Connexin 43 as a Mediator of Androgen and Estrogen-induced Protection against Oxidative Stress in Astrocytes

Purpose: Connexin 43 (Cx43) is a transmembrane protein highly expressed in astrocytes, whose expression and/or channel permeability can impact cell viability in the presence of oxidative stress. Androgens and estrogens are also known to protect against oxidative stress. However, it remains unknown whether these hormones alter Cx43 channel expression or permeability as a mediator of their protective effects. Here, we propose that the androgen, dihydrotestosterone (DHT) and the estrogens, E2 and 3betadiol (the latter being a metabolite of DHT), protect against oxidative stress in astrocytes through the regulation of Cx43 expression and/or permeability. Hypothesis: Reducing the permeability of Cx43 gap junctions or Cx43 hemichannels enhances the protective efficacy of E2, DHT, or 3betadiol against oxidative stress in cerebral cortical astrocytes. Methods: Using cortical astrocytes from neonatal female C57/Bl6 mice and the astrocytic C6 glioma cell line, we assessed changes in Cx43 mRNA expression, Cx43 permeability, and cell viability following treatment with 100 nM E2, DHT, or the DHT metabolite, 5-alpha-androstane-3-beta,17-beta-diol (3betadiol), and compared it to the effect of the vehicle (DMSO) control, in the presence or absence of the metabolic and oxidative stressor, iodoacetic acid. Changes in Cx43 mRNA expression relative to GAPDH were assessed by RTPCR; The MTT assay was used to assess cell viability under conditions of metabolic/oxidative stress, with or without the pharmacological manipulation of Cx43 channels; changes in Cx43 hemichannel permeability were assessed using the Ethidium Bromide dye uptake assay. Results & Conclusions: While only E2 transiently increased astrocyte Cx43 mRNA expression, E2, DHT, 3betadiol each blocked IAA induced increase in Cx43 hemichannel permeability to an extent similar to the hemichannel antagonist Boldine. Given that Boldine-induced hemichannel blockade significantly protected cells from oxidative stress, hemichannel blockade may also be a component of these steroids’ protective mechanisms. These data indicate that enhanced Cx43 hemichannel permeability not only inhibits the protective effects of E2 or DHT, but that the protective effects of E2 and DHT may be mediated, at least in part, by the regulation of Cx43 hemichannel permeability.