Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A 2 (sPLA 2 ). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA 2 . Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A 2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA 2 is a consequence of increased order of membrane lipids.Under normal conditions, healthy cell membranes resist catalysis by secretory phospholipase A 2 (sPLA 2 ) 1 (1-4). However, they may become susceptible under circumstances that cause alteration of membrane physical properties (1-4). Previous studies using artificial membranes demonstrated that alterations that increase susceptibility generally increase the anionic charge of the outer leaflet, increase bilayer curvature, and/or decrease interactions among neighboring phospholipids (5-9). In some cases, enhanced susceptibility of artificial membranes depends on an increase in the order of the phospholipids (8, 10 -14). These changes increase susceptibility by augmenting the binding of sPLA 2 and/or by improving access of membrane phospholipids to the active site of the enzyme (5-12, 15, 16).It is not known whether the properties that induce susceptibility to sPLA 2 in artificial membranes also contribute to the vulnerability of biological membranes to attack by the enzyme. In order to address this issue, we manipulated various properties of erythrocyte membranes by preparing different types of ghosts as explained in the accompanying particle (17). We found that the factors that determined the degree of susceptibility were increased exposure of phosphatidylserine, an anionic phospholipid, and increased membrane order. These interpretations agreed with those from studies of susceptibility using artificial membranes (5-16). The next question, then, is whether these same factors are important in the hydrolysis of intact cells by sPLA 2 under conditions at which they have become susceptible such as in the presence...
Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A 2 (sPLA 2 ) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA 2 activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA 2 was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.
Cerebral blood flow ensures delivery of nutrients, such as glucose, to brain sites with increased metabolic demand. However, little is known about rapid glucose dynamics at discrete locations during neuronal activation in vivo. Acute exposure to many substances of abuse elicits dopamine release and neuronal activation in the striatum; however, the concomitant changes in striatal glucose remain largely unknown. Recent developments have combined fast-scan cyclic voltammetry with glucose oxidase enzyme modified carbon-fiber microelectrodes to enable the measurement of glucose dynamics with subsecond temporal resolution in the mammalian brain. This work evaluates several waveforms to enable the first simultaneous detection of endogenous glucose and dopamine at single recording sites. These molecules, one electroactive and one non-electroactive, were found to fluctuate in the dorsal striatum in response to electrical stimulation of the midbrain and systemic infusion of cocaine/ raclopride. The data reveal the second-by-second dynamics of these species in a striatal microenvironment, and directly demonstrate the coupling of glucose availability with increased metabolic demand. This work provides a foundation that will enable detailed investigation of local mechanisms that regulate the coupling of cerebral blood flow with metabolic demand under normal conditions, and in animal studies of drug abuse and addiction.
L-DOPA has been the gold standard for symptomatic treatment of Parkinson’s disease. However, its efficacy wanes over time as motor complications develop. Very little is known about how L-DOPA therapy affects the dynamics of fluctuating dopamine concentrations in the striatum on a rapid timescale (seconds). Electrochemical studies investigating the effects of L-DOPA treatment on electrically evoked dopamine release have reported conflicting results with significant variability. We hypothesize that the uncertainty in the electrochemical data is largely due to electrode fouling caused by polymerization of L-DOPA and endogenous catecholamines on the electrode surface. Thus, we have systematically optimized the procedure for fabricating cylindrical, Nafion-coated, carbon-fiber microelectrodes. This has enabled rapid and reliable detection of L-DOPA’s effects on striatal dopamine signaling in intact rat brain using fast-scan cyclic voltammetry. An acute dose of 5 mg/kg L-DOPA had no significant effect on dopamine dynamics, demonstrating the highly efficient regulatory mechanisms at work in the intact brain. In contrast, administration of 200 mg/kg L-DOPA significantly increased the amplitude of evoked dopamine release by ~200%. Overall, this work describes a reliable tool that allows a better measure of L-DOPA augmented dopamine release in vivo, measured using fast-scan cyclic voltammetry. It provides a methodology that improves the stability and performance of the carbon fiber microelectrode when studying the molecular mechanisms underlying L-DOPA therapy, and also promises to benefit a wide variety of studies because Nafion is so commonly used in electroanalytical chemistry.
Exposure of human erythrocytes to elevated intracellular calcium causes fragments of the cell membrane to be shed as microvesicles. This study tested the hypothesis that microvesicle release depends on microscopic membrane physical properties such as lipid order, fluidity, and composition. Membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the activity of the trans-membrane phospholipid transporter, scramblase. Microvesicle release was enhanced by increasing the experimental temperature. Reduction in membrane cholesterol content by treatment with methyl-beta-cyclodextrin also facilitated vesicle shedding. Inhibition of scramblase with R5421 impaired vesicle release. These data were interpreted in the context of membrane characteristics assessed previously by fluorescence spectroscopy with environment-sensitive probes such as laurdan, diphenylhexatriene, and merocyanine 540. The observations supported the following conclusions: 1) calcium-induced microvesicle shedding in erythrocytes relates more to membrane properties detected by diphenylhexatriene than by the other probes; 2) loss of trans-membrane phospholipid asymmetry is required for microvesicle release.PACS Codes: 87.16.dj, 87.16.dt.
Background: Skull diversity in the neotropical leaf-nosed bats (Phyllostomidae) evolved through a heterochronic process called peramorphosis, with underlying causes varying by subfamily. The nectar-eating (subfamily Glossophaginae) and blood-eating (subfamily Desmondontinae) groups originate from insect-eating ancestors and generate their uniquely shaped faces and skulls by extending the ancestral ontogenetic program, appending new developmental stages and demonstrating peramorphosis by hypermorphosis. However, the fruit-eating phyllostomids (subfamilies Carollinae and Stenodermatinae) adjust their craniofacial development by speeding up certain developmental processes, displaying peramorphosis by acceleration. We hypothesized that these two forms of peramorphosis detected by our morphometric studies could be explained by differential growth and investigated cell proliferation during craniofacial morphogenesis. Results: We obtained cranial tissues from four wild-caught bat species representing a range of facial diversity and labeled mitotic cells using immunohistochemistry. During craniofacial development, all bats display a conserved spatiotemporal distribution of proliferative cells with distinguishable zones of elevated mitosis. These areas were identified as modules by the spatial distribution analysis. Ancestral state reconstruction of proliferation rates and patterns in the facial module between species provided support, and a degree of explanation, for the developmental mechanisms underlying the two models of peramorphosis. In the long-faced species, Glossophaga soricina, whose facial shape evolved by hypermorphosis, cell proliferation rate is maintained at lower levels and for a longer period of time compared to the outgroup species Miniopterus natalensis. In both species of studied short-faced fruit bats, Carollia perspicillata and Artibeus jamaicensis, which evolved under the acceleration model, cell proliferation rate is increased compared to the outgroup. Conclusions: This is the first study which links differential cellular proliferation and developmental modularity with heterochronic developmental changes, leading to the evolution of adaptive cranial diversity in an important group of mammals.
Electrochemical monitoring of non-electroactive species requires a biosensor that is stable and selective, with sensitivity to physiological concentrations of targeted analytes. We have combined glucose oxidase-modified carbon-fiber microelectrodes with fast-scan cyclic voltammetry for real-time measurements of glucose fluctuations in brain tissue. Work presented herein quantitatively compares three approaches to enzyme immobilization on the microelectrode surface-physical adsorption, hydrogel entrapment, and entrapment in electrospun nanofibers. The data suggest that each of these methods can be used to create functional microbiosensors. Immobilization of glucose oxidase by physical adsorption generates a biosensor with poor sensitivity to glucose and unstable performance. Entrapment of glucose oxidase in poly(vinyl alcohol) nanofibers generates microbiosensors that are effective for glucose measurements over a large linear range, and that may be particularly useful when targeting glucose concentrations in excess of 3 mm, such as in blood. Hydrogel entrapment is the most effective in terms of sensitivity and stability. These microbiosensors can be used for simultaneous monitoring of glucose and dopamine in real time. The findings outlined herein should be applicable to other oxidase enzymes, and thus they are broadly important for the development of new tools for real-time measurements of fluctuating molecules that are not inherently electroactive.
Recent studies have described a role for lactate in brain energy metabolism and energy formation, challenging the conventional view that glucose is the principle energy source for brain function. To date, lactate dynamics in the brain are largely unknown, limiting insight into function. We addressed this by developing and characterizing a lactate oxidase-modified carbon-fiber microelectrode coupled with fast-scan cyclic voltammetry. This new tool boasts a sensitivity for lactate of 22 ± 1 nA•mM −1 and LOD of 7.0 ± 0.7 μM. The approach has enabled detection of rapid lactate fluctuations with unprecedented spatiotemporal resolution as well as excellent stability, selectivity, and sensitivity. The technology was characterized both in vitro and in vivo at discrete recording sites in rat striatum. We provide evidence that striatal lactate availability increases biphasically in response to electrical stimulation of the dopaminergic midbrain in the anesthetized rat. This new tool for real-time detection of lactate dynamics promises to improve understanding of how lactate availability underscores neuronal function and dysfunction.
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