Biomedical subjects

M Tsacopoulos

Publications and source records attributed to M Tsacopoulos.

At least 37 records · Page 2Linked to original sources

PubMed1993

Microinjection of L-lactate in the preretinal vitreous induces segmental vasodilation in the inner retina of miniature pigs.

PURPOSE: The authors investigated the hypothesis that the retinal vasomotor effect of acute hypoxia is mediated by lactate. METHODS: Retinal vasomotor arteriolar response was measured in the intact eyes of miniature pigs after systemic administration and after local preretinal juxta-arteriolar microinjection of lactate. RESULTS: Injection of L-lactate (physiologically produced lactate) into the systemic circulation decreased the arterial blood pH but did not dilate the retinal arterioles. By contrast, microinjections of L-lactate (0.5 mol/l, pH 2) into the juxta-arteriolar vitreous induced a reversible segmental vasodilation of 32 +/- 4% (standard deviation). This vasodilation did not depend on periarteriolar pH lowering because microinjections of a 0.5 mol/l L-lactate at neutral pH also dilated segmentally the retinal arterioles (37 +/- 5.5%). The effect of lactate was stereospecific because microinjections of the isomer D-lactate (0.5 mol/l, pH 2) did not affect the arteriolar caliber (P = 0.63). Perfusion of the eye with the cyclo-oxygenase inhibitor indomethacin, through cannulization of the sublingual artery, caused a generalized reversible arteriolar vasoconstriction of 51 +/- 9.8% but did not inhibit the segmental vasodilator effect of locally microinjected L-lactate. CONCLUSIONS: It is known that acute hypoxia in the isolated retina causes an increase in lactate production. In the intact eye, there is a retinal vasodilation, which is not inhibited by indomethacin. Hence, it was concluded that retinal, but not blood, lactate is a possible mediator of the acute hypoxia-induced vasodilation.

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PubMed1992

Glucose metabolism in freshly isolated Müller glial cells from a mammalian retina.

Glucose metabolism was studied in isolated retinal Müller glial cells from the juvenile guinea pig. Cells, once enzymatically isolated and purified, were identified by morphological criteria, positive vimentin immunoreactivity, and histochemical staining for glycogen. Purified suspensions of Müller cells were obtained in quantities sufficient for biochemical analysis (approximately 2 x 10(5)/pair of retinas) and light microscopic autoradiography. In bicarbonate-buffered Ringer's medium containing 3H-2-deoxyglucose and no glucose, greater than or equal to 80% of the glucose analogue taken up intracellularly by Müller cells was phosphorylated to 3H-2-deoxyglucose-6-phosphate. In autoradiographs, this non-metabolized product provided visual evidence of glucose phosphorylation: the distribution of cell grains mirrored the morphology of individual Müller cells in situ. Exposure to the glycolytic inhibitor iodoacetate (500 microM) caused an 85% decrease in adenosine triphosphate (ATP) content; concomitantly, 3H-2-deoxyglucose-6-phosphate decreased by 90% and paralleled a dramatic decrease of cell labelling in autoradiographs, while levels of 3H-2-deoxyglucose did not change. In the continual absence of glucose, glycogen content decreased with time and this decrease was slowed by 36% in the presence of iodoacetate. This indicated that, in control conditions, glycosyl units from glycogen sustain cellular metabolism, and hence 3H-2-deoxyglucose phosphorylation. 3H-2-deoxyglucose-6-phosphate concentration was 43-fold less than that of ATP in the control conditions so that depletion of ATP during iodoacetic acid (IAA)-blocked glycolysis was not due to hexokinase activity. These results demonstrate that this preparation is adequate for quantitative studies of glucose metabolism at the cellular and molecular level in an important metabolic compartment of the mammalian retina.

Adenosine Triphosphate↗

PubMed1992

[Profile of pO2. I. Profile of transretinal pO2 in hypoxia].

Transretinal PO2 measurements during systemic hypoxia, made during variations of the PO2 by steps of 10 mmHg between 120 and 30 mmHg, have shown that the PO2 values measured at the inner-retina up to half the thickness of the retina remained stable. On the other hand, those measured at the choroid and at the outer-retina decreased in a linear manner according to the variations of PaO2. These results suggest a regulation in the retinal blood flow, allowing the PO2 to remain stable for the different steps of hypoxia studied. On the other hand, the values measured at the choroid and at the outer-retina showed the absence of regulation in the choroidal blood flow during hypoxia. Systemic hypoxia may affect the metabolism of the photoreceptors and the pigmentary epithelium.

Animals↗

PubMed1992

[Metabolic factors of vasomotor regulation of the inner retina].

Lactic acid, the end metabolic product of anaerobic glycolysis is probably the mediator of the hypoxia induced vasodilation on retinal arterioles. In this study we explored the mechanisms of the retinal vasomotor effect of this metabolite by performing preretinal juxtaarteriolar pulsatile pressure microinjections on the intact eye of anesthetized and artificially ventilated miniature pigs. Microinjections of the levorotatory isomer L-lactic acid (pH: 2) induced a local maximal dilation of retinal arterioles. This vasodilator effect, like that of systemic hypoxia, was not mediated by the release of prostaglandins. Preretinal pulsatile pressure juxtaarteriolar microinjections of neutral-pH solution of L-lactic acid also induced a segmental retinal arteriolar dilation. In contrast, microinjections of the dextrorotatory isomer D-lactic acid (pH: 2, solution), which is not produced by the retina, did not affect significantly the arteriolar diameter. Consequently, the vasodilator effect of lactic acid does not depend on periarteriolar pH modification and probably interferes with retinal metabolism since only the natural levorotatory metabolite is recognized.

Animals↗

PubMed1991

Metabolic signaling between photoreceptors and glial cells in the retina of the drone (Apis mellifera).

Experimental evidence showing metabolic interaction and signaling between photoreceptors-neurons and glial cells of the honeybee drone retina is presented. In this tissue [3H]2-deoxyglucose ([3H]2DG) in the dark and during repetitive light stimulation is phosphorylated to [3H]2-deoxyglucose-6P ([3H]2DG-6P) almost exclusively in the glial cells. Hence, stimulus-induced changes in the rate of formation of [3H]2DG-6P occurs predominantly in the glial cells. Repetitive stimulation of the photoreceptors with light flashes induced about a 47% rise in the rate of formation of [3H]2DG-6P in the glial cells and this effect is probably due to the activation of hexokinase. The potent inhibitor of glycolysis iodoacetic acid (IAA), inhibited this phosphorylation by about 75%. Probably this was largely due to an about 70% decrease of adenosine triphosphate (ATP). Exposure of the retina to IAA suppressed the transient rise in oxygen consumption (delta QO2) in the photoreceptors and subsequently the light-induced receptor potential. This indicates that the supply of a glycolytic substrate by glial cells to the photoreceptors is greatly reduced by IAA. Anoxia, by rapidly suppressing QO2, abolished the receptor potential of the photoreceptors and caused a rapid drop of about 50% in the ATP content of the retina. At the same time the formation of [3H]2DG-6P was inhibited by about 30%. This indicates that respiring photoreceptors send a metabolic signal to glial cells which is suppressed by anoxia.

3-O-Methylglucose↗

PubMed1991

Separation of nucleotides in homogenates of octopus retina by ion-pair reversed-phase liquid chromatography and identification by mass spectrometry.

An isocratic ion-pair reversed-phase liquid chromatographic method has been developed for the determination of thirteen nucleotides including cyclic AMP and cyclic GMP. The resolution capability of this method was evaluated successfully using homogenates of octopus retina, the aim being to elucidate the role of nucleotides (particularly ADP and ATP) in the control of oxidative metabolism. To overcome the inherent lack of specificity of ultraviolet detection we used the coupling of liquid chromatography with mass spectrometry, via a thermospray interface, to confirm the identity of the nucleotides of interest in the biological samples.

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PubMed1991

Glial (Müller) cells take up and phosphorylate [3H]2-deoxy-D-glucose in mammalian retina.

[3H]2-Deoxy-D-glucose-6-PO4 ([3H]2DG-6P) was visualised at the level of single cells in freeze-dried guinea pig retinal sections by in vitro light microscopic autoradiography after incubation with [3H]2-deoxy-D-glucose ([3H]2DG). In the dark, autoradiographs revealed heterogeneous labeling within individual retinal layers. Labeling, representing [3H]2DG-6P, was preferentially located over Müller (glial) cells. Labelling over identified neurones in the inner nuclear layer, in contrast, was scarce and over ganglion cells was exceptional. Our observations indicate that Müller cells in the mammalian retina phosphorylate [3H]2DG to [3H]2DG-6P, the first step in glycolysis.

Animals↗

PubMed1991

The light-induced increase of carbohydrate metabolism in glial cells of the honeybee retina is not mediated by K+ movement nor by cAMP.

The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur essentially in glia, whereas O2 consumption occurs in the photoreceptors. After [3H] glucose loading of superfused retinal slices, light stimulation induced a significant rise in [3H] glycogen turnover in the glia. This occurs without a concomitant covalent modification of glycogen enzymes. Probably only an increase or a decrease of the availability of [3H] glycosyls that are incorporated into glycogen is necessary. As only photoreceptors are directly excitable by light, we searched for a signal that stimulates glycogen metabolism in the glia. Although K+ in extracellular space and glia increases after repetitive light stimulation, increasing bath K+ in the dark did not mimic the metabolic effects of light, despite an equivalent increase of K+ in the extracellular space and glia. We subsequently explored the role of cAMP, a universal intracellular second messenger. Exposure of retinal slices to the adenylate-cyclase activator forskolin induced an expected increase in the rate of formation of cAMP, but only partially mimicked the metabolic effects of light. Furthermore, light stimulation failed to induce a rise in the rate of formation of cAMP. We conclude that in this nervous system, without synapses, neither K+ nor cAMP mediates the effect of light stimulation on intraglial glucose metabolism.

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PubMed1990

A method for measuring the oxygen consumption of photoreceptor cells in the steady state and after a brief stimulation by light.

The rate of oxygen consumption (QO2) in living tissue cannot be directly measured but may be estimated by mathematically modelling the diffusion of oxygen in the tissue and measuring the local partial pressure of oxygen (PO2). The retina of arthropods contains only two types of cells, photoreceptor and glial cells, which are regularly distributed. Because of this simple structure, simple models of diffusion can be used to estimate the QO2 of the tissue, both in steady state and after a brief stimulation by light. We used a model of diffusion in a plane sheet to calculate the QO2 in a slice of honeybee drone retina, which contains a few thousand cells. We then modified the method slightly and used a model with spherical symmetry to calculate the QO2 in the cluster of three photoreceptor cells of the barnacle and in the single ventral photoreceptor cells of Limulus.

Animals↗

PubMed1990

Diffusion of O2 in normal and ischemic retinas of anesthetized miniature pigs in normoxia and hyperoxia.

Transretinal PO2 profiles were recorded with O2-sensitive microelectrodes in the normal retina and in ischemic retinal foci induced by the occlusion of a retinal branch vein with argon laser photocoagulation in anesthetized miniature pigs. In the normal retina there are two PO2 gradients: one from the inner retina and the other from the choroid, both directed toward the middle of the retina. Both PO2 gradients persisted during hyperoxia. Thus, even in hyperoxia, the choroid does not supply the whole thickness of the normal retina with O2. Preretinal and transretinal PO2 measurements in ischemic inner retinal foci showed the existence of two PO2 gradients in steady-state systemic normoxia, as did those in the normal retina. This finding indicates that even in ischemia the choroid does not supply O2 to the inner retina; as a result, tissue hypoxia is maintained. During systemic hyperoxia, the intraretinal PO2 measurements in the ischemic foci showed only one gradient going from the choroid toward the inner retina. This gradient indicates that under these conditions, the choroid can supply O2 to the entire thickness of the ischemic retina. Extending a previously formulated hypothesis, we propose that in the ischemic retina as opposed to the normal retina, hyperoxia does not induce an increase in the O2 consumption of the outer retina. This suggestion could explain the rise in PO2 in the inner ischemic retina during hyperoxia.

Animals↗

PubMed1990

Scatter photocoagulation restores tissue hypoxia in experimental vasoproliferative microangiopathy in miniature pigs.

Experimental retinal branch vein occlusion using argon laser photocoagulation in miniature pigs induced the development of ischemic retinal territories associated with preretinal neovascularization. Preretinal partial pressure of oxygen (PO2) measurements on the ischemic territories, using O2-sensitive microelectrodes, established that the ischemic retinal areas were hypoxic. Scatter photocoagulation of these ischemic hypoxic territories restores the local PO2 to the normal values within 2 weeks. Hence, the reported inhibitory effect of photocoagulation on the development of retinal neovascularization could be due to a reversal effect on tissue hypoxia.

Animals↗

PubMed1990

Experimental retinal branch vein occlusion in miniature pigs induces local tissue hypoxia and vasoproliferative microangiopathy.

In miniature pigs, retinal veins were experimentally occluded using argon laser coagulation. Microvascular modifications leading to retinal hemorrhages and retinal edema were observed some hours after the occlusion. These lesions resolved progressively within 3 weeks after the occlusion, but in most cases ischemic retinal territories persisted. Preretinal partial pressure of oxygen (PO2) measurements, using double barrelled O2-sensitive microelectrodes, showed that all the ischemic areas were indeed hypoxic. In half of the experiments, preretinal and intravitreal new vessels grew on the ischemic territories. Tissue hypoxia appears to be a key step in triggering neovascularization. However, the critical level of hypoxia was not determined.

Animals↗

PubMed1990

[Changes in the concentration of K+ induced by a flash of light in the preretinal vitreous of the anesthesized minipig].

When measured with double-barreled ion-selected microelectrodes, delta K+0 is found to be heterogeneously distributed over the retinal surface. A transient fall (less than or equal to 200 microM) in delta K+0 near an arteriole wall and a transient increase (less than or equal to 500 microM) in zones free of arterioles were observed during light stimulation. In view of these findings the authors assume that the former is due to K+ uptake and transport by perivascular astrocytes, and that the latter is due to K+ siphoning by Müller cells.

Animals↗

PubMed1990

The increase of oxygen consumption after a flash of light is tightly coupled to sodium pumping in the lateral ocellus of barnacle.

In the lateral ocellus of the barnacle, we have tested the hypothesis that the transient increase of oxygen consumption (delta QO2) induced by light results from an increase in the rate of Na+ pumping. With a Na(+)-sensitive microelectrode, we measured the intracellular concentration of Na+ (Nai) in the photoreceptor cells. Nai was 17.6 +/- 1.2 mM (SE; n = 18) in darkness and it increased transiently by 10-20 mM after an 80-ms flash of intense light. The increase of Nai recovered in about the same time as the delta QO2, and the Na+/O2 ratio was 19.2 +/- 3.8 (SE; n = 6). Removing Na+ from the bath caused the delta QO2 to decrease by 79 +/- 3% (SE; n = 5). Exposure to 25 microM ouabain inhibited Na+ pumping and abolished the delta QO2. Removal of K+ from the bathing solution inhibited Na+ pumping in darkness, but mostly shortened the duration of the delta QO2; with a K(+)-sensitive microelectrode, we measured pericellular [K+] and found that it increased after the flash for about the same time as the delta QO2. Increasing Na+ pumping in darkness by reintroducing K+ in the bath or by injecting Na+ into one of the photoreceptor cells induced a delta QO2. Finally, intracellular injection of adenosine diphosphate and inorganic phosphate (ADP + Pi), the metabolic products of ATP splitting by the Na+ pump, also induced a delta QO2 in darkness. We conclude that all the results obtained are consistent with the formulated hypothesis.

Adenosine Diphosphate↗

PubMed1989

Diffusion of O2 in the retina of anesthetized miniature pigs in normoxia and hyperoxia.

Intraretinal oxygen tension (pO2) and local electroretinogram (ERG) were simultaneously measured in miniature pigs using double-barreled recess type microelectrodes. Transretinal pO2 profiles were recorded during normoxia and hyperoxia in areas close to (juxta-arteriolar) or far from (intervascular) retinal arterioles. In normoxia, in both areas, the pO2 decreased from the inner retina and the choroid towards the middle of the retina. In the inner retina the gradient of the juxta-arteriolar pO2 profile was steeper than that of the intervascular profile. This characteristic persisted during the breathing of 100% O2. Analysis of the pO2 profiles shows that, even in hyperoxia, the choroid cannot supply O2 to the whole retina. The results also support the conclusions of previous work (Riva, Pournaras and Tsacopoulos, 1986) indicating that in the normal retina it is not the O2 diffusing from the choroid to the retinal arterioles the induces vasoconstriction of these vessels. In the miniature pig this constriction appears to maintain inner retina tissue pO2 at a constant level during hyperoxia. From the pO2 transretinal profiles and previously published choroidal O2 fluxes and pO2 values near retinal vessels an explanatory working hypothesis is formulated according to which O2 consumption (qO2) of the outer retina increases during hyperoxia in the miniature pig.

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PubMed1988

Activation of mitochondrial oxidative metabolism by calcium ions in Limulus ventral photoreceptor.

Cells regulate their metabolic energy production to meet the requirements of their energy consuming activities. For most animal cells the prime site of energy production, in the form of ATP, is the mitochondrion. Extensive in vitro studies of isolated mitochondria have provided detailed information about the specific biochemical reactions involved in energy production. At present there is a debate about whether respiration in excitable cells is controlled by the availability of ADP to the mitochondrion and/or by calcium ions. Using the large ventral photoreceptor of the horseshoe crab (Limulus polyphemus) we describe a method for measuring the transient increase in the mitochondrial O2 consumption (delta QO2) following a flash of light of a single photoreceptor. We then show that this delta QO2 results in part from a rise in the intracellular concentration of calcium (Cai).

Animals↗

PubMed1988

Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate.

The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy[3H]glucose convert this glucose analogue to 2-deoxy[3H]glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O2 and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system.

Animals↗

PubMed1988

Light-induced oxygen consumption in Limulus ventral photoreceptors does not result from a rise in the intracellular sodium concentration.

Illumination of Limulus ventral photoreceptors leads to an increase in the intracellular concentration of sodium, [Na+]i, and to an increase in the consumption of O2 (delta QO2). After a 1-s light flash, it takes approximately 480 s for [Na+]i to return to within 10% of its preillumination level, whereas delta QO2 takes approximately 90 s. Thus, the delta QO2 is complete long before [Na+]i has returned to its resting level. Pressure injection of Na+ into the cell in order to elevate [Na+]i to the same levels as attained by illumination causes a rise in [Na+]i that returns to baseline with the same time course as the light-induced rise in [Na+]i. However, the injection of Na+ does not lead to an increase of the consumption of O2. We conclude that activation of the Na pump by a rise in [Na+]i is not a factor involved in the light-induced activation of O2 consumption in these cells.

Animals↗