Phosphoinositide Metabolism in the Retina: Localization to Horizontal Cells and Regulation by Light and Divalent Cations

Isolated retinas from Xenopus laevis incorporated greater amounts of [3H]inositol and 32Pi into phosphoinositides when incubated in light than did control retinas incubated in the dark. Inositol was primarily incorporated into phosphatidylinositol (83-86%), while phosphate labeled the polyphosphoinositides (72-79%). The incorporation of radioactive glycerol, serine, choline, or ethanolamine into retinal lipids was unaffected by light. Following incubation with [3H]inositol, the cell type involved in the light response was identified by light and electron microscope autoradiography to be the horizontal cell. These results are consistent with a classic phosphatidylinositol effect in the retina. An interesting feature of this response is that the stimulus (light) is received in the photoreceptor cell and the effect is manifest in the horizontal cell.     

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)     

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.     

The Influence of Specific Phospholipids on the Interaction of Hexokinase with the Outer Mitochondrial Membrane

Repeated washing of a brain mitochondrial fraction results in a progressive decrease in the proportion of mitochondrially bound hexokinase that can be solubilized during a subsequent incubation with glucose-6-phosphate (glucose-6-P). Phospholipids removed during the washing procedure can be added back to washed mitochondria, resulting in enhancement of the solubilization by glucose-6-P. Column and thin-layer chromatographic methods have been used to isolate and identify active phospholipids. Additional studies were performed with purified lipids obtained commercially. Both lysophospholipids and acidic phospholipids were active in enhancing solubilization of hexokinase by glucose-6-P. Phospho-inositides, particularly diphosphoinositide, were quite effective, raising the possibility that the actively metabolized phosphoinositides may be involved in regulation of hexokinase binding in vivo.     

Polyamine Localization and Biosynthesis in Chemically Fractionated Rat Retina

For elucidation of polyamine localization and biosynthesis in various cell types of rat retina, the putrescine, spermidine, and spermine contents as well as the ornithine decarboxylase and S-adenosylmethionine decarboxylase activities have been measured in retinal cell layers obtained by the selective cytotoxic action of iodoacetate on photoreceptor cells and of monosodium glutamate on higher-order retinal neurons. A notable depletion only in spermine content was associated with loss of the visual cell layer. Total ornithine decarboxylase and S-adenosylmethionine decarboxylase activities per retina were significantly lower in all chemically fractionated tissue, but loss of the photoreceptor layer produced the greatest decrease. The specific activities of these enzymes did not show marked changes in rat retinas deprived of inner neurons. The data support the suggestions that polyamine synthesis, storage, and catabolism have different distributions in the retinal layers and that the spermine levels and the high value of the spermine/spermidine molar ratio might depend essentially on the proportion of rods to cones.     

In Vitro Metabolism of Mevalonic Acid in the Bovine Retina

Bovine retinas were incubated with 3RS-[5-3H]-mevalonic acid under conditions similar to those previously shown to support opsin biosynthesis in vitro. TLC of the total lipids indicated the formation of numerous radiolabeled components, including sterols, hydrocarbons, and "fatty acid-like material." The nonsaponifiable lipids were analyzed by TLC, GLC, and chromatography on columns of silicic acid-Super Cel, silica gel G-Super Cel-silver nitrate, and alumina-Super Cel-silver nitrate. The major nonsaponifiable components had the chromatographic properties of squalene and "methylated sterols" (i.e., C30, C29, and C28 monohydroxy sterols). Cholesterol represented no more than 1% of the total radioactivity in the nonsaponifiable lipid fraction. The "fatty acid-like material" was derivatized with diazomethane, and the resulting methyl esters were analyzed by GLC before and after catalytic hydrogenation. The radioactivity did not correspond to the normal fatty acids endogenous to the retina, but rather had the chromatographic properties of C15 and C20 isoprenoid acids. These results obtained with intact retinas are consistent with our previous observations concerning mevalonic acid metabolism in cell-free homogenates of bovine retinas.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

猫视网膜年龄相关的形态学变化

取老年猫(12龄,3～3．5kg)和青年猫(1～3龄,2～2．5kg)各4只的视网膜,经4％多聚甲醛处理后,用H．E．染色以显示视网膜结构,Nissl染色显示神经节细胞,免疫组织化学ABC法染色以显示星形胶质细胞特征性标志物胶质纤维酸性蛋白(GFAP)的阳性反应细胞的分布。显微镜下观察测量视网膜厚度,计数神经节细胞、GFAP免疫反应阳性细胞数。与青年猫比较,老年猫视网膜总厚度以及外核层、外网状层、内核层和内网状层厚度均显著减小;神经节细胞层的细胞密度显著下降;GFAP免疫反应阳性细胞显著增加,GFAP阳性细胞阳性反应强,胞体明显膨胀,突起稠密粗大。推测在衰老过程中视网膜细胞有神经元丢失现象,可能是造成视觉功能衰退的重要原因之一;视网膜星形胶质细胞的功能增强可能会延缓衰老。     

猫外层视网膜和脉络膜的年龄相关变化

取老年猫(12龄,2.5~3 kg)和青年猫(1~3龄,2~2.5 kg)各4只的视网膜,经4%多聚甲醛处理后用H.E染色以显示视网膜和脉络膜的结构。光学显微镜下观察感光细胞层、玻璃膜(Bruch’s membrane)结构的变化,计数色素上皮层(retinal pigment epithelium,RPE)细胞数、脉络膜毛细血管数,测量玻璃膜、脉络膜厚度,脉络膜毛细血管之间的距离。结果显示,与青年猫比较,老年猫视网膜感光细胞层结构杂乱;色素上皮细胞数显著下降;玻璃膜厚度无显著变化,出现较多碎片;脉络膜厚度明显变薄,脉络膜毛细血管数显著减小,脉络膜毛细血管之间的距离显著增大。推测老年猫脉络膜的退化可能是导致玻璃膜、色素上皮层的退化,进而导致感光细胞的功能衰退的重要原因。     

Noradrenaline, a Transmitter Candidate in the Retina

The occurrence, metabolism, uptake, and release of noradrenaline were studied in the bovine retina with the following results. (1) Small amounts of noradrenaline occur in the retina and are restricted to the area corresponding to the inner nuclear and plexiform layers. (2) Retinal tissue can metabolise [¹⁴C]dopamine to form quantities of [¹⁴C]noradrenaline. (3) [¹⁴C]Noradrenaline can also be partly metabolised to form [¹⁴C]normetanephrine. (4) When bovine retinas were incubated with 5 × 10^-7 M-[³H]noradrenaline for 20 min and processed for autoradiography, most of the label was associated with apparent nerve processes in the inner plexiform layer. Biochemical analysis showed that more than 95% of the label was noradrenaline. (5) [¹⁴C]Noradrenaline uptake saturated with increasing noradrenaline concentrations and followed Michaelis-Menten kinetics. This uptake could be accounted for by two processes, a high-affinity system with a K_m1 of 5 × 10^-8 M and a V_max1 of 0.193 pmol/mg/10 min and a low-affinity system with a K_m2 of 6.3 × 10^-5 M and a V_max2 of 0.109 nmol/mg/10 min. (6) Noradrenaline uptake was strongly dependent on temperature and sodium, less dependent on potassium, and independent of calcium and magnesium ions. (7) Centrally acting drugs, such as desipramine, imipramine, desmethylimipramine, and amitriptyline, inhibited noradrenaline uptake by more than 55% at the concentration of 5 × 10^-5 M. These drugs at the same concentration diminished dopamine uptake by less than 30%. (8) Noradrenaline uptake is stereospecific, the (-) isomer having a greater affinity for the uptake sites than the (+) isomer. (9) [¹⁴C]Noradrenaline in the retina could be released by increasing the external potassium concentration. This release was calcium-dependent and was blocked by 20 mM-cobalt chloride. The present studies could be interpreted as supporting the idea that noradrenaline acts as a transmitter in the retina.     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

Incorporation of [1-14C]Palmitic Acid into Neutral Lipids and Phospholipids of Rat Cerebral Cortex In Vitro

Abstract: Incorporation of [1-¹⁴C]palmitic acid into neutral lipids and phospholipids of rat cerebral cortex was examined in vitro in normal Krebs-Ringer bicarbonate buffer containing 3% (wthol) albumin and 0.75 mM palmitic acid. Under standard assay conditions, radioactivity in the triacylglycerol fraction increased rapidly during the first 30 min, and then decreased after 60 min, with corresponding increase in radioactivity in phosphatidyl choline, phosphatidyl ethanolamine, and a fraction of phosphatidyl inositol plus phosphatidyl serine. Diacylglycerol was shown to be an intermediate metabolite. Radioactivity increased in triacylglycerol, and decreased in phosphatidyl choline and phosphatidyl ethanolamine throughout incubation under NZ gas. In the fraction of phosphatidyl inositol plus phosphatidyl serine, radioactivity decreased after 30 min during incubation under N, gas. A possible acylation-deacylation cycle, in which triacylglycerol could be a source of free fatty acids for phospholipids, is discussed.     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

缺氮介导的莱茵衣藻油脂合成过程的内参及标志物蛋白质的筛选鉴定

微藻被认为是最有潜力的生物能源原料之一,了解油脂合成机理、提升油脂合成的效率是重要的生物学问题.莱茵衣藻缺氮胁迫是油脂合成机理研究的模式系统,组学研究已经积累了大量的数据,但针对莱茵衣藻缺氮介导的油脂合成过程的内参及标志物蛋白质还鲜有报道.本研究对莱茵衣藻进行了对照和缺氮胁迫培养,比较了多个时间点(0、1、2、4和6d)在2种处理条件下的培养物表型、细胞密度、油脂含量以及总蛋白质含量的变化等特征.结果显示:莱茵衣藻细胞在受到缺氮胁迫后表现为培养物颜色由绿变黄;A750和细胞计数结果显示细胞生长趋于停滞;尼罗红染色定量实验鉴定到油脂含量的显著升高;考马斯亮蓝染色实验检测到总蛋白质含量降低.以20个莱茵衣藻蛋白质为候选,利用蛋白质印迹技术(Western blot,WB)检测了其在不同处理和不同时间点的表达特征变化,通过计算总蛋白质和候选蛋白质含量的皮尔森相关系数(Pearson’s correlation coefficient, PCC)筛选了莱茵衣藻缺氮胁迫的内参蛋白质,发现Histone H3、 RBCL(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit)和BCR1(biotin carboxylase,ACCase complex 1)在对照和缺氮胁迫条件下均与总蛋白质含量变化呈现极显著或显著正相关,所以被选作内参蛋白质.进而通过比较候选蛋白质的平均相对倍率变化(average relative fold change, ARF),鉴定了莱茵衣藻缺氮胁迫的标志物蛋白质,发现ATPs-β(ATP synthase CF1beta subunit)、 GAP2(glyceraldehyde 3-phosphate dehydratase 2)和RMT1(rubisco large subunit N-methyltransferase 1)蛋白质的ARF值分别为180.59、52.90和12.48,明显高出其他蛋白质,由此把它们选做缺氮胁迫的标志物蛋白质.接下来,对缺氮胁迫早期(0、2、4、8、12、18、24和48 h)的样品进行蛋白质印迹法分析,发现可检测的ATPs-β、GAP2和RMT1的缺氮诱导条带出现的时间分别是8、18和12 h.综上可以认为,在所有候选蛋白质中,ATPs-β是出现最早且变化幅度最大的缺氮处理标志物蛋白质.本研究鉴定的内参和标志物蛋白质对了解缺氮应答及油脂合成机理会有所帮助,所积累的蛋白质表达信息可供研究同行参考.     

Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments – Toward the Self-Reproduction of Minimal Cells

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes – like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Changes in the Redox State in the Retina and Brain During the Onset of Diabetes in Rats

Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio.Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD⁺/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Biosynthesis of the bisbenzylisoquinoline alkaloid,tetrandrine

The incorporation of (±)-coclaurine, (±)-norcoclaurine, (±)-N-methylcoclaurine and didehydro-N-methyleoclaurinium iodide into tetrandrine in Cocculus laurifolius has been studied and specific utilization of (±)-N-ethylcoclaurine demonstrated. The evidence indicates that tetrandrine is formed in the plants by oxidative dimerization of N-methylcoclaurine. Double labelling experiment with (±)-N- [¹⁴C]-methyl- [1-³H]-coclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into tetrandrine. Parallel feedings of (+)-(S)- and (?)-(R)-N-methylcoclaurines showed that the stereospecificity is maintained in the biosynthesis of tetrandrine from the 1-benzylisoquinoline precursor.     

Lipids in digestive gland of Littorina saxatilis rudis (Maton) and in daughter sporocysts of Microphallus similis (Jäg. 1900)

Parasitism by sporocysts was associated with a slight decrease in triglycerides and fatty acids in host digestive gland cells suggesting that the parasite digests, absorbs, and metabolises fats of host origin. An increase in phospholipids and a marked increase in the incorporation of acetate-1-¹⁴C in parasitised digestive glands may be the result of attempted cell regeneration. The parasite had less monoglycerides, triglycerides, and fatty acids but took up more palmitate-1-¹⁴C than the host. Sterols and sterol esters are more concentrated and more readily synthesised in the parasite than in the host. The relatively high level of phospholipids in the former may be related to number of cercariae within the sporocysts.     

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.