FEZF2 and AIRE1: An Evolutionary Trade-off in the Elimination of Auto-reactive T Cells in the Thymus.

Autoimmune Regulator 1 (AIRE1) and Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2) play pivotal roles in orchestrating the expression of tissue-restricted antigens (TRA) to facilitate the elimination of autoreactive T cells. AIRE1's presence in the gonads of various vertebrates has raised questions about its potential involvement in gene expression control for germline cell selection. Nevertheless, the evolutionary history of these genes has remained enigmatic, as has the rationale behind their apparent redundancy in vertebrates. Furthermore, the origin of the elimination process itself has remained elusive. To shed light on these mysteries, we conducted a comprehensive evolutionary analysis employing a range of tools, including multiple sequence alignment, phylogenetic tree construction, ancestral sequence reconstruction, and positive selection assessment. Our investigations revealed intriguing insights. AIRE1 homologs emerged during the divergence of T cells in higher vertebrates, signifying its role in this context. Conversely, FEZF2 exhibited multiple homologs spanning invertebrates, lampreys, and higher vertebrates. Ancestral sequence reconstruction demonstrated distinct origins for AIRE1 and FEZF2, underscoring that their roles in regulating TRA have evolved through disparate pathways. Furthermore, it became evident that both FEZF2 and AIRE1 govern a diverse repertoire of genes, encompassing ancient and more recently diverged targets. Notably, FEZF2 demonstrates expression in both vertebrate and invertebrate embryos and germlines, accentuating its widespread role. Intriguingly, FEZF2 harbors motifs associated with autophagy, such as DKFPHP, SYSELWKSSL, and SYSEL, a process integral to cell selection in invertebrates. Our findings suggest that FEZF2 initially emerged to regulate self-elimination in the gonads of invertebrates. As organisms evolved toward greater complexity, AIRE1 likely emerged to complement FEZF2's role, participating in the regulation of cell selection for elimination in both gonads and the thymus. This dynamic interplay between AIRE1 and FEZF2 underscores their multifaceted contributions to TRA expression regulation across diverse evolutionary contexts.

Susceptibility to Pentylenetetrazole-Induced Seizures in Mice with Distinct Activity of the Endogenous Opioid System.

Currently, pharmacotherapy provides successful seizure control in around 70% of patients with epilepsy; however, around 30% of cases are still resistant to available treatment. Therefore, effective anti-epileptic therapy still remains a challenge. In our study, we utilized two mouse lines selected for low (LA) and high (HA) endogenous opioid system activity to investigate the relationship between down- or upregulation of the opioid system and susceptibility to seizures. Pentylenetetrazole (PTZ) is a compound commonly used for kindling of generalized tonic-clonic convulsions in animal models. Our experiments revealed that in the LA mice, PTZ produced seizures of greater intensity and shorter latency than in HA mice. This observation suggests that proper opioid system tone is crucial for preventing the onset of generalized tonic-clonic seizures. Moreover, a combination of an opioid receptor antagonist-naloxone-and a GABA receptor agonist-diazepam (DZP)-facilitates a significant DZP-sparing effect. This is particularly important for the pharmacotherapy of neurological patients, since benzodiazepines display high addiction risk. In conclusion, our study shows a meaningful, protective role of the endogenous opioid system in the prevention of epileptic seizures and that disturbances in that balance may facilitate seizure occurrence.

The Role of Opioid Receptor Antagonists in Regulation of Blood Pressure and T-Cell Activation in Mice Selected for High Analgesia Induced by Swim Stress.

Opioid peptides and their G protein-coupled receptors are important regulators within the cardiovascular system, implicated in the modulation of both heart and vascular functions. It is known that naloxone-an opioid antagonist-may exert a hypertensive effect. Recent experimental and clinical evidence supports the important role of inflammatory mechanisms in hypertension. Since opioids may play a role in the regulation of both blood pressure and immune response, we studied these two processes in our model. We aimed to evaluate the effect of selective and non-selective opioid receptor antagonists on blood pressure and T-cell activation in a mouse model of high swim stress-induced analgesia. Blood pressure was measured before and during the infusion of opioid receptor antagonists using a non-invasive tail-cuff measurement system. To assess the activation of T-cells, flow cytometry was used. We discovered that the non-selective antagonism of the opioid system by naloxone caused a significant elevation of blood pressure. The selective antagonism of µ and κ but not δ opioid receptors significantly increased systolic blood pressure. Subsequently, a brief characterization of T-cell subsets was performed. We found that the blockade of µ and δ receptors is associated with the increased expression of CD69 on CD4 T-cells. Moreover, we observed an increase in the central memory CD4 and central memory CD8 T-cell populations after the δ opioid receptor blockade. The antagonism of the µ opioid receptor increased the CD8 effector and central memory T-cell populations.

Mouse CCL9 Chemokine Acts as Tumor Suppressor in a Murine Model of Colon Cancer.

Colorectal cancer is the third most frequently diagnosed cancer in the world. Despite extensive studies and apparent progress in modern strategies for disease control, the treatment options are still not sufficient and effective, mostly due to frequently encountered resistance to immunotherapy of colon cancer patients in common clinical practice. In our study, we aimed to uncover the CCL9 chemokine action employing the murine model of colon cancer to seek new, potential molecular targets that could be promising in the development of colon cancer therapy. Mouse CT26.CL25 colon cancer cell line was used for introducing lentivirus-mediated CCL9 overexpression. The blank control cell line contained an empty vector, while the cell line marked as CCL9+ carried the CCL9-overexpressing vector. Next, cancer cells with empty vector (control) or CCL9-overexpressing cells were injected subcutaneously, and the growing tumors were measured within 2 weeks. Surprisingly, CCL9 contributed to a decline in tumor growth in vivo but had no effect on CT26.CL25 cell proliferation or migration in vitro. Microarray analysis of the collected tumor tissues revealed upregulation of the immune system-related genes in the CCL9 group. Obtained results suggest that CCL9 reveals its anti-proliferative functions by interplay with host immune cells and mediators that were absent in the isolated, in vitro system. Under specific study conditions, we determined unknown features of the murine CCL9 that have so far bee reported to be predominantly pro-oncogenic.

Investigation of the Molecular Evolution of Treg Suppression Mechanisms Indicates a Convergent Origin.

Regulatory T cell (Treg) suppression of conventional T cells is a central mechanism that ensures immune system homeostasis. The exact time point of Treg emergence is still disputed. Furthermore, the time of Treg-mediated suppression mechanisms' emergence has not been identified. It is not yet known whether Treg suppression mechanisms diverged from a single pathway or converged from several sources. We investigated the evolutionary history of Treg suppression pathways using various phylogenetic analysis tools. To ensure the conservation of function for investigated proteins, we augmented our study using nonhomology-based methods to predict protein functions among various investigated species and mined the literature for experimental evidence of functional convergence. Our results indicate that a minority of Treg suppressor mechanisms could be homologs of ancient conserved pathways. For example, CD73, an enzymatic pathway known to play an essential role in invertebrates, is highly conserved between invertebrates and vertebrates, with no evidence of positive selection (w = 0.48, p-value < 0.00001). Our findings indicate that Tregs utilize homologs of proteins that diverged in early vertebrates. However, our findings do not exclude the possibility of a more evolutionary pattern following the duplication degeneration−complementation (DDC) model. Ancestral sequence reconstruction showed that Treg suppression mechanism proteins do not belong to one family; rather, their emergence seems to follow a convergent evolutionary pattern.

Factors regulating the differences in frequency of infiltration of Th17 and Treg of the blood-brain barrier.

Controlling CD4+ immune cell infiltration of the brain is a leading aim in designing therapeutic strategies for a range of neuropathological disorders such as multiple sclerosis, Alzheimer's disease, and depression. CD4+ T cells are a highly heterogeneous and reprogrammable family, which includes various distinctive cell types such as Th17, Th1, and Treg cells. Interestingly Th17 and Treg cells share a related transcriptomic profile, where the TGFß-SMADS pathway plays a fundamental role in regulating the differentiation of both of these cell types. However, Th17 could be highly pathogenic and was shown to promote inflammation in various neuropathological disorders. Conversely, Treg is anti-inflammatory and is known to inhibit Th17. It could be noticed that Th17 frequencies of infiltration of the blood-brain barrier in various neurological disorders are significantly upregulated. However, Treg infiltration numbers are significantly low. The reasons behind these contradicting observations are still unknown. In this perspective, we propose that the difference in the T-cell receptor repertoire diversity, diapedesis pathways, chemokine expression, and mechanical properties of these two cell types could be contributing to answering this intriguing question.

Investigation of Mutated in Colorectal Cancer (MCC) Gene Family Evolution History Indicates a Putative Role in Th17/Treg Differentiation.

The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 but not MCC2 plays a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species' genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into the MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs' emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1, but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for MCC1. In the case of MCC2, we found that the "modifier of rudimentary" motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis.

The Journey of Cancer Cells to the Brain: Challenges and Opportunities.

Cancer metastases into the brain constitute one of the most severe, but not uncommon, manifestations of cancer progression. Several factors control how cancer cells interact with the brain to establish metastasis. These factors include mediators of signaling pathways participating in migration, infiltration of the blood-brain barrier, interaction with host cells (e.g., neurons, astrocytes), and the immune system. Development of novel therapies offers a glimpse of hope for increasing the diminutive life expectancy currently forecasted for patients suffering from brain metastasis. However, applying these treatment strategies has not been sufficiently effective. Therefore, there is a need for a better understanding of the metastasis process to uncover novel therapeutic targets. In this review, we follow the journey of various cancer cells from their primary location through the diverse processes that they undergo to colonize the brain. These processes include EMT, intravasation, extravasation, and infiltration of the blood-brain barrier, ending up with colonization and angiogenesis. In each phase, we focus on the pathways engaging molecules that potentially could be drug target candidates.

Gold (III) Derivatives in Colon Cancer Treatment.

Cancer is one of the leading causes of morbidity and mortality worldwide. Colorectal cancer (CRC) is the third most frequently diagnosed cancer in men and the second in women. Standard patterns of antitumor therapy, including cisplatin, are ineffective due to their lack of specificity for tumor cells, development of drug resistance, and severe side effects. For this reason, new methods and strategies for CRC treatment are urgently needed. Current research includes novel platinum (Pt)- and other metal-based drugs such as gold (Au), silver (Ag), iridium (Ir), or ruthenium (Ru). Au(III) compounds are promising drug candidates for CRC treatment due to their structural similarity to Pt(II). Their advantage is their relatively good solubility in water, but their disadvantage is an unsatisfactory stability under physiological conditions. Due to these limitations, work is still underway to improve the formula of Au(III) complexes by combining with various types of ligands capable of stabilizing the Au(III) cation and preventing its reduction under physiological conditions. This review summarizes the achievements in the field of stable Au(III) complexes with potential cytotoxic activity restricted to cancer cells. Moreover, it has been shown that not nucleic acids but various protein structures such as thioredoxin reductase (TrxR) mediate the antitumor effects of Au derivatives. The state of the art of the in vivo studies so far conducted is also described.

Mechanisms Underlining Inflammatory Pain Sensitivity in Mice Selected for High and Low Stress-Induced Analgesia-The Role of Endocannabinoids and Microglia.

In this work we strived to determine whether endocannabinoid system activity could account for the differences in acute inflammatory pain sensitivity in mouse lines selected for high (HA) and low (LA) swim-stress-induced analgesia (SSIA). Mice received intraplantar injections of 5% formalin and the intensity of nocifensive behaviours was scored. To assess the contribution of the endocannabinoid system, mice were intraperitoneally (i.p.) injected with rimonabant (0.3-3 mg/kg) prior to formalin. Minocycline (45 and 100 mg/kg, i.p.) was administered to investigate microglial activation. The possible involvement of the endogenous opioid system was investigated with naloxone (1 mg/kg, i.p.). Cannabinoid receptor types 1 and 2 (Cnr1, Cnr2) and opioid receptor subtype (Oprm1, Oprd1, Oprk1) mRNA levels were quantified by qPCR in the structures of the central nociceptive circuit. Levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled with the mass spectrometry method (LC-MS/MS). In the interphase, higher pain thresholds in the HA mice correlated with increased spinal anandamide and 2-AG release and higher Cnr1 transcription. Downregulation of Oprd1 and Oprm1 mRNA was noted in HA and LA mice, respectively, however no differences in naloxone sensitivity were observed in either line. As opposed to the LA mice, inflammatory pain sensitivity in the HA mice in the tonic phase was attributed to enhanced microglial activation, as evidenced by enhanced Aif1 and Il-1ß mRNA levels. To conclude, Cnr1 inhibitory signaling is one mechanism responsible for decreased pain sensitivity in HA mice in the interphase, while increased microglial activation corresponds to decreased pain thresholds in the tonic inflammatory phase.

Adera2.0: A Drug Repurposing Workflow for Neuroimmunological Investigations Using Neural Networks.

Drug repurposing in the context of neuroimmunological (NI) investigations is still in its primary stages. Drug repurposing is an important method that bypasses lengthy drug discovery procedures and focuses on discovering new usages for known medications. Neuroimmunological diseases, such as Alzheimer's, Parkinson's, multiple sclerosis, and depression, include various pathologies that result from the interaction between the central nervous system and the immune system. However, the repurposing of NI medications is hindered by the vast amount of information that needs mining. We previously presented Adera1.0, which was capable of text mining PubMed for answering query-based questions. However, Adera1.0 was not able to automatically identify chemical compounds within relevant sentences. To challenge the need for repurposing known medications for neuroimmunological diseases, we built a deep neural network named Adera2.0 to perform drug repurposing. The workflow uses three deep learning networks. The first network is an encoder and its main task is to embed text into matrices. The second network uses a mean squared error (MSE) loss function to predict answers in the form of embedded matrices. The third network, which constitutes the main novelty in our updated workflow, also uses a MSE loss function. Its main usage is to extract compound names from relevant sentences resulting from the previous network. To optimize the network function, we compared eight different designs. We found that a deep neural network consisting of an RNN neural network and a leaky ReLU could achieve 0.0001 loss and 67% sensitivity. Additionally, we validated Adera2.0's ability to predict NI drug usage against the DRUG Repurposing Hub database. These results establish the ability of Adera2.0 to repurpose drug candidates that can shorten the development of the drug cycle. The workflow could be download online.

Cardiovascular Effects Mediated by HMMR and CD44.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The most dangerous life-threatening symptoms of CVD are myocardial infarction and stroke. The causes of CVD are not entirely clear, and new therapeutic targets are still being sought. One of the factors involved in CVD development among vascular damage and oxidative stress is chronic inflammation. It is known that hyaluronic acid plays an important role in inflammation and is regulated by numerous stimuli, including proinflammatory cytokines. The main receptors for hyaluronic acid are CD44 and RHAMM. These receptors are membrane proteins that differ in structure, but it seems that they can perform similar or synergistic functions in many diseases. Both RHAMM and CD44 are involved in cell migration and wound healing. However, their close association with CVD is not fully understood. In this review, we describe the role of both receptors in CVD.

Disulfiram Abrogates Morphine Tolerance-A Possible Role of µ-Opioid Receptor-Related G-Protein Activation in the Striatum.

One of the key strategies for effective pain management involves delaying analgesic tolerance. Early clinical reports indicate an extraordinary effectiveness of off-label disulfiram-an agent designed for alcohol use disorder-in potentiating opioid analgesia and abrogation of tolerance. Our study aimed to determine whether sustained µ-opioid signaling upon disulfiram exposure contributes to these phenomena. Wistar rats were exposed to acute and chronic disulfiram and morphine cotreatment. Nociceptive thresholds were assessed with the mechanical Randal-Selitto and thermal tail-flick tests. µ-opioid receptor activation in brain structures important for pain processing was carried out with the [35S]GTPγS assay. The results suggest that disulfiram (12.5-50 mg/kg i.g.) augmented morphine antinociception and diminished morphine (25 mg/kg, i.g.) tolerance in a supraspinal, opioid-dependent manner. Disulfiram (25 mg/kg, i.g.) induced a transient enhancement of µ-opioid receptor activation in the periaqueductal gray matter (PAG), rostral ventromedial medulla (RVM), hypothalamus, prefrontal cortex and the dorsal striatum at day 1 of morphine treatment. Disulfiram rescued µ-opioid receptor signaling in the nucleus accumbens and caudate-putamen 14 days following morphine and disulfiram cotreatment. The results of this study suggest that striatal µ-opioid receptors may contribute to the abolition of morphine tolerance following concomitant treatment with disulfiram.

Hypertensive Effect of Downregulation of the Opioid System in Mouse Model of Different Activity of the Endogenous Opioid System.

The opioid system is well-known for its role in modulating nociception and addiction development. However, there are premises that the endogenous opioid system may also affect blood pressure. The main goal of the present study was to determine the impact of different endogenous opioid system activity and its pharmacological blockade on blood pressure. Moreover, we examined the vascular function in hyper- and hypoactive states of the opioid system and its pharmacological modification. In our study, we used two mouse lines which are divergently bred for high (HA) and low (LA) swim stress-induced analgesia. The obtained results indicated that individuals with low endogenous opioid system activity have higher basal blood pressure compared to those with a hyperactive opioid system. Additionally, naloxone administration only resulted in the elevation of blood pressure in HA mice. We also showed that the hypoactive opioid system contributes to impaired vascular relaxation independent of endothelium, which corresponded with decreased guanylyl cyclase levels in the aorta. Together, these data suggest that higher basal blood pressure in LA mice is a result of disturbed mechanisms in vascular relaxation in smooth muscle cells. We believe that a novel mechanism which involves endogenous opioid system activity in the regulation of blood pressure will be a promising target for further studies in hypertension development.

6-Acetyl-5-hydroxy-4,7-dimethylcoumarin derivatives: Design, synthesis, modeling studies, 5-HT1A, 5-HT2A and D2 receptors affinity.

Molecular docking studies using appropriate 5-HT1A, 5-HT2A and D2 receptors models were used to design sixteen new 5-hydroxycoumarin derivatives with piperazine moiety (3-18). The microwave radiation have been used to synthesize them and their structures have been confirmed using mass spectrometry, 1H and 13C NMR. All newly prepared derivatives were evaluated for their 5-HT1A, 5-HT2A and D2 receptor affinity. Seven of the synthesized derivatives showed very high affinities to 5-HT1A receptor (3-4.0 nM, 6-4.0 nM, 7-1.0 nM, 9-6.0 nM, 15-4.3 nM, 16-1.0 nM, 18-3.0 nM) and one of them showed high affinities to 5-HT2A receptor (16-8.0 nM). In the case of the D2 receptor none of the tested derivatives showed high affinity. Compounds 7 and 16 were identified as potent antagonists of the 5-HT1A receptor as shown by the [35S]GTPcS binding assay but they didn't show any antidepressant effect at the single dose tested (10 mg/kg) in the tail suspension tests.

Ethanol consumption following mild traumatic brain injury is related to blood-brain barrier permeability.

Several preclinical and clinical studies that deal with the neuropathological consequences of mild traumatic brain injury (mTBI) have focused on unraveling its effect on ethanol drinking behavior. Previous reports describe changes in ethanol consumption, both in animal models of mTBI as well as in patients, after concussive brain injury. However, the neurobiological mechanisms underlying this phenomenon are still poorly understood. In the present study, we used a unique model of mouse lines divergently selected for high (HA) or low (LA) swim stress-induced analgesia to examine the effect of mTBI on ethanol drinking behavior. In comparison with LA mice, their HA counterparts exhibited increased blood-brain barrier (BBB) permeability, lower basal alcohol preference, and lower level of stress-induced ethanol intake. Here, we showed that mTBI attenuates voluntary ethanol intake in LA, but not in HA mice. Interestingly, BBB disruption after mannitol infusion also decreases the level of ethanol drinking behavior in this line. We conclude that in alcohol-preferring LA mice, BBB disruption as a consequence of mTBI attenuates ethanol consumption. Our results suggest that the innate level of BBB integrity plays a pivotal role in regulation of ethanol consumption in mice showing differential endogenous opioid system activity.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors.

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

5-HT1A and 5-HT2A receptors affinity, docking studies and pharmacological evaluation of a series of 8-acetyl-7-hydroxy-4-methylcoumarin derivatives.

In this work we describe the synthesis, docking studies and biological evaluation of a focused library of novel arylpiperazinyl derivatives of 8-acetyl-7-hydroxy-4-methylcoumarin. The new compounds were screened for their 5-HT1A and 5-HT2A receptor affinity. Among the evaluated compounds, six displayed high affinities to 5-HT1A receptors (4a-0.9 nM, 6a-0.5 nM, 10a-0.6 nM, 3b-0.9 nM, 6b-1.5 nM, 10b-1 nM). Compound 6a and 10a bearing a bromo- or methoxy- substituent in ortho position of the piperazine phenyl ring, were identified as potent antagonists of the 5-HT1A receptors. In the tail suspension test, mice injected with 6a showed a dose-dependent increase in depressive-like behavior that was related to a decrease in locomotor activity. Compound 10a did not decrease or prolong immobility time nor did it affect home cage activity. Molecular docking studies using 5-HT1A and 5-HT2A homology models revealed structural basis of the high affinity of ortho-substituted derivatives and subtle changes in amino acid interactions patterns depending on the length of the alkyl linker.

Central antinociceptive effect of tapentadol is increased by nitric oxide synthase inhibitors.

Nitric oxide synthases (NOSs) have been shown to participate in the mechanism of the antinociceptive action of tapentadol. The results obtained in this study indicate that tapentadol administered intrathecally at a range of doses (30-100 µg) increased nociceptive thresholds in the Randall-Selitto and tail-flick tests in rats; however, this effect was significant only for the higher doses. After intracerebroventricular administration of tapentadol at the same dose range, an antinociceptive effect was observed only in response to mechanical stimuli. In coadministration studies, L-N-nitro arginine (L-NOArg) - a nonselective NOS inhibitor as well as selective inhibitors: 7-Nitroindazole (7-NI), L-N(1-iminoethyl)lysine (L-NIL) or N-(1-iminoethyl)-L-ornithine (L-NIO) for the respective neuronal, inducible, and endothelial NOSs enhanced the antinociceptive activity of intrathecally administered tapentadol in the Randall-Selitto test and to a lesser extent in the tail-flick test. A similar, although less pronounced effect of intracerebroventricular tapentadol was also observed after previous administration of NOS inhibitors in the Randall-Selitto test, but not in the tail-flick test. In conclusion, neuronal NOS, inducible NOS, and endothelial NOS influence the antinociceptive action of tapentadol at the spinal level and to a much lesser extent at the supraspinal level.

Association between the A107V substitution in the δ-opioid receptors and ethanol drinking in mice selected for high and low analgesia.

Experimental evidence suggests that endogenous opioids play an important role in the development of ethanol addiction. In this study, we employed two mouse lines divergently bred for opioid-mediated stress-induced analgesia. In comparison with HA (high analgesia line) mice, LA (low analgesia line) mice, having lower opioid receptor system activity, manifest enhanced basal as well as stress-induced ethanol drinking. Here, we found that recently discovered C320T transition in exon 2 of the δ-opioid receptor gene (EU446125.1), which results in an A107V substitution (ACA23171.1), leads to higher ethanol preference in CT mice compared with CC homozygotes. This genetic association is particularly evident under chronic mild stress (CMS) conditions. The interaction between stress and ethanol intake was significantly stronger in HA than in LA mice. Ethanol almost completely attenuated the pro-depressive effect of CMS (assessed with the tail suspension test) in both the CC and CT genotypes in the HA line. In the LA mice, a lack of response to ethanol was observed in the CC genotype, whereas ethanol consumption strengthened depressive-like behaviours in CT individuals. Our results suggest that constitutively active A107V substitution in δ-opioid receptors may be involved in stress-enhanced vulnerability to ethanol abuse and in the risk of ethanol dependence.