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  • 1
    Electronic Resource
    Electronic Resource
    Connexin43 in rat pituitary: localization at pituicyte and stellate cell gap junctions and within gonadotrophs (1993)
    Springer
    Histochemistry and cell biology 100 (1993), S. 53-64 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunohistochemical methods were employed to investigate the cellular and ultrastructural localization of the gap junction protein connexin43 (Cx43) in rat pituitary. Western blots of pituitary homogenates probed with anti-Cx43 antibodies showed the presence of Cx43 in both anterior and posterior pituitary lobes. By light microscopy (LM), Cx43-immunoreactive (Cx43-IR) puncta were found in all areas of the posterior lobe, but at greater concentrations in peripheral regions of this structure. By electron microscopy (EM), immunogold labelling for Cx43 was seen at gap junctions between thin cytoplasmic processes of pituicytes. No immunoreactivity was detected in the intermediate lobe. The anterior lobe contained puncta similar to but more sparsely scattered than those in the posterior lobe, and by EM analysis these were demonstrated to correspond to labelled gap junctions between stellate cells. In addition, anti-Cx43 antibodies produced intracellular labelling in a small percentage of endocrine cells, which were distributed throughout the anterior lobe and determined by double immunostaining methods to be cells containing luteinizing hormone. By EM, labelling within these cells was associated with predominantly large secretory granules and other loosely organized organelles. The results indicate that gap junctions in the pituitary are composed of Cx43 and that this or a related protein may have a novel intracellular function within gonadotrophs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00268878
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  • 2
    Electronic Resource
    Electronic Resource
    Rat Brain Adenosine Deaminase After 2′-Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2′-[3H]Deoxycoformycin Ligand Binding (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 58 (1992), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2′-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r= 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1992.tb09739.x
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  • 3
    Electronic Resource
    Electronic Resource
    Phosphorylated Forms of Connexin43 Predominate in Rat Brain: Demonstration by Rapid Inactivation of Brain Metabolism (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 62 (1994), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The gap junction protein connexin43 (Cx43) has been reported to exist as several phosphorylated forms migrating at ˜43 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as an unphosphorylated 41-kDa form. In brain, Cx43 is expressed predominantly in astrocytes and is also expressed in several other cell types. Whereas the phosphorylated forms of Cx43 predominate in heart, several studies have indicated that high levels of the unphosphorylated form of Cx43 are present in brain. Various experiments in this report indicate that the 41-kDa molecular form in brain is a postmortem dephosphorylation product of phosphorylated Cx43. In rats killed by cranial high-energy microwave irradiation leading to rapid inactivation of brain metabolism, Cx43 in cerebral cortex was present almost exclusively as the 43-kDa phosphorylated form. Rapid dissection of brain followed by heat treatment or inclusion of phosphatase inhibitors during tissue homogenization also largely prevented the conversion of the 43-to the 41-kDa form. The 41-kDa species was generated after alkaline phosphatase digestion of the 43-kDa material obtained by immunoprecipitation from microwave-irradiated brain. Immunolabeling patterns and relative regional levels of Cx43 as seen by immunohistochemical and western blot detection were the same whether or not metabolism to the 41-kDa species was prevented. In developing rat brain, Cx43 levels in frontal cortex and brainstem increased with age, but the degree of dephosphorylation of the 43-to the 41-kDa form was greater at earlier ages in the brainstem. It appears that brain contains a phosphatase that may be involved in modulating the phosphorylation state of Cx43 and thus may regulate intercellular communication via astrocytic gap junctions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1994.62062394.x
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  • 4
    Electronic Resource
    Electronic Resource
    Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 62 (1994), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: [3H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCI) and low (200 mM KCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca2+, Mg2+, β,γ-methylene-adenosine 5′-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent KD (nM) and Bmax (fmol/mg of protein) values of 2.4 and 34, respectively, for the high-affinity site and 19.9 and 157, respectively, for the low-affinity site. Unlabeled titration/equilibrium measurements gave a single high-affinity site with a KD value of 1.9 nM and a Bmax value of 95 fmol/mg of protein. The apparent KD value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca2+ (KD/Ca2+= 14 nM), inhibited by Mg2+ (IC60= 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with a KD value similar to and a Bmax value 1.8-fold greater than those obtained for the low-affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the KD value was fivefold lower, whereas the Bmax value was unaffected. The KD value derived from association and dissociation analysis was 2.4-fold greater in 200 mM KCI compared with 1.0 M KCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg2+ inhibited binding was increased by 3.8-fold, whereas the affinity of the activation site for Ca2+ was reduced by 13-fold. Addition of caffeine in the presence of low salt increased the affinity of Ca2+ activation by 1.7-fold. The inhibitory effect of Mg2+ on [3H]-ryanodine binding in the presence of 200 mM KCI was reversed by AMP-PCP and caffeine with apparent EC50 values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1994.62062340.x
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  • 5
    Electronic Resource
    Electronic Resource
    Ontogenesis of Adenosine Deaminase Activity in Rat Brain (1987)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 48 (1987), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1-day-old rats, the highest activity was seen in olfactory bulbs (550 ± 15 nmol/mg protein/30 min) and this was 4.5-fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 ± 5 μM) were not significantly different from that observed in adult rats (38 ± 7 μM), whereas the Vmax values decreased significantly from 339 ± 9 to 108 ± 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes. The complex changes in ADA activity during ontogenesis suggest an important role of ADA at very early stages of development as well as in specific regions of the adult rat brain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1987.tb13139.x
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  • 6
    Electronic Resource
    Electronic Resource
    2′-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 54 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: 2′-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 μM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 μg/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the 〉95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5–50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68–78% of control values in brain regions with normally high levels of activity and by 44–59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb01945.x
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  • 7
    Electronic Resource
    Electronic Resource
    Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve (2002)
    Oxford, UK : Blackwell Science, Ltd
    European journal of neuroscience 16 (2002), S. 0 
    Details
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) ‘rosettes’ and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1460-9568.2002.02149.x
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  • 8
    Electronic Resource
    Electronic Resource
    Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition (2000)
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 12 (2000), S. 0 
    Details
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti-Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30 min after hypoxia. This was preceded by a 77% reduction in GJIC 15 min after hypoxia, indicating that reduced GJIC occurs prior to Cx43 dephosphorylation. Hypoxia caused a reduction in punctate immunostaining (epitope masking) at cell–cell contacts with one anti-Cx43 antibody, and increased labelling with another antibody (13–8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)-1 and PP-2A with okadaic acid or calyculin A had little effect on hypoxia-induced Cx43 dephosphorylation. Inhibition of PP-2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro are similar to those seen after ischaemia in vivo, and that inhibition of protein phosphatase protects astrocytes from hypoxia-induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1460-9568.2000.00162.x
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  • 9
    Electronic Resource
    Electronic Resource
    A brain slice model for in vitro analyses of astrocytic gap junction and connexin43 regulation: actions of ischemia, glutamate and elevated potassium (2000)
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 12 (2000), S. 0 
    Details
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Brain slices prepared from adult rats and maintained for up to 3 h in vitro were used to investigate the effects of pharmacological treatments on the phosphorylation state, immunolabelling characteristics and ultrastructural localization of astrocytic gap junctions and connexin43 (Cx43). Slices deprived of glucose/oxygen to mimic ischemia or those exposed to 1 mm glutamate for 1 h exhibited Cx43 dephosphorylation, epitope masking and gap junction internalization as revealed by Western blotting and Cx43 immunolocalization with various antibodies. Treatment with 15 mm K+ caused Cx43 dephosphorylation without junction internalization. The effects of glutamate and K+ were completely blocked by the N-methyl-d-aspartate (NMDA) glutamate receptor antagonist 2-amino-5-phosphonovalerate (APV), which acts largely on neuronal NMDA receptors, suggesting neuronal mediation of glial gap junction responses to these treatments. Astrocytes contained a dephosphorylated form of Cx43 with a typical migration profile at 41 kDa as well as novel, apparently dephosphorylated or partially phosphorylated, forms migrating at 43 kDa. These results indicate that slices prepared from adult brain can serve as a convenient model to investigate the molecular basis and receptor-mediated mechanisms underlying astrocytic Cx43 responses that have been observed in vivo following cerebral ischemia or neural activation. These processes can be related in part to neuronal regulation of astrocytic gap junctional coupling state, which is also amenable to analysis in brain slices.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1460-9568.2000.01331.x
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  • 10
    Electronic Resource
    Electronic Resource
    Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia (1998)
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 10 (1998), S. 0 
    Details
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1460-9568.1998.00253.x
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