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

Glaucoma is a leading cause of blindness worldwide, characterized by retinal ganglion cell degeneration and damage to the optic nerve. We investigated the non-image forming visual system in an experimental model of glaucoma in rats induced by weekly injections of chondroitin sulphate (CS) in the eye anterior chamber. Animals were unilaterally or bilaterally injected with CS or vehicle for 6 or 10 weeks. In the retinas from eyes injected with CS, a similar decrease in melanopsin and Thy-1 levels was observed. CS injections induced a similar decrease in the number of melanopsin-containing cells and superior collicular retinal ganglion cells. Experimental glaucoma induced a significant decrease in the afferent pupil light reflex. White light significantly decreased nocturnal pineal melatonin content in control and glaucomatous animals, whereas blue light decreased this parameter in vehicle- but not in CS-injected animals. A significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was observed in glaucomatous animals. General rhythmicity and gross entrainment appear to be conserved, but glaucomatous animals exhibited a delayed phase angle with respect to lights off and a significant increase in the percentage of diurnal activity. These results indicate the glaucoma induced significant alterations in the non-image forming visual system.

Effect of neurosteroids on the retinal gabaergic system and electroretinographic activity in the golden hamster.

It has been established that neurosteroids can either inhibit or enhance GABA(A) receptor activity. Although GABA is the main inhibitory neurotransmitter in the mammalian retina, the effects of neurosteroids on retinal GABAergic activity have not been investigated. The aim of this work was to study the neurochemical and electroretinographic effects of neurosteroids in the golden hamster. On one hand, pregnenolone sulfate inhibited and allotetrahydrodeoxycorticosterone increased GABA-induced [36Cl]- uptake in neurosynaptosomes. On the other hand, in whole retinas, pregnenolone sulfate increased, whereas allotetrahydrodeoxycorticosterone decreased high potassium-induced [3H]GABA release. The effect of both neurosteroids on GABA release was Ca2+-dependent, as in its absence release was not altered. The intravitreal injection of pregnenolone sulfate or vigabatrin (an irreversible inhibitor of GABA degradation) significantly decreased scotopic b-wave amplitude, whereas the opposite effect was evident when bicuculline or allotetrahydrodeoxycorticosterone were injected. A protein with a molecular weight close to that of hamster adrenal cytochrome P450 side-chain cleavage (P450scc) was detected in the hamster retina. P450scc-like immunoreactivity was localized in the inner nuclear and the ganglion cell layers. These results indicate that neurosteroids significantly modulate retinal GABAergic neurotransmission and electroretinographic activity. In addition, the selective localization of P450scc suggests that neurosteroid biosynthesis might occur only in some layers of 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 alpha-(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.

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day.

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa.

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.

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 V(max) of retinal NOS without changes in the K(m). On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L-arginine influx. A decrease in the V(max) of L-arginine uptake was observed in the presence of melatonin, whereas the K(m) 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 adenosine on melatonin and morepinephrine release in rat pineal explants

Con el objeto de estudiar el efecto de la adenosina sobre la producción de melatonina, se incubaron durante 6 h explantos de pineales de rata con adenosina o 2-cloroadenosina 10**-4 M en presencia y ausencia de noradrenalina (NE) (5 x 10**-5 M). El contenido de melatonina en las glándulas pineales y en los medios se determinó por RIA. Tanto adenosina como 2-cloroadenosina estimularon la producción de melatonina entre 3 y 4 veces e incrementaron aquella inducida por NE en un 30-40%. El agregado de adenosina deaminasa previo a NE redujo el incremento en la liberación de melatonina inducido por NE en un 40-46%. El tratamiento con 2-cloroadenosina contrarrestó dicho efecto de la enzima. La adenosina y su agonista Al, ciclohexiladenosina (CHA), disminuyeron en un 20-22% la liberación del neurotransmisor-3H inducida por estímulo despolarizante de K+ en pineales de rata preincubadas con 3H-NE. Estos resultados sugieren que la adenosina modula mecanismos pineales pre- y post-sinápticos

Effect of adenosine on melatonin and morepinephrine release in rat pineal explants

Con el objeto de estudiar el efecto de la adenosina sobre la producción de melatonina, se incubaron durante 6 h explantos de pineales de rata con adenosina o 2-cloroadenosina 10**-4 M en presencia y ausencia de noradrenalina (NE) (5 x 10**-5 M). El contenido de melatonina en las glándulas pineales y en los medios se determinó por RIA. Tanto adenosina como 2-cloroadenosina estimularon la producción de melatonina entre 3 y 4 veces e incrementaron aquella inducida por NE en un 30-40%. El agregado de adenosina deaminasa previo a NE redujo el incremento en la liberación de melatonina inducido por NE en un 40-46%. El tratamiento con 2-cloroadenosina contrarrestó dicho efecto de la enzima. La adenosina y su agonista Al, ciclohexiladenosina (CHA), disminuyeron en un 20-22% la liberación del neurotransmisor-3H inducida por estímulo despolarizante de K+ en pineales de rata preincubadas con 3H-NE. Estos resultados sugieren que la adenosina modula mecanismos pineales pre- y post-sinápticos (AU)