Ruth E. Rosenstein

Melatonin increases in vivo GABA accumulation in rat hypothalamus, cerebellum, cerebral cortex and pineal gland

The effect of melatonin on in vivo gamma-aminobutyric acid (GABA) accumulation in several brain regions was determined by measuring the increase of GABA levels following inhibition of GABA transaminase. A single melatonin injection (25-300 micrograms/kg) augmented significantly, by 17-20%, GABA accumulation in the hypothalamus and caused a dose-dependent increase of this parameter in the pineal gland. Significant rises of GABA accumulation were found in the cerebellum and cerebral cortex after administering 100-300 and 300 micrograms/kg of melatonin, respectively.

Diurnal variations of benzodiazepine binding in rat cerebral cortex: Disruption by pinealectomy

In a previous work, pinealectomy was found to depress benzodiazepine (BZP) receptor binding in cerebral cortex membranes of rats killed at noon. In order to assess the effect of pineal removal on diurnal variations of BZP binding site concentration and affinity, groups of intact, pinealectomized, or sham‐ pinealectomized rats (subjected to surgery 2 wk earlier) were killed at six different time intervals during the 24‐h cycle. BZP binding was assessed by Scatchard analysis of 3H‐flunitrazepam high‐affinity binding to cerebral cortex membranes. In intact and sham‐pinealectomized rats, a maximum in BZP receptor concentration was found at midnight. Pinealectomy blunted the nocturnal peak of receptor concentration and caused a significant depression of binding site number at noon. No changes in the affinity of the binding sites for the radioligand were detected as a function of time of day or following surgery. In a doseresponse experiment for melatonin ability to restore the depressed BZP receptor concentration of cerebral cortex membranes of pinealectomized rats killed at noon, a minimal effective dose of 25 μg/kg body weight was obtained. These results further support a link between pineal activity and brain BZP receptors in rats.

Effect of glaucoma on the retinal glutamate/glutamine cycle activity

Glutamate‐induced excitotoxicity has been proposed to mediate the death of retinal ganglion cells in glaucoma. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Thus, the aim of the present work was to study the retinal glutamate/glutamine activity in eyes with hypertension induced by intracameral injections of hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 10 weeks of treatment, glutamate and glutamine uptake and release were assessed using [3H]‐glutamate and [3H]‐glutamine as radioligands, respectively. In addition, glutamine synthetase activity was assessed by a spectrophotometric assay, whereas glutaminase activity was measured through the conversion of [3H]‐glutamine to [3H]‐glutamate. At 3 weeks of treatment with HA, a significant decrease (P<0.01) in glutamate uptake and glutamine synthetase activity was observed. Glutamine uptake and release, as well as glutaminase activity, were significantly increased (P<0.01) in eyes injected with HA for 3 weeks compared with vehicle‐injected eyes, whereas [3H]‐glutamate release did not change in hypertensive eyes. Only the changes in glutamine synthetase activity persisted at 10 weeks of treatment with HA. These results indicate a significant alteration in the retinal glutamate/glutamine cycle activity in hypertensive eyes. Since these changes preceded both functional and histological alterations induced by ocular hypertension, these results support the involvement of glutamate in glaucomatous neuropathy.

Melatonin site and mechanism of action: Single or multiple?

ABSTRACT: By affecting the entrainment pathways of the biologic clock, melatonin has a major influence on the circadian and seasonal organization of vertebrates. In addition, a number of versatile functions that far transcend melatonin actions on photoperiodic time measurement and circadian entrainment have emerged. Melatonin is a free radical scavenger and antioxidant and it has a significant immunomodulatory activity, being presumably a major factor in an organisms defense toxic agents and invading organisms. Besides affecting specific receptors in cell membranes to exert its effects, the interaction of melatonin with nuclear receptor sites and with intracellular proteins, like calmodulin or tubulin‐associated proteins, as well as the direct antioxidant effects of melatonin, may explain many general functions of the pineal hormone.

Physiological concentrations of melatonin inhibit the nitridergic pathway in the Syrian hamster retina.

In the present work, the effect of melatonin on the hamster retinal nitridergic pathway was examined. When the retinas were incubated in the presence of low concentrations (1 pM–10 nM) of melatonin for 15 min, a significant decrease of nitric oxide synthase (NOS) activity was observed. However, when crude retinal homogenates were preincubated with melatonin for 15 min, no changes in NOS activity were detected, despite the fact that under the same conditions trifluoperazine, a calmodulin inhibitor, significantly decreased enzymatic activity. Kinetic analysis showed that melatonin decreased the Vmax of retinal NOS without changes in the Km. On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L‐arginine influx. A decrease in the Vmax of L‐arginine uptake was observed in the presence of melatonin, whereas the Km remained unchanged. Melatonin significantly inhibited the accumulation of cyclic guanosine monophosphate (cGMP) levels induced by both L‐arginine and sodium nitroprusside (SNP). In summary, the present results indicate that melatonin could be a potent inhibitor of the retinal nitridergic pathway.

Melatonin as a therapeutic tool in ophthalmology: implications for glaucoma and uveitis

Abstract:  Several lines of evidence support the view that increased free radical generation and altered nitric oxide (NO) metabolism play a role in the pathogenesis of highly prevalent ocular diseases, such as glaucoma and uveitis. Data are discussed indicating that melatonin, being an efficient antioxidant that displays antinitridergic properties, has a promising role in the treatment of these ocular dysfunctions. Melatonin synthesis occurs in the eye of most species, and melatonin receptors are localized in different ocular structures. In view of the fact that melatonin lacks significant adverse collateral effects even at high doses, the application of melatonin could potentially protect ocular tissues by effectively scavenging free radicals and excessive amounts of NO generated in the glaucomatous or uveitic eye.

Effect of nitroxyl on human platelets function

There is a growing body of evidence on the role of nitric oxide (NO) in human platelet physiology regulation. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO-), because it is formed by a number of diverse biochemical reactions. The aim of the present study was to comparatively analyze the effect of HNO and NO on several functional parameters of human platelets. For this purpose, sodium trioxodinitrate (Angelis salt,AS) and sodium nitroprusside (SNP) were used as HNO and NO releasers, respectively. BothAS and SNP significantly inhibited platelet aggregation and ATP release induced by different agonists and adrenaline. AS or SNP did not modify the expression of platelet glycoproteins (Ib, IIb-IIIa, la-IIa, IV), whereas they substantially decreased the levels of CD62P, CD63 and of PAC-1 (a platelet activated glycoprotein IIb/IIIa epitope) after the stimulation with ADP. AS and SNP significantly increased cGMP accumulation in a 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ)-sensitive manner. However, while L-cysteine reduced the effect of AS, it increased the effect of SNP on this parameter. Accordingly, a differential effect of L-cysteine was observed on the antiaggregatory effect of both compounds. In summary, these results indicate that HNO is an effective inhibitor of human platelet aggregation.

Melatonin: a novel neuroprotectant for the treatment of glaucoma

Abstract:  Glaucoma is a leading cause of blindness. Although ocular hypertension is the most important risk factor, several concomitant factors such as elevation of glutamate and decrease in gamma‐aminobutyric acid (GABA) levels, disorganized NO metabolism, and oxidative damage could significantly contribute to the neurodegeneration. The aim of this report was to analyze the effect of melatonin on retinal glutamate clearance, GABA concentrations, NO synthesis, and retinal redox status, as well as on functional and histological alterations provoked by chronic ocular hypertension induced by intracameral injections of hyaluronic acid (HA) in the rat eye. In normal retinas, melatonin increased glutamate uptake, glutamine synthase activity, GABA turnover rate, glutamic acid decarboxylase activity, superoxide dismutase activity, and reduced glutathione (GSH) levels, whereas it decreased NOS activity, L‐arginine uptake, and lipid peroxidation. To assess the effect of melatonin on glaucomatous neuropathy, weekly injections of HA were performed in the eye anterior chamber. A pellet of melatonin was implanted subcutaneously 24 hr before the first injection or after six weekly injections of HA. Melatonin, which did not affect intraocular pressure (IOP), prevented and reversed the effect of ocular hypertension on retinal function (assessed by electroretinography) and diminished the vulnerability of retinal ganglion cells to the deleterious effects of ocular hypertension. These results indicate that melatonin could be a promissory resource in the management of glaucoma.

Nitrosation of melatonin by nitric oxide: a computational study

Melatonin is being increasingly promoted as a therapeutic agent for the treatment of jet lag and insomnia, and is an efficient free radical scavenger. We have recently characterized a product for the reaction of melatonin with nitric oxide (NO), N‐nitrosomelatonin. In the present work, reaction pathways with N1, C2, C4, C6 and C7 as possible targets for its reaction with NO that yield the respective nitroso derivatives have been investigated using semiempirical AM1 computational tools, both in vacuo and aqueous solution. Specifically, two different pathways were studied: a radical mechanism involving the hydrogen atom abstraction to yield a neutral radical followed by NO addition, and an ionic mechanism involving addition of nitrosonium ion to the indolic moiety. Our results show that the indolic nitrogen is the most probable site for nitrosation by the radical mechanism, whereas different targets are probable considering the ionic pathway. These results are in good agreement with previous experimental findings and provide a coherent picture for the interaction of melatonin with NO.

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [3H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 nM) significantly increased [3H]glutamate uptake, and this effect persisted in a Ca2+‐free medium. On the other hand, melatonin significantly increased [3H]glutamate release in retinas excised at 2400 h, but this effect was Ca2+ sensitive. Melatonin significantly increased 45Ca2+ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [3H]glutamate uptake, [3H]glutamate release, or 45Ca2+ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8‐bromoguanosine 3′,5′‐cyclic monophosphate and 2′‐O‐dibutyrylguanosine 3′,5′‐cyclic monophosphate, significantly increased [3H]glutamate uptake, [3H]glutamate release, and 45Ca2+ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.