Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.
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ABSTRACT:
SUBMITTER: Yeung SM
PROVIDER: S-EPMC1148596 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected.
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Project description:Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.
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Project description:The amino acid sequence of the rat adenosine A2A receptor and the atomic coordinates of bacteriorhodopsin were combined to generate a three-dimensional model for the adenosine A2A receptor. This model consists of seven amphipathic alpha-helices, forming a pore that is rather hydrophilic compared to the hydrophobic outside of the protein. Subsequently, a highly potent and selective ligand for this receptor, 2-(cyclohexylmethylidinehydrazino)adenosine (SHA 174), was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Subsequently, the deduced binding site was used to model other potent ligands, including 8-(3-chlorostyryl)caffeine, a new A2-selective antagonist, that could all be accommodated consistent with earlier biochemical and pharmacological findings. Finally, some thoughts on how adenosine receptor activation might proceed are put forward, based on structural analogies with the enzyme family of serine proteases.
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Project description:1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50-80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K(m)(ATP) 0.60mm, K(m)(glucose) 0.042mm; peak II, K(m)(ATP) 0.66mm, K(m)(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K(m)(ATP) 0.56mm, K(m)(glucose) 0.048mm; peak II, K(m)(ATP) 0.68mm, K(m)(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.
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Project description:Traumatic brain injury (TBI) initiates a complex sequence of destructive and neuroprotective cellular responses. The initial mechanical injury is followed by an extended time period of secondary brain damage. Due to the complicated pathological picture a better understanding of the molecular events occurring during this secondary phase of injury is needed. This study was aimed at analysing gene expression patterns following cerebral cortical contusion in rat using high throughput microarray technology with the goal of identifying genes involved in an early and in a more delayed phase of trauma, as genomic responses behind secondary mechanisms likely are time-dependent. Keywords: Traumatic brain injury, genomic response
2005-10-01 | GSE3396 | GEO
Project description:BACKGROUND AND PURPOSE:Inactivation of the gene for adenosine A2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell-specific pathogenic effects of A2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A2A receptors after thromboembolic stroke improves post-stroke outcomes via down-regulation of inflammation. EXPERIMENTAL APPROACH:Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post-stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood-brain barrier (BBB) leakage, and oedema assessment. Anti-inflammatory effects of silencing the gene for A2A receptors or pharmacological antagonism of these receptors were assessed in vitro. KEY RESULTS:Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a-/- ) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2a?VEC ) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down-regulating cleaved caspase 1 and IL-1? expression. CONCLUSIONS AND IMPLICATIONS:Specific inactivation of endothelial A2A receptors mitigated ischaemic brain injury and improved post-stroke outcomes, at least partly, through anti-inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke.
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Project description:Guanyl-5'-yl imidodiphosphate (p[NH]ppG) stimulated a rapid phospholipase C-mediated breakdown of exogenously added phosphatidylinositol 4,5-bisphosphate (PIP2) in rat cerebral-cortical membranes, with half-maximal activation at approx. 33 microM. NaF stimulated phospholipase C activity, with half-maximal activation at 0.5 mM. Stimulation of phospholipase C activity by NaF exhibited pH optima at approx. 5.5 and 7.0, with the stimulatory activity at pH 7.0 greater than that at pH 5.5. With p[NH]ppG, only stimulation at pH 7.0 was observed. Neither p[NH]ppG nor NaF stimulated hydrolysis of added phosphatidylinositol (PI) or phosphatidylinositol 4-phosphate (PIP). Mg2+ (0.5 mM) potentiated p[NH]ppG-stimulated breakdown of PIP2. Ca2+ increased basal and p[NH]ppG-stimulated breakdown of PIP2. PI breakdown was stimulated only by high Ca2+ concentrations and was unaffected by p[NH]ppG at any Ca2+ concentration examined. These results indicate that, in cerebral-cortical membranes, activation of phospholipase C by guanine nucleotides or fluoride directly increases a phospholipase C activity which specifically hydrolyses PIP2.
| S-EPMC1147949 | biostudies-other
Project description:The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes.
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Project description:BackgroundNeurocognitive investigations suggest that conscious sensory perception depends on recurrent neuronal interactions among sensory, parietal, and frontal cortical regions, which are suppressed by general anesthetics. The purpose of this work was to investigate if local interactions in sensory cortex are also altered by anesthetics. The authors hypothesized that desflurane would reduce recurrent neuronal interactions in cortical layer-specific manner consistent with the anatomical disposition of feedforward and feedback pathways.MethodsSingle-unit neuronal activity was measured in freely moving adult male rats (268 units; 10 animals) using microelectrode arrays chronically implanted in primary and secondary visual cortex. Layer-specific directional interactions were estimated by mutual information and transfer entropy of multineuron spike patterns within and between cortical layers three and five. The effect of incrementally increasing and decreasing steady-state concentrations of desflurane (0 to 8% to 0%) was tested for statistically significant quadratic trend across the successive anesthetic states.ResultsDesflurane produced robust, state-dependent reduction (P = 0.001) of neuronal interactions between primary and secondary visual areas and between layers three and five, as indicated by mutual information (37 and 41% decrease at 8% desflurane from wakeful baseline at [mean ± SD] 0.52 ± 0.51 and 0.53 ± 0.51 a.u., respectively) and transfer entropy (77 and 78% decrease at 8% desflurane from wakeful baseline at 1.86 ± 1.56 a.u. and 1.87 ± 1.67 a.u., respectively). In addition, a preferential suppression of feedback between secondary and primary visual cortex was suggested by the reduction of directional index of transfer entropy overall (P = 0.001; 89% decrease at 8% desflurane from 0.11 ± 0.18 a.u. at baseline) and specifically, in layer five (P = 0.001; 108% decrease at 8% desflurane from 0.12 ± 0.19 a.u. at baseline).ConclusionsDesflurane anesthesia reduces neuronal interactions in visual cortex with a preferential effect on feedback. The findings suggest that neuronal disconnection occurs locally, among hierarchical sensory regions, which may contribute to global functional disconnection underlying anesthetic-induced unconsciousness.
| S-EPMC8190972 | biostudies-literature
Project description:BackgroundBilayer collagen membranes are routinely used in guided bone/tissue regeneration to serve as osteoconductive scaffolds and prevent the invasion of soft tissues. It is recommended to place the membranes with their dense layer towards the soft tissue and their porous layer towards the bony defect area. However, evidence supporting this recommendation is lacking. This study aimed to determine whether the alignment of bilayer collagen membranes has an effect on bone regeneration.MethodsIn two groups of ten male Sprague-Dawley rats each, a 5-mm calvarial defect was created. Thereafter, the defect was randomly covered with a bilayer, resorbable, pure type I and III collagen membrane placed either regularly or upside-down (i.e., dense layer towards bone defect). After 4 weeks of healing, micro-computed tomography (μCT), histology, and histomorphometry of the inner cylindrical region of interest (4.5 mm in diameter) were performed to assess new bone formation and the consolidation of the collagen membrane in the defect area.ResultsQuantitative μCT showed similar bone volume (median 8.0 mm3, interquartile range 7.0-10.0 vs. 6.2 mm3, 4.3-9.4, p = 0.06) and trabecular thickness (0.21 mm, 0.19-0.23 vs. 0.18 mm, 0.17-0.20, p = 0.03) between upside-down and regular placement, both leading to an almost complete bony coverage. Histomorphometry showed comparable new bone areas between the upside-down and regularly placed membranes, 3.9 mm2 (2.7-5.4) vs. 3.8 mm2 (2.2-4.0, p = 0.31), respectively. Both treatment groups revealed the same regeneration patterns and spatial distribution of bone with and without collagen fibers, as well as residual collagen fibers.ConclusionsOur data support the osteoconductive properties of collagen membranes and suggest that bone regeneration is facilitated regardless of membrane layer alignment.
| S-EPMC8180471 | biostudies-literature