María I. Keller Sarmiento

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 increases cGMP and decreases cAMP levels in rat medial basal hypothalamus in vitro

Measurement of cyclic nucleotide accumulation in rat medial basal hypothalamus (MBH) incubated in vitro with methoxyindoles indicated that melatonin (10(-8) M or greater) increased significantly cGMP and depressed cAMP levels. Only the effect on MBH cAMP was shared by 5-methoxytryptophol which exhibited a greater activity than melatonin. 6-Fluoromelatonin (10(-7) M) increased cGMP, whereas the effect of 6-hydroxymelatonin was not significant. Both 6-fluoro- and 6-hydroxymelatonin (10(-5) M) depressed MBH cAMP accumulation while only 6-fluoromelatonin affected it at 10(-7) M concentrations. These data suggest that melatonin and 5-methoxytryptophol affect differently cyclic nucleotide content of MBH at physiological concentrations.

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.

Neuroendocrine integrative mechanisms in mammalian pineal gland: Effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland.

Summary1.To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10–1000 nM NPY in the presence or absence of 10µM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA).2.NPY (10–1000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1000 nM), enhanced NE stimulation of melatonin production.3.NPY (1000 nM) impaired3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with3H-NE.4.These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.

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.

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.

Effect of experimental glaucoma on the non-image forming visual system.

J. Neurochem. (2011) 117, 904–914.

Evidence for local synthesis of melatonin in golden hamster retina.

Daily variations in melatonin content of retinas of pinealectomized and sham-operated golden hamsters were studied. Melatonin content showed significant daily variations with maximal values at night (i.e. early in the night in pinealectomized hamsters and late at night in sham-operated animals). Moreover, mean retinal melatonin levels augmented significantly after pinealectomy. In vitro the augmented melatonin levels found in retinas incubated in darkness for 8 h was suppressed by exposure to light, indicating the ability of hamster retina to regulate melatonin synthesis in isolated conditions. Taken together, the in vivo and in vitro results support daily variations of melatonin content of exclusive retinal origin.

Therapeutic efficacy of melatonin in reducing retinal damage in an experimental model of early type 2 diabetes in rats

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25 mg/kg). One day or 3 wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 wk of treatment, animals which received a sucrose‐enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a‐wave, b‐wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.