牛蛙视网膜诱导型一氧化氮合酶免疫组化定位

用免疫组织化学方法研究了诱导型一氧化氮酶（iNOS)在牛蛙视网膜中的表达。结果显示,在正常状态视网膜中,无长突细胞呈弱阳性反应;节细胞层、双极细胞,水平细胞和光感受器内段呈阴性反应,在暗适应状态下,神经节细胞,内核层的无长突细胞呈强阳性反应;一些双极细胞,水平细胞和光感受器内段呈弱阳性反应,提示NO主要在暗适应状态下参与视网膜的信息传递过程。     

Labeling of Retina and Optic Tectum Phospholipids in Chickens Exposed to Light or Dark

The labeling of retina ganglion cell and optic tectum phospholipids was determined in chickens given an intraocular injection of 32P and then either exposed to light or maintained in the dark. Significantly higher labeling was found in the optic tectum phospholipids of light-exposed compared with dark-maintained animals after 3-24 h of labeling. In the ganglion cells, the labeling of phospholipids increased in dark with respect to light at 15 and 30 min of labeling; from 60 min to 24 h, the labeling of phospholipids was significantly higher in light with respect to dark, even if the precursor pool showed a higher labeling in dark at all times studied. When labeling was allowed to proceed in the dark for 30 min and then half of the animals were exposed to light for 15 min, the labeling of ganglion cell phospholipids of light-exposed animals was significantly higher than those of animals kept in the dark. No individual phospholipid accounted for the differences observed in the labeling of the total phospholipid pool. These results are interpreted as an increase in the biosynthesis of phospholipids in the ganglion cell somas in light with respect to dark.     

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.     

Physiologic diversity and development of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate numerous nonvisual phenomena, including entrainment of the circadian clock to light-dark cycles, pupillary light responsiveness, and light-regulated hormone release. We have applied multielectrode array recording to characterize murine ipRGCs. We find that all ipRGC photosensitivity is melanopsin dependent. At least three populations of ipRGCs are present in the postnatal day 8 (P8) murine retina: slow onset, sensitive, fast off (type I); slow onset, insensitive, slow off (type II); and rapid onset, sensitive, very slow off (type III). Recordings from adult rd/rd retinas reveal cells comparable to postnatal types II and III. Recordings from early postnatal retinas demonstrate intrinsic light responses from P0. Early light responses are transient and insensitive but by P6 show increased photosensitivity and persistence. These results demonstrate that ipRGCs are the first light-sensitive cells in the retina and suggest previously unappreciated diversity in this cell population.     

Aromatic L-Amino Acid Decarboxylase Is Modulated by D1 Dopamine Receptors in Rat Retina

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina increases when animals are placed in a lighted environment from the dark. The increase of activity can be inhibited by administering the selective dopamine D1 receptor agonist SKF 38393, but not the selective D2 agonist quinpirole, or apomorphine. Conversely, in the dark, enzyme activity can be enhanced by administering the selective D1 antagonist SCH 23390 or haloperidol, but not the selective D2 antagonist (-)-sulpiride. Furthermore, in animals exposed to room light for 3 h, the D1 agonist SKF 38393 reduced retinal AAAD activity, and this effect was prevented by prior administration of SCH 23390. In contrast, quinpirole had little or no effect when administered to animals in the light. Kinetic analysis indicated that the apparent Vmax for the enzyme increases with little change in the apparent Km for the substrate 3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. We suggest that dopamine released in the dark tonically occupies D1 receptors and suppresses AAAD activity. When the room light is turned on, D1 receptors are vacated and selective D1 agonists can either prevent the rise of AAAD or reverse light-enhanced AAAD activity.     

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.
The phospholipid composition of rat retina did not differ significantly from that of calf retina and the different conditions of preparation and incubation did not modify the distributions.
The specific radioactivities of the different phospholipids of calf and rat retina, incubated in the presence of ³²P, distinguished in both species two groups of components characterized by the rate of labelling. Phosphatidic acid (PA) and inositol glycerophospholipids (PI) belonged to the first group and showed the highest uptake of labelled phosphorus; the second group, comprising choline glycerophospholipids (PC), serine glycerophospholipids (PS), sphingomyelin (SP), ethanolamine glycerophospholipids (PE) and cardiolipin (CL) showed low incorporation activities. Only SP was labelled differently in rat and calf retina. With the exception of PS, there was no evidence for the influence of light on the turnover of individual phospholipids. The finding that PS showed higher specific radioactivities when adaptation and incubation proceeded in the dark, seems to be of interest and needs further study.     

Optic nerve integrity is required for light to affect retina ganglion cell gangliosides

The labeling of retina ganglion cell and optic tectum gangliosides after an intraocular injection ofN-[³H]acetylmannosamine ([³H]ManNAc) is higher in chickens exposed to light than in those maintained in darkness (1,2). In the present work we studied whether the signal for the higher labeling of ganglion cells in light originates in the photoreceptor layer or comes from the nerve terminal. For this purpose the labeling of ganglion cell gangliosides was determined in light and dark in chickens with one optic nerve severed. The results showed that the effect of light occurred only in the eye normally connected to the optic tectum. In the eye with its optic nerve severed, no difference was observed between the labeling of gangliosides in animals in light and dark, having both groups the labeling values of the normal eyes exposed to light. The results indicate that the information that decreases labeling in darkness or accelerates it in light originates in the nerve terminal.Special Issue dedicated to Prof. Edwardo De Robertis.     

Effect of light on the labeling of optic tectum gangliosides after an intraocular injection of N-[3H]acetylmannosamine.

Axonally transported gangliosides from retina were more labeled in the optic tectum of chickens exposed to light compared to those maintained in the dark. No differences were observed between the labeling of retinal gangliosides from the two groups. These results indicate that light modifies either the labeling of ganglion cell gangliosides or their axonal transport.     

c-fos基因在内皮素-1促肺泡Ⅱ型细胞表面活性物质合成中的作用

应用反义技术探讨ｃ－ｆｏｓ基因ＥＴ－１调控肺泡Ⅱ型细胞（ＡＴⅡ）表面活性物质（ＰＳ）合成的胞内信号转导中的作用,结果显示：（１）内皮素－１（ＥＴ－１）可提高ＡＴⅡ细胞的＾３Ｈ－胆碱掺入。（２）蛋白激酶Ｃ（ＰＫＣ）激活剂ＰＭＡ可使ＡＴⅡ细胞的＾３Ｈ－胆碱掺入量增加,ＰＫＣ抑制剂Ｈ７可抑制ＥＴ－１的促ＰＳ合成效应。（３）ＥＴ－１和ＰＭＡ可显著提高Ｆｏｓ蛋白表达量,Ｈ７和ｃ－ｆｏｓ反义寡核苷酸（ＯＤＮ）     

Aromatic l-Amino Acid Decarboxylase Activity of the Rat Retina Is Modulated In Vivo by Environmental Light Maria Hadjiconstantinou

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina is low in animals placed in the dark. When the room lights are turned on, activity rises for almost 3 h and reaches values that are about twice the values found in the dark. A study of the kinetics of the enzyme revealed that the apparent Km values for L-3,4-dihydroxyphenylalanine and pyridoxal-5'-phosphate were unchanged in light- and dark-exposed animals, whereas the Vmax increased in the light. Treating the animals with cycloheximide before exposure to light prevented the increase of enzyme activity. Immunotitration with antibodies to AAAD suggested that more enzyme molecules are present in the light than in the dark. When the room lights are turned off AAAD activity drops rapidly at first and then more slowly, suggesting that at least two processes are responsible for the fall of enzyme activity. Exposure to short periods of dark followed by light results in a rapid increase of AAAD activity. Mixing homogenates from light- and dark-exposed rats results in activity values that are less than expected, suggesting the presence of an endogenous inhibitor(s). These studies demonstrate that AAAD activity is modulated in vivo.     

Effect of Light on the Metabolism of Lipids in the Rat Retina

The effect of light on the in vitro incorporation of a variety of radioactive precursors into glycerolipids was tested in isolated retinas of albino rats. There was an increase in the incorporation of [2-3H]myo-inositol, 32Pi, [2-3H]glycerol, and [methyl-3H]choline into retinal phospholipids in light compared to that in darkness. [2-3H]myo-Inositol was incorporated primarily into phosphatidylinositol. 32Pi was incorporated primarily into the phosphoinositides, although there were significant increases in the specific activities of all retinal phospholipids in light compared to those in darkness. Likewise, [2-3H]glycerol incorporation into all retinal phospholipids and diglycerides was greater in light than in the dark. There was no effect of light on the incorporation of [2-3H]ethanolamine into phosphatidylethanolamine or of [3-3H]serine into phosphatidylserine, although these phospholipids were labeled to a greater extent in light with [2-3H]glycerol. There was no effect of light on the incorporation of [3H]palmitic acid into diglycerides and phospholipids, with the exception of phosphatidylinositol. Light also had no effect on the uptake of [2-3H]glycerol, [2-3H]inositol, or [methyl-3H]choline into the retina. We conclude from these studies that light stimulates the phosphoinositide effect in the rat retina. Although some of the results are consistent with a stimulation of de novo synthesis of all lipid classes, our studies with [3H]palmitate, [2-3H]ethanolamine, and [3-3H]serine do not support this conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

光和暗对埃及蟾蜍(BUFO REGULARIS)发育中的视网膜的影响(英文)

本文研究了埃及蟾蜍(Bufo regularis Reuss)从早期幼虫到变态结束,光和暗对视网膜的影响。所观察到的对光和暗反应的变化限于色素上皮和光感受细胞。实验动物分四组:1)在亮处固定的对照(CL);2)在暗处固定的对照(CD);3)连续养在暗处的动物(DD);4)连续受光照的动物(LL)。在CD和DD组动物,黑色素颗粒在色素上皮细胞突起(PEP)中的分布限于光感受细胞顶部间之外周区(巩膜方位)。与此相反。在CL和LL组动物,大量黑色素颗粒则分散在视觉细胞外节段和椭球段间色素上皮细胞突起之向心端位置(玻璃体方位)。在这种动物,上皮细胞色素对光和暗反应发生的光机械运动出现在肢芽期以前。新变态小蟾蜍DD组眼球,与其他三组比较起来,色素上皮层相当厚并含有大的脂肪滴。在较晚期,只有DD组动物的一些杆细胞外节段呈现退化现象。其次,这一组中,由于连续缺少光照的结果,眼球之锥细胞数目有明显减少。四组动物的视网膜上也观察到锥细胞的细胞核位置略有不同。     

Regulation of ganglion cell production by Notch signaling during retinal development

Although progenitor cells in developing vertebrate retina are capable of producing all retinal cell types, they are competent to produce only certain cell types at a given time, and this competence changes as development progresses. We asked whether a change in progenitor cell competence is primarily responsible for ending production of a specific cell type, the retinal ganglion cell. Reducing Notch expression using an antisense oligonucleotide in vitro or in vivo increased ganglion cell genesis. The antisense treatment could reinitiate ganglion cell genesis after it had terminated in a region of the retina, but only for a brief period. The failure of the Notch antisense treatment to reinitiate ganglion cell production after this period was not due to the lack of receptor or ligand expression, as both Notch-1 and Delta-1 were still expressed. The failure of the Notch antisense treatment to reinitiate ganglion cell production is consistent with the suggestion that the intrinsic competence of progenitor cells changes as development progresses. Because reducing Notch signaling can reinitiate ganglion cell production for a brief period after ganglion cell production has normally ceased, it appears that ganglion cell production initially ends in a region of the retina because of cell-cell interactions and not because progenitor cells lose the competence to make ganglion cells. Notch signaling appears to temporarily prevent production of ganglion cells in a region, while some other signal must initiate a change in progenitor cell competence, thus permanently ending the possibility of further ganglion cell production.     

Modulation of Retinal Aromatic l-Amino Acid Decarboxylase via α2 Adrenoceptors

Aromatic L-amino acid decarboxylase (AAAD) activity of the rat retina increases when animals are placed in a lighted environment from the dark. The rise of activity can be inhibited by administering alpha 2 adrenoceptor agonists. In the dark, the enzyme activity can be made to increase by administering alpha 2 adrenoceptor antagonist drugs. Kinetic analysis indicates that the maximum velocity of the enzyme increases with little change of the Km for the substrate L-3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. The rise of activity in the light and in the dark after alpha 2 antagonists can be blocked by administering cycloheximide, suggesting that protein synthesis is needed for the response. We speculate that epinephrine released in the dark from a subpopulation of retinal amacrine cells onto alpha 2 receptors suppresses AAAD activity that is associated with dopaminergic amacrines.     

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

We have previously shown that platelet-activating factor (PAF), a naturally occurring lipid mediator of cell-to-cell communication, was produced by 3-day-old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)-insensitive cholinephosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT-insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT-insensitive cholinephosphotransferase and acetyltransferase activities were present in 8-day-old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT-insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT-insensitive cholinephosphotransferase, but not acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-dependent Structural Change of Chicken Retinal Cryptochrome4

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells. We further developed several monoclonal antibodies to the carboxyl-terminal extension of cCRY4 and localized cCRY4 protein with immunohistochemistry. Consistent with the results of in situ hybridization, cCRY4 immunoreactivity was found in visual pigment cells and cells located at the inner nuclear layer and the retinal ganglion cell layer. Among the antibodies, one termed C1-mAb had its epitope within the carboxyl-terminal 14-amino acid sequence (QLTRDDADDPMEMK) and associated with cCRY4 in the retinal soluble fraction more strongly in the dark than under blue light conditions. Immunoprecipitation experiments under various light conditions indicated that cCRY4 from the immunocomplex formed in the dark dissociated from C1-mAb during blue light illumination as weak as 25 μW/cm² and that the release occurred with not only blue but also near UV light. These results suggest that cCRY4 reversibly changes its structure within the carboxyl-terminal region in a light-dependent manner and operates as a photoreceptor or magnetoreceptor with short wavelength sensitivity in the retina.     

Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light-dark Cycle

Nitrogenase of the non-heterocystous nitrogen-fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe-protein, Component I) and dinitrogenase reductase (Fe-protein, Component II). O. limosa was grown diazotrophically under an alternating light-dark cycle (16–8h light-dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light-dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm^?2 thin section were detected. Nitrate-grown cells did not contain nitrogenase label. Western blot analysis of the Fe-protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti-MoFe-protein antiserum revealed a single band of 56 kDa which was present throughout the light-dark cycle. Nitrate-grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light-dark cycle but that the Fe-protein is modified (inactive form) during the light period when nitrogenase activity is absent.     

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.     

Differential expression of c-fos mRNA in the rat neocortex by in situ hybridization

The c-fos mRNA expression pattern in rat neocortex, was determined in the rat kept in a 12:12 light/dark cycle, in constant dark, or in constant light by in situ hybridization. At the beginning of the light period, c-fos mRNA was induced both in the neocortex and suprachiasmatic nucleus (SCN). Transiently increased c-fos mRNA expression was detected from 0830 to 0900 and soon declined to basal levels. Immediately prior to the beginning of the dark period, c-fos mRNA expression also increased and remained elevated in the neocortex following the dark period. In the constant dark group, c-fos mRNA expression showed no transient elevation at the beginning of the light period. On the other hand, c-fos mRNA expression in the constant light group increased during their subjective dark period as well as normal light/dark cycle. These results demonstrate a circadian pattern of c-fos mRNA expression in the neocortex which is similar to that observed previously in the inner and outer nuclear layers of the retina.