PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

The PACAP/PAC1 Receptor System and Feeding

Pituitary adenylyl cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon superfamily. PACAP is present in two forms (PACAP-38 and PACAP-27) and binds to three guanine-regulatory (G) protein-coupled receptors (PAC1, VPAC1, and VPAC2). PACAP is expressed in the central and peripheral nervous systems, with high PACAP levels found in the hypothalamus, a brain region involved in feeding and energy homeostasis. PAC1 receptors are high-affinity and PACAP-selective receptors, while VPAC1 and VPAC2 receptors show a comparable affinity to PACAP and VIP. PACAP and its receptors are expressed in the central and peripheral nervous systems with moderate to high expression in the hypothalamus, amygdala, and other limbic structures. Consistent with their expression, PACAP is involved in several physiological responses and pathological states. A growing body of literature suggests that PACAP regulates food intake in laboratory animals. However, there is no comprehensive review of the literature on this topic. Thus, the purpose of this article is to review the literature regarding the role of PACAP and its receptors in food intake regulation and to synthesize how PACAP exerts its anorexic effects in different brain regions. To achieve this goal, we searched PubMed and reviewed 68 articles regarding the regulatory action of PACAP on food intake. Here, we present the literature regarding the effect of exogenous PACAP on feeding and the role of endogenous PACAP in this process. We also provide evidence regarding the effect of PACAP on the homeostatic and hedonic aspects of food intake, the neuroanatomical sites where PACAP exerts its regulatory action, which PACAP receptors may be involved, and the role of various signaling pathways and neurotransmitters in hypophagic effects of PACAP.

Emotionality vs. Other Biobehavioural Traits: A Look at Neurochemical Biomarkers for Their Differentiation

This review highlights the differential contributions of multiple neurochemical systems to temperament traits related and those that are unrelated to emotionality, even though these systems have a significant overlap. The difference in neurochemical biomarkers of these traits is analysed from the perspective of the neurochemical model, Functional Ensemble of Temperament (FET) that uses multi-marker and constructivism principles. Special attention is given to a differential contribution of hypothalamic–pituitary hormones and opioid neuropeptides implicated in both emotional and non-emotional regulation. The review highlights the role of the mu-opioid receptor system in dispositional emotional valence and the role of the kappa-opioid system in dispositional perceptual and behavioural alertness. These opioid receptor (OR) systems, microbiota and cytokines are produced in three neuroanatomically distinct complexes in the brain and the body, which all together integrate dispositional emotionality. In contrast, hormones could be seen as neurochemical biomarkers of non-emotional aspects of behavioural regulation related to the construction of behaviour in fast-changing and current situations. As examples of the role of hormones, the review summarised their contribution to temperament traits of Sensation Seeking (SS) and Empathy (EMP), which FET considers as non-emotionality traits related to behavioural orientation. SS is presented here as based on (higher) testosterone (fluctuating), adrenaline and (low) cortisol systems, and EMP, as based on (higher) oxytocin, reciprocally coupled with vasopressin and (lower) testosterone. Due to the involvement of gonadal hormones, there are sex and age differences in these traits that could be explained by evolutionary theory. There are, therefore, specific neurochemical biomarkers differentiating (OR-based) dispositional emotionality and (hormones-based) body’s regulation in fast-changing events. Here we propose to consider dispositional emotionality associated with OR systems as emotionality in a true sense, whereas to consider hormonal ensembles regulating SS and EMP as systems of behavioural orientation and not emotionality.

Ectodysplasin A/Ectodysplasin A Receptor System and Their Roles in Multiple Diseases

Ectodysplasin A (EDA) is a member of the tumor necrosis factor (TNF) family of ligands that was initially reported to induce the formation of various ectodermal derivatives during normal prenatal development. EDA exerts its biological activity as two splice variants, namely, EDA-A1 and EDA-A2. The former binds to the EDA receptor (EDAR), resulting in the recruitment of the intracellular EDAR-associated death domain (EDARADD) adapter protein and the activation of the NF-κB signaling pathway, while the latter binds to a different receptor, EDA2R, also known as X-linked ectodermal dysplasia receptor (XEDAR). Inactivation mutation of the EDA gene or the genes coding for its receptors can result in hypohidrosis ectodermal dysplasia (HED), a condition that is characterized by oligotrichosis, edentulosis or oligodontia, and oligohidrosis or anhidrosis. Recently, as a new liver factor, EDA is gradually known and endowed with some new functions. EDA levels were observed to be upregulated in several metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance. In addition, EDA and its receptors have been implicated in tumor pathogenesis through the regulation of tumor cell proliferation, apoptosis, differentiation, and migration. Here, we first review the role of EDA and its two-receptor system in various signaling pathways and then discuss the physiological and pathological roles of EDA and its receptors.

Role of Nociceptin/Orphanin FQ-NOP Receptor System in the Regulation of Stress-Related Disorders

Nociceptin/orphanin FQ (N/OFQ) is a 17-residue neuropeptide that binds the nociceptin opioid-like receptor (NOP). N/OFQ exhibits nucleotidic and aminoacidics sequence homology with the precursors of other opioid neuropeptides but it does not activate either MOP, KOP or DOP receptors. Furthermore, opioid neuropeptides do not activate the NOP receptor. Generally, activation of N/OFQ system exerts anti-opioids effects, for instance toward opioid-induced reward and analgesia. The NOP receptor is widely expressed throughout the brain, whereas N/OFQ localization is confined to brain nuclei that are involved in stress response such as amygdala, BNST and hypothalamus. Decades of studies have delineated the biological role of this system demonstrating its involvement in significant physiological processes such as pain, learning and memory, anxiety, depression, feeding, drug and alcohol dependence. This review discusses the role of this peptidergic system in the modulation of stress and stress-associated psychiatric disorders in particular drug addiction, mood, anxiety and food-related associated-disorders. Emerging preclinical evidence suggests that both NOP agonists and antagonists may represent a effective therapeutic approaches for substances use disorder. Moreover, the current literature suggests that NOP antagonists can be useful to treat depression and feeding-related diseases, such as obesity and binge eating behavior, whereas the activation of NOP receptor by agonists could be a promising tool for anxiety.

Post-Translational Modification of HMGB1 Disulfide Bonds in Stimulating and Inhibiting Inflammation

High mobility group box 1 protein (HMGB1), a highly conserved nuclear DNA-binding protein, is a “damage-associated molecular pattern” molecule (DAMP) implicated in both stimulating and inhibiting innate immunity. As reviewed here, HMGB1 is an oxidation-reduction sensitive DAMP bearing three cysteines, and the post-translational modification of these residues establishes its proinflammatory and anti-inflammatory activities by binding to different extracellular cell surface receptors. The redox-sensitive signaling mechanisms of HMGB1 also occupy an important niche in innate immunity because HMGB1 may carry other DAMPs and pathogen-associated molecular pattern molecules (PAMPs). HMGB1 with DAMP/PAMP cofactors bind to the receptor for advanced glycation end products (RAGE) which internalizes the HMGB1 complexes by endocytosis for incorporation in lysosomal compartments. Intra-lysosomal HMGB1 disrupts lysosomal membranes thereby releasing the HMGB1-transported molecules to stimulate cytosolic sensors that mediate inflammation. This HMGB1-DAMP/PAMP cofactor pathway slowed the development of HMGB1-binding antagonists for diagnostic or therapeutic use. However, recent discoveries that HMGB1 released from neurons mediates inflammation via the TLR4 receptor system, and that cancer cells express fully oxidized HMGB1 as an immunosuppressive mechanism, offer new paths to targeting HMGB1 for inflammation, pain, and cancer.

The PACAP/PAC1 Receptor System and Feeding

Pituitary adenylyl cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon superfamily. PACAP is present in two forms, PACAP-38 and PACAP-27, and binds to three guanine-regulatory (G) protein-coupled receptors (PAC1, VPAC1, and VPAC2). PACAP is expressed in the central and peripheral nervous systems with high PACAP levels found in the hypothalamus, a brain region involved in feeding and energy homeostasis. PAC1 receptors are high-affinity and PACAP-selective receptors, while VPAC1 and VPAC2 receptors show a comparable affinity to PACAP and VIP. PACAP and its receptors are expressed in the central and peripheral nervous systems, with moderate to high expression in the hypothalamus, amygdala, and other limbic structures. Consistent with their expression, PACAP is involved in several physiological responses and pathological states. A growing body of literature suggests that PACAP regulates food intake in laboratory animals. However, there is no comprehensive review of the literature on this topic. Thus, the purpose of this article is to review the literature regarding the role of PACAP and its receptors in food intake regulation and to synthesize how PACAP exerts its anorexic effects in different brain regions. To achieve this goal, we searched PubMed and reviewed 68 articles regarding the regulatory action of PACAP on food intake. Here, we present the literature regarding the effect of exogenous PACAP on feeding and the role of endogenous PACAP in this process. We also provide evidence regarding the effect of PACAP on the homeostatic and hedonic aspects of food intake, the neuroanatomical sites where PACAP exerts its regulatory action, which PACAP receptors may be involved, and the role of various signaling pathways and neurotransmitters in hypophagic effects of PACAP.

Repeated Ethanol Exposure Alters DNA Methylation Status and Dynorphin/Kappa-Opioid Receptor Expression in Nucleus Accumbens of Alcohol-Preferring AA Rats

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, −3a, and −3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.

Somatostatin and Its Receptor System in Colorectal Cancer

Somatostatin (SST)/somatotropin release-inhibiting factor (SRIF) is a well-known neuropeptide, widely distributed in the central and peripheral nervous systems, that regulates the endocrine system and affects neurotransmission via interaction with five SST receptors (SST1-5). In the gastrointestinal tract, the main SST-producing cells include intestinal enteroendocrine cells (EECs) restricted to the mucosa, and neurons of the submucosal and myenteric plexuses. The action of the SRIF system is based on the inhibition of endocrine and exocrine secretion, as well as the proliferative responses of target cells. The SST1–5 share common signaling pathways, and are not only widely expressed on normal tissues, but also frequently overexpressed by several tumors, particularly neuroendocrine neoplasms (NENs). Furthermore, the SRIF system represents the only peptide/G protein-coupled receptor (GPCR) system with multiple approved clinical applications for the diagnosis and treatment of several NENs. The role of the SRIF system in the histogenesis of colorectal cancer (CRC) subtypes (e.g., adenocarcinoma and signet ring-cell carcinoma), as well as diagnosis and prognosis of mixed adenoneuroendocrine carcinoma (MANEC) and pure adenocarcinoma, is poorly understood. Moreover, the impact of the SRIF system signaling on CRC cell proliferation and its potential role in the progression of this cancer remains unknown. Therefore, this review summarizes the recent collective knowledge and understanding of the clinical significance of the SRIF system signaling in CRC, aiming to evaluate the potential role of its components in CRC histogenesis, diagnosis, and potential therapy.

Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists

The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, have been developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45127/SRI-45128 as inverse agonists. Then, these compounds were evaluated in vitro for their binding affinity by radioligand binding and functional activity by 35S-GTPγS coupling and cAMP accumulation in cells expressing the human DOR. All three compounds demonstrated high binding affinity and selectivity at the DOR, and all three displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45127/SRI-45128). Together, these results demonstrate that we have designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.