Ca2+ regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca2+ ([Ca2+](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca2+](i) was in the 500nM range but the responses disappeared with larger [Ca2+](i) transients. Ca2+-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase 11, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca2+-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca2+](i), triggers hemichannel opening, not by a direct action of Call on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca2+](i), and acting to restrain cellular ATP loss. (C) 2009 Elsevier Ltd. All rights reserved.