[Experimental branch vein occlusion: the effect of carbogen breathing on preretinal PO2]. / Occlusion veineuse rétinienne expérimentale: effet de l'inhalation de carbogène sur la PO2 prérétinienne.

PURPOSE: To evaluate the variations in preretinal PO2 in normal and in ischemic postexperimental branch vein occlusion (BRVO) retinal areas during normoxia, hyperoxia (100% O2) and carbogen (95% O2, 5% CO2) breathing. METHODS: Preretinal PO2 measurements were obtained in intervascular retinal areas far from the retinal vessels of 13 anesthetized miniature pigs with oxygen-sensitive microelectrodes (10 microm tip diameter) introduced through the vitreous cavity by a micromanipulator. The microelectrode tip was placed at 50 microm from the vitreoretinal interface in the preretinal vitreous. PO2 was measured continuously for 10 minutes in systemic normoxia, hyperoxia (100% O2 breathing) and carbogen (95% O2, 5% CO2) breathing. A BRVO was induced with an argon green laser, and oxygen measurements were repeated in normoxia, hyperoxia and carbogen breathing. RESULTS: In hyperoxia, preretinal PO2 remained almost constant in both normal retinas (DeltaPO2=1.33 mmHg +/- 3.39; n=13) and ischemic retinas (DeltaPO2=3.73 mmHg +/- 2.84; n=8), although systemic PaO2 significantly increased. Carbogen breathing induced a significant increase in systemic PaO2 and PaCO2. Furthermore, it significantly increased preretinal PO2: DeltaPO2=23.05 mmHg +/- 17.06 (n=12) in normal retinas, and DeltaPO2=22.54 mmHg +/- 5.96 (n=6) in ischemic retinal areas. CONCLUSIONS: Systemic hyperoxia does not increase preretinal PO2 significantly in normal and ischemic post-BRVO retinal areas of miniature pigs, as hyperoxia induces a decrease in the retinal blood flow. Carbogen breathing significantly increases preretinal PO2 in normal and in ischemic post-BRVO retinal areas. This effect is probably due to the vasodilatation of the retinal arterioles induced by the intravascular PaCO2 increase.

Angiogenesis of liver metastases: role of sinusoidal endothelial cells.

PURPOSE: Tumor-induced angiogenesis requires migration and remodeling of endothelial cells derived from pre-existing blood vessels. Vascular endothelial growth factor is the growth factor most closely implicated in the development of neovessels in colon cancer. However, vascular endothelial growth factor-specific receptors flt-1 and KDR mRNA expression are absent in normal sinusoid vessels surrounding vascular endothelial growth factor-producing secondary hepatic tumors. Thus, the potential role of sinusoidal endothelial cells in the mechanism of neovessel formation within liver metastatic carcinomas remains unclear. The purpose of this study was to determine whether sinusoidal endothelial cells are involved in tumor angiogenesis in a syngeneic model of liver metastases from colorectal cancer. METHODS: Sinusoidal endothelial cells were identified by fluorescence microscopy after uptake of acetylated low density lipoprotein labeled with a fluorescent probe (dioctadecylindocarbocyanine). One hundred microliters of dioctadecylindocarbocyanine acetylated low density lipoprotein were injected intraportally at the start of experiment in BD IX rats. Two days later, intraportal injection of 10(7) DHD K12, a chemically induced colon carcinoma cell line, was performed in syngeneic BD IX rats. Animals were killed one week later and the livers were processed for routine histologic examination and immunohistochemistry using the rat endothelial cell antigen-1 monoclonal antibody. RESULTS: In normal parenchyma fluorescence was associated with sinusoidal cells but not with endothelium of large blood vessels. Thus, specific acetylated low density lipoprotein uptake allowed histological differentiation of sinusoidal endothelial cells from other large-vessel endothelial cells present in the hepatic parenchyma. In tumor-bearing liver a spatial gradient of fluorescence was generated. Labeled cells accumulated at the periphery of the metastases. When tumors grow beyond 200 microm, neovessel formation was observed; there was an invasion of fluorescent-labeled cells from the periphery, which were arranged in a tubular formation within neoplasia. CONCLUSION: In liver metastases tumor vessels are lined with sinusoidal endothelial cells. Identification of a specific cell type involved in the formation of the stromal compartment of tumors has important implications. Sinusoidal endothelial cells express well-characterized surface receptors and differ morphologically and metabolically from large-vessel endothelia. They should be considered as attractive targets for future and existing antiangiogenic strategies directed against the stromal compartment of liver metastases.

Mechanisms of glutamate metabolic signaling in retinal glial (Müller) cells.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

Trafficking of molecules and metabolic signals in the retina.

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.

The nutritive function of glia is regulated by signals released by neurons.

The idea of a metabolic coupling between neurons and astrocytes in the brain has been entertained for about 100 years. The use recently of simple and well-compartmentalized nervous systems, such as the honeybee retina or purified preparations of neurons and glia, provided strong support for a nutritive function of glial cells: glial cells transform glucose to a fuel substrate taken up and used by neurons. Particularly, in the honeybee retina, photoreceptor-neurons consume alanine supplied by glial cells and exogenous proline. NH4+ and glutamate are transported into glia by functional plasma membrane transport systems. During increased activity a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Quantitative biochemistry showed that this is due to activation of glycolysis in glial cells by the direct action of NH4+ and of glutamate, probably on the enzymatic reactions controlled by phosphofructokinase alanine aminotransferase and glutamate dehydrogenase. This activation leads to a massive increase in the production and release of alanine by glia. This constitutes an intracellular signal and it depends upon the rate of conversion of NH4+ and of glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells. Alanine and alpha-ketoglutarate are released extracellularly and then taken up by neurons where they contribute to the maintenance of the mitochondrial redox potential. This signaling raises the novel hypothesis of a tight regulation of the nutritive function of glia.

Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell.

Glial cells transform glucose to a fuel substrate taken up and used by neurons. In the honeybee retina, photoreceptor neurons consume both alanine supplied by glial cells and exogenous proline. Ammonium (NH4+) and glutamate, produced and released in a stimulus-dependent manner by photoreceptor neurons, contribute to the biosynthesis of alanine in glia. Here we report that NH4+ and glutamate are transported into glia and that a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Biochemical measurements indicate that this is attributable to activation of glycolysis in glial cells by the direct action of NH4+ and glutamate on at least two enzymatic reactions: those catalyzed by phosphofructokinase (PFK; ATP:D-fructose-6-phosphotransferase, EC2.7.1.11) and glutamate dehydrogenase (GDH; L-glutamate:NAD oxidoreductase, deaminating; EC1.4.1.3). This activation leads to an increase in the production and release of alanine by glia. This signaling, which depends on the rate of conversion of NH4+ and glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells, raises the novel possibility of a tight regulation of the nutritive function of glia.

An electrochemical method for making enzyme microsensors. Application to the detection of dopamine and glutamate.

A novel method of microbiosensor fabrication is described. It is based on the electrochemical polymerization of an enzyme-amphiphilic pyrroleammonium solution on the surface of a microelectrode in the absence of supporting electrolyte. By trapping glutamate oxidase (GMO) or polyphenol oxidase (PPO) in such polypyrrole films, we made microbiosensors for the amperometric determination of glutamate or dopamine, respectively. The response of the GMO microelectrode to glutamate was based on the amperometric detection of the enzymically generated hydrogen peroxide at 0.6 V vs SCE. The detection limit and sensitivity of this microbiosensor were 1 µM and 32 mA M(-1) cm(-2), respectively. The response of the PPO microelectrode to dopamine was based on the amperometric detection of the enzymically generated quinoid product at -0.2 V. The calibration range for dopamine measurement was 5 × 10(-8)-8 × 10(-5) M and the detection limit and sensitivity were 5 × 10(-8) M and 59 mA M(-1) cm(-2), respectively.

Kinetics of oxygen consumption and light-induced changes of nucleotides in solitary rod photoreceptors.

We made simultaneous measurements of light-induced changes in the rate of oxygen consumption (QO2) and transmembrane current of single salamander rod photoreceptors. Since the change of PO2 was suppressed by 2 mM Amytal, an inhibitor of mitochondrial respiration, we conclude that it is mitochondrial in origin. To identify the cause of the change of QO2, we measured, in batches of rods, the concentrations of ATP and phosphocreatine (PCr). After 3 min of illumination, when the QO2 had decreased approximately 25%, ATP levels did not change significantly; in contrast, the amount of PCr had decreased approximately 40%. We conclude that either the light-induced decrease of QO2 is not caused by an increase in [ATP] or [PCr], or that the light-induced change of [PCr] is highly heterogeneous in the rod cell.

Nitric oxide controls arteriolar tone in the retina of the miniature pig.

PURPOSE: Experimental evidence indicates that the retinal microcirculation is mainly controlled by factors released from the tissue surrounding the arterioles. This study explores whether nitric oxide (NO), a possible factor, is released in the retina and controls the arteriolar tone. METHODS: Using a NO microprobe, the authors measured [NO] in the preretinal vitreous of miniature pigs as a function of distance from the retinal surface. Additionally, the NO-synthase inhibitor nitro-L-arginine was pressure injected. Finally, the retinal pool size of arginine and its biosynthesis from 14C(U)-glucose were biochemically assessed on retinal tissue and acutely isolated Müller cells. RESULTS: At the retinal surface, [NO] measured 6 to 9 microM, and, in the vitreous, it fell to zero approximately 180 microns away from the retina. Therefore, NO is degraded faster in the vitreous (65 to 80 microM.minute-1) than in aqueous solution. Light flicker stimulation of the dark-adapted retina induced a reversible increase of [NO] (approximately 1.6 microM). Preretinal juxta-arteriolar microinjections of nitro-L-arginine (0.6 mM) induced a segmental and reversible arteriolar vasoconstriction of 45%; in contrast, intravenous infusion of nitro-L-arginine had no measurable effect on arteriolar diameter. The retinal pool size of arginine was small (< or = 200 microM), but there was an important rate of arginine biosynthesis in Müller cells. CONCLUSIONS: These results strongly suggest that cells in the retina, other than endothelial cells, produce and release NO, which in turn controls the basal dilating arteriolar tone in the inner retina.

Lactate released by Müller glial cells is metabolized by photoreceptors from mammalian retina.

The nature of fuel molecules trafficking between mammalian glial cells and neurons was explored using acute retinal cell preparations of solitary Müller glial cells, Müller cells still attached to photoreceptors (the "cell complex"), and solitary photoreceptors. 14C-Molecules in the cell complex, Müller cells, and respective baths were quantitated following 30 min incubation in bicarbonate-buffered Ringer's solution carrying 5 mM 14C(U)-glucose, and substrate preference by solitary photoreceptors was assessed by measuring 14CO2 production. Müller cells alone metabolized 14C-glucose predominantly to carbohydrate intermediates, while the presence of photoreceptors raised proportionately the amount of radiolabeling in amino acids. 14C-Lactate was the major carbohydrate found in the bath. However, in the presence of photoreceptors, its amount was 70% less than that for Müller cells alone. This decrease matched the expected production of 14CO2 by photoreceptor oxidative metabolism and was antagonized by the addition of unlabeled lactate. Moreover, while solitary photoreceptors consumed both exogenous 14C-lactate and 14C-glucose, lactate was a better substrate for their oxidative metabolism. In the cell complex, the metabolism of amino acids increased and illumination affected primarily glutamate and glutamine production: the specific activity of glutamate changed in parallel with that of lactate, and that of glutamine increased by eightfold in darkness. These results demonstrate transfer of lactate from Müller cells to photoreceptors and underscore a photoreceptor-dependent modulation of lactate and amino acid metabolism. We propose that net production and release of lactate by Müller cells serves to maintain their glycolysis elevated and to fuel mitochondrial oxidative metabolism and glutamate resynthesis in photoreceptors.

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.

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.

Kinetics of oxygen consumption after a flash of light in the lateral ocellus of the barnacle.

Until recently, polarographic methods for measuring the time course of transient changes in the rate of oxygen consumption (DeltaQO(2)) have been applied only to tissue preparations containing thousands of cells. Here, we describe DeltaQO(2) measurements on the lateral ocellus of the barnacle (Balanus eburneus) which contains only three photoreceptor cells. The decrement of partial pressure of oxygen (DeltaPO(2)) elicited by an 80 ms flash of light was measured near the cells with a microelectrode and the DeltaQO(2) was calculated from the DeltaPO(2) using a model of diffusion with spherical symmetry. As shown by mathematical simulation, the exact shape of the preparation is not crucial for our measurements of the time course of the DeltaQO(2). For a given DeltaQO(2), the model describes correctly the attenuation of the DeltaPO(2) measured at increased distances from the preparation. To know more about the mechanisms controlling the DeltaQO(2), we compared it with the electrical response of the photoreceptor cells: both responses have a similar spectral dependence, but only the DeltaQO(2) was abolished by a 10-min exposure to 50 muM dinitrophenol or to 3 mM amytal. We conclude that the DeltaQO(2) reflects an increase in mitochondrial respiration and that it is initiated by the phototransformation of rhodopsin, as was already found in the honeybee drone retina (Dimitracos and Tsacopoulos, 1985; Jones and Tsacopoulos, 1987).

Effect of laser photocoagulation on oxygenation of the retina in miniature pigs.

A large area of the posterior pole of the retina of the miniature pig was photocoagulated, and 2-3 wk later the PO2 in the preretinal vitreous was mapped with O2-sensitive microelectrodes. In the control retina, the PO2 was highly heterogeneous being much higher close to an artery than opposite an intervascular zone. After photocoagulation, PO2 opposite an intervascular zone was found to be significantly increased. A quantitative histologic analysis showed that in the photocoagulated areas more than 28% of the outer retina was destroyed. The authors conclude from these results that photocoagulation, by partially destroying the pigment epithelium-photoreceptor complex, causes an increase of the oxygenation of the retina.

Oxygen uptake occurs faster than sodium pumping in bee retina after a light flash.

When neurones are active there is an entry of Na+, which must subsequently be pumped out, and an increase in their oxygen consumption rate (Qo2). The Na+ pump derives its energy from ATP, splitting it into ADP and Pi, and it has reasonably been proposed that the changes in concentrations of ATP, ADP and Pi lead to a stimulation of the O2 consumption by the mitochondria and hence to a restoration of the stock of ATP. Here we present evidence suggesting that Qo2 must be controlled differently in the retinal photoreceptor cells of the honeybee drone. Stimulation of drone photoreceptors with a flash of light causes an entry of Na+ (ref. 4) and a transient increase in Qo2 that indicates respiration of the right order of magnitude to provide ATP to pump the Na+ out. We report intracellular recordings of changes in intracellular sodium (Nai+) and potassium (Ki+) in response to single light flashes and have compared the time course of extra oxygen consumption (delta Qo2) with these ion changes and other indices of Na+ pumping. We found that the time course of pumping seems to lag behind the time course of delta Qo2. It follows that the mitochondrial respiration must be stimulated by some signal which is generated earlier than the rise in ADP produced by the Na+ pump.

Kinetics of oxygen consumption after a single flash of light in photoreceptors of the drone (Apis mellifera).

The time course of the rate of oxygen consumption (QO2) after a single flash of light has been measured in 300-micrometers slices of drone retina at 22 degrees C. To measure delta QO2(t), the change in QO2 from its level in darkness, the transients of the partial pressure of O2 (PO2) were recorded with O2 microelectrodes simultaneously in two sites in the slice and delta QO2 was calculated by a computer using Fourier transforms. After a 40-ms flash of intense light, delta QO2, reached a peak of 40 microliters O2/g.min and then declined exponentially to the baseline with a time constant tau 1 = 4.96 +/- 0.49 s (SD, n = 10). The rising phase was characterized by a time constant tau 2 = 1.90 +/- 0.35 s (SD, n = 10). The peak amplitude of delta QO2 increased linearly with the log of the light intensity. Replacement of Na+ by choline, known to decrease greatly the light-induced transmembrane current, caused a 63% decrease of delta QO2. With these changes, however, the kinetics of delta QO2 (t) were unchanged. This suggest that the recovery phase is rate-limited by a single reaction with apparent first-order kinetics. Evidence is provided that suggests that this reaction may be the working of the sodium pump. Exposure of the retina to high concentrations of ouabain or strophanthidin (inhibitors of the sodium pump) reduced the peak amplitude of delta QO2 by approximately 80% and increased tau 1. The increase of tau 1 was an exponential function of the time of exposure to the cardioactive steroids. Hence, it seems likely that the greatest part of delta QO2 is used for the working of the pump, whose activity is the mechanism underlying the rate constant of the descending limb of delta QO2 (t).

Diffusion and consumption of oxygen in the superfused retina of the drone (Apis mellifera) in darkness.

Double-barreled O2 microelectrodes were used to study O2 diffusion and consumption in the superfused drone (Apis mellifera) retina in darkness at 22 degrees C. Po2 was measured at different sites in the bath and retinas. It was found that diffusion was essentially in one dimension and that the rate of O2 consumption (Q) was practically constant (on the macroscale) down to Po2 s less than 20 mm Hg, a situation that greatly simplified the analysis. The value obtained for Q was 18 +/- 0.7 (SEM) microliter O2/cm3 tissue . min (n = 10), and Krogh's permeation coefficient (alpha D) was 3.24 +/- 0.18 (SEM) X 10(-5) ml O1/min . atm . cm (n = 10). Calculations indicate that only a small fraction of this Q in darkness is necessary for the energy requirements of the sodium pump. the diffusion coefficient (D) in the retina was measured by abruptly cutting off diffusion from the bath and analyzing the time-course of the fall in Po2 at the surface of the tissue. The mean value of D was 1.03 +/- 0.08 (SEM) X 10(-5) cm2/s (n = 10). From alpha D and D, the solubility coefficient alpha was calculated to be 54 +/- 4.0 (SEM) microliter O2 STP/cm3 . atm (n = 10), approximately 1.8 times that for water.