Daniel A. Sáenz

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.

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.

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.

Therapeutic Effect of Melatonin in Experimental Uveitis

Uveitis is a common ophthalmic disorder that can be induced in hamsters by a single intravitreal injection of bacterial lipopolysaccharide (LPS). To examine the therapeutic effects of melatonin on uveitis, a pellet of melatonin was implanted subcutaneously 2 hours before the intravitreal injection of either vehicle or LPS. Both 24 hours and 8 days after the injection, inflammatory responses were evaluated in terms of i) the integrity of the blood-ocular barrier, ii) clinical signs, iii) histopathological studies, and iv) retinal function. Melatonin reduced the leakage of proteins and cells in the anterior segment of LPS-injected eyes, decreased clinical signs such as dilation of the iris and conjunctival vessels, and flare in the anterior chamber, and protected the ultrastructure of the blood-ocular barrier. A remarkable disorganization of rod outer segment membranous disks was observed in animals injected with LPS, whereas no morphological changes in photoreceptor outer segments were observed in animals treated with melatonin. Furthermore, melatonin prevented a decrease in LPS-induced electroretinographic activity. In addition, melatonin significantly abrogated the LPS-induced increase in retinal nitric-oxide synthase activity, tumor necrosis factor-alpha, and nuclear factor kappaB p50 and p65 subunit levels. These results indicate that melatonin prevents the clinical, biochemical, histological, ultrastructural, and functional consequences of experimental uveitis, likely through a nuclear factor kappaB-dependent mechanism, and support the use of melatonin as a new therapeutic strategy for the treatment of uveitis.

Circadian activity of the GABAergic system in the golden hamster retina

Daily changes in gamma-aminobutyric acid (GABA) turnover rate were studied in the golden hamster retina. This parameter showed significant variations throughout the light-dark cycle, with minimal values during the day. Retinal glutamic acid decarboxylase (GAD) activity was higher at midnight than at noon. Moreover, [3H]GABA binding significantly varied throughout the 24-h cycle, with maximal values during the day. Saturation studies performed at 12:00 and 24:00 h indicated that the maximal concentration of [3H]GABA binding sites (Bmax) was significantly higher at noon, whereas the dissociation constant (Kd) remained unchanged. High K+-induced GABA release was significantly higher at midnight than at midday. Daily variations in retinal GABA turnover rate, GABA release, and in its specific binding persisted in golden hamsters exposed to constant darkness. In summary, these results support the idea of a circadian clock-controlled GABAergic activity in the hamster retina.

Effect of melatonin on the retinal glutamate/glutamine cycle in the golden hamster retina

Glutamate is the main excitatory neurotransmitter in the retina, but it is neurotoxic when present in excessive amounts. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Since melatonin has been shown to be neuroprotective in several systems, in the present report, its effect on the glutamate/glutamine cycle activity was examined in the golden hamster retina. Melatonin (0.1–10 nM) significantly increased retinal glutamine synthetase activity but it did not affect L‐glutamine release. A characterization of the hamster retinal L‐ glutamine uptake mechanism was performed. This mechanism was partly Na+‐dependent, and it was significantly inhibited by 2‐aminobicyclo (2, 2, 1) heptane 2‐carboxylic acid (BCH, a selective antagonists for the L‐type system) and by α‐(methylamino)‐isobutyric acid (MeAIB, substrate characteristic for the A ‐type transporter) suggesting the coexistence of these transport systems in the hamster retina. Melatonin (0.1–10 nM) significantly increased total glutamine uptake as well as the BCH and the MeAIB‐insensitive transporters activity. On the other hand, melatonin significantly decreased retinal glutaminase activity. On the basis of these results, it might be presumed that hamster retinal glutamate/glutamine cycle activity is regulated by physiological concentrations of melatonin. Furthermore, these findings suggest that a treatment with melatonin could be considered as a new approach to handling glutamate‐mediated neuronal degeneration.

Effect of bacterial lipopolysaccharide on ischemic damage in the rat retina.

PURPOSE The purpose of this study was to investigate whether bacterial lipopolysaccharide (LPS) induces ischemic preconditioning in the rat retina, and, if so, whether nitric oxide (NO) is involved in this process. METHODS Rats were intravitreously injected with different doses of LPS (0.1, 1, or 5 microg) in one eye and vehicle in the contralateral eye 24 hours before retinal ischemia induced by increasing intraocular pressure to 120 mm Hg for 40 or 60 minutes. Subsequently, 7 or 14 days after ischemia, the rats were subjected to electroretinography and histologic analysis. One group of animals received intraperitoneal injections of NOS inhibitors, N-nitro-L-arginine methyl ester (L-NAME) aminoguanidine or N-(3-(aminomethyl)benzyl)acetamidine (W1400) before the injection of LPS or vehicle. Retinal nitric oxide synthase (NOS) activity was assessed through the conversion of (3)H-L-arginine to (3)H-L-citrulline. RESULTS One microgram (but not 0.1 or 5 microg) LPS afforded significant morphologic and functional protection in eyes exposed to ischemia-reperfusion injury. The beneficial effect of LPS was reversed by treatment with L-NAME, aminoguanidine, or W1400. LPS (1 and 5 microg, but not 0.1 microg) significantly increased retinal NOS activity. CONCLUSIONS These results indicate that LPS provides retinal protection against ischemia-reperfusion injury in a dose-dependent manner, probably through an inducible NOS-dependent mechanism.

Photodynamic inactivation of Gram-positive bacteria employing natural resources.

The aim of this paper was to investigate a collection of plant extracts from Argentina as a source of new natural photosensitizers (PS) to be used in Photodynamic Inactivation (PDI) of bacteria. A collection of plants were screened for phototoxicity upon the Gram-positive species Staphylococcus epidermidis. Three extracts turned out to be photoactive: Solanum verbascifolium flower, Tecoma stans flower and Cissus verticillata root. Upon exposure to a light dose of 55J/cm(2), they induced 4, 2 and 3logs decrease in bacterial survival, respectively. Photochemical characterisation of S. verbascifolium extract was carried out. PDI reaction was dependent mainly on singlet oxygen and to a lesser extent, on hydroxyl radicals, through type II and I reactions. Photodegradation experiments revealed that the active principle of the extract was not particularly photolabile. It is noticeable that S. verbascifolium -PDI was more efficient under sunlight as compared to artificial light (total eradication vs. 4 logs decrease upon 120min of sunlight). The balance between oxidant and antioxidant compounds is likely to be masking or unmasking potential PS of plant extracts, but employing the crude extract, the level of photoactivity of S. verbascifolium is similar to some artificial PS upon exposure to sunlight, demonstrating that natural resources can be employed in PDI of bacteria.

Bacterial lipopolysaccharide protects the retina from light-induced damage.

J. Neurochem. (2012) 122, 392–403.

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.