1,721,001 research outputs found
Identification and functional binding analysis of GPR23/! LPA4 as a candidate G protein-coupled receptor for Guanosine.
No full textSeveral studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine
Distinct pattern of Connexin gene expression during skeletal muscle regeneration in the adult rat.
No full textAim: The aim of present work was to test the hypothesis that Cx37, Cx39, Cx40, Cx43 and Cx45 expression could be regulated in adult regenerating skeletal muscle in response to injury promoting activation of satellite cells involved in myofibers repair and regeneration.
Methods: Using in situ hybridization and immunohistochemistry procedures we examined the spatial and temporal expression pattern of above listed connexins in the regenerating gastrocnemious muscle following a mechanical injury.
Results: Cx43 and Cx45 mRNA were up-regulated very early, by 3 hour following muscle injury, and were localised in satellite cells, M-cadherin positive cells, distributed around the area of lesion. Three days after lesion a large number of Cx43 and Cx45 mRNA labelled cells were found inside the area of lesion where they become myoblasts, myogenin positive, and participate to muscle regeneration. By contrast, Cx39, not expressed in control muscle, appears in myogenin positive cells 3 days following muscle lesion and its expression was restricted to the area of lesion. Cx39 mRNA was mainly localised in myoblast myogenin positive cells forming clusters or rows of closely apposed cell nuclei committed to form myotubes. Cx40 mRNA labelled cells were observed within 24 hours from injury in the endothelial cells of blood vessels around the area of lesion. After 3 days these cells were localised inside the area of lesion where they co-express myogenin and start to form myotubes. By contrast, Cx37 mRNA expression appeared by 24 hrs from lesion and never was colocalised in myogenin positive cells, but it was involved in new blood vessels formation. All transcripts examined reached a peak of expression between 7-9 days from injury and then progressively declined, being undetectable at 4-5 weeks from injury.
Conclusion:
Taken together these results support the suggestion that, in regenerating skeletal muscle several connexins may be differentially involved in communication of myogenic committed cells during the process of cell proliferation, aggregation and fusion to form new myotubes
Regulation of Connexin gene expression during skeletal muscle regeneration in the adult rat.
No full textIn the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24 h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesion-forming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24 h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofiber
Characterization of metabotropic glutamate receptors negatively linked to adenylyl cyclase in brain slices.
No full textGuanosine-Induced Antiproliferative Effects Are Modulated by GPCR Expression in Human Glioma and Melanoma Cell Lines.
No full textGuanosine-Induced Antiproliferative Effects Are Modulated by GPCR Expression in Human Glioma and Melanoma Cell Lines
Antiproliferative effects of guanine-based purines and expression of a candidate receptor.
No full text
Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures
No full textDNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in cultur
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