Regina P. Markus

Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites.

he development of malarial parasites is a complex process involving both intracellular and extracellular phases. Intraerythrocytic maturation proceeds through well-defined stages, termed rings, trophozoites and schizonts. In vivo, transition to a new stage and invasion of new erythrocytes are highly synchronous. The timing of these processes varies between Plasmodium species, but is always a multiple of 24 h. The simultaneous appearance of billions of individual parasites in the bloodstream may represent an efficient evolutionary strategy to escape the defence mechanisms of the host. The synchronicity of these processes is rapidly lost in culture, indicating the possible involvement of a host-derived signal, although the nature of this signal is presently unknown. Here we show that the hormone melatonin modifies the development of malarial parasites in vitro, that in vivo surgical ablation of the pineal gland leads to reduced synchronicity in the maturation process of Plasmodium, an effect that is reversed upon treatment with melatonin, and that in vivo inhibition of melatonin receptors mimics the effect of pinealectomy. We also demonstrate that melatonin, through activation of specific receptors coupled to phospholipase C activation, causes release of Ca from the intracellular stores of Plasmodium grown in vitro. We therefore propose that circadian changes in melatonin concentration in the host represent a key signal that controls synchronous maturation of Plasmodium in vivo. In search of a host-derived signal that undergoes circadian changes in mammals (and other vertebrates), we considered melatonin as a potential candidate. Melatonin is synthesized and released by the pineal gland during darkness, and this hormone is thought to participate in regulation of circadian rhythms in many eukaryotes, including vertebrates, invertebrates, higher plants and dinoflagellates. Not only does melatonin release exhibit a circadian rhythm, but the molecule is also sufficiently hydrophobic to cross T

Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B(4) induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT(2) and MT(3) melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

The Immune-Pineal Axis: A Shuttle between Endocrine and Paracrine Melatonin Sources

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-α 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-α 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.

Effect of TNF-α on the melatonin synthetic pathway in the rat pineal gland: basis for a ‘feedback’ of the immune response on circadian timing

Abstract:  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‐α (TNF‐α) 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‐α. This inhibition was transient, and a preincubation of TNF‐α 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.

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B

Abstract:  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 μm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 μm), the proteasome inhibitor, N‐acetyl‐leucinyl‐leucinyl‐norleucinal‐H (ALLN, 12.5 μm) or the antagonist of the nuclear factor kappa B (NFκB), pyrrolidinedithiocarbamate (PDTC, 12.5 μm). 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 μm 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 NFκB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline‐induced melatonin and NAS production through GR inhibition of NFκB 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.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Abstract:  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.

Pineal melatonin and the innate immune response: the TNF-α increase after cesarean section suppresses nocturnal melatonin production

Abstract:  The nocturnal surge of melatonin is the endocrine expression of the circadian system and is essential for organizing the timing of various endogenous processes. Previous works suggest that, in the beginning of a defense response, the increase in circulating tumor necrosis factor‐α (TNF‐α) leads to a transient block of nocturnal melatonin production and promotes a disruption of internal time organization. In the present paper, the concentration of melatonin and cytokines [TNF‐α, interferon‐γ (IFN‐γ), interleukin (IL)‐2, IL‐4, IL‐5, IL‐10, IL‐12] in the colostrum (postdelivery day 3) and in the milk (postdelivery days 10, 15, 20 and 30) obtained at midday and midnight from mothers who gave birth by vaginal or cesarean section were compared. The nocturnal melatonin surge observed 3 days after vaginal delivery was absent after cesarean section. IL‐12 presented no daily variation in either case, while daily variations in IFN‐γ, IL‐10, IL‐4 and IL‐5 were observed after vaginal delivery and cesarean section. On the other hand, the increase in TNF‐α after cesarean section resulted in suppression of the nocturnal melatonin surge. Daily variation of IL‐2 was only observed after recovery of the nocturnal melatonin surge, 30 days after cesarean section. The present paper supports the hypothesis of a cross‐talk between the pineal gland and the immune system, which could represent a putative immune–pineal axis.

Intracellular Peptides as Natural Regulators of Cell Signaling

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 μm) 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 α-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.

Injury switches melatonin production source from endocrine (pineal) to paracrine (phagocytes) - melatonin in human colostrum and colostrum phagocytes.

Abstract:  A large number of data show that melatonin has immunomodulatory properties and is produced by immunocompetent cells; also, some evidence suggests a ‘feedback’ of the activated immune system on the pineal gland. In this paper, we studied immune–pineal interactions in colostrum obtained from healthy puerperae and mothers with mastitis taking into account that, (a) melatonin levels in milk reflects pineal activity and (b) colostrum quiescent mononuclear and polymorphonuclear phagocytes from healthy mothers in culture are adequate for evaluating the ability of immunocompetent cells to produce melatonin. Here we compared the diurnal and nocturnal melatonin levels in colostrum from healthy puerperae and mothers with mastitis; this is a unique noninvasive model for determining pineal activity in the proinflammatory phase of a defense response. In addition, we determined the ‘in vitro’ production of melatonin by colostrum immunocompetent cells stimulated by enteropathogenic Escherichia coli or zymosan. Suppression of nocturnal melatonin rise in mothers with mastitis was highly correlated with increased tumor necrosis factor‐α (TNF‐α) secretion. This result, interpreted taking into account the presence of the transcription factor nuclear factor kappa B in pineal gland, suggest that the proinflammatory cytokine can inhibit nocturnal pineal melatonin production. On the other hand, stimulated, but not quiescent, immunocompetent cells secreted in the colostrum produced melatonin in vitro. In addition, this production ceases after bacteria killing. These results suggest that during the response to an injury the production of melatonin can be transiently shifted from an endocrine (pineal) to a paracrine (immunocompetent cells) source.

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrPC) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrPC extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrPC engagement induces an increase in intracellular Ca2+ levels. This effect was not detected in PrPC-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrPC. Using a best candidate approach to test for potential channels involved in Ca2+ influx evoked by STI1-PrPC, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrPC-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrPC and allowed reconstitution of signaling by PrPC-STI1 interaction. These results indicate that STI1 can interact with the PrPC·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.