Blocked gap junctional coupling increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures.
Gap junctional communication is likely one means by which neurons can endure glutamate cytotoxicity associated with CNS insults (i.e. ischemia). To examine this neuroprotective role of gap junctions, we employed gap junctional blockers to neuronal and astrocytic co-cultures during exposure to a high concentration of extracellular glutamate. Co-cultures were treated with the blocking agents carbenoxolone (CBX; 25 microM), 18alpha-glycyrrhetinic acid (AGA; 10 microM), vehicle or the inactive blocking analogue glycyrrhizic acid (GZA; 25 microM). Twenty-four hours following the insult, cell mortality was analyzed and quantified by the release of lactate dehydrogenase (LDH) into the media, the cells' inability to exclude propidium iodide, and terminal dUTP nick end labeling (TUNEL). Measurement of LDH release revealed that the glutamate insult was detrimental to the co-cultures when gap junctions were blocked with CBX and AGA. Based on propidium iodide and TUNEL labeling, the glutamate insult caused significant cell death compared to sham vehicle and mortality was amplified in the presence of CBX and AGA. Since blockers were not themselves toxic and did not affect astrocytic uptake of glutamate, it is likely that blocked gap junctions lead to the increased glutamate cytotoxicity. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.
Ozog
Mark A
MA
Department of Anatomy & Cell Biology, The University of Western Ontario, London, Canada.
Siushansian
Ramin
R
Naus
Christian C G
CC
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015415:Biomarkers; D002229:Carbenoxolone / Q000737:chemistry / Q000494:pharmacology; D002450:Cell Communication / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology*; D006034:Glycyrrhetinic Acid / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism
D015415:Biomarkers; D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D007770:L-Lactate Dehydrogenase; D002229:Carbenoxolone; D006034:Glycyrrhetinic Acid