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PubMed · 7779401

A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer.

G S Goldberg·J F Bechberger·C C Naus
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Abstract

We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.

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CarbenoxoloneCarbocyaninesCell CommunicationCell LineCell Line, TransformedConnexin 43FluoresceinsFluorescent DyesGap JunctionsGlycyrrhetinic AcidMicroscopy, FluorescenceOncogene Protein pp60(v-src)StereoisomerismSuccinatesJournal ArticleResearch Support, Non-U.S. Gov't

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G S Goldberg, J F Bechberger, C C Naus. 1995. A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer.. https://pubmed.ncbi.nlm.nih.gov/7779401/

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Gap junctions are required for trophoblast proliferation in early human placental development.

Little is known about the role of gap junctional intercellular communication (GJIC) in human trophoblast differentiation, particularly during the formation of extravillous trophoblast (EVT) cell columns and their subsequent differentiation into invasive cells. We have identified transcripts for five connexin gap junction proteins in the early human placenta (Cx32, Cx37, Cx40, Cx43 and Cx45). Of these, Cx40 and Cx45 proteins immunolocalize to EVT in anchoring cell columns. Cx40 expression is prominent in the anchoring column throughout the first trimester of pregnancy (6-14 weeks gestation). We used first trimester placental villous explant cultures to determine the functional significance of the inhibition of GJIC in EVT cell proliferation and differentiation using two known GJIC inhibitors, carbenoxolone (CBX) and heptanol. The morphology of EVT outgrowths changed dramatically upon GJIC-blockade, from compact and organized outgrowths into a scattered group of rounded individual trophoblast cells, reminiscent of an early invasive phenotype. Furthermore, the inhibition of GJIC in placental explants by CBX or heptanol induced a switch away from the proliferative and towards an invasive EVT phenotype, as evident from (a) the loss of the proliferation marker Ki67 and (b) an increase in the invasive marker alpha1 integrin. We also utilized antisense oligonucleotides to inhibit Cx40 protein expression in placental explants. Cx40 antisense treatment also resulted in the abolishment of outgrowth EVT cell proliferation (as determined by Ki67 immunostaining). Together, these results suggest that gap junctions composed particularly of Cx40 channels are required for the proliferation of EVT cells in anchoring cell columns, and that a loss of GJIC contributes to differentiation to the invasive EVT phenotype.

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