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Evaluation of Connexin 43 Redistribution and Endocytosis in Astrocytes Subjected to Ischemia/Reperfusion or Oxygen-Glucose Deprivation and Reoxygenation

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ABSTRACT

Connexin 43 (Cx43) is the major component protein in astrocytic gap junction communication. Recent studies have shown the cellular processes of gap junction internalization and degradation, but many details remain unknown. This study investigated the distribution of Cx43 and its mechanism after ischemic insult. Astrocyte culture system and a model of ischemia/reperfusion (IR) or oxygen-glucose deprivation and reoxygenation (OGDR) were established. Cx43 distribution was observed by laser scanning confocal microscopy under different cultivation conditions. Western blot and RT-PCR assays were applied to quantify Cx43 and MAPRE1 (microtubule-associated protein RP/EB family member 1) expression at different time points. The total number of Cx43 was unchanged in the normal and IR/OGDR groups, but Cx43 particles in the cytoplasm of the IR/OGDR group were significantly greater than that of the normal group. Particles in the cytoplasm were significantly fewer after endocytosis was blocked by dynasore. There was no difference among the groups at each time point regarding protein or gene expression of MAPRE1. We concluded that internalization of Cx43 into the cytoplasm occurred during ischemia, which was partially mediated through endocytosis, not by the change of Cx43 quantity. Moreover, internalization was not related to microtubule transport.

No MeSH data available.


Representative western blot of Cx43 in the normal and at different time points of IR/OGDR groups. (a) The first stripe was Cx43 protein; the other was β-actin as the internal stripe. (b) Cx43 protein relative expression. There was no significant difference among the groups at each time point (P = 0.307). (c) Representative graph of fluorescence intensity value of Cx43 in normal and IR/OGDR 6 h (saline/CBX) groups. There was no significant difference among the three groups (P = 0.208).
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fig1: Representative western blot of Cx43 in the normal and at different time points of IR/OGDR groups. (a) The first stripe was Cx43 protein; the other was β-actin as the internal stripe. (b) Cx43 protein relative expression. There was no significant difference among the groups at each time point (P = 0.307). (c) Representative graph of fluorescence intensity value of Cx43 in normal and IR/OGDR 6 h (saline/CBX) groups. There was no significant difference among the three groups (P = 0.208).

Mentions: We made the quantitative analysis of Cx43 in the normal and at different time points of IR/OGDR groups. As shown in Figures 1(a) and 1(b), the total amount of Cx43 was unchanged under normal and IR/OGDR conditions (P = 0.307). We analyzed the fluorescence intensity of Cx43 in astrocytes by Image-Pro Plus 7.0 Software and drew the graph by GraphPad Prism 6.0 software. As shown in Figure 1(c), the total level of Cx43 of astrocytes was unchanged among groups; only distribution occurred. This was consistent with the conclusion of Figures 1(a) and 1(b).


Evaluation of Connexin 43 Redistribution and Endocytosis in Astrocytes Subjected to Ischemia/Reperfusion or Oxygen-Glucose Deprivation and Reoxygenation
Representative western blot of Cx43 in the normal and at different time points of IR/OGDR groups. (a) The first stripe was Cx43 protein; the other was β-actin as the internal stripe. (b) Cx43 protein relative expression. There was no significant difference among the groups at each time point (P = 0.307). (c) Representative graph of fluorescence intensity value of Cx43 in normal and IR/OGDR 6 h (saline/CBX) groups. There was no significant difference among the three groups (P = 0.208).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5382357&req=5

fig1: Representative western blot of Cx43 in the normal and at different time points of IR/OGDR groups. (a) The first stripe was Cx43 protein; the other was β-actin as the internal stripe. (b) Cx43 protein relative expression. There was no significant difference among the groups at each time point (P = 0.307). (c) Representative graph of fluorescence intensity value of Cx43 in normal and IR/OGDR 6 h (saline/CBX) groups. There was no significant difference among the three groups (P = 0.208).
Mentions: We made the quantitative analysis of Cx43 in the normal and at different time points of IR/OGDR groups. As shown in Figures 1(a) and 1(b), the total amount of Cx43 was unchanged under normal and IR/OGDR conditions (P = 0.307). We analyzed the fluorescence intensity of Cx43 in astrocytes by Image-Pro Plus 7.0 Software and drew the graph by GraphPad Prism 6.0 software. As shown in Figure 1(c), the total level of Cx43 of astrocytes was unchanged among groups; only distribution occurred. This was consistent with the conclusion of Figures 1(a) and 1(b).

View Article: PubMed Central - PubMed

ABSTRACT

Connexin 43 (Cx43) is the major component protein in astrocytic gap junction communication. Recent studies have shown the cellular processes of gap junction internalization and degradation, but many details remain unknown. This study investigated the distribution of Cx43 and its mechanism after ischemic insult. Astrocyte culture system and a model of ischemia/reperfusion (IR) or oxygen-glucose deprivation and reoxygenation (OGDR) were established. Cx43 distribution was observed by laser scanning confocal microscopy under different cultivation conditions. Western blot and RT-PCR assays were applied to quantify Cx43 and MAPRE1 (microtubule-associated protein RP/EB family member 1) expression at different time points. The total number of Cx43 was unchanged in the normal and IR/OGDR groups, but Cx43 particles in the cytoplasm of the IR/OGDR group were significantly greater than that of the normal group. Particles in the cytoplasm were significantly fewer after endocytosis was blocked by dynasore. There was no difference among the groups at each time point regarding protein or gene expression of MAPRE1. We concluded that internalization of Cx43 into the cytoplasm occurred during ischemia, which was partially mediated through endocytosis, not by the change of Cx43 quantity. Moreover, internalization was not related to microtubule transport.

No MeSH data available.