Gap‐junction blocker carbenoxolone differentially enhances NMDA‐induced cell death in hippocampal neurons and astrocytes in co‐culture

Abstract

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca 2+ level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca 2+ rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca 2+ rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pretreatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBXinduced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.

The P2X7 receptor–pannexin-1 complex decreases muscarinic acetylcholine receptor–mediated seizure susceptibility in mice

“…Like connexins, Panx1 and P2X7R contribute to Ca 2+ wave propagation by transfer of IP3 and ATP-induced ATP release independent of extracellular Ca 2+ (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Indeed, CBX accelerates NMDA-induced membrane depolarization with a concomitant rise in intracellular Ca 2+ in vitro (34). Although the high dosage of CBX was needed because of its partial exclusion by the blood-brain barrier (35,36), the present data also showed that CBX increased PILO-induced seizure susceptibility in WT mice.…”

Carbenoxolone Enhances TRAIL-Induced Apoptosis Through the Upregulation of Death Receptor 5 and Inhibition of Gap Junction Intercellular Communication in Human Glioma

“…7). It is possible that CBX treatment increased the production of ROS [43], which induced phosphorylation of Cx43 and the subsequent inhibition of GJ through MAPK signaling [50]. Along the same line, ROS-induced DR5 upregulation most probably occurs through activation of c-Jun N-terminal kinase ( JNK) [51], which increased the expression of DR5 and the subsequent recruitment of Fas-associated death domain to form the DISC [52] and activation of downstream active caspases.…”

“…Taken together, our results on the inhibition of dye transfer and downregulation of Cx43 in CBX-treated cells suggested that the enhancement in cell death observed upon treatment with CBX and TRAIL occurred through a GJ-dependent manner. However, it is difficult to rule out GJ-independent cell death because CBX was also shown to induce mitochondrial depolarization and oxidative stress through the production of reactive oxygen species (ROS) in neurons [43]. Generation of ROS subsequently activates mitogen-activated protein kinase (MAPK) and DR expression to initiate downstream death signals [44].…”

Detection of de- and hyperpolarization of mitochondria of cultured astrocytes and neurons by the cationic fluorescent dye rhodamine 123

“…This we have already seen for the analysis of the mitochondrial potential as a result of P2Y receptor activation in astrocytes and neurons and of gap junction inhibition in hippocampal mixed cultures (Zündorf et al, 2007).…”