Certain aspects of acetylcholine metabolism in teleost retina

Acetylcholine (ACh) synthesis and release from isolated superfused retina of the teleostEugerres plumieri has been studied under different physiological conditions. The retinas were superfused with Krebs-Ringer solutions containing [¹⁴C]choline and the extracellular space of 32% was determined by [³H]inulin. The retina accumulates choline (Ch) from the superfusion medium and this process is mediated by a high affinity transport system with aK _m of 1.82 M. The incorporated Ch is mainly utilized for the synthesis of ACh. The ACh content of the light-adapted retina is not significantly different from that of a dark-adapted one. However, the release of [¹⁴C]ACh from the light-adapted retina was 52% higher as compared to the release from the dark-adapted retina. Flicker stimulation induced a larger increase in ACh release, than from either light or dark adapted retina, proportional to flicker frequency. The results suggest that changes in ACh utilization were related to the function of cellular units responsible for light changes transduction rather than light detection.In partial fulfillment of a MSc degree.     

Adrenergic neurons in teleost retina

Summary The adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.The research reported in this document has been sponsored by USPHS Grant No. 06092 and by a Research Professorship from Research to Prevent Blindness, Inc. to A.M.L. and by the Swedish Medical Research Council (B69-14X-712-04C and B68-14X-2321-01).     

Age-related changes of glycogen metabolism in human fetal heart

The changes in the activities of three important glycogen metabolising enzymes, viz. glycogen synthetase, glycogen phosphorylase and alpha-D-glucosidase, along with glycogen content have been measured in adult human heart and human fetal heart collected at 13-36 weeks of gestation. At an early period, particularly 13-16 weeks of gestational age, the activity of glycogen synthetase and glycogen content were found to be maximum. However the activity of glycogen phosphorylase remained constant throughout the gestation and that of alpha-D-glucosidase showed a peak at 25-28 weeks of gestation, thereby indicating that fetal heart tissue has the capacity to utilise glycogen for energy.     

Ultrastructural localization of endogenous calcium in the teleost retina

Summary The ultrastructural localization of endogenous calcium in the retina of adult cichlid fishOreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electronspectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.     

Microtubules in cone myoid elongation in the teleost retina

The myoids of retinal cone cells of the blue-striped grunt (Haemulon sciurus) undergo significant elongation during dark adaptation of the retina. Longitudinally oriented microtubules are present in myoids both before and after elongation. Injection of colchicine into the vitreous of the eye in vivo disrupts the microtubules in the myoids and prevents dark-adaptive myoid elongation. Counts of microtubules in transverse sections along the lengths of elongating myoids show that there is a uniform decrease in the number of microtubules at any one point along the myoid as the myoid elongates. The magnitude of the decrease is proportional to the extent of the elogation. The product of the mean myoid microtubule number (determined from counts at progressive intervals along the myoid) and the myoid length remains essentially constant during myoid elongation, indicating that the total quantity of microtubules in the myoid does not increase with elogation. Serial section tracings of the microtubules along the myoids suggest that individual microtubules do not extend the length of the myoid and that the myoid microtubular apparatus consists of bundles of overlapping shorter microtubules. We propose that elongation of the myoid is accompanied by sliding redistribution of microtubules along the length of the myoid, and that the sliding may be generated by interaction between microtubules in regions where they closely overlap in bundles. We find no evidence for the involvement of discrete, electron-dense microtubular organizing centers in myoid elogation.     

Electrophysiological and metabolic responses of the isolated teleost retina to changes in po2, and temperature

• 1.1. Comparisons of electrophysiological responses (ERG) were made between two different in vitro preparations of teleost retina.
• 2.2. The ERG was independent of temperature over the normal environmental range.
• 3.3. The Q₁₀ demonstrated temperature independence between 5 and 20°C.
• 4.4. The electrical response of the isolated retina was found to be independent of partial pressures of oxygen at levels above 250 mm Hg.

     

Adrenaline stimulation of phospholipid metabolism in adipocyte ghosts

The incorporation of long-chain fatty acids into phospholipids has been detected in adipocyte ghosts that were incubated with [1-14 C] stearic, [1-14 C] linoleic or [1-14 C] arachidonic acid. Adrenaline and adenosine activated this incorporation within 15 s of exposure of the ghosts to the hormones and the response was dose dependent. Maximum incorporation of labelled linoleic acid occurred at 10(-5) M adrenaline and 10(-7) M adenosine. The alpha-agonist phenylephrine and the beta-agonist isoproterenol were also shown to stimulate the incorporation of fatty acid in a dose dependent manner. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were each labelled preferentially with linoleic or arachidonic acid. p-Bromophenacylbromide, quinacrine and centrophenoxine inhibited the adrenaline-stimulated incorporation of fatty acids into ghost membrane phospholipids, and p-bromophenacylbromide also reduced the activation of adenylate cyclase by adrenaline. NaF, an activator of adenylate cyclase, like adrenaline, stimulated the incorporation of linoleic acid into ghost membrane phospholipids.     

Pentylenetetrazole induced changes in cerebellar energy metabolism

Pentylenetetrazole was administered to Swiss-Albino mice, producing clonic-tonic seizures. Other groups were pretreated with one of the three anticonvulsants: phenytoin, clonazepam, or sodium valproate. Mice were sacrificed during the preseizure (1 minute) stage and at the onset of clonic-tonic seizures (2 minutes). Glucose, glycogen, ATP, and phosphocreatine were measured in layers of the parietal cortex and cerebellar vermis. Cortical metabolites were unchanged, or increased slightly, suggesting decreased utilization. In both cerebellar layers, glucose and glycogen were significantly decreased, and phosphocreatine was decreased in the molecular layer. These results indicate a regionally selective effect for pentylenetetrazole on cerebral energy metabolites. Pretreatment with anticonvulsants reduced the severity of the seizure, and eliminated the effect of pentylenetetrazole on glucose and glycogen.     

Control of glycogen content in retina: allosteric regulation of glycogen synthase

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.     

Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.     

Structural changes induced in glycogen phosphorylase b by the binding of glucose and caffeine

The allosteric inhibitors glucose and caffeine cause significant structural alterations in glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1). Both cause a masking of two sulfhydryl groups and a reduction of binding affinity for AMP. Caffeine produces an alteration in the microenvironment of the binding site for 1-anilin-naphthalene-8-sulfonate, resulting in a decrease of quantum yield of fluorescence and a change in spectral distribution. The binding of glucose is exothermic with an enthalpy of binding of -6.0 kcal/mol. Glucose causes a change in the molecular ellipticity in the pyridoxal-5'-phosphate region. The implications of these results are discussed.     

Copper influenced changes of lipid metabolism in the tissues of freshwater teleost Labeo rohita (Hamilton).

Modulations in the lipid metabolism in the gill, liver, muscle and brain of freshwater teleost Labeo rohita exposed to 1, 2 and 3 days to lethal (1.2 mg/l) and 1, 15 and 30 days to sublethal (0.24 mg/l) concentrations of copper were studied. The total lipids decreased and there was an increase in the free fatty acids, glycerol and lipase activity in the organs studied at lethal concentration of copper. The degree in these shifts increased over time of exposure (1 less than 2 less than 3 days). In sublethal concentration, the levels of total lipids, free fatty acids, glycerol and lipase activity increased in all the four organs and the shifts followed two different trends during the exposure periods, 1 less than 15 less than 30 days in total lipids and 1 greater than 15 greater than 30 days in the other parameters. Among the organs, in both concentration media the changes in the lipid metabolism were in the order liver greater than gill greater than muscle greater than brain.     

Changes in enzyme activity of glycogen and hexose metabolism during oocyte maturation in a teleost,Misgurnus fossilis L.

Summary The oocyte at the end of oogenesis, mature egg and developing embryo of the loachMisgurnus fossilis L.) are characterized by indentical enzyme profiles of the Embden-Meyerhof chain, pentose phosphate cycle and key gluconeogenic enzymes. However, the carbohydrate metabolism in the oocyte differs substantially from that in the embryo.Oocyte maturation is followed by a complete loss of hexokinase (EC2.7.1.1),2-fold decrease of glycogen synthetase (EC 2.4.1.11) and 10-fold increase of glycogen phosphorylase (EC2.4.1.1) activity. This process is correlated with a gradual decrease of the ATP/(ADP+AMP) ratio from 41 to 21 and increase of the Fructose-6-Phosphate/Fructose-1,6-Diphosphate ratio from 0.27 to 2.0. Thus, oocyte maturation involves a number of changes in control mechanisms resulting in cessation of glycogen accumulation and a transition of carbohydrate metabolism from gluconeogenesis to glycogenolysis.

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Zusammenfassung Die Oozyte und das reife Ei vonMisgurnus fossilis L. besitzen die gleiche Enzymzusammensetzung der Embden-Meyerhof Wege, unterscheiden sich aber wesentlich in den Enzymen des Metabolismus des Glykogens und der Hexosemonophosphate. Beim Reifen der Oozyte erfolgt eine 10fache Zunahme der Aktivität der Phosphorylase (EC 2.4.1.1.), völliger Verlust der Hexokinase (EC 2.7.1.1.) und eine wesentliche Abnahme der Glycogensynthetase (EC 2.4.1.11).Das Verhältnis ATP/(ADP+AMP)geht von 4l auf 2-1 zurück, dasjenige von Fructose-6-Phosphat/Fructose-1,6-Diphosphat nimmt von 0,27 auf 2,0 zu.Änderungen der regulatorischen Mechanismen beim Reifen der Oozyte setzen die Geschwindigkeit der Glykogensynthese und Glukoneogenese scharf herab, unterbinden die Ausnutzung der freien Glukose bei der Glykolyse und sichert eine allmähliche Zunahme der Glykogenolyse während der frühen Entwicklung des Embryos.

     

Endogenous control of spinule formation in horizontal cells of the teleost retina

Summary The processes of horizontal cells invaginating teleost cone pedicles are studded with small finger-like projections which are present only in the light-adapted state. The aim of this study was to investigate whether the formation and degradation of these so-called spinules, which are thought to function as feed-back synapses onto the cones, is endogenously controlled.Three types of experiment were carried out involving fish entrained to a 12 h light/dark cycle: 1) The number of spinules was determined in goldfish at various times during exposure to either constant darkness (36 h) or constant light (57 h). 2) The time course of spinule formation and degradation in goldfish was investigated following exposure to light or darkness at various times during the light/dark cycle. 3) The time course of flash-induced spinule formation in tench following dark adaptation at noon was compared to that following dark adaptation at midnight.The results of these experiments show that spinule formation and degradation are partially under endogenous control but that they need light for full expression. This endogenous rhythm is reflected in the time courses of spinule formation and breakdown during different phases of the light/dark cycle.