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In mammal's pineal glands, ATP interacts with the high-affinity P2Y 1 and the low-affinity P2X7 receptors. ATP released from sympathetic nerve terminals potentiates noradrenaline-induced serotonin N-acetyltransferase (Snat) transcription, N-acetylserotonin (NAS), and melatonin (MLT) synthesis. Circulating melatonin impairs the expression of adhesion molecules in endothelial cells, blocking the migration of leukocytes. Acute defence response induced by pathogen- and danger/damage-associated molecular patterns (PAMPs and DAMPs) triggers the NF-κB pathway in pinealocytes and blocks the transcription of Snat. Therefore, the darkness hormone is not released, and neutrophils and monocytes migrate to the lesion sites. ATP released in high amounts from apoptotic and death cells was considered a DAMP, and the blockage of P2X7 receptors was tested as a new class of drugs for treating brain damage. However, this is not a simple equation. High ATP injected in a lateral ventricle blocked MLT, but not NAS, synthesis as it impairs the transcription of acetyl serotonin N-methyltransferase. NAS is released in the plasma and the cerebral spinal fluid. NAS also blocks the rolling and adhesion of leukocytes to endothelial cells. Otherwise, it is metabolised specifically in each brain area to provide the requested concentration of MLT as a neuroprotector. As observed in physiological conditions, high extracellular ATP, different from the other DAMPs, reports the environmental light/dark cycle rhythm because NAS substitutes MLT as the nocturnal chemical indicator, the darkness hormone. Thus, blocking the P2X7R should not be considered a universal therapy for improving acute strokes, as MLT and ATP are partners in health and disease., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The pineal gland is a key player in surveillance and defense responses. In healthy conditions, nocturnal circulating melatonin (MEL) impairs the rolling and adhesion of leukocytes to the endothelial layer. Fungi, bacteria, and pro-inflammatory cytokines block nocturnal pineal MEL synthesis, facilitating leukocyte migration to injured areas. ATP is a cotransmitter of the noradrenergic signal and potentiates noradrenaline (NAd)-induced MEL synthesis via P2Y 1 receptor (P2Y 1 R) activation. Otherwise, ATP low-affinity P2X7 receptor (P2X7R) activation impairs N-acetylserotonin (NAS) into MEL conversion in NAd incubated pineals. Here we mimicked a focal increase of ATP by injecting low (0.3 and 1.0 µg) and high (3.0 µg) ATP in the right lateral ventricle of adult rats. Nocturnal pineal activity mimicked the in culture data. Low ATP doses increased MEL output, while high ATP dose and the P2X7R agonist BzATP (15.0-50.0 ng) increased NAS pineal and blood content. In the brain, the response was structure-dependent. There was an increase in cortical and no change in cerebellar MEL. These effects were mediated by changes in the expression of coding genes to synthetic and metabolizing melatonergic enzymes. Thus, the pineal gland plays a role as a first-line structure to respond to the death of cells inside the brain by turning NAS into the darkness hormone., Competing Interests: Acknowledgements KSS - Data acquisition, analysis, interpretation, and writing the manuscript; CLQ - Data acquisition, revising the manuscript; SMM - Data acquisition, revising the manuscript; ZSF - Concept/design, data acquisition, interpretation, and writing the manuscript; RPM - Coordinated the project and funding, concept/design, data interpretation, writing the manuscript; We gratefully acknowledge to Dr. Pedro A Fernandes to the contribution in discussing data and protocols, to Dr. Débora R Fior-Chadi and Prof. Lucile Maria Floeter-Winter (IB/USP) to the lab and equipment sharing and to Debora Aparecida Moura for the technical assistance. All authors declare that they have no conflict of interest and approve the manuscript. Funding sources This work was supported by the São Paulo Research Foundation (FAPESP 2019/03348-4), Conselho Nacional de Desenvolvimento Cientí́fico e Tecnológico (CNPq 140274/2018-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – (Finance Code 001, 2019/03348-4)., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

Increased plasma glucocorticoids (corticosterone - CORT, in amphibians) and melatonin (MEL) are associated with the daily activity phase and with environmental darkness, respectively. Besides, CORT and MEL also play pivotal immunomodulatory roles in several vertebrates. Herein we described the daily profile of plasma MEL and CORT for Rhinella icterica toads in captivity. Thereafter, we investigated the effects of lipopolysaccharide (LPS)-induced systemic inflammation on the production of CORT and MEL in the R. icterica . Captive toads showed CORT and MEL diurnal variation typical of nocturnal species, with increased values for CORT at ZT12 (18 h) and MEL peak at ZT18 (24 h). LPS-induced hormonal changes included increased plasma CORT and decreased ocular and plasma MEL when compared to those from toads treated with saline 2 h post-injection. Our results demonstrated the presence of a diurnal CORT and MEL variation in toads. We also showed the crosstalk between CORT and MEL during the toad's systemic inflammation in response to an immune challenge with LPS. Additionally, our results demonstrated that anuran eyes' MEL production might be regulated during the inflammatory processes.

Melatonin is a highly conserved molecule found in prokaryotes and eukaryotes that acts as the darkness hormone, translating environmental lighting to the whole body, and as a moderator of innate and acquired defense, migration, and cell proliferation processes. This review evaluates the importance of pineal activity in monitoring PAMPs and DAMPs and in mounting an inflammatory response or innate immune response. Activation of the immune-pineal axis, which coordinates the pro-and anti-inflammatory phases of an innate immune response, is described. PAMPs and DAMPs promote the immediate suppression of melatonin production by the pineal gland, which allows leukocyte migration. Monocyte-derived macrophages, important phagocytes of microbes, and cellular debris produce melatonin locally and thereby initiate the anti-inflammatory phase of the acute inflammatory response. The role of locally produced melatonin in organs that directly contact the external environment, such as the skin and the gastrointestinal and respiratory tracts, is also discussed. In this context, as resident macrophages are self-renewing cells, we explore evidence indicating that, besides avoiding overreaction of the immune system, extra-pineal melatonin has a fundamental role in the homeostasis of organs and tissues.

Environmental pollution in the form of particulate matter <2.5 μm (PM 2.5 ) is a major risk factor for diseases such as lung cancer, chronic respiratory infections, and major cardiovascular diseases. Our goal was to show that PM 2.5 eliciting a proinflammatory response activates the immune-pineal axis, reducing the pineal synthesis and increasing the extrapineal synthesis of melatonin. Herein, we report that the exposure of rats to polluted air for 6 hours reduced nocturnal plasma melatonin levels and increased lung melatonin levels. Melatonin synthesis in the lung reduced lipid peroxidation and increased PM 2.5 engulfment and cell viability by activating high-affinity melatonin receptors. Diesel exhaust particles (DEPs) promoted the synthesis of melatonin in a cultured cell line (RAW 264.7 cells) and rat alveolar macrophages via the expression of the gene encoding for AANAT through a mechanism dependent on activation of the NFκB pathway. Expression of the genes encoding AANAT, MT1, and MT2 was negatively correlated with cellular necroptosis, as disclosed by analysis of Gene Expression Omnibus (GEO) microarray data from the human alveolar macrophages of nonsmoking subjects. The enrichment score for antioxidant genes obtained from lung gene expression data (GTEx) was significantly correlated with the levels of AANAT and MT1 but not the MT2 melatonin receptor. Collectively, these data provide a systemic and mechanistic rationale for coordination of the pineal and extrapineal synthesis of melatonin by a standard damage-associated stimulus, which activates the immune-pineal axis and provides a new framework for understanding the effects of air pollution on lung diseases., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA + macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aim: The nocturnal production of melatonin by the pineal gland is triggered by sympathetic activation of adrenoceptors and may be modulated by immunological signals. The effect of glucocorticoids on nocturnal melatonin synthesis is controversial; both stimulatory and inhibitory effects have been reported. During pathophysiological processes, an increased sympathetic tonus could result in different patterns of adrenoceptor activation in the pineal gland. Therefore, in this investigation, we evaluated whether the pattern of adrenergic stimulation of the pineal gland drives the direction of the glucocorticoid effect on melatonin production., Methods: The corticosterone effect on the pineal hormonal production induced by β-adrenoceptor or β+α1-adrenoceptor activation was evaluated in cultured glands. We also investigated whether the in vivo lipopolysaccharide (LPS)-induced inhibition of melatonin is dependent on the interaction of glucocorticoids and the α1-adrenoceptor in adrenalectomized animals and on the in vivo blockade of glucocorticoid receptors (GRs) or the α1-adrenoceptor., Results: Corticosterone potentiated β-adrenoceptor-induced pineal melatonin synthesis, whilst corticosterone-dependent inhibition was observed when melatonin production was induced by β+α1-adrenoceptors agonists. The inhibitory effect of corticosterone is mediated by GR, as it was abolished in the presence of a GR antagonist. Moreover, LPS-induced reduction in melatonin nocturnal plasma content was reversed by adrenalectomy and by antagonizing GR or α1-adrenoceptors., Conclusions: The dual effect of corticosterone on pineal melatonin synthesis is determined by the activation pattern of adrenoceptors (β or β+α1) in the gland during GR activation, suggesting that increased activation of the sympathetic system and the hypothalamic-pituitary-adrenal axis are necessary for the control of melatonin production during defense responses., (© 2016 S. Karger AG, Basel.)

The hippocampus is a brain region that is rich in nicotinic acetylcholine receptors (nAChRs), especially the α7 subtype. The hippocampus is severely affected in disorders that have a neuroinflammatory component, such as Alzheimer's disease, Parkinson's disease, and schizophrenia. Previous studies demonstrated both in vivo and in vitro that nicotine inhibits immunological responses, including those that are triggered by the inflammatory agent lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria. The present study investigated whether chronically administered nicotine interferes with the nuclear binding of nuclear factor-κB (NF-κB) and the expression of LPS-induced inflammatory response genes. The results indicated that chronic nicotine administration (0.1mg/kg, s.c., 14days) inhibited the LPS-induced nuclear binding of NF-κB and mRNA expression levels of Tnf, Il1b, Nos2, and Tlr4. The presence of both the selective α7 nAChR antagonist methyllycaconitine (MLA; 5.0mg/kg i.p., 14days) and the nonselective nAChR antagonist mecamylamine (Meca; 1.0mg/kg, s.c., 14days) reversed the inhibitory effects of nicotine. These results suggest that the chronic activation of α7- and α x β y -containing nAChRs reduces acute inflammatory responses in the brain., (Copyright © 2016. Published by Elsevier B.V.)

Schistosomiasis is caused by an intravascular parasite and linked to phenotypic changes in endothelial cells that favor inflammation. Endothelial cells express P2Y1 receptors (P2Y1R), and their activation by ADP favors leukocyte adhesion to the endothelial monolayer. We aimed to evaluate the influence of schistosomiasis upon endothelial purinergic signaling-mediated leukocyte adhesion. Mesenteric endothelial cells and mononuclear cells from control and Schistosoma mansoni-infected mice were used in co-culture. P2Y1R levels were similar in both groups. Basal leukocyte adhesion was higher in the infected than in the control group; leukocyte adhesion increased after treatment with the P2Y1R agonist 2-MeSATP in both groups, though it only marginally increased in the infected group. Pre-incubation with the selective P2Y1R antagonist MRS2179 (0.3μM) prevented the agonist effect. However, in the infected group it also reduced the basal leukocyte adhesion, suggesting endothelial cell pre-activation. The endothelial expressions of NTPDases 2 and 3 were significantly increased in the infected group, increasing extracellular ATP hydrolysis and ADP formation by endothelial cells. Therefore, mesenteric endothelial cells are primed by schistosomiasis to a pro-inflammatory phenotype characterized by an increased expression of NTPDases 2 and 3, favoring ADP accumulation and mononuclear cell adhesion, possibly contributing to mesenteric inflammation and schistosomiasis morbidity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice.

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Melatonin, the darkness hormone, synchronizes several physiological functions to light/dark cycle. Besides the awake/sleep cycle that is intuitively linked to day/night, daily variations in memory acquisition and innate or acquired immune responses are some of the major activities linked to melatonin rhythm. The daily variation of these complex processes is due to changes in specific mechanisms. In the last years we focused on the influence of melatonin on the expression and function of nicotinic acetylcholine receptors (nAChRs). Melatonin, either "in vivo" or "in vitro", increases, in a selective manner, the efficiency of alpha-bungarotoxin (alpha-BTX)-sensitive nAChRs. Melatonin's effect on receptors located in rat sympathetic nerve terminals, cerebellum, skeletal muscle and chick retina, was tested. We observed that melatonin is essential for the development of alpha-BTX-sensitive nAChRs, and important for receptor maintenance in aging models. Taking into account that both melatonin and alpha-7 nAChRs (one of the subtypes sensitive to alpha-BTX) are involved in the development of Alzheimer's disease, here we discuss the possibility of a therapeutic strategy focused on both melatonin replacement and its potential association with cholinergic drugs., (Copyright 2010 Elsevier Inc. All rights reserved.)

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h:12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information.

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NFkappaB and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NFkappaB and AP-1. Activation of NFkappaB and AP-1 depended on phospholipase C and protein kinase A. Activation of NFkappaB also depended on protein kinase C. Isoprenaline activated both NFkappaB and AP-1, and phenylephrine activated NFkappaB and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 microm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Background and Purpose: We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells., Experimental Approach: Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA., Key Results: RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats., Conclusions and Implications: Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

Background and Purpose: We have previously shown that melatonin inhibits bradykinin-induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin., Experimental Approach: RT-PCR assays were performed with rat cultured endothelial cells. The putative effect of melatonin upon arteriolar tone was investigated by intravital microscopy while NO production by endothelial cells in vitro was assayed by fluorimetry, and intracellular Ca(2+) measurements were assayed by confocal microscopy., Key Results: No expression of the mRNA for the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase, or for the melatonin MT(2) receptor was detected in microvascular endothelial cells. Melatonin fully inhibited L-NAME-sensitive bradykinin-induced vasodilation and also inhibited NO production induced by histamine, carbachol and 2-methylthio ATP, but did not inhibit NO production induced by ATP or alpha, beta-methylene ATP. None of its inhibitory effects was prevented by the melatonin receptor antagonist, luzindole. In nominally Ca(2+)-free solution, melatonin reduced intracellular Ca(2+) mobilization induced by bradykinin (40%) and 2-methylthio ATP (62%) but not Ca(2+) mobilization induced by ATP., Conclusions and Implications: We have confirmed that melatonin inhibited NO production both in vivo and in vitro. In addition, the melatonin effect was selective for some G protein-coupled receptors and most probably reflects an inhibition of Ca(2+) mobilization from intracellular stores.

The time course of the innate immunological response involves a pro-inflammatory phase followed by an anti-inflammatory phase. Pro-inflammatory responses serve as a defense against several stressor conditions, and sequential processes that shut down these responses are necessary to avoid exacerbation or the development of chronic diseases. In the present review, we put together recent data that show that the pineal gland is a player in bidirectional control of the inflammatory response. Healthy organisms stay in standby mode, ready to react. The nocturnal melatonin surge impairs the rolling and adherence of leukocytes to endothelial layers, limiting cell migration, and stimulates nocturnal production of IL-2 by T helper lymphocytes, exerting an immunostimulatory effect. Otherwise, the release of TNF-alpha from activated macrophages suppresses the nocturnal melatonin surge, allowing a full cell migration and inhibiting IL-2 production. In sequence, activated mononuclear and polymorphonuclear cells produce melatonin in a paracrine manner at the site of injury, which scavenges free radicals and collaborates to resolve the inflammatory response. The sequential diminution of TNF-alpha production is followed by the recovery of the nocturnal melatonin surge and IL-2 production. In summary, the immune-pineal axis, implicated in the sequential involvement of the melatonin produced by the pineal gland and immune-competent cells, is an integral participant of the innate immune response., (Copyright 2007 S. Karger AG, Basel.)

A retino-hypothalamic-sympathetic pathway drives the nocturnal surge of pineal melatonin production that determines the synchronization of pineal function with the environmental light/dark cycle. In many studies, melatonin has been implicated in the modulation of the inflammatory response. However, scant information on the feedback action of molecules present in the blood on the pineal gland during the time course of an inflammatory response is available. Here we analyzed the effect of tumor necrosis factor-alpha (TNF-alpha) and corticosterone on the transcription of the Aa-nat, hiomt and 14-3-3 protein genes in denervated pineal glands of rats stimulated for 5 hr with norepinephrine, using real-time reverse transcription-polymerase chain reaction. The transcription of Aa-nat, a gene encoding the key enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N-acetylserotonin, was inhibited by TNF-alpha. This inhibition was transient, and a preincubation of TNF-alpha for more than 24 hr had no detectable effect. In fact, a protein(s) transcribed, later on, as shown by cycloheximide, was responsible for the reversal of the inhibition of Aa-nat transcription. In addition, corticosterone induced a potentiation of norepinephrine-induced Aa-nat transcription even after 48 hr of incubation. These data support the hypothesis that the nocturnal surge in melatonin is impaired at the beginning of an inflammatory response and restored either during the shutdown of an acute response or in a chronic inflammatory pathology. Here, we introduce a new molecular pathway involved in the feedback of an inflammatory response on pineal activity, and provide a molecular basis for understanding the expression of circadian timing in injured organisms.

In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N-acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm-1.0 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). Corticosterone potentiated noradrenaline-induced melatonin and NAS production in a bell-shaped manner. The increase in NAS (12.9 +/- 2.7, n=6 versus 34.3 +/- 8.3 ng per pineal) and melatonin (16.3 +/- 2.0, n=6 versus 44.3 +/- 12.9 ng per pineal) content induced by 1 microm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]-dexamethasone binding (0.30 +/- 0.09 pmol/mg protein) and corticosterone inhibition of NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

Pineal gland G-protein coupled P2Y(1) receptors potentiate noradrenaline-induced N'-acetylserotonin production, a long term response which occurs after 5 h incubation. In the current study we show that a short-term effect of stimulation of P2Y(1) receptors is the increase in extracellular acidification rate (ECAR), which is mediated by an increase in intracellular calcium concentration ([Ca(2+)](i)). The pD(2) values for ATP (3.06 +/- 0.12)-induced ECAR increase was significantly smaller (p < 0.01) than that for ADP (3.64 +/- 0.18), 2MeSATP (3.56 +/- 0.02) and 2MeSADP (3.65 +/- 0.13). The selective P2Y(1) receptor antagonists A3'P-5'P and A3'P-5'PS inhibited the increase in ECAR-induced by ADP. Clamping [Ca(2+)](i) with BAPTA (30 and 50 micromol/l) led to inhibition of ADP-induced increase in ECAR. Agonist and antagonist data indicate P2Y(1) activation leads to a [Ca(2+)](i)-dependent acidification of the extracellular medium., (Copyright 2003 S. Karger AG, Basel)

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.

ATP is coreleased with noradrenaline in several noradrenergic synapses, and P2-like receptors were shown to be present in rat pineal glands. A new method of functional investigation was developed to assess the importance of both transmitters (noradrenaline and ATP) in eliciting the synthesis of melatonin and its precursor N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) through transmural electrical field stimulation of cultured pineal glands. Incubation with the beta-adrenoceptor antagonist propranolol (>10(-7) M) blocked almost completely the production of N'-acetyl-5-HT, whilst the P2 receptor antagonists pyridoxalphosphate-6 azophenyl-2',4'-disulfonic acid (PPADS, >3x10(-6) M) and suramin (>10(-6) M) blocked it partially. These findings indicate a physiologically relevant role for the purinergic cotransmission in this system.

1. The effects of noradrenaline, ATP, adenylyl-imidodiphosphate (AMP-PNP), adenosine, alpha,beta-methylene-ATP and the P2-purinoceptor antagonist, suramin on N'-acetyl-5-hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2. Noradrenaline (3 nM-1 microM) increased N'-acetyl-5-hydroxytryptamine production as measured both in the gland and the culture medium. 3. In noradrenaline (10 nM)-stimulated pineal glands, ATP (0.03 nM-1 mM) or AMP-PNP (0.1 microM-1 mM) increased N'-acetyl-5-hydroxytryptamine production in a concentration-dependent manner. 4. Alpha,beta-Methylene-ATP at the concentration of 0.1 mM, but not 3 microM, attenuated the enhancement by ATP (0.1 mM) of noradrenaline (10 nM)-induced N'-acetyl-5-hydroxytryptamine production. 5. Suramin (0.1 mM) blocked the potentiating effect of ATP (0.1 mM), but not the potentiating effect of adenosine (0.1 mM) in glands incubated with noradrenaline (10 nM). 6. These findings suggest that the rat pineal gland possesses P2-purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N'-acetyl-5-hydroxytryptamine production.

Monotherapy is the policy for management of patients with epilepsy. With increasing knowledge of the biology of epilepsy and of the modes of action of antiepileptic drugs (AEDs), this concept must be reevaluated. When monotherapy fails to control seizures, subsequent treatment should be based on "rational pharmacology," taking into consideration the mode of action of the drugs, to provide improved efficacy with maintained tolerance and ease of administration. Introduction of vigabatrin (VGB) as a new AED calls for just such a reevaluation. VGB is an enzyme-activated irreversible inhibitor of gamma-aminobutyric acid (GABA)-transaminase that increases brain and cerebrospinal (CSF) GABA concentrations in animals and humans. It has limited efficacy in the classic animal seizure screening tests, but in many clinical studies has halved the incidence of seizures in approximately 50% of patients, especially those with partial epilepsies. We evaluated the efficacy of VGB in "socially integrated and active outpatients" as a likely subset to demonstrate any advantage of rational polytherapy. The criteria for this evaluation included the effects on seizure frequency, patient tolerability, and cognitive performance in a battery of psychometric tests. Fourteen of the 19 patients (73%) completing the study had > 50% reduction in seizure frequency, and 10 of 19 (52%) had > 70% reduction in seizure frequency. Tolerability appeared good; somnolence was the most frequent adverse event. Three patients complained of a worsening of their seizures, 1 with an increase in frequency and 2 with development of myoclonic jerks not previously reported.(ABSTRACT TRUNCATED AT 250 WORDS)

Cognitive function of patients on monotherapy specific for their epileptic syndrome has been studied infrequently. We evaluated 7 patients with symptomatic localised epilepsies (SEL) on phenytoin aged 30 +/- 12 (mean +/- standard deviation) years, 8 with idiopathic generalised epilepsies on sodium valproate aged 18 +/- 4 years, 16 with SEL on carbamazepine aged 28 +/- 11 years, and 35 healthy controls aged 27 +/- 11 years. All subjects were of normal intelligence, educated appropriately to age, and led productive lives in the community. Two of the patients on carbamazepine and one on valproate had less than five partial, absence or myoclonic seizures monthly, the remaining were controlled. Carbamazepine serum concentrations were 12 +/- 5 micrograms/ml, phenytoin were 23 +/- 7, and valproate were 62 +/- 23 (mean +/- sd). Tests included immediate recall and recognition for pictures, Stroop test, delayed recall and recognition of pictures. Patients on phenytoin and valproate performed significantly worse than controls on immediate recall, and patients on carbamazepine performed significantly worse than controls in Stroop test (p < 0.01). The results indicate relatively minor effects of the epileptic syndromes and of phenytoin, carbamazepine and valproate on cognition of patients with controlled epilepsy leading productive lives in the community. We conclude that the cognitive deficit found in chronic epileptic patients on poly-therapeutic drug regimen must be multifactorial, and that future studies need to control for all possible variables in order to achieve meaningful results.