Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture

Jorge E. Contreras, Helmut A. Sánchez, Eliseo Eugenin, Dina Speidel, Martin Theis, Klaus Willecke, Feliksas F. Bukauskas, Michael V L Bennett, Juan C. Sáez

Research output: Contribution to journalArticle

423 Citations (Scopus)

Abstract

Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18α-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La3+ also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.

Original languageEnglish (US)
Pages (from-to)495-500
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number1
DOIs
StatePublished - Jan 8 2002
Externally publishedYes

Fingerprint

Connexin 43
Gap Junctions
Astrocytes
Communication
Coloring Agents
Cell Death
Ischemia
Lipoxygenases
Glycyrrhetinic Acid
Octanols
Free Radical Scavengers
Ethidium
Inhibition (Psychology)
Dextrans
L-Lactate Dehydrogenase
Arachidonic Acid
Adenosine Triphosphate
Cell Membrane
Ions
Membranes

Keywords

  • Astroglia
  • Cx43
  • Dye coupling
  • Dye uptake
  • Phosphorylation

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. / Contreras, Jorge E.; Sánchez, Helmut A.; Eugenin, Eliseo; Speidel, Dina; Theis, Martin; Willecke, Klaus; Bukauskas, Feliksas F.; Bennett, Michael V L; Sáez, Juan C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 1, 08.01.2002, p. 495-500.

Research output: Contribution to journalArticle

Contreras, Jorge E. ; Sánchez, Helmut A. ; Eugenin, Eliseo ; Speidel, Dina ; Theis, Martin ; Willecke, Klaus ; Bukauskas, Feliksas F. ; Bennett, Michael V L ; Sáez, Juan C. / Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 1. pp. 495-500.
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AU - Speidel, Dina

AU - Theis, Martin

AU - Willecke, Klaus

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AU - Sáez, Juan C.

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