regulates dopamine D2 receptor and mRNA in the nucleus accumbens Shell.

Orexins-A (OrxA) and -B (OrxB) neuropeptides are synthesized by a group of neurons located in the lateral hypothalamus and adjacent perifornical area, which send their projections to the mesolimbic dopaminergic (DAergic) system including ventral tegmental area and nucleus accumbens (NAc), where orexin receptors are expressed. NAc plays a central role in reward-seeking behavior and drug abuse. NAc-neurons express dopamine-1 (D1R) and dopamine-2 (D2R) receptors. Orexins bind to their two cognate G-protein-coupled receptors, orexin-receptor type-1 (Orx1R) and type-2 (Orx2R). Orexin receptor signaling is involved in behaviors such as motivation and addiction. Orexin-containing neurons modulate DAergic activity that is key in synaptic plasticity induced by addictive drugs. However, the effect of OrxA on expression and content of DAergic receptors in NAc is unknown. The purpose of this study was to investigate whether OrxA can alter gene expression and protein levels of D1R/D2R in NAc. Gene expression was evaluated by real-time PCR analysis and protein levels by western blot in rats. The results show that intracerebroventricular (i.c.v.) injection of OrxA increases both gene transcription and protein content of D2R but fails to modify D1R. This effect was also confirmed with OrxA infusion in NAc/Shell. Our results demonstrate for the first time that OrxA induces up-regulation of gene and protein of D2R in NAc. These findings support the hypothesis that OrxA modulates the DAergic transmission and this may serve to understand how orexin signaling enhances DA responses at baseline conditions and in response to psychostimulants.

Examining the Role of Histaminergic, Orexinergic, and Cannabinergic Systems in Redox Regulation in Gastric Adenocarcinoma.

Histaminergic, orexinergic, and cannabinoid systems play a role in both physiologic and oncogenic mechanisms in digestive tissues. These three systems are important mediators of tumor transformation, as they are associated with redox alterations, which are key aspects in oncological disorders. The three systems are known to promote alterations in the gastric epithelium through intracellular signaling pathways, such as oxidative phosphorylation, mitochondrial dysfunction, and increased Akt, which might promote tumorigenesis. Histamine promotes cell transformation through redox-mediated alterations in the cell cycle, DNA repair, and immunological response. The increase in histamine and oxidative stress generates angiogenic and metastatic signals through the VEGF receptor and H2R-cAMP-PKA pathway. Immunosuppression in the presence of histamine and ROS is linked to a decrease in dendritic and myeloid cells in gastric tissue. These effects are counteracted by histamine receptor antagonists, such as cimetidine. Regarding orexins, overexpression of the Orexin 1 Receptor (OX1R) induces tumor regression through the activation of MAPK-dependent caspases and src-tyrosine. OX1R agonists are candidates for the treatment of gastric cancer by stimulating apoptosis and adhesive interactions. Lastly, cannabinoid type 2 (CB2) receptor agonists increase ROS, leading to the activation of apoptotic pathways. In contrast, cannabinoid type 1 (CB1) receptor agonists decrease ROS formation and inflammation in gastric tumors exposed to cisplatin. Overall, the repercussion of ROS modulation through these three systems on tumor activity in gastric cancer depends on intracellular and/or nuclear signals associated with proliferation, metastasis, angiogenesis, and cell death. Here, we review the role of these modulatory systems and redox alterations in gastric cancer.

Intra-accumbal orexin-1 receptor inhibition prevents the anxiolytic-like effect of ethanol and leads to increases in orexin-A content and receptor expression.

Alcohol use is frequently associated with mood disorders. Similarly, individuals suffering from these disorders have a higher risk of developing alcoholism. Several reports have implicated orexin signaling in different behaviors related to alcohol consumption, whereas antagonists block these actions. However, the involvement of orexin-1-receptor (Orx1R) in ethanol-induced anxiolysis remains relatively unexplored. The purpose of this study was to investigate whether intra-accumbal inhibition of Orx1R blocks the anxiolytic-like effect of ethanol and to determine if ethanol administration modifies orexin-A content and Orx1R expression in the nucleus accumbens (NAc). The elevated-plus-maze test (EPM-test) was used to measure anxiety; orexin-A content and Orx1R expression were determined by enzyme-immunoassay and western blot, respectively. The results showed that the pretreatment with a selective antagonist of Orx1R, SB-334867 (SB, 3 µg/side), prevents the anxiolytic-like behavior induced by acute ethanol (2.5 g/kg). SB-334867 per se had no effect on anxiety levels. Pretreatment with SB-334867 followed by ethanol (SB + Et) increased orexin-A content and Orx1R levels in the NAc in comparison to the groups that only received ethanol (V + Et) or SB-334867 (SB + S). Ethanol treatment significantly augmented Orx1R expression but not the peptide content. The increase in orexin-A observed in SB + Et animals could be due in part to the inhibition of Orx1R, since SB-334867 prevents the binding of orexin-A to the receptor. This increase in orexin-A may, in turn, induce an up-regulation of receptor. Other possible explanations were discussed. In general, these findings suggest that orexin-A contributes largely to expression of ethanol-induced anxiolytic-like effect through the signaling of Orx1R in the NAc.

Orexin-A promotes EEG changes but fails to induce anxiety in rats.

Orexins (OXs) system has been suggested to play a key role in regulate processes related to arousal, including anxious behaviors. However, until now, the contribution of OXs in anxiogenic-like effects has not been completely clear, particularly in rats, whose results are not yet conclusive in behavioral-tests such as elevated-plus-maze test (EPM-test). The goal of this study was to explore the anxiogenic-like effect induced by orexin-A (OX-A) using two different paradigms; the EPM-test and simultaneously a quantitative index in vivo, the cortical-electroencephalographic-(EEG)-record. This index proposes that a low-frequency domain EEG, particularly 0.5-5-Hz (delta and low portion of theta-waves), is a key indicator to evaluate anxiety levels. We also explored whether the anxious effect of OX-A could be altered by an antagonist of dopamine-D2-receptor (D2R) sulpiride (SUL). Our results showed that intracerebroventricular (i.c.v.) injection of a low dose of OX-A (140 pmol) did not increase anxiety levels in rats. On the other hand, cortical-EEG-activity showed only a decrease in delta-spectral-power but no changes in theta-potency. These data suggest that the reduction in delta-power induced by OX-A only keeps the animals awake and alert without changes in anxiety levels.

Ethanol-induced changes in proenkephalin mRNA expression in the rat nigrostriatal pathway.

Endogenous opioid systems have been suggested to play a key role in ethanol reinforcement mechanisms and alcohol-drinking behavior. Ethanol induces differential alterations in opioid peptide expression in brain areas of the reward circuits, which may be linked to the reinforcing effects of ethanol. In addition, ethanol-induced alterations in opioidergic nigrostriatal transmission could be involved in brain sensitivity to ethanol and play a role in addictive processes. The aim of this work was to study the effects of acute ethanol administration on proenkephalin (proenk) mRNA expression in the rat substantia nigra and caudate-putamen (CP) for up to 24 h post treatment. Male Wistar rats received ethanol (2.5 g/kg) or distilled water by intragastric administration, and proenk mRNA expression was studied by in situ hybridization and densitometry. Ethanol transiently increased proenk mRNA expression in the CP 1 h after drug administration. Proenk mRNA levels remained elevated 2 h post treatment in the anterior-medial and medial-posterior regions of the CP. In contrast, ethanol decreased proenk mRNA expression in the substantia nigra pars compacta and pars reticulata 2 h after drug exposure. Alterations in enkephalin expression in the substantia nigra and CP in response to ethanol exposure could be involved in the mechanisms underlying brain sensitivity to the drug.

Changes in Proenkephalin mRNA expression in forebrain areas after amphetamine-induced behavioural sensitization.

Acute and repeated psychostimulant administration induces a long-lasting enhanced behavioural response to a subsequent drug challenge, known as behavioural sensitization. This phenomenon involves persistent neurophysiological adaptations, which may lead to drug addiction. Brain dopaminergic pathways have been implicated as the main neurobiological substrates of behavioural sensitization, although other neurotransmitters and neuromodulators may also participate. In order to investigate a possible involvement of opioid systems in amphetamine (AMPH) behavioural sensitization, we studied the AMPH-induced changes in Proenkephalin (Pro-Enk) mRNA expression in forebrain areas in both drug-naïve and AMPH-sensitized rats. Male Sprague-Dawley rats were sensitized to AMPH by means of a single AMPH (1 mg/kg s.c.) injection and the same dose was injected 7 days later to assess the expression of sensitization. Pro-Enk mRNA levels were evaluated by in situ hybridization in coronal brain sections. AMPH injection induced an increase in Pro-Enk mRNA expression in the nucleus accumbens and the medial-posterior caudate-putamen in drug-naïve rats. Challenge with AMPH to rats injected 1 week earlier with AMPH induced motor sensitization and increased and decreased Pro-Enk mRNA expression in the prefrontal cortex and the anterior medial caudate-putamen, respectively. Our results suggest that alterations in cortical and striatal enkephalinergic systems could contribute to the expression of AMPH behavioural sensitization.

Ethanol exposure differentially alters pro-enkephalin mRNA expression in regions of the mesocorticolimbic system.

RATIONALE: Opioid peptides have been suggested to play a major role in ethanol reinforcement mechanisms and alcohol drinking behaviour. However, in non-selected strains of rodents, it is not known whether opioid biosynthesis is a critical event in these processes. OBJECTIVE: The aim of this work was to study the effects of a high dose of ethanol (2.5 g/kg body weight) on pro-enkephalin (pro-enk) mRNA expression in brain regions of the mesocorticolimbic system for up to 24 h after drug administration. MATERIALS AND METHODS: Male Wistar rats were administered with ethanol (2.5 g/kg body weight) or distilled water and were killed 30 min, 1, 2, 4, 8 or 24 h after treatment. Coronal brain sections (20 mu) were obtained and pro-enk mRNA expression was studied by in situ hybridization and densitometry. RESULTS: Acute ethanol administration induced a transient decrease and increase in pro-enk mRNA expression in the ventral tegmental area (33.2%) and prefrontal cortex (26.5%) 2 and 4 h after treatment, respectively. In contrast, ethanol induced prolonged increases in pro-enk mRNA expression in the core and shell regions of the nucleus accumbens, with different kinetics. Maximal effects were observed 2 h after ethanol exposure (core, 70.0%; shell, 60.0%). CONCLUSIONS: Our results indicate that enkephalin expression in regions of the rat mesocorticolimbic system is differentially altered by acute ethanol treatment and suggest that enkephalins may play a key role in ethanol reinforcement mechanisms.

Mediobasal hypothalamic and adenohypophyseal TRH-degrading enzyme (PPII) is down-regulated by zinc deficiency.

Thyrotropin-releasing hormone (TRH) synthesized in hypothalamic paraventricular nucleus directs hypothalamus-pituitary-thyroid (HPT) axis function, regulating thyrotropin release from adenohypophysis and thyroid hormones serum concentration. Pyroglutamyl aminopeptidase II (PPII), a Zn-dependent metallopeptidase located in adenohypophysis and medial-basal-hypothalamus degrades TRH released from the median eminence and participates in HPT axis function by regulating TRH-induced thyrotropin release from adenohypophysis. It is unknown whether dietary Zn deficiency down-regulates PPII. Our aim was to compare adenohypohyseal and medial-basal-hypothalamic PPII activity and expression of adult rats fed a Zn-deficient diet (2ppm) throughout their lifespan (DD), prenatally (DC) or after weaning (CD) vs. that of animals fed a control diet (20ppm:CC). Female rats consumed a Zn-deficient or control diet from two weeks before gestation and up to the end of lactation. We analyzed adenohypophyseal and medial-basal-hypothalamic PPII activity of dams and male offspring when adults; its relation to median eminence TRH, serum thyrotropin, leptin and thyroid hormones concentration. Offspring ate the same diet as their dams (CC, DD) or were switched from dietary regime after weaning (CD, DC) and until 2.5 months of age. DD males showed decreased adenohypophyseal and medial-basal-hypothalamic PPII activity, along with high thyrotropin serum concentration. Post-weaning Zn-deficiency (CD) decreased PPII activity only in adenohypophysis and increased thyrotropin circulating levels. Zn-replenishment (DC) normalized PPII activity in both regions and serum thyrotropin concentration. Adenohypophyseal PPII activity decreased and prolactin levels increased in Zn-deficient dams. We concluded that long-term changes in dietary Zn down-regulate PPII activity independently of T3, increasing thyrotropin serum concentration, overall resembling sub-clinical hypothyroidism.

[3H]DPDPE binding to delta opioid receptors in the rat mesocorticolimbic and nigrostriatal pathways is transiently increased by acute ethanol administration.

Dopaminergic transmission in the mesolimbic and nigrostriatal pathways plays a key role in the reinforcement mechanisms and brain sensitivity to ethanol, respectively. Ethanol reinforcement and high alcohol drinking behaviour have been postulated to be partially mediated by a neurobiological mechanism involving the ethanol-induced activation of the endogenous opioid system. Activation of opioid neural pathways by ethanol may include alterations in the processing, release and/or the receptor binding of opioid peptides. The aim of this work was to investigate the effects of acute ethanol administration on delta opioid receptors in the rat mesocortical, meso-accumbens and nigrostriatal pathways by quantitative receptor autoradiography, using [(3)H] (2-D-penicillamine, 5-D-penicillamine)-enkephalin as radioligand. A significant increase in [(3)H] (2-D-penicillamine, 5-D-penicillamine)-enkephalin binding was observed in the substantia nigra pars reticulata 1 h after ethanol treatment. Two hours after drug exposure, ligand binding was significantly increased in the frontal and prefrontal cortices, the core and shell regions of the nucleus accumbens, and in the anterior-medial and medial-posterior regions of the caudate-putamen. In contrast, ligand binding was significantly decreased in the posterior region of the caudate-putamen 30 min after ethanol administration. The observed effects may reflect ethanol-induced changes in ligand binding affinity and/or in receptor density. Our results suggest that transitory changes in delta opioid receptors with different kinetic patterns may be involved in ethanol reinforcement and brain sensitivity to the drug. Ethanol-induced delta receptor up- and down-regulation mechanisms may participate in modulation of dopaminergic transmission in the mesocorticolimbic and nigrostriatal pathways.

Acute ethanol administration differentially alters enkephalinase and aminopeptidase N activity and mRNA levels in regions of the nigrostriatal pathway.

Opioid peptides play a key role in ethanol reinforcement and may also represent important determinants in brain sensitivity to ethanol through modulation of nigrostriatal dopaminergic activity. Regulation of opioid levels by peptidase-degrading enzymes could be relevant in ethanol's actions. The aim of this work was to study the acute ethanol (2.5 g/kg) effects on the activity and mRNA expression of enkephalinase (NEP) and aminopeptidase N (APN) in the rat substantia nigra (SN) and the anterior-medial (amCP) and medial-posterior (mpCP) regions of the caudate-putamen (CP). Enzymatic activities were measured by fluorometric assays and mRNA expression by reverse transcriptase polymerase chain reaction. Acute ethanol administration differentially altered peptidase activities and mRNA expression with different kinetics. Ethanol increased and decreased NEP mRNA levels in the SN and amCP, respectively, but produced biphasic effects in the mpCP. APN mRNA levels were increased by ethanol in all brain regions. Ethanol induced a transient and long-lasting increase in NEP (mpCP) and APN (amCP) activities, respectively. Peptidase activities were not changed by ethanol in the SN. Our results indicate that striatal NEP and APN are important ethanol targets. Ethanol-induced changes in these neuropeptidases in the CP could contribute to the mechanisms involved in brain sensitivity to ethanol.

Activity and expression of enkephalinase and aminopeptidase N in regions of the mesocorticolimbic system are selectively modified by acute ethanol administration.

Opioid peptides play a key role in ethanol reinforcement and alcohol drinking behavior. However, regulation of opioid levels by peptidase-degrading activities in ethanol's actions in brain is still unclear. The aim of this work was to study the acute effects of ethanol (2.5 g/kg) on enkephalinase (NEP) and aminopeptidase N (APN) activities and expression in regions of the mesocorticolimbic system, as well as on corticosterone levels in serum for up to 24 h after administration. Enzymatic activities were measured by fluorometric assays, mRNA's expression by reverse transcriptase polymerase chain reaction (RT-PCR) and corticosterone levels by radioimmunoassay. Acute ethanol administration modified peptidase activity and expression with different kinetics. Ethanol induced a transitory increase and decrease in NEP and APN activities in the frontal cortex (FC) and ventral tegmental area (VTA), whereas only increases in these activities were observed in the nucleus accumbens (NAcc). Ethanol induced an increase in NEP mRNA in the FC and decreases in APN mRNA in the FC and NAcc. In contrast, ethanol produced biphasic effects on both enzymes expression in the VTA. Corticosterone levels were not changed by ethanol. Our results suggest that NEP and APN could play a main role in ethanol reinforcement through regulation of opioid levels in mesolimbic areas.

Role of mu and delta opioid receptors in alcohol drinking behaviour.

The dopaminergic mesolimbic system plays a key role in the mechanisms of reinforcement elicited by alcohol (ethanol) and other drugs of abuse. Numerous lines of evidence indicate that ethanol reinforcement mechanisms involve, at least partially, the ethanol-induced activation of the endogenous opioid system. Ethanol may alter opioidergic transmission at different levels, including the biosynthesis, release, and degradation of opioid peptides, as well as binding of endogenous ligands to opioid receptors. Several studies suggest that mu and delta opioid receptors play a major role in ethanol reinforcement and dependence. These studies implicate enkephalins and beta-endorphin as physiological mediators of ethanol's actions in the brain. In this review we describe the pharmacological characteristics of opioid receptors and their distribution in brain, as well as the major functions of their endogenous ligands. Thereafter, we present evidence supporting the participation of mu and delta opioid receptors in ethanol reinforcement mechanisms and high alcohol drinking behaviour. The use of opioid receptor agonists and antagonists, as well as ethanol-preferring selected rodents and knockout mice, has contributed to understand the role of mu and delta receptors in these processes. The effects of ethanol on binding of selective ligands to opioid receptors in different experimental models are also reviewed. The relevance of opioid receptors in human alcoholism is further evidenced by the association of mu receptor polymorphisms with ethanol dependence. The clinical implication of these findings is discussed regarding the differential responses observed in some alcoholic patients to treatment with opioid receptor antagonists such as naltrexone.