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

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.


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Representative confocal images of Cx43 distribution under different conditions at 6 h after reperfusion (red, Cx43; green, GFAP; blue, Hoechst 33342; magnification was 400x, resp.). (a) Under normal cultivation conditions, Cx43 was mainly distributed in the cytoplasmic membrane, and its density was higher in the adjacent cell membrane joint. A small amount of scattered Cx43 has been circulated in the cytoplasm with a few particles distributed in the nucleus. (b) Under IR/OGDR conditions with physiological saline, Cx43 was distributed in the membrane, cytoplasm, and nucleus, with the highest content in the cytoplasm. These vesicles of different sizes and shapes were observed in the cytoplasm. The total number of particles of this group was almost the same as that of the normal group (P = 0.761), but the cytoplasm particles of this group were significantly more than those of the normal group (P = 0.019). (c) Under IR/OGDR conditions with CBX intervention, Cx43 was distributed in the membrane, cytoplasm, and nucleus but mainly in the cytoplasm. There was no significant difference between the CBX intervention and saline control group (P = 0.217).
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fig2: Representative confocal images of Cx43 distribution under different conditions at 6 h after reperfusion (red, Cx43; green, GFAP; blue, Hoechst 33342; magnification was 400x, resp.). (a) Under normal cultivation conditions, Cx43 was mainly distributed in the cytoplasmic membrane, and its density was higher in the adjacent cell membrane joint. A small amount of scattered Cx43 has been circulated in the cytoplasm with a few particles distributed in the nucleus. (b) Under IR/OGDR conditions with physiological saline, Cx43 was distributed in the membrane, cytoplasm, and nucleus, with the highest content in the cytoplasm. These vesicles of different sizes and shapes were observed in the cytoplasm. The total number of particles of this group was almost the same as that of the normal group (P = 0.761), but the cytoplasm particles of this group were significantly more than those of the normal group (P = 0.019). (c) Under IR/OGDR conditions with CBX intervention, Cx43 was distributed in the membrane, cytoplasm, and nucleus but mainly in the cytoplasm. There was no significant difference between the CBX intervention and saline control group (P = 0.217).

Mentions: We examined the distribution of Cx43 with a laser-scanning confocal microscope under normal and IR/OGDR conditions. As shown in Figures 2(a) and 2(b), it was found that there was no difference in the total amount of Cx43 (P = 0.761) between the normal and IR/OGDR groups; however, the cytoplasm particles of the IR/OGDR group were significantly greater in number than that of the normal group (P = 0.019). Furthermore, some vesicles of different sizes and shapes appeared in the cytoplasm of the IR/OGDR group, which were obviously distinct from the endoplasmic reticulum and Golgi apparatus transport vesicles in the normal group. Under IR/OGDR conditions with CBX intervention, which was shown in Figure 2(c), Cx43 redistribution was not affected (P = 0.217) compared with the saline control group.


Evaluation of Connexin 43 Redistribution and Endocytosis in Astrocytes Subjected to Ischemia/Reperfusion or Oxygen-Glucose Deprivation and Reoxygenation
Representative confocal images of Cx43 distribution under different conditions at 6 h after reperfusion (red, Cx43; green, GFAP; blue, Hoechst 33342; magnification was 400x, resp.). (a) Under normal cultivation conditions, Cx43 was mainly distributed in the cytoplasmic membrane, and its density was higher in the adjacent cell membrane joint. A small amount of scattered Cx43 has been circulated in the cytoplasm with a few particles distributed in the nucleus. (b) Under IR/OGDR conditions with physiological saline, Cx43 was distributed in the membrane, cytoplasm, and nucleus, with the highest content in the cytoplasm. These vesicles of different sizes and shapes were observed in the cytoplasm. The total number of particles of this group was almost the same as that of the normal group (P = 0.761), but the cytoplasm particles of this group were significantly more than those of the normal group (P = 0.019). (c) Under IR/OGDR conditions with CBX intervention, Cx43 was distributed in the membrane, cytoplasm, and nucleus but mainly in the cytoplasm. There was no significant difference between the CBX intervention and saline control group (P = 0.217).
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fig2: Representative confocal images of Cx43 distribution under different conditions at 6 h after reperfusion (red, Cx43; green, GFAP; blue, Hoechst 33342; magnification was 400x, resp.). (a) Under normal cultivation conditions, Cx43 was mainly distributed in the cytoplasmic membrane, and its density was higher in the adjacent cell membrane joint. A small amount of scattered Cx43 has been circulated in the cytoplasm with a few particles distributed in the nucleus. (b) Under IR/OGDR conditions with physiological saline, Cx43 was distributed in the membrane, cytoplasm, and nucleus, with the highest content in the cytoplasm. These vesicles of different sizes and shapes were observed in the cytoplasm. The total number of particles of this group was almost the same as that of the normal group (P = 0.761), but the cytoplasm particles of this group were significantly more than those of the normal group (P = 0.019). (c) Under IR/OGDR conditions with CBX intervention, Cx43 was distributed in the membrane, cytoplasm, and nucleus but mainly in the cytoplasm. There was no significant difference between the CBX intervention and saline control group (P = 0.217).
Mentions: We examined the distribution of Cx43 with a laser-scanning confocal microscope under normal and IR/OGDR conditions. As shown in Figures 2(a) and 2(b), it was found that there was no difference in the total amount of Cx43 (P = 0.761) between the normal and IR/OGDR groups; however, the cytoplasm particles of the IR/OGDR group were significantly greater in number than that of the normal group (P = 0.019). Furthermore, some vesicles of different sizes and shapes appeared in the cytoplasm of the IR/OGDR group, which were obviously distinct from the endoplasmic reticulum and Golgi apparatus transport vesicles in the normal group. Under IR/OGDR conditions with CBX intervention, which was shown in Figure 2(c), Cx43 redistribution was not affected (P = 0.217) compared with the saline control group.

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.


Related in: MedlinePlus