Francisco Vives

Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b.w) was i.v. injected 6 h before rats were killed and melatonin (10–60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, γ‐glutamyl‐transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b.w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS‐induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose‐dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b.w., 58–65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS‐induced endotoxemia in rats.—Crespo, E., Macías, M., Pozo, D., Escames, G., Martín, M., Vives, F., Guerrero, J. M., Acuña‐Castroviejo, D. Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide‐induced multiple organ dysfunction syndrome in rats. FASEB J. 13, 1537–1546 (1999)

Cell protective role of melatonin in the brain.

Abstract: In recent years an increasing amount of data has been published involving melatonin in the control of brain function. The pineal gland exerts a depressive influence on CNS excitability. This activity is linked to melatonin, since pharmacological doses of the hormone prevent seizures in several animal models. In addition, melatonin also has analgesic properties in these species. However, the sites and mechanism of melatonin action are not known. A role for the pineal gland and its hormone melatonin as a homeostatic system controlling brain excitability has been proposed, and GABA‐containing neurons may be involved in some central action of melatonin. There is evidence supporting a role of melatonin in the regulation of the GABA‐benzodiazepine receptor complex, and it appears that melatonin potentiates this inhibitory neurotransmitter system in brain. Melatonin does not bind to GABA or benzodiazepine binding sites themselves, because in vitro binding data showed that melatonin is a weak competitor of benzodiazepine binding in brain membranes at concentrations greater than 10−5 M. The effect of melatonin on brain activity also involves the participation of corticotropic and opioid peptides, and the existence of an opioid‐antiopioid homeostatic system is proposed, with the GABA‐benzodiazepine receptor complex as an effector. Moreover, the interaction of melatonin with corticotropic peptides and mitochondrial benzodiazepine receptors may result in a participation of neurosteroids in the control of GABA activity and function. The most recently available data from biochemical and electrophysiological studies support the possibility that the anticonvulsant and depressive effects of melatonin on neuron activity may depend on its antioxidant and antiexcitotoxic roles, acting as a free radical scavenger and regulating brain glutamate receptors. The full characterization of the nuclear melatonin receptor explains the genomic effects of melatonin, opening a new perspective regarding actions and roles of melatonin as a cellular protector.

Comparative effects of melatonin, l‐deprenyl, Trolox and ascorbate in the suppression of hydroxyl radical formation during dopamine autoxidation in vitro

Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinsons disease (PD). Inhibitors of monoamine oxidase B (MAO‐B) have been used in the treatment of PD and at least one of them, i.e., deprenyl, also displays antioxidant activity. Dopamine (DA) autoxidation produces reactive oxygen species implicated in the loss of dopaminergic neurons in the nigrostriatal pathway. In this study we compared the effects of melatonin with those of deprenyl and vitamins E and C in preventing the hydroxyl radical (•OH) generation during DA oxidation. The rate of production of 2,3‐dihydroxybenzoate (2,3‐DHBA) in the presence of salicylate, an •OH scavenger, was used to detect the in vitro generation of •OH during iron‐catalyzed oxidation of DA. The results showed a dose‐dependent effect of melatonin, deprenyl and vitamin E in counteracting DA autoxidation, whereas vitamin C had no effect. Comparative analyses between the effect of these antioxidants showed that the protective effect of melatonin against DA autoxidation was significantly higher than that of the other compounds tested. Also, when melatonin plus deprenyl were added to the incubation medium, a potentiation of the antioxidant effect was found. These findings suggest that antioxidants may be useful in brain protection against toxicity of reactive oxygen species produced during DA oxidation, and melatonin, alone or in combination with deprenyl, may be an important component of the brains antioxidant defenses to protect it from dopaminergic neurodegeneration.

Modification of Nitric Oxide Synthase Activity and Neuronal Response in Rat Striatum by Melatonin and Kynurenine Derivatives

Tryptophan is mainly metabolized in the brain through methoxyindole and kynurenine pathways. The methoxyindole pathway produces (among other compounds) melatonin, which displays inhibitory effects on human and animal central nervous systems, including a significant attenuation of excitatory, glutamate‐mediated responses. The kynurenine pathway produces kynurenines that interact with brain glutamate‐mediated responses. Nitric oxide (NO) increases glutamate release, and melatonin and kynurenines may act via modification of NO synthesis. In the present study, the effects of melatonin and four synthetic kynurenines were studied on the activity of rat striatal nitric oxide synthase (NOS) and on the response of rat striatal neurons to sensorimotor cortex (SMCx) stimulation, a glutamate‐mediated response. Melatonin inhibited both NOS activity and the striatal glutamate response, and these effects were dose‐related. Compound A (2‐acetamide‐4‐(3‐methoxyphenyl)‐4‐oxobutyric acid) did not inhibit NOS activity but inhibited the striatal response similarly to melatonin. Compound B (2‐acetamide‐4‐(2‐amino‐5‐methoxyphenyl)‐

Plasma α-synuclein in patients with Parkinson's disease with and without treatment

Alpha‐synuclein (α‐syn) is an intracellular protein with a high tendency to aggregation. It is the major component of Lewy bodies and may play a key role in the pathogenesis of Parkinsons disease (PD). α‐Syn is also released by neurons and can be detected in biological fluids, such as plasma. The purpose of this study was to determine whether plasma α‐syn concentrations are elevated in newly diagnosed PD patients before treatment (nontreated PD group, ntPD; n = 53) and to compare them with concentrations in PD patients with at least 1 year of specific treatment (tPD; n = 42) and in healthy controls (n = 60). Plasma α‐syn concentrations in the ntPD and tPD groups were similar and significantly higher than in healthy controls. In conclusion, α‐syn was elevated early in the development of PD and specific PD treatment did not change plasma α‐syn levels.

Melatonin-dopamine interaction in the striatal projection area of sensorimotor cortex in the rat.

The excitatory response to motor cortex stimulation of 201 striatal neurones was recorded electrophysiologically to test the effects of melatonin (aMT) and/or D1 and D2 antagonists. Iontophoresis of aMT attenuated the excitatory response in 68.5% of neurones, with a latency of 2–4 min and enhanced the excitatory response in 11.9% of the neurones; 19.6% showed no change in response. Iontophoresis of sulpiride (D2 antagonist) produced an immediate increase in the excitatory response in 62.8% of neurones, an attenuation in 2.3% and no change in the response of 34.9%. The ejection of sulpiride counteracted the aMT-dependent inhibition of the excitatory response of striatal neurones. SCH-23390 (D1 antagonist) iontophoresis had no significant effect. The results show that the same striatal units may be driven by aMT and D2 receptors. However, the significant difference in the latency of the responses suggests that the effects of these two substances are mediated by different receptor/intracellular messengers.

Changes in brain amino acids and nitric oxide after melatonin administration in rats with pentylenetetrazole‐induced seizures

Abstract: We examined the effect of melatonin on brain levels of amino acids and nitric oxide (NO) after pentylenetetrazole (PTZ)‐induced seizures in rats. Animals were treated with melatonin (10–160 mg/kg, i.p.) 30 min before PTZ administration (100 mg/kg, s.c.), and were killed 3 hr later. At the dose of 80 mg/kg, melatonin significantly increased the latency (5.7–12.7 min) and decreased the duration (31.2–18.4 s) of the first seizure, reducing PTZ induced mortality from 87.5 to 25%. After kill, brains were removed and neurotransmitters and nitrite levels measured in prefrontal cortex (PF), parieto‐temporal cortex (PF), striatum (ST), hippocampus (HP) and brain stem (BS) by high performance liquid chromatography. PTZ treatment increased glutamine levels in all brain areas studied, without changes in glutamate, gamma‐amino butyric acid (GABA) and glycine. Aspartate and taurine increased in PF and PT and in HS and PT, respectively. Melatonin administration displayed a dose‐dependent effect. At doses of 10–40 mg/kg, melatonin counteracted the PTZ‐induced glutamine increase and reduced both glutamate and asparatate levels in the studied areas, with minor changes in GABA and glycine content. At doses of 80 and 160 mg/kg, the levels of glutamine, and glutamate, and to a lesser extent aspartate increased, whereas serine levels did not change. These two doses of melatonin also increased taurine, GABA and glycine in most brain areas studied. Treatment with melatonin (40–160 mg/kg) significantly decreased nitrite content in PT cortex, ST and BS areas of epileptic rats, without changes in the other brain regions. The results suggest that the anticonvulsant property of melatonin involves a modulation of both brain amino acids and NO production.

Plasma lipid peroxidation in sporadic Parkinson's disease. Role of the l-dopa

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinsons disease (PD). There are several methods to measure oxidative stress, being lipid peroxidation (LPO) one of the most frequently used. Endogenous plasma LPO was determined by a spectrofluorimetric method in fifty two patients with sporadic PD and in forty controls. To know the maximum capacity of lipids to peroxidate, LPO was also measured after co-incubation with Fe2+/H2O2 (exogenous LPO). All PD patients were taken L-dopa and the effect of this treatment on LPO levels was additionally studied. Urine catecholamines and their main metabolites were also analyzed, and their possible correlation to LPO statistically studied. Endogenous plasma LPO levels were 33% higher in PD group than in control group (P<0.001). Exogenous plasma or oxidizability was also higher in PD patients compared to controls (20%, P<0.05). The intake of L-dopa was negatively dose-related to endogenous and exogenous plasma LPO. In conclusion, plasma of PD patients has elevated levels of LPO and also is more prone to peroxidation than that in the control group. The results also suggest an antioxidant effect of L-dopa.

Calcium-dependent effects of melatonin inhibition of glutamatergic response in rat striatum.

The effects of melatonin, amlodipine, diltiazem (l‐type Ca2+ channel blockers) and ω‐conotoxin (N‐type Ca2+ channel blocker) on the glutamate‐dependent excitatory response of striatal neurones to sensory‐motor cortex stimulation was studied in a total of 111 neurones. Iontophoresis of melatonin produced a significant attenuation of the excitatory response in 85.2% of the neurones with a latency period of 2 min. Iontophoresis of either l‐ or N‐type Ca2+ channel blocker also produced a significant attenuation of the excitatory response in more than 50% of the recorded neurones without significant latency. The simultaneous iontophoresis of melatonin + amlodipine or melatonin + diltiazem did not increase the attenuation produced by melatonin alone. However, the attenuation of the excitatory response was significantly higher after ejecting melatonin + ω‐conotoxin than after ejecting melatonin alone. The melatonin–Ca2+ relationship was further supported by iontophoresis of the Ca2+ ionophore A‐23187, which suppressed the inhibitory effect of either melatonin or Ca2+ antagonists. In addition, in synaptosomes prepared from rat striatum, melatonin produced a decrease in the Ca2+ influx measured by Fura‐2AM fluorescence. Binding experiments with [3H]MK‐801 in membrane preparations from rat striatum showed that melatonin did not compete with the MK‐801 binding sites themselves although, in the presence of Mg2+, melatonin increased the affinity of MK‐801. The results suggest that decreased Ca2+ influx is involved in the inhibitory effects of melatonin on the glutamatergic activity of rat striatum.

Effects of agonists and antagonists of D1 and D2 dopamine receptors on self-stimulation of the medial prefrontal cortex in the rat.

The possible participation of D1 versus D2 dopamine receptors in mediating dopaminergic neurotransmission of self-stimulation (SS) in the medial prefrontal cortex (MPC) of the rat was studied neuropharmacologically. Intracerebral as well as intraperitoneal injections of agonists and antagonists of dopamine receptors were used in this study. In all experiments performed with systemic injections, spontaneous motor activity (SM) was measured parallel to self-stimulation behavior as control for non specific effects of the drugs. Intracranial injections were done unilaterally serving SS of the contralateral side (not injected or injected with 0.9% NaCl) as control in the same animals. Spiroperidol and pimozide were used as D1-D2 dopamine antagonists, while sulpiride was used as a specific D2 antagonist. Apomorphine was used as D1-D2 agonist, while bromocriptine and lergotrile were used at doses in which these ergot drugs are considered predominantly D2 agonists. Sulpiride, intraperitoneally or intracerebrally injected at the same locus at which the stimulating electrode was located produced no effect on SS. On the contrary, the D1-D2 antagonists, spiroperidol and pimozide intraperitoneally or intracerebrally injected produced a dose-dependent decrease on SS. On the basis of these data it is suggested, that the dopamine neurotransmission involved in SS of the MPC is mediated via D1 dopamine receptors. This suggestion is further emphasized by the results obtained with the agonists, apomorphine, bromocriptine and lergotrile. Apomorphine produced a dose-related decrease on SS and a decrease at lower doses and an increase at higher doses on SM. Bromocriptine and lergotrile had, on the contrary, no effect on SS and a dose-related decrease on SM.