Regulation of connexin43 and astrocytic gap junctional intercellular communication in the central nervous system
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The objective of this project was to determine the functional states of astrocytic gap junctions under physiological and pathological conditions by analyzing the expression, localization, phosphorylation and immunorecognition of a major astrocytic gap junction protein, connexin43 (Cx43). These studies were aided by antibody 13-8300 that selectively recognizes the non-phosphorylated form, but not the multiply phosphorylated forms of Cx43 in several cell types in vitro and in vivo. The failure of 13-8300 to recognize phosphorylated Cx43 is likely due to blockade of phosphate groups, suggesting that the epitope recognized by 13-8300 contains an early phosphorylation site. Non-phosphorylated Cx43 was seen primarily in he cytoplasm, whereas phosphorylated Cx43 was seen at gap junctions as well as in the cytoplasm. Sciatic nerve stimulation induced preferential dephosphorylation of junctional Cx43 in spinal cord astrocytes, suggesting that junctional Cx43 is more vulnerable to dephosphorylation than cytoplasmic Cx43 and that astrocytic gap junctional intercellular communication (GJIC) can be regulated by neuronal activity. Dephosphorylation of astrocytic Cx43 was also seen in ischemic rat brain. Thus, Cx43 dephosphorylation may represent a common mechanism of the regulation of astrocytic GJIC under physiological and pathological conditions. Mild brain ischemia induced rapid and reversible Cx43 dephosphorylation, whereas severe ischemia led to total removal of Cx43 gap junctions in the lesion center surrounded by a zone of dephosphorylated Cx43 in the penumbral region, indicating distinct functional states of astrocytes in these regions. Reactive astrocytes appear in injured rat CNS at a later survival time and in the vicinity of senile and amyloid plaques in human Alzheimer's disease brain. These cells may express Cx43 and form gap junctions, indicating the re-establishment of GJIC in damaged tissue. Chemical hypoxia induced immediate reduction of astrocytic GJIC in vitro, which was followed by massive Cx43 dephosphorylation. Inhibition of Cx43 dephosphorylation by calcineurin inhibitors led to partial preservation of GJIC, indicating that Cx43 dephosphorylation is related to further reduction of GJIC in hypoxic astrocytes. These results suggest that astrocytes regulate GJIC in a programmed manner under physiological and pathological conditions.