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  • 1
    ISSN: 1573-6903
    Keywords: A1 adenosine receptor ; subcellular fractionation ; myelin ; 5′-nucleotidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [3H]R-PIA binding, the activity of adenosine metabolizing enzymes (5′-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A1 adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A1 adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5′-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.
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  • 2
    ISSN: 1573-6903
    Keywords: Myelin ; 5′-nucleotidase ; myelin isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2′,3′-cyclic nucleotide 3′-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5′-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH: cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5′-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.
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  • 3
    ISSN: 1573-6903
    Keywords: 5′-nucleotidase ; myelin ; myelin subfractions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and 5′-nucleotidase (5′N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5′N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5′N activity maxima suggest that myelin may be a predominant locus of 5′N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5′N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5′N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5′N activity were studied, and the results also suggest the intrinsic nature of 5′N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5′N activity, but not for the solubilization of this enzyme.
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  • 4
    ISSN: 0730-2312
    Keywords: brain cortical membranes ; receptor modulation ; trypsin ; binding capacity ; co-solubilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that (1) there is co-solubilization of receptor and phospholipids; (2) the phospholipid requirements are different for the coupled and the uncoupled receptor; (3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and (4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.
    Additional Material: 4 Ill.
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  • 5
    ISSN: 1432-1912
    Keywords: Adenosine recetpor ; Antagonist kinetic components ; Pig brain ; DPCPX
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The results described in this paper show, for the first time, that At adenosine receptors can have two kinetic components for the binding of the antagonist [3H]DPCPX. At low ionic strength (≤ 42mmo1/l), dissociation of [3H]DPCPX bound to A1 receptors fitted better to a two kinetic components model than to a one kinetic component model. The kinetic constants were consistent with comparable Kd values for the two components of the antagonist binding, and therefore these two components cannot be distinguished by saturation isotherm analysis.
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins.
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a. ligand CGS-21680 was low. In contrast, the binding of 5′-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX 〉 NECA 〉 chloroadenosine 〉 R-PIA 〉 theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Adenosine A1 receptors (A1Rs) and adenosine A2A receptors (A2ARs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A2ARs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A2ARs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A2AR-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A2AR agonists but not by A1R agonists. PKA activation is therefore necessary for A2AR-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A2A receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.
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  • 9
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Gene expression throughout the different stages of Alzheimer's disease was analysed in samples from cerebral cortex. The gene encoding the voltage-gated potassium channel Kv3.4 was already overexpressed in early stages of the disease, and in advanced stages Kv3.4 was present at high levels in neurodegenerative structures. This subunit regulates delayed-rectifier currents, which are primary determinants of spike repolarization in neurones. In unique samples from a patient with Alzheimer's disease whose amount of amyloid plaques was decreased by β amyloid immunization, Kv3.4 was overexpressed. The channel subunit was expressed in the neuropil, in the remaining conventional plaques in the frontal cortex and in collapsed plaques in the orbitary cortex. Therefore, amyloid deposition in plaques does not seem to be responsible for the increase in Kv3.4 levels. Nevertheless, Kv3.4 up-regulation is related to amyloid pathology, given that transgenic mice with the Swedish mutation of amyloid precursor protein showed increased expression of Kv3.4. Up-regulation of voltage-gated potassium channel subunits alters potassium currents in neurones and leads to altered synaptic activity that may underlie the neurodegeneration observed in Alzheimer's disease. Thus, Kv3.4 likely represents a novel therapeutic target for the disease.
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.
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