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.

The photic regulation of heme oxygenase (HO) activity was examined in the golden hamster retina. This enzymatic activity was significantly higher at midday than at midnight. When the hamsters were placed under constant darkness for 48 h and killed at subjective day or at subjective night, the differences in HO activity disappeared. Western blot analysis showed no differences in HO levels among these time points. Dopamine significantly increased this activity in retinas excised at noon or at midnight, with a higher sensitivity at night. The effect of dopamine was reversed by SCH 23390 but not by spiperone and clozapine and it was not reproduced by quinpirole. In vitro, the increase in HO activity found in retinas incubated under light for 1 h was significantly reduced by SCH 23390. Two cAMP analogs increased HO activity and their effect, as well as the effect of dopamine was blocked by H-89, a protein kinase A (PKA) inhibitor. Tin protoporphyrin IX, an HO inhibitor, significantly decreased cGMP accumulation with maximal effects during the day. Low concentrations of bilirubin decreased retinal thiobarbituric acid substances levels (an index of lipid peroxidation) in basal conditions and after exposing retinal cells to H 2 O 2 . These results suggest that hamster retinal HO activity is regulated by the photic stimulus, probably through a dopamine/cAMP/PKA dependent pathway.

One of the limiting steps in the regulation of nitric oxide (NO) synthesis is the availability of its precursor, L-arginine, which depends on the presence of a speci®c uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospeci®c, saturable, and monophasic, with an apparent K m of 56.1 2.0 lM and a maximum velocity of 36.0 2.8 pmol/mg prot/min. The basic amino acids L-lysine and L-ornithine but not D-arginine or the nitric oxide synthase inhibitors, N x -nitro-L-arginine methyl ester and N x -nitro-Larginine impaired L-arginine in¯ux. Preincubation with L-lysine for 1 h prior to the transport assay signi®cantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of V max at midnight than at midday. When the hamsters were placed under constant darkness or constant light for 48 h and killed at equivalent time points, representing subjective day and subjective night, the differences in L-arginine in¯ux disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were signi®-cantly higher at midnight than at midday. L-Arginine signi®-cantly increased cGMP accumulation in a time-dependent manner, with maximal effects during the night. Based on these results, it might be presumed that hamster retinal L-arginine uptake is regulated by the photic stimulus.