Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets

The preparation of HEMA-MPC films for ocular drug delivery

There is a need to prolong drug residence time using a biocompatible formulation in the subconjunctival space after surgery to treat glaucoma. Drug releasing discs were prepared with 2-(hydroxyethyl)methacrylate (HEMA) and 2-methacryloyl-oxyethyl phosphorylcholine (MPC). The ratio of bound water (Wb) to free water (Wf) ratio increased from 1:0.3 to 1:6.8 with increasing MPC (0 to 50%, w/w). The optimal balance between water content, SR and mechanical strength were obtained with 10% MPC (w/w) hydrogels. Water-alcohol mixtures were examined to facilitate loading of poorly soluble drugs, and they showed greater hydrogel swelling than either water or alcohol alone. The SR was 1.2 ± 0.02 and 3.3 ± 0.1 for water and water:ethanol (1:1) respectively. HEMA-MPC (10%) discs were loaded with dexamethasone using either water:ethanol (1:1) or methanol alone. Drug release was examined in an outflow rig model that mimics the subconjunctival space in the eye. Dexamethasone loading increased from 0.3 to 1.9 mg/disc when the solvent was changed from water:ethanol (1:1) to methanol with the dexamethasone half-life (t½) increasing from 1.9 to 9.7 days respectively. These encouraging results indicate that HEMA-MPC hydrogels have the potential to sustain the residence time of a drug in the subconjunctival space of the eye

Neuropeptide S: a novel regulator of pain-related amygdala plasticity and behaviors.

Amygdala plasticity is an important contributor to the emotional-affective dimension of pain. Recently discovered neuropeptide S (NPS) has anxiolytic properties through actions in the amygdala. Behavioral data also suggest antinociceptive effects of centrally acting NPS, but site and mechanism of action remain to be determined. This is the first electrophysiological analysis of pain-related NPS effects in the brain. We combined whole cell patch-clamp recordings in brain slices and behavioral assays to test the hypothesis that NPS activates synaptic inhibition of amygdala output to suppress pain behavior in an arthritis pain model. Recordings of neurons in the laterocapsular division of the central nucleus (CeLC), which serves pain-related amygdala output functions, show that NPS inhibited the enhanced excitatory drive [monosynaptic excitatory postsynaptic currents (EPSCs)] from the basolateral amygdala (BLA) in the pain state. As shown by miniature EPSC analysis, the inhibitory effect of NPS did not involve direct postsynaptic action on CeLC neurons but rather a presynaptic, action potential-dependent network mechanism. Indeed, NPS increased external capsule (EC)-driven synaptic inhibition of CeLC neurons through PKA-dependent facilitatory postsynaptic action on a cluster of inhibitory intercalated (ITC) cells. NPS had no effect on BLA neurons. High-frequency stimulation (HFS) of excitatory EC inputs to ITC cells also inhibited synaptic activation of CeLC neurons, providing further evidence that ITC activation can control amygdala output. The cellular mechanisms by which EC-driven synaptic inhibition controls CeLC output remain to be determined. Administration of NPS into ITC, but not CeLC, also inhibited vocalizations and anxiety-like behavior in arthritic rats. A selective NPS receptor antagonist ([d-Cys(tBu)(5)]NPS) blocked electrophysiological and behavioral effects of NPS. Thus NPS is a novel tool to control amygdala output and pain-related affective behaviors through a direct action on inhibitory ITC cells

Corneal guttata associated with the corneal dystrophy resulting from a βig-h3 R124H mutation

AIMS—To investigate the frequency of corneal guttata in patients with a corneal dystrophy resulting from an Arg124His (R124H) mutation of βig-h3 gene.
METHODS—Slit lamp examination was performed on 30 eyes with corneal dystrophy from a genetically confirmed βig-h3 R124H mutation and on 50 age matched control eyes. The stage of the corneal dystrophy was classified as stage 0, I, or II and the degree of guttata was classified as none, mild, or severe. Specular microscopic examinations were performed to evaluate the morphology of the corneal endothelium.
RESULTS—Slit lamp examination disclosed the presence of corneal guttata in 21 eyes (70%) of the 30 eyes with the corneal dystrophy, but in only one (2%) of the 50 eyes in the age matched control group (p<0.001, χ(2) with Yates's correction). Of the 12 eyes with stage I βig-h3 R124H corneal dystrophy, seven had no corneal guttata and five had a mild degree of guttata. Of the 18 eyes with stage II, the degree of guttata was none in two, mild in nine, and severe in seven. The degree of corneal guttata was significantly related to the stage of the corneal dystrophy (p<0.0001, Kruskul-Wallis test ANOVA on ranks). There was no significant differences between eyes with βig-h3 R124H corneal dystrophy and normal eyes in cell density, coefficient of variation, and cell hexagonality of corneal endothelium.
CONCLUSION—Corneal guttata are one of the characteristics of the corneal dystrophy resulting from βig-h3 R124H mutation.


Phase-Dependent Formation of Coherent Interface Structure between PtO₂ and TiO₂ and Its Impact on Thermal Decomposition Behavior

This study investigated the thermal decomposition behaviors of platinum oxide (PtO₂) nanoparticles deposited on polycrystalline TiO₂ in different crystal phases. The dissociation of PtO₂ to metallic platinum in air occurred at 400 °C on anatase TiO₂ (Pt/TiO₂-A), but required 650 °C or higher on rutile TiO₂ (Pt/TiO₂-R). The higher thermal stability of PtO₂ on rutile TiO₂ is caused by thermodynamic effect and rather than kinetic effect. In contrast to the thermodynamic prediction, metallic Pt (Pt⁰) on TiO₂-R was reversibly oxidized to PtO₂ (Pt⁴⁺) at 650 °C. This behavior was attributed to the coherent interface structure formed by strong interactions between PtO₂ and rutile TiO₂, as revealed by combined extended X-ray adsorption spectroscopy (EXAFS) and density functional theory (DFT) studies. At the optimized interface structure, between the (100) planes of α-PtO₂ and rutile TiO₂, the interface formation energy was −17.04 kJ mol^–1 Å^–2 versus −9.84 kJ mol^–1 Å^–2 in the anatase TiO₂ model. The larger interface formation energy provides a stabilizing effect against PtO₂ dissociation. Therefore, the widely used Pt-loaded rutile TiO₂ typifies the interfacial interactions under an oxidizing atmosphere, which differ from the strong metal–support interactions prevailing under a reducing atmosphere