title: Differential connexin expression, gap junction intercellular coupling, and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons.
abstract: Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.
authors.affiliation: Neural Regeneration Laboratory, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Sherri
authors.initials: S
authors.lastname: Boucher
authors.forename: Steffany A L
authors.initials: SA
authors.lastname: Bennett
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002450:Cell Communication; D002454:Cell Differentiation / Q000187:drug effects; D000072259:Connexin 26; D017630:Connexins / Q000096:biosynthesis*; D017629:Gap Junctions / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D018243:Teratocarcinoma; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000502:physiology
chemical_list: D000970:Antineoplastic Agents; D017630:Connexins; C000607042:GJB2 protein, human; D012333:RNA, Messenger; D000072259:Connexin 26; D014212:Tretinoin