SHORT COMMUNICATION Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition W. E. I. Li and J. I. Nagy Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7 Keywords: calcineurin, dye-coupling, glia, hypoxia, protein phosphatase. Abstract The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti-Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30min after hypoxia. This was preceded by a 77%reductioninGJIC15minafterhypoxia,indicatingthatreducedGJICoccurspriortoCx43dephosphorylation.Hypoxiacauseda reduction in punctate immunostaining (epitope masking) at cell±cell contacts with one anti-Cx43 antibody, and increased labelling with another antibody (13±8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)-1 and PP-2A with okadaic acid or calyculin A had little effect on hypoxia-induced Cx43 dephosphorylation. Inhibition of PP-2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro aresimilartothoseseenafterischaemia in vivo,andthatinhibition of protein phosphatase protects astrocytes from hypoxia-induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state. Introduction Astrocytes in the central nervous system express the gap junctional proteins connexin43 (Cx43) and connexin30 (Cx30), and are extensively linked by gap junctions (Nagy & Rash 2000). Junctional channels allow cell-to-cell passage of ions and small molecules, which is thought to help coordinate the activity of astrocytes (Nagy & Dermietzel, 2000). Astrocytic gap junctional intercellular communication (GJIC) may be a dynamic process subject to regulation by a variety of factors (Giaume & McCarthy, 1996). One mechanism for regulation is connexin phosphorylation, which has been extensively studied in cells coupled by Cx43 (Bruzzone etal., 1996; Goodenough etal., 1996). Several protein kinases act on Cx43 and a number of serine phosphorylation sites havebeenidenti®edinthisconnexin(Warn-Cramer etal.,1996;Saez etal., 1997). Cx43 phosphorylation is correlated with alterations in junctionalcoupling(Warn-Cramer etal.,1998)anditsdephosphoryl- ation is correlated with either decreased GJIC (Godwin etal., 1993; Oelze etal., 1995) or increased channel conductance (Moreno etal., 1994). In cultured astrocytes exposed to chemical hypoxia, Cx43 dephosphorylation occurred in conjunction with reduced GJIC (Cotrina etal., 1998). We have reported that the vast proportion of Cx43 in brain and spinal cord is phosphorylated in vivo and that it undergoes dephosphorylation during brain extraction, in response to ischaemic injury and after peripheral nerve stimulation (Hossain etal., 1994; Li etal., 1998; Li and Nagy, 2000). This dephosphorylation was often accompanied by altered recognition of Cx43 (epitope masking) by various antibodies. Relationships between astrocytic GJIC and Cx43 dephosphorylation in vivo are not yet clear, but may be important for understanding how astrocytes regulate their junctional coupling during neural activity or after CNS injury. To address this issue, we investigated whether Cx43 in cultured astrocytes exhibits responses to injury similar to those seen in vivo and we examined the contribution of various phosphatases to hypoxia-induced alterations in Cx43 phosphorylation state and astrocytic coupling status. Materials and methods Antibodies Two well characterized polyclonal anti-Cx43 antibodies (designated 18Aand71±0700)andamonoclonalanti-Cx43antibody(designated 13±8300) were used in this study. Antibodies were obtained from Zymed Laboratories, South San Franscico, CA. Antibody 13±8300 was previously shown by us to detect selectively a dephosphorylated form of Cx43 in Western blots of several tissues and cultured cells (Nagy etal., 1997; Li etal., 1998; Li and Nagy, 2000). Cell culture Astrocytes from cerebral cortices of 2-day-old decapitated Sprague± Dawley rats were cultured in Dulbecco's modi®ed essential medium supplemented with F12 nutrient and 10% fetal calf serum as Correspondence: Professor J. I. Nagy, as above. E-mail: Nagyji@ms.umanitoba.ca Received 10 April 2000, accepted 25 April 2000 European Journal of Neuroscience, Vol. 12, pp. 2644±2650, 2000 Ó Federation of European Neuroscience Societies