Localization of both type 2 angiotensin II receptors and a non-angiotensin II binding site by [125 I] CGP42112 in rat brain stem

Abstract not availabl

Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

<div><p>Background</p><p>Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke.</p><p>Results</p><p>Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; <i>P</i><0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia.</p><p>Conclusion</p><p>Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.</p></div

Expression levels of GDE, and activity of GP and PKA are decreased during ischemic stroke.

<p>(<b>A</b>) Representative immunoblot showing expression levels of GDE in contralateral and ipsilateral hemispheres of sham and stroke brains at different time points following stroke induction. Expression level of GDE was normalized against β-Actin protein. Lower panel: Quantification data of GDE expression level. Data are represented as mean ±SD, n = 6; ^*<i>P</i><0.05 vs contralateral hemispheres (6 hr and 24 hr). (<b>B</b>) Representative immunoblot of phosphorylation level of GP in sham and stroke rat brains. Immunoblot analysis data (lower panel) as mean ±SD, n = 6, ^*<i>P</i><0.05 vs contralateral hemispheres of stroke brains (6 hr and 24 hr). (<b>C</b>) GP enzyme activity in sham and stroke rat brains. Data are mean ±SD, n = 6 rats for each group. ^#<i>P</i> = 0.001 vs sham ipsilateral and ^*<i>P</i> = 0.003 vs stroke contralateral hemisphere (24 hr). (<b>D</b>) PKA activity in sham and stroke brains [mean ±SD, n = 6;^#<i>P</i> = 0.003 (24 hr) vs sham ipsilateral and ^*<i>P</i> = 0.004 (6 hr) and 0.003 (24 hr) vs stroke contralateral hemispheres]. (<b>E</b>) Representative immunoblot of p-PKA substrate of sham and stroke brains. Phosphorylation level was normalized against β-Actin protein [mean ±SD, n = 5; ^*<i>P</i> = 0.003 (6 hr) and 0.002 (24 hr)]. (^*<i>P</i> =  Student's t-test and ^#<i>P</i> =  one-way ANOVA followed by Tukey's <i>post hoc</i> test).</p

Elevated glycogen levels protect astrocytes during hypoxia.

<p>(<b>A</b>) Glycogen levels in astrocytes treated with either DAB (1 mM) or CP-316819 (10 µM) or DMSO for 24 hr. Data are presented as mean ±SD, n = 5; ^*<i>P</i><0.05. MTT assay showing cell viability in astrocytes treated with either CP-316819 (<b>B</b>) or DAB (<b>C</b>) for 24 hr and exposed to hypoxia for indicated time points. (Data are mean ±SD, n = 5; ^#<i>P</i><0.05). LDH assay in astrocytes treated with either CP-316819 (<b>D</b>) or DAB (<b>E</b>) for 24 hr and exposed to hypoxia. (Data are mean ±SD, n = 5; ^#<i>P</i><0.05). (<b>F</b>) Representative images of live (green) and dead (red) cells in astrocytes exposed to hypoxia for 24 hr. Astrocytes were treated with CP-316819, DMSO or DAB for 24 hr prior to hypoxia. (<b>G</b>) Ratio of live to dead cells in astrocytes treated with either CP-316819 or DAB and exposed to hypoxia for 241hr. Data are mean ±SD, n = 5; ^#<i>P</i><0.05. (^*<i>P</i> =  Student's t-test and ^#<i>P</i> =  one-way ANOVA followed by Tukey's <i>post hoc</i> test).</p

Inhibition of PKA activity in cultured cerebellar astrocytes increases glycogen accumulation.

<p>(<b>A</b>) PKA activity in cerebellar astrocytes treated with different concentrations of H89 for 24 hr [mean ±SD, n = 5; ^*<i>P</i><0.05 compared with DMSO (vehicle) treated astrocytes]. (<b>B</b>) Representative immunoblot of p-PKA substrate in astrocytes treated with either DMSO or H89 for 24 hr. Phosphorylation level was normalized against β-Actin protein, the analysis is shown in the lower panel (n = 5; ^*<i>P</i><0.05). Glycogen levels in astrocytes treated with H89 (<b>C</b>) and Rp-8-Cl-cAMPs (<b>D</b>) (mean ±SD, n = 5; ^*<i>P</i><0.05). (<b>E</b>) Immunoblot of pGP in H89 treated astrocytes. Quantification, shown in the lower panel, was performed using total GP protein. Data are represented as mean ±SD, n = 5; ^*<i>P</i><0.05. (<b>F</b>) GP activity in H89 treated astrocytes. Data are mean ±SD, n = 5; ^*<i>P</i><0.05 vs DMSO treated astrocytes. (^*<i>P</i> =  Student's t-test).</p

Primers used in real-time RT-PCR.

<p>Primers used in real-time RT-PCR.</p

Glycogen is accumulated in stroke-affected brain.

<p>(<b>A</b>) A representative sample image generated from an unstained section collected between 1.2 mm to −2.8 mm bregma from an ET-1-induced stroke rat brain. Stroke-induced damage is shown to be located predominately around the site of ET-1-induced vasoconstriction, referred to as the ‘core' ischemic infarct (arrow sign) in the ipsilateral hemisphere. (<b>B</b>) Glycogen concentration in contralateral and ipsilateral hemispheres of control (sham) and stroke brains. Values are expressed as mean ±SD, n = 5 rats for each group. ^#<i>P</i> and ^*<i>P</i><0.05 compared to sham ipsilateral and stroke contralateral hemispheres, respectively. Expression levels of GBE (<b>C</b>) and GS (<b>D</b>) in contralateral and ipsilateral hemispheres of sham and stroke rat brains between 1 hr and 24 hr. Expression levels were normalized against β-Actin (middle panel). Quantification of this data is shown in the lower panel. Data are mean ±SD, n = 5. (<b>E</b>) GS activity in sham and stroke rat brains at different time points post-stroke. Data are represented as mean ±SD; n = 5. (^*<i>P</i>  =  Student's t-test and ^#<i>P</i> =  one-way ANOVA followed by Tukey's <i>post hoc</i> test).</p

Glycogen is accumulated in astrocytes during hypoxia.

<p>(<b>A</b>) Glycogen levels in rat primary cerebellar astrocytes and cortical neurons. Data are expressed as mean ±SD of 5 independent cultures. (<b>B</b>) Glycogen levels in rat cerebellar astrocytes kept under hypoxic conditions for 0 (control), 1, 3, 6 and 24 hr as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097570#s2" target="_blank">Materials and Methods</a> section. Glycogen levels were measured following 24 hr incubation in normoxic condition. Values are expressed as mean ±SD, n = 5; ^#<i>P</i><0.05 vs control astrocytes. (<b>C</b>) PKA activity in astrocytes exposed to hypoxic conditions. (Data are mean ±SD, n = 5; ^#<i>P</i><0.05 compared with control astrocytes). (<b>D</b>) Representative immunoblot of p-PKA substrate immunoblot analysis in astrocytes maintained in hypoxic conditions for the indicated time periods. Phosphorylation levels were normalized against β-Actin protein, the analysis is shown in the lower panel (mean ±SD, n = 5; ^*<i>P</i><0.05). (<b>E)</b> Representative immunoblot of pGP in astrocytes incubated under hypoxic conditions. Quantification of phosphorylation levels was performed using the total level of GP protein. Quantification data (lower panel) are represented as mean ±SD, n = 5; ^*<i>P</i><0.05. (<b>F</b>) GP activity in astrocytes during hypoxia. Data are mean ±SD, n = 5; ^#<i>P</i><0.05 vs control astrocytes. (<b>G</b>) Representative immunoblot showing expression level of GDE in astrocytes exposed to hypoxic condition for indicated time points. Expression of GDE was normalized against β-Actin protein. Data are represented as mean ±SD, n = 5;^*<i>P</i><0.05 vs control astrocytes. GS protein expression (<b>H</b>) and GS enzymatic activity (<b>I</b>) in hypoxic astrocytes. Data are represented as mean ±SD, n = 5. (^*<i>P</i> =  Student's t-test and ^#<i>P</i> =  one-way ANOVA followed by Tukey's <i>post hoc</i> test).</p

Signaling model of PKA's involvement in impaired glycogen metabolism during ischemic stroke.

<p>Within astrocytes under physiological conditions, the binding of an agonist to its receptor leads to the activation of G-proteins (α, β and γ subunits), which in turn activate adenylyl cyclase (AC). Active AC then converts ATP to cAMP that binds to the regulatory subunits of tetrameric PKA. This releases free catalytic subunits that phosphorylate phosphorylase kinase b to convert it to active phosphorylase kinase a. Active phosphorylase kinase then phosphorylates GP<i>b</i> to GP<i>a</i> initiating glycogen breakdown to provide energy for associating neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097570#pone.0097570-Rossi1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097570#pone.0097570-Brown2" target="_blank">[59]</a>. Inhibition of PKA activity during ischemic conditions, however suppresses this downstream signaling cascade that is essential for glycogen metabolism leading to the accumulation of glycogen in astrocytes in the stroke-affected brain.</p

Primary and secondary antibodies used in immunoblots.

<p>Primary and secondary antibodies used in immunoblots.</p