Photic regulation of L-arginine uptake in the golden hamster retina

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 specific uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospecific, saturable, and monophasic, with an apparent of 56.1 +/- 2.0 microM 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(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine impaired L-arginine influx. Preincubation with L-lysine for 1 h prior to the transport assay significantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of Vmax 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 influx disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were significantly higher at midnight than at midday. L-Arginine significantly 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.     

Endophthalmitis by Aspergillus fumigatus after retina detachment

A fungal infection in the right eye after retina detachment on an immunocompetent patient is reported. After surgery, she developed an infection that was empirically treated with antibiotics and corticoids. Later the patient developed another retina and choroid detachment. The infection evolved to endophthalmitis and a sample was sent to the microbiology laboratory, where Aspergillus fumigatus was isolated. In spite of treatment with intravenous and intravitreous amphotericin B, the eye was eventually removed by enucleation.     

Sodium-independent synaptosomal uptake of leucine

The uptake of leucine by rat brain synaptosomes was investigated in a Na+-free medium. It was found that leucine was transported into synaptosomes by 2 uptake processes which were both extremely sensitive to inhibition by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These 2 components were distinguishable from each other by kinetic analysis and by their relative sensitivity to inhibition by selected amino acids.     

Functional homogeneity of leucine pool in retina cells

Data on leucine metabolism in isolated rabbit retina are examined for evidence, for or against, a common intracellular pool of free leucine. Data include values for: concentrations, transport rates, degradative metabolism and protein incorporation of labelled leucine measured over a wide range of concentrations; protein incorporation of labelled threonine, measured simultaneously; and an indirect measurement of protein breakdown. The fall in labelled leucine incorporation into protein, when medium leucine was reduced below 100 microM, corresponded closely with the fall in intracellular specific activity predicted from rate of influx of labelled leucine from medium and rate of release of unlabelled leucine from protein breakdown. Protein incorporation of labelled leucine competed with decarboxylation and outward transport and reduced the free intracellular leucine in about the amounts predicted for a common pool. Implications for measurements using labelled amino acid are discussed.     

Changes in laser-irradiated retina in the first 24 h after irradiation.

Retinas of laser-irradiated mice were studied in the first 24 h after irradiation. Decrease in Na+ and K+ concentrations and in phagosome number and increase in pyknotic cells were observed several hours after irradiation.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

High-affinity taurine uptake in developing retina

A specific system for taurine transport is present at the early stages of development in both chick and rat retinas. The results obtained with taurine analogs indicate a high degree of specificity of taurine uptake. Two transport systems were detected for the adult rat retina: a high-affinity (K _m 21 M) and a low-affinity transport system (K _m 312 M). On the other hand, in the adult chick retina, only a low-affinity transport system (K _m 580 M) could be detected. Nevertheless, embryo chick retina accumulated [³H]taurine by two different kinetic mechanisms withK _m s of 242 M and 21 M for the low- and high-affinity processes, respectively. Taurine uptake systems were absolutely Na⁺ dependent. The sodium-dependence curve for taurine uptake was sigmoid. These mechanisms appear not to be mediated by a Na⁺ cotransport system. In spite of the differences observed in taurine uptake in both species, in each of them it closely parallels the changes brought about by the morphological and functional maturation of the retina.     

Changes to the viscoelastic properties of brain tissue after traumatic axonal injury

While it has been shown that repetitive mild brain injuries can cause cumulative damage to the brain, changes to the mechanical properties of brain tissue at large deformations were also noted in the literature. The goal of this study was to show that the viscoelastic properties of brain tissue significantly change after traumatic axonal injury (TAI). An impact acceleration model was used to create TAI in the rat brainstem which was quantified with an immunohistochemistry technique at the ponto-medullary junction (PmJ) and pyramidal decussation (PDx). The viscoelastic properties at these two points with and without preconditioning were characterized using an indentation technique combined with finite element analysis and a comparison was made between injured and uninjured specimens, which revealed statistically significant reduction in the instantaneous elastic force at PDx where the brain tissue sustained a significantly higher level of injury. The result of this study can be used to characterize a damage function for the brain tissue undergoing large deformation.     

Ethanolamine inhibits choline uptake in the isolated hamster heart

The effect of exogenous ethanolamine on phosphatidylcholine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [Me-3H]choline in the presence of 0.05-0.5 mM ethanolamine. Incorporation of label into phosphatidylcholine was decreased 26-63% at 0.1-0.5 mM ethanolamine. Similar decreases in the labelling of the metabolites of the CDP-choline pathway were observed at these ethanolamine concentrations. The observed decrease in phosphatidylcholine labelling at 0.1-0.5 mM ethanolamine was attributed to an inhibition of labelled choline uptake by ethanolamine. The inhibitory role of ethanolamine to choline uptake was examined by comparison to hemicholinium-3. Both compounds inhibited choline uptake in a competitive manner. Intracellular choline, phosphocholine and CDP-choline concentrations were not altered under all experimental conditions. It can be concluded that exogenous ethanolamine has no immediate effect on the rate of phosphatidylcholine biosynthesis in the isolated hamster heart. The reduced labelling of phosphatidylcholine in the presence of ethanolamine is a direct result of the reduction of labelled choline taken up by the heart.     

Pharmacological properties of glycine uptake in the developing rat retina

A pharmacological characterization of glycine transport was performed in the rat retina at different postnatal ages. The uptake of 3H-glycine increased during the first 2 weeks of postnatal age, reaching maximum values at 12 days; then it decreased sharply to the adult values. We found a Na+ -dependent and high-affinity transport system with a Km of 100 microM. The Na+ Hill coefficient for glycine uptake was 1.76 +/- 0.07. Although glycine uptake was insensitive to staurosporine and phorbol ester, it was reduced 40-50% by sarcosine and ALX5407. Besides, amoxapine inhibited glycine uptake by 40 and 70% in adult and immature retina, respectively. These results suggest that the Glyt1 transporter was concentrated in the nerve terminals. In addition to the presence of Glyt1 in the retina, our results provided evidence of the occurrence of Glyt2 and/or another isoform of glycine transporter, which might have had a role in the retina development.     

Changes in the composition of the hamster zona pellucida after fertilization in vivo but not in vitro

Hamster zonae pellucidae were obtained from follicular oocytes, superovulated eggs, and eggs fertilized in vivo or in vitro. Zonae were labelled with N-succinimidyl-3(4-hydroxy,5-[125I]iodophenyl)propionate, and compared on single- and two-dimensional SDS-PAGE. Single-dimensional electrophoresis showed considerable differences between zona categories in the amount of label that they incorporated; follicular zonae incorporated the least label and zonae from eggs fertilized in vivo the most. On two-dimensional electrophoresis, polypeptides from 3 of the 4 zona categories migrated into 4 major groups: two of these groups each with Mr 150,000-250,000 were within the Mr range of ZP1, and two others, at Mr 90,000 and 55,000, appeared to be analogous to ZP2 and ZP3, respectively. The fourth zona category (zonae from eggs fertilized in vivo) showed a changed polypeptide profile as well as incorporating the most label; one of the polypeptides, Mr 150,000-250,000, was undetectable, but a train of Mr 70,000-90,000 polypeptides and a discrete polypeptide at Mr 20,000 were new. Since this changed profile did not occur in zonae from superovulated eggs, or in zonae from eggs fertilized in vitro, a synergism between oviductal factors and factors from the spermatozoon or egg, or both, towards the zona in vivo is indicated.     

Effect of GABA on melatonin content in golden hamster retina

The effect of GABA on melatonin content in vitro was studied in the golden hamster retina. GABA significantly increased melatonin levels in a dose-dependent manner, its effect being reversed by a GABA(A) receptor antagonist, bicuculline, but not by saclofen, a GABA(B) antagonist. Moreover, an equimolar concentration of muscimol, a GABA(A) receptor agonist, significantly increased retinal melatonin content, whereas baclofen, a GABA(B) receptor agonist, was ineffective. The darkness-induced increase in melatonin content in vitro was inhibited by bicuculline, whereas saclofen was ineffective. Retinal GABA turnover rate was significantly higher at midnight than at midday. GABA significantly decreased cyclic AMP and increased cyclic GMP accumulation in the golden hamster retina. The effect of GABA on both nucleotide levels was reversed by bicuculline, but baclofen had no effect. Cyclic GMP analogues (i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate) significantly increased retinal melatonin content in vitro. Taken together, these results support the hypothesis that GABA may be important for the "dark message" in the hamster retina.     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

Peroxidase uptake by photoreceptor terminals of the skate retina

The photoreceptors of dark-adapted skate retinas bathed in a Ringer solution containing horseradish peroxidase (HRP) incorporate the tracer into membrane-bound compartments within the synaptic terminal of the cell; after 1 or 2 h of incubation, approx. 10-38% of the synaptic vesicles were labeled. The receptors appeared to be functioning normally throughout the incubation period, since electrical potentials of normal amplitude could be elicited in response to dimphotic stimuli. However, it was possible to block the uptake of peroxidase by a regimen of light adaptation that effectively suppressed light-induced activity in the electroretinogram. If, during incubation with peroxidase, retinas were exposed at 10-min intervals to an intense 1-ms flash from a xenon discharge tube, the receptor terminals were almost completely devoid of peroxidase; fewer than 2% of the vesicles were labeled. The suppression of HRP uptake could also be achieved in dark-adapted retinas by adding magnesium to the bathing solution, suggesting that calcium is necessary for transmitter release from vesicles in the receptor terminals. These findings are consistent with the view that vertebrate photoreceptors discharge a neurotransmitter in darkness, and that light decreases the release of this substance. It seems likely that the incorporation of peroxidase into vesicles of physiologically active receptor terminals reflects a mechanism for the retrieval of vesicle membrane after exocytosis.     

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.     

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.