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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 53 (1989), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Membranes prepared from either neuronal or glial cultures contain α2-adrenergic receptors as determined by the characteristics of [3H]yohimbine ([3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 ± 1.35 nM (n = 10) for neuronal cultures and 18.42 ± 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a 5max of 1.6 ± 0.33 pmol/mg protein (n = 10) compared with 0.143 ± 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for α2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the α2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynan-thine, or propranolol. The agonists showed the same pattern with the α2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog, 5′-guanylyl-imidodiphos-phate, were able to lower the affinity of the α2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of α2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain α-adrenoceptors.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Neuronal cells in primary culture from the brains of normotensive, Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats express angiotensin II type 1 (AT1) receptors. Treatment of WKY rat brain cultures with a phorbol ester, phorbol 12-myristate 13-acetate (PMA), causes a time-and dose-dependent increase in the levels of an ˜2.3-kb AT1 receptor mRNA transcript. A maximal stimulation of 4.5-fold in the AT1 receptor mRNA transcript level is observed with 200 nM PMA in 4 h and is blocked by 1 μM staurosporine. Forskolin also increases the AT1 receptor mRNA levels in WKY rat brain neurons in a time-and dose-dependent manner, and a 4.5-fold stimulation is achieved with 50μM forskolin in 4 h. The stimulatory effects of both PMA and forskolin are completely abolished by coincubation of neuronal cultures with 1 μM actinomycin D. In addition, nuclear run-on assay indicated an increase in the transcription of AT1 receptor mRNA in WKY rat brain neurons treated with either PMA or forskolin. Both PMA and forskolin also stimulate levels of AT1 receptor mRNA in neuronal cultures from brain of the SH rat. The degree of stimulation in these cultures is comparable to that in WKY rat brain neurons. These observations show that although the basal AT1 receptor gene expression is significantly higher in SH rat brain neurons compared with WKY rat brain neurons, the protein kinase C-and protein kinase A-responsive stimulation is not altered. These data suggest a possible involvement of protein kinase C and protein kinase A response elements in AT1 receptor gene expression.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 60 (1993), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The type 1 angiotensin II (All) receptor (AT1-R) has been implicated in the physiological actions mediated by All in the brain. In view of the reported hyperactivity of the brain All system in the spontaneously hypertensive rat (SHR), we compared the expression of AT,-R mRNAs in the brains of normotensive [Wistar Kyoto (WKY)] and SHR animals. Northern blot analysis showed about three- and ∼20-fold increases in the levels of AT1-R mRNAs from the hypothalamus and brainstem areas, respectively, of the SHR compared with the WKY rat brain. This was attributable to greater levels of both AT,1A- and AT,1B-R mRNA subtypes in these areas from the SHR. These observations suggest that increased All receptor levels in SHR brain may, in part, be a result of increased expression of the AT1-R gene.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 47 (1986), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract The cellular characteristics of the β-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (−)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (−)-isoproterenol 〉 (−)-epinephrine = (−)-norepinephrine 〉 (+)-isoproterenol was observed. 5′-GuanylyI-imidodiphosphate reduced the affinity of (−)-isoproterenol for the β-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (−)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible β-blocker bromoacetylalprenololmentane (2 μM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of β-adrenoreceptors in both cell types are the β−1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the β-adrenoreceptor.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 47 (1986), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4–8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hyper tensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by α1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving α1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific α1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at α1-adrenergic receptors, which is a novel finding.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Recent studies have suggested a role for an inhibitory guanine nucleotide binding (Gi) protein and protein (serine/threonine) phosphatase 2A (PP2A) in the angiotensin II type 2 (AT2) receptor-mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of angiotensin II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Angiotensin II elicited time (30 min–24 h)- and concentration (10 nM-1 µM)-dependent increases in PP2A activity in these cells, an effect mimicked by the AT2 receptor ligand CGP-42112A. These effects of angiotensin II and CGP-42112A involve AT2 receptors, because they were inhibited by the AT2 receptor-selective ligand PD 123,319 (1 µM) but not by the angiotensin II type 1 receptor antagonist losartan (1 µM). Furthermore, the stimulatory effects of angiotensin II and CGP-42112A on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (200 ng/ml; 24 h), indicating the involvement of a Gi protein. These effects of angiotensin II and CGP-42112A appear to be via activation of PP2A, and western blot analyses revealed no effects of either peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a cellular target modified following neuronal AT2 receptor activation.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar-Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of α1-adrenergic receptors. Binding of a selective α1 antagonist, [125I]2-[β-(4-hydroxy-3-iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75–80% specific, rapid, and time-dependent although the binding was 1.5–2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin 〈 phentolamine 〈 yohimbine 〈 phenylephrine 〈 norepinephrine 〈 propranolol, suggesting that [125I]-HEAT bound selectively to α1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of α1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an α1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of α1-adrenergic receptors. In conclusion, we demonstrated that the expression of α1-adrenergic receptors in neuronal cultures of SH rat brain was increased compared with neuronal cultures from normotensive controls. We suggest that the elevated number of these receptors in neurons from the brains of SH rats is an intrinsic property of these cells and therefore provides an etiological basis for the predisposition of these animals to the hypertensive state.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1–100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II-stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 µM) inhibited the Ang II-stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 µM) did not inhibit the Ang II-stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II-stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca2+-free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 µM), cyclopiazonic acid (10 µM), and ryanodine (10 µM), but not caffeine (1–10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 µM) and nifedipine (10 µM); in contrast, gadolinium (100 µM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 60 (1993), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μM) to the culture dishes for 25 min in Mg2+-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 μM and 50 μM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Previous studies have suggested that protein ki-nase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.
    Type of Medium: Electronic Resource
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