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Up-regulation of TREK-2 potassium channels in cultured astrocytes requires de novo protein synthesis: relevance to localization of TREK-2 channels in astrocytes after transient cerebral ischemia.

Rivera-Pagán AF, Rivera-Aponte DE, Melnik-Martínez KV, Zayas-Santiago A, Kucheryavykh LY, Martins AH, Cubano LA, Skatchkov SN, Eaton MJ - PLoS ONE (2015)

Bottom Line: Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions.By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions.Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.

No MeSH data available.


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TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.Cortical astrocytes were treated with different protein synthesis inhibitors (1μg/mL cycloheximide, 300nM emetine, 5μg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).
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pone.0125195.g002: TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.Cortical astrocytes were treated with different protein synthesis inhibitors (1μg/mL cycloheximide, 300nM emetine, 5μg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).

Mentions: Increases in protein synthesis may occur together with or independent from changes in mRNA synthesis [27–29]. Since we observed no effect of hypoxia/hypoglycemic conditions on TREK-2 mRNA levels, we examined the possibility that up-regulation of TREK-2 channels is due to De novo protein synthesis. If increased TREK-2 protein levels require De novo synthesis of TREK-2, then inhibitors of protein synthesis should obviate differences in TREK-2 protein levels between control astrocytes and astrocytes exposed to hypoxia/hypoglycemia. To assess protein synthesis inhibition, astrocytes were treated with different protein synthesis inhibitors (300nM emetine, 1μg/mL cycloheximide, or 5μg/mL puromycin) and then exposed to control or hypoxia/hypoglycemic conditions for 24 hours. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. We found that TREK-2 up-regulation was blocked by pharmacological blockade using protein synthesis inhibitors (Fig 2). In control cells, ischemia increased TREK-2 levels measured as relative chemiluminescence by 1.76 fold ± 0.15 SEM. The relative chemiluminescence determined for the ischemic treatment in the presence of protein synthesis inhibitors was 1.15 ± 0.10, 1.04 ±. 074, 1.23 ± 0.22 SEM for emetine, cycloheximide and puromycin, respectively. This indicates that up-regulation of TREK-2 protein requires De novo protein synthesis. An additional alternative possibility is that ischemia could decrease the rate of TREK-2 protein degradation, which would also result in an increase in protein content. Therefore, we tested this possibility.


Up-regulation of TREK-2 potassium channels in cultured astrocytes requires de novo protein synthesis: relevance to localization of TREK-2 channels in astrocytes after transient cerebral ischemia.

Rivera-Pagán AF, Rivera-Aponte DE, Melnik-Martínez KV, Zayas-Santiago A, Kucheryavykh LY, Martins AH, Cubano LA, Skatchkov SN, Eaton MJ - PLoS ONE (2015)

TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.Cortical astrocytes were treated with different protein synthesis inhibitors (1μg/mL cycloheximide, 300nM emetine, 5μg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401746&req=5

pone.0125195.g002: TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.Cortical astrocytes were treated with different protein synthesis inhibitors (1μg/mL cycloheximide, 300nM emetine, 5μg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).
Mentions: Increases in protein synthesis may occur together with or independent from changes in mRNA synthesis [27–29]. Since we observed no effect of hypoxia/hypoglycemic conditions on TREK-2 mRNA levels, we examined the possibility that up-regulation of TREK-2 channels is due to De novo protein synthesis. If increased TREK-2 protein levels require De novo synthesis of TREK-2, then inhibitors of protein synthesis should obviate differences in TREK-2 protein levels between control astrocytes and astrocytes exposed to hypoxia/hypoglycemia. To assess protein synthesis inhibition, astrocytes were treated with different protein synthesis inhibitors (300nM emetine, 1μg/mL cycloheximide, or 5μg/mL puromycin) and then exposed to control or hypoxia/hypoglycemic conditions for 24 hours. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. We found that TREK-2 up-regulation was blocked by pharmacological blockade using protein synthesis inhibitors (Fig 2). In control cells, ischemia increased TREK-2 levels measured as relative chemiluminescence by 1.76 fold ± 0.15 SEM. The relative chemiluminescence determined for the ischemic treatment in the presence of protein synthesis inhibitors was 1.15 ± 0.10, 1.04 ±. 074, 1.23 ± 0.22 SEM for emetine, cycloheximide and puromycin, respectively. This indicates that up-regulation of TREK-2 protein requires De novo protein synthesis. An additional alternative possibility is that ischemia could decrease the rate of TREK-2 protein degradation, which would also result in an increase in protein content. Therefore, we tested this possibility.

Bottom Line: Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions.By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions.Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.

No MeSH data available.


Related in: MedlinePlus