Gap junctions are expressed widely among astrocytes of the adult mammalian brain and are required for astroctic calcium signaling. We have found that forced expression of the gap junction protein, Cx43, in an otherwise gap junction-deficient cell line, C6 glioma, results not only in functional coupling, but also in a morphological phenotypic transformation from minimally interactive, nonadhesive cells into epithelial monolayers and in organization of the actin cytoskeleton. Accompanying this morphological transformation is the monolayer's acquisition of competence in propagating intercellular calcium waves. Past studies have assumed that these calcium waves depended upon the diffusion of Ca2+ and/or IP3 across gap junctions channels. However we found that in our Cx43-Transfectants as well as in astrocytic cultures, Ca2+ waves were actually insensitive to gap junction inhibition in uncoupling concentrations, but instead required the activation of ATP- sensitive P2-purinergic receptors. This suggested that CA2+ waves might be propagated by ATP release and activation of purinergic receptors, rather than by the transcellular diffusion of secondary messengers. Indeed, Cx43' cells released substantially more ATP when stimulated with CA2+ mobilizing agents than did their null-transfected controls, and their release of ATP required an intact cytoskeleton. Our intention is to define the role of actin organization in inter-astrocytic signaling, and to test the hypothesis that CA/2+ waves in gap junction-coupled cells are propagated by autocrine ATP signaling, rather than by transit of intracellular messengers across gap junctions. In addition, we will test the hypothesis that the phenotypic transformation observed in gap junction-coupled cells is a result of intrinsic adhesive properties of gap junction hemichannels, and that expression of connexin proteins is thus sufficient for the initiation of homotypic cell-cell contact. That has led us to ask: `. Do gap junctions modulate cytoskeletal organization? Is the cytoskeletal organization of coupled cells dictated or organized by gap junction formation? 2. Does gap junction-dictated cytoskeletal assembly regulate intercellular calcium signaling? 3. Is Ca/2+ signaling effected through ATP release? 4. Can connexins act as adhesion molecules? Do gap junction hemichannels function as macromolecular adhesive complexes? Defining the pathways by which astrocytes adhere to and signal among one another, as well as to their neuronal partners, will provide a basis for the directed manipulation of neuro-glial signaling, both in health and disease. In particular, the astroctic syncytium, in its capacity as a modulating influence upon neuronal excitability, may prove an especially fertile target for therapeutic intervention in such pathologic derangements as ischemic spreading depression, migraine and epilepsy.
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