The oxidation products of melatonin derivatives exhibit acetylcholinesterase and butyrylcholinesterase inhibitory activity

Abstract:  It is already well documented that melatonin exhibits strong antioxidant properties. It traps several reactive oxygen species including singlet oxygen, peroxyl and hydroxyl radicals. Also, peroxynitrite-induced reactions are inhibited by melatonin. The oxidation of melatonin by singlet molecular oxygen [O₂ (¹Δ_g)] may produce cyclic 3-hydroxymelatonin whose structure we have already studied. In this investigation we report on the synthesis of several melatonin analogues having a carbamate substituent instead of the methoxy group at 5 position of the indole ring. These compounds behave analogously to melatonin with respect to singlet oxygen and produce the corresponding cyclic 3-hydroxymelatonin analogues. The structures of the products were investigated with spectral methods and X-ray crystallography. The compounds obtained possess the 2,3,8,8a-tetrahydropyrrolo[2,3-b]indole heterocyclic system which is a structural motif characteristic of alkaloids, physostigmine and phenserine, that are potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors used in the Alzheimer's disease treatment. We measured the inhibitory activity of the obtained compounds against AChE and BChE from human erythrocytes and serum. In the case of the compounds having a phenylcarbamate and methoxyphenylcarbamate substituents, the inhibitory activity (IC₅₀) ranged from 0.252 ± 0.033 to 3.804 ± 0.581 μ m . Other compounds were less active and showed rather complex interactions with the structure–activity relationship in need of further investigation.     

Congenital Methaemoglobinaemia due to NADH Methaemoglobin Reductase Deficiency: Successful Treatment with Oral Riboflavin

S ummary . A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromato-graphy. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (α²⁺β²⁺)₂, 29.6% half-oxidized forms, 20.9% (α+β²+)₂ and 8.7% (α²⁺β⁺)₂, and 3% full-oxidized methaemoglobin (α⁺β⁺)₂. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.     

Existence and function of opioid receptors on nammalian pinealocytes

Abstract: Previous studies in our laboratories have identified a single population of opioid receptors in bovine pineal gland, which we have chosen to characterize further on pinealocytes isolated from the cow and rat pineal gland. The bovine pinealocytes isolated by trypsinization or mechanical manipulation revealed receptor density (B_max) values of 206.95 ± 131.15 and 220.34 ± 11.80 fmol/mg protein, respectively, and dissociation equilibrium constant (K_d) values of 1.93 ± 0.48 and 1.96 ± 0.21 nM, respectively. The rat pinealocytes cultured for 7 days exhibited a [³H]diprenorphine binding site of 56 fmol/10⁶ cells. Morphine (100 μM) enhanced the activity of N-acetyltransferase and the level of melatonin in rat pineal gland in culture incubated for 21 hr. The results of these studies suggest that opioidergic receptors exist on pinealocytes and they are involved in stimulating the activity of N-acetyltransferase and the synthesis of melatonin, thereby regulating the physiology of mammalian pineal gland.     

The melatonin metabolite N1-acetyl-5-methoxykynuramine is a potent singlet oxygen scavenger

Abstract:  Singlet oxygen was generated by means of rose bengal under irradiation by visible light. N ¹-acetyl-5-methoxykynuramine (AMK) was rapidly destroyed by this reactive oxygen species, whereas its formylated precursor, N ¹-acetyl- N ²-formyl-5-methoxykynuramine (AFMK), was remarkably inert. At photon fluence rates of 1400  μ mol photons/m²s, and using 20  μ m rose bengal, most of initially 0.2 m m AMK was destroyed within 2 min, whereas AFMK remained practically unchanged for much longer periods of time. Competition experiments with other scavengers revealed the following order of reactivity towards singlet oxygen: diazabicyclo-[2,2,2]-octane (DABCO) << imidazole < 4-ethylphenol <  N _α-acetylhistidine < histidine < melatonin < AMK, the last one being about 150 times more effective than DABCO. Contrary to the oxidation in free radical-generating systems, AMK did not form adducts with the tyrosine side chain fragment, 4-ethylphenol, under the influence of singlet oxygen. In UV-exposed cells (keratinocytes, plant cells) it is likely to be more rapidly destroyed by singlet oxygen than formed from AFMK.     

(β-Adrenergic receptor subtypes in human pineal gland

Abstract: The secretion of melatonin by the pineal has been promoted as a direct monitor of adrenergic function in depressive illness. However, discrepant findings have been reported, possibly reflecting a complex adrenergic regulation of pineal output. In order to clarify the anatomical localization and relative density of β-adrenergic receptors and their subtypes in human pineal, quantitative autoradiographic analysis was conducted of β-adrenergic receptors in postmortem specimens using the high affinity radioligand ¹²⁵I-pindolol. Dense specific binding was found throughout the gland. β₁,-adrenergic receptors were more numerous, but β₂-receptors were present in an overlapping anatomical distribution with β₁-receptors.     

Melatonin impairs NADPH oxidase assembly and decreases superoxide anion production in microglia exposed to amyloid-beta1-42

Abstract:  Melatonin shows significant protective effects in Alzheimer's disease (AD) models in vitro and in vivo; these effects are related to its function as an antioxidant. The source of reactive oxygen species (ROS) generation in the AD brain is primarily the amyloid-β (Aβ)- activated microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, the effects of melatonin on the activation of NADPH oxidase remain unclear. In the present study, the cultures of microglia were incubated in the presence of fibrillar Aβ_1–42, which induces the assembly and the activation of NADPH oxidase, and triggers the production of superoxide anion-derived ROS. Pretreatment of microglia with melatonin dose-dependently prevents the activation of NADPH oxidase and decreases the production of ROS. Melatonin inhibits the phosphorylation of the p47 ^phox subunit of NADPH oxidase via a PI3K/Akt-dependent signalling pathway, blocks the translocation of p47 ^phox and p67 ^phox subunit to the membrane, down-regulates the binding of p47 ^phox to gp91 ^phox , and impairs the assembly of NADPH oxidase. Our data offer new insights into the mechanism of inhibiting ROS generation by melatonin in Aβ-activated microglia. Inhibition of ROS production indirectly might be the underlying mechanism for the neuroprotection by melatonin in the AD brain.     

Characterization and localization of delta opioid binding sites in the bovine pineal gland

Abstract: Opioid binding sites in the bovine pineal were characterized using the highly selective delta opioid agonist ³H-[D-Pen², pCl-Phe⁴, D-Pen⁵] enkephalin (DPDP(Cl)E). Pineal membranes possess a single class of high affinity binding sites for this delta ligand (K_d= 0.26 nM; B_max= 250 fmol/mg protein). The specific opioid antagonist naloxone dose dependently inhibited ³H-DPDP(C1)E binding, confirming that this ligand is indeed binding to opioid receptors. The delta selective ligands deltorphin and [D-Pen^2,5] enkephalin (DPDPE) were much more potent than the mu selective compounds dermorphin and [D-Ala², MePhe⁴ Gly⁵-ol]enkephalin (DAMGO) in inhibiting ³H-DPDP(Cl)E binding. These results demonstrate that in bovine pineal membranes, DPDP(Cl)E binds to delta opioid sites. Autoradiographic studies showed a uniform distribution of ³H-DPDP(Cl)E binding over the bovine pineal in the sections we analyzed. This distribution suggests that delta opioid binding sites are associated with pinealocytes which account for the majority of cell types in the pineal. However, it is not possible to rule out that these receptors may also be associated with other cell types which are present in the bovine pineal. The density and widespread distribution of delta opioid receptors supports the hypothesis that endogenous opioid peptides directly modulate pineal function.     

Five haplotypes in Black β-thalassaemia heterozygotes: three are associated with high and two with low Gγ values in fetal haemoglobin

Genotypes at seven different polymorphic restriction sites (5'to the ° gene, at the ^G γ, at the ^A γ, at the Ψβ , 3'to the Ψβ , at the β , and 3'to the β genes) were analysed by restriction endonuclease mapping of the DNA from 66 Black β -thalassaemia heterozygotes from Georgia and several of their normal relatives. Five different haplotypes were observed. Three of these were associated with high ^G γ values in the small amount of Hb F (0.8-8.3%) present in the blood of these patients and two with low ^G γ values. One haplotype [- + - ++++] that occurred on two of every three β thalassaemia chromosomes was associated with high ^G γ levels, and is the same as that found in some Black SS patients also having high ^G γ values (Gilman & Huisman, 1984). Two others [- ++ - + - +] and [−+−−+++] were also associated with high ^G γ, while two [−−−−+++] and [+−−−−++] were associated with low ^G γ. Variation in haematological data, mainly MCV and MCH values, was found to be caused in part by the type of β -thalassaemia (defined by its haplotype) and by the presence of an additional α-thalassaemia-2 heterozygosity or homozygosity.     

Association between angiotensin-converting enzyme gene polymorphisms and exercise performance in patients with COPD

Background and objective:   Recent studies have shown that polymorphisms of the angiotensin-converting enzyme (ACE) gene are closely associated with pulmonary disorders. The ACE gene is involved in the regulation of inflammatory reactions to lung injury, respiratory drive, erythropoiesis and tissue oxygenation. The hypothesis for this study was that the ACE gene may be associated with the ventilatory response to exercise and the aerobic work efficiency of skeletal muscle in patients with COPD.
Methods:   Sixty-one Chinese Han COPD patients and 57 healthy control subjects performed incremental cardiopulmonary exercise testing on a cycle ergometer. ACE genotypes were determined using PCR amplification.
Results:   Resting lung function and blood gas index were not significantly different among the three ACE genotype COPD groups. Similarly, there were no significant differences in AT, maximal O₂ uptake, maximal O₂ pulse, maximal dyspnoea index, ventilatory response (ΔVE/ΔVCO₂), O₂ cost of ventilation (VO₂/W/VE), end-tidal partial pressure of carbon dioxide at maximal exercise and maximal SaO₂ among the three ACE genotype COPD patients. Maximal work load and aerobic work efficiency were higher in the COPD group with the II genotype than in those with the ID or DD genotype. There were no significant differences in resting lung function and cardiopulmonary exercise testing parameters among the three ACE genotype control groups.
Conclusions:   The ACE gene may be involved in the regulation of skeletal muscle aerobic work efficiency, but is not associated with the ventilatory responses to exercise in COPD patients.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

A role for bursa fabricii and bursin in the ontogeny of the pineal biosynthetic activity in the chicken

Abstract: The tripeptide bursin (Lys-His-Gly-NH₂) is a B cell differentiation hormone derived from the bursa fabricii. The latter is a cloacal diverticulum and the site of B lymphocyte differentiation and selection in aves; also the bursa fabricii is involved in endocrine functions. Herein we demonstrate that in the chicken, the bursa fabricii and bursin are crucial to the ontogeny of both the pineal response to antigenic challenge and pineal circadian synthetic activity. In early embryonically bursectomized chickens, the plasma melatonin response to immunization by porcine thyroglobulin (Tg) was abolished. Also, the amplitudes of both plasma melatonin and pineal N-acetyltransferase (NAT) circadian rhythms were reduced by 50%, whereas the activity of hydroxyindole-O-methyltransferase (HIOMT) remained unchanged. Conversely, administration of either minute amounts (100 pg, 100 fg) or highly dilute (5 × 10⁻²⁷ g) bursin, with the exception of a highest dose (100 μg), to bursaless embryos induced recovery of normal antigen-induced melatonin response and normal amplitudes of melatonin and NAT rhythms. These findings establish that early in embryonic life, the bursa fabricii and its derived signal (bursin) are essential for normal development of pineal synthetic activity and underline the efficacy of very dilute bursin as an informative signal.     

N-acetyl-serotonin (normelatonin) and melatonin protect neurons against oxidative challenges and suppress the activity of the transcription factor NF-κB

Abstract: It is now well established that the formation of free radicals and oxidative stress-induced neuronal cell death can be involved in various neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. The pineal hormone melatonin has been suggested to be a neuroprotective antioxidant. To better understand the molecular mechanism of this activity, we compared the ability of melatonin and its precursor, N-acetyl-serotonin (normelatonin), to protect human neuroblastoma SK-N-MC cells and primary cerebellar granular neurons against oxidative stress. We found that normelatonin and melatonin have differential neuroprotective effects depending on the neuronal cell type. Normelatonin was more protective against hydrogen peroxide (H₂O₂) and glutamate-induced cell death in SK-N-MC cells compared to melatonin which was more effective to protect primary cerebellar granular neurons against the toxicity of H₂O₂, glutamate and N-methyl-D-aspartate when compared to normelatonin. At the molecular level, we tested the capacity of normelatonin and melatonin to inhibit the oxidative stress-induced NF-κB activation in both neuronal systems. Whereas normelatonin was more potent in the suppression of the activation of NF-κB by H₂O₂ in SK-N-MC cells compared to melatonin, no apparent differences in the extent of suppression could be detected in primary neurons. Normelatonin's and melatonin's neuroprotective activity in SK-N-MC neuroblastoma cells may be mediated by the suppression of NF-κB activation.     

The Pasteur effect in human platelets: implications for storage and metabolic control

Summary. The Pasteur effect and the associated acidosis have long been considered a major cause of platelet death during storage. We have investigated this phenomenon using a defined platelet preparation and a system whereby the oxidative and glycolytic contributions to total ATP production can be measured over a range of oxygen concentrations from saturating (pO₂=158mmHg) to anoxic (pO₂=OmmHg). Platelets do not show a Pasteur effect until the pO₂ decreases to & 2'OmmHg, whereupon lactate production increases 1-5-fold. The Pasteur effect is therefore not a likely cause of platelet death during storage where pO₂ in a storage bag typically drops to no less than 50mmHg. The data also have implications for the role of oxygen diffusion in oxidative metabolism, and for the compensatory nature of the Pasteur effect. As platelets are relatively small cells, and the onset of the Pasteur effect occurs at a relatively low oxygen concentration, diffusion may limit the rate of oxygen consumption in most other (larger) cells. The Pasteur effect is only fully compensative if the P/O₂ ratio used for the calculations is lower than the conventional one. Since recent research strongly suggests that the conventional P/O₂ ratio is too high, examples of fully compensative Pasteur effects may be more common than the literature suggests.     

Interaction between the Glucose-6-Phosphate Dehydrogenase Deficiency and Thalassaemia Genes at Phenotype Level

Summary. No significant differences were observed in the mean values of Hb A₂ levels and red cell indices between G6PD^- and G6PD+β thalassaemia carriers apart from the MCV, which was significantly higher in β thalassaemia G6PD^- subjects, but still in the thalassaemia carrier range. No difference was seen between G6PD+ and G6PD^-α thalassaemia carriers. G6PD+β thalassaemia carriers show a significant increase in G6PD levels expressed as activity per g of Hb and to lesser extent as activity per number of red cells x 10⁹; in G6PD+α thalassaemia carriers this increase is statistically significant only when the enzyme levels are expressed as activity per g of Hb. G6PD^-β thalassaemia carriers had enzyme levels higher than non-thalassaemic G6PD^- subjects only when the activity is expressed per g of Hb. G6PD activity was found to be increased in G6PD+ and G6PD^- Hb H disease patients.     

Epinephrine test and plasma elastase as diagnostic tools in a patient with CD3+ large granular lymphocyte proliferation

Large granular lymphocytes (LGL) proliferation is characterized by expansion of cytotoxic lymphocytes and associated with neutropenia. In a case of CD3⁺ LGL-proliferation the epinephrine stimulation test (EST) induced a striking elevation of CD3⁺, CD8⁺, CD57⁺ LGL in peripheral blood from 2.7 x 10⁹/1 to 20 x 10⁹/1 and might be an additional diagnostic tool in patients with normal or low absolute numbers of circulating LGL. After treatment with steroids, plasma elastase - a marker of neutrophil destruction - decreased from 162 to 40μg/1 (normal < 47μg/1) which correlated well with a simultaneous increase in peripheral neutrophil counts from 0.14 to 1.0 x 10⁹/1. This finding supports the hypothesis that neutropenia in CD3⁺ LGL proliferation is due to neutrophil destruction, possibly mediated by LGL.     

Exogenous melatonin entrains rhythm and reduces amplitude of endogenous melatonin: An in vivo microdialysis study

Abstract: The circadian rhythm of melatonin production was studied using on-line, in vivo microdialysis in the rat pineal gland. With this technique it was possible to record a pronounced melatonin rhythm with very high time resolution. Three phase-markers of the rhythm were calculated from the data, indicating increase (IT₅₀), decrease (DT₅₀) and amplitude of the rhythm. Comparing these phase markers led to several conclusions. Entrainment of the rhythm under constant darkness was performed with melatonin administration at different circadian stages [circadian time (CT) 8 and CT12] and for different periods of time (2 weeks and 4 weeks). Also, entrainment was established by applying 15 min light pulses at CTO. Entrainment of IT₅₀ with melatonin partially uncoupled it from DT₅₀. Four weeks entrainment in constant darkness (DD) caused a phase-delay in DT₅₀ of 2.2 hr. Entrainment of IT₅₀ with light at CTO for 2 weeks in DD caused a phase-advance in DT₅₀ of 1.3 hr. The entrainment with melatonin was restricted to a narrow window for melatonin to be applied, since injections at CT8 did not result in entrainment. Exogenous melatonin reduced the amplitude of the rhythm of endogenous melatonin. This effect was not circadian time dependent, since administration at CT8 for 2 weeks and at CT12 for 4 weeks resulted in a highly significant decrease. Light did not seem to have an effect on the amplitude. The data presented here provide us with new information about the nature of entrainment by melatonin. Since the present development of melatonergic agents for clinical use focuses on the entrainment capacity, effects of these compounds on amplitude of circadian rhythms needs to be addressed. In vivo microdialysis seems to be a good technique for that.     

Specific binding of 2-[125I]iodomelatonin by rat spleen crude membranes: Day-night variations and effect of pinealectomy and continuous light exposure

Abstract: Melatonin binding sites were characterized in rat spleen crude membranes. The specific binding of 2-[¹²⁵I]iodomelatonin by spleen crude membranes fulfills all the criteria for binding to a receptor site. Thus, binding was dependent on time and temperature, stable, specific, and increased under constant light exposure and after pinealectomy. In competition studies, the specific binding of 2-[¹²⁵I]iodomelatonin to spleen crude membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that the data were compatible with the existence of two classes of binding sites: a high affinity site with a K_d of 0.53 nM and a binding capacity of 2.52 pM, and a low-affinity site with a K_d of 374 nM and binding capacity of 820 pM. Moreover, binding of 2-[^l25I]iodomelatonin exhibited day-night variations with the highest binding observed late during the light period, and the lowest binding was observed late at night. However, binding of 2-[¹²⁵I]iodomelatonin to membranes remained high when animals were kept under light exposure at night. Results support the hypothesis of a regulatory role of melatonin on the immune system in which melatonin downregulates its own binding site.     

Increases in Volume, Fluid Content, and Lens Weight of Eyes Following Systemic Administration of Melatonin

Melatonin's effects were studied in male golden hamsters ( Mesocricetus auratus ) distributed among five surgical groups (nonoperated, sham-pinealectomized, sham-pinealectomized plus black plastic shielding of the pineal region, pinealectomized, and pinealectomized plus black plastic shielding of the pineal region) and three injection groups (vehicle only, 25 μg melatonin, and 2,500 μg melatonin). Injections (s.c.) were daily for 28 d at L₁₁ to L_11.75 in a (light:dark) L:D 14:10 artificial photoperiod. Animals (N = 112) were killed and dissected on the day after the last injection (at 55–65 d of age). None of rhe surgical procedures affected weights of eyes or their parts, nor did they influence the effects of administered melatonin on the eyes. Melatonin caused an increase in absolute and relative eye weight and an increase in fluid content of intraocular space. The magnitudes of these effects were positively related to melatonin dose. These same eyes had a progressively lower weight of nonlenticular tissues with low to high doses of melatonin, probably in relation to greater fluid content, and suspected increase in intraocular pressure. Lens wet and dry weights were significantly greater in animals receiving melatonin, but only at the high dose. These actions of melatonin are likely to be direct and are shown to not require the presence of the pineal. Experiments of other designs are suggested in order to determine whether the effects of the low, near physiological, dose of melatonin represent physiological actions of endogenous melatonin, synthesized and released within the eye. However, effects of large doses of melatonin on the eye are still noteworthy in relation to interpretation of experiments employing such dosages, and of disease states involving changes in intraocular pressure.     

Putative melatonin receptors in the male guinea pig kidney

Abstract: The direct action of pineal melatonin on the renal system is supported by our demonstration of 2-[¹²⁵I]iodomelatonin binding sites in the male guinea pig kidney. Scatchard analyses and Hill coefficients revealed a single type of binding site with an equilibrium dissociation constant (Kd) of 22.3 ±1.6 pmol/1 and a maximum binding density (Bmax) of 0.99 ±0.03 fmol/mg protein (n = 7) at mid-light. There was no significant difference in the Kd and Bmax values between kidney tissues collected at the middle of light and dark periods. The pharmacological profile of these 2-[¹²⁵I]iodomelatonin binding sites indicated high specificity for melatonin, 2-iodomelatonin and 6-chloromelatonin while kinetic studies generated a Kd value of 28.4 ±7.3 pmol/1 (n = 5) which was comparable to that determined from Scatchard transformations. Our results suggest that these binding sites are stable, reversible, saturable, specific, and of high affinity. Regional distribution study showed that specific binding of 2-[¹²⁵I]iodomelatonin was 8-fold higher in the cortical region than that in the medullary region. Studies of subcellular distribution showed that 59.3% of binding sites were localized in crude nuclear fractions followed by crude mitochondrial fractions (22.3%) and crude microsomal fractions (18.3%) with no detectable binding in cytosolic fractions. Our present findings suggest the presence of putative melatonin receptors in the guinea pig kidney, which support the hypotheses of melatonin-regulated renin secretion together with renal excretory functions via melatonin receptors.     

Antigonadal effects of two novel melatonin analogues in adult Djungarian hamsters

Abstract: This study examined the effects of two novel melatonin analogues in adult Djungarian hamsters housed under long photoperiod (LD 16:8). Daily injection (10 μg, s.c.) of either melatonin, 5-methoxy N-butanoyltryptamine (bMT), or 5-methyl N-butanoyltryptamine (5-MebT) 3 hr before lights off for 8 weeks led to a significant decrease in paired testis weight compared to vehicle-injected controls. The reduction in testis weight was of similar magnitude with melatonin and bMT, but 5-MebT was not as effective. The affinity of the analogues was determined in competition experiments using chicken brain membranes and 2-[¹²⁵I]iodomelatonin (2-[¹²⁵I]aMT). Replacing the N-acetyl side-chain of melatonin with an N-butanoyl group increased affinity for the chicken brain 2-[¹²⁵I]aMT binding site, but exchanging the 5-methoxy group of melatonin for a 5-methyl group reduced affinity. These studies show that these analogues not only inhibit 2-[¹²⁵I]aMT binding in vitro but also mimic melatonin's antigonadal activity in vivo.