During pregnancy, the trophoblast develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. As the exchange of molecules through gap junctions is considered to play a role in the control of cell and tissue differentiation, the cell to cell diffusion of a fluorescent dye was investigated in human trophoblastic cells differentiating in culture. The fluorescence recovery after photobleaching technique was used to estimate the transfer of 6-carboxyfluorescein from contiguous cellular elements into photobleached cells. Fluorescence recovery follows a slow exponential time course when the cell to cell exchange process is rate limited by the presence of gap junctional channels between contiguous cells, contrasting with a much faster step-like course in the case of fusion of the plasma membranes. In the presence of 10% fetal calf serum, Percoll-purified cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 24-48 h after plating. During this in vitro differentiation, fluorescence recoveries after photobleaching with a time course typical for gap junctions were observed between aggregated cytotrophoblastic cells, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous syncytiotrophoblasts. The maximum percentage of gap junctional coupling occurs on the fourth day. This fluorescence recovery is attributed to the diffusion of dye through gap junctions, because it can be interrupted by exposure to a known junctional uncoupler (3 mM heptanol). The effects of hCG on this gap junctional communication during trophoblast differentiation were investigated. In the presence of 500 mIU/ml hCG in the culture medium, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days of culture vs. 4 days in control medium. Moreover, the diffusion rate constant k (the inverse value of the time constant measured on recovery curves) was also significantly increased in the presence of chorionic hormone. It is concluded that during trophoblast differentiation, the development of a cell to cell communication through gap junctions precedes the formation of a morphological syncytium by cell fusion. This gap junctional communication is promoted by hCG. Furthermore, our study confirms the differentiating role and the autocrine action of hCG in the physiology of the trophoblast.

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The kinetics of the reversible interruption of gap junction communication by the aliphatic alcohol heptanol and the possible mediation of an increase of the cytosolic Ca2+ concentration have been investigated in pairs of myocytes dissociated from neonatal rat ventricles and cultured for 2-3 days. Junctional communication was estimated by measuring either the cell-to-cell electrical conductance with a double whole-cell voltage-clamp method, or the rate constant of dye diffusion with the fluorescence recovery after photo-bleaching (gap FRAP) technique. Electrical coupling was seen to be abruptly interrupted (in less than 0.5 s) by heptanol (1-3 mM). The cytosolic Ca2+ concentration was not affected, even at a saturating heptanol concentration. Heptanol removal allowed a gradual re-opening of gap junctional channels, as shown by the recovery curve of the cell-to-cell conductance, which is 90% complete within 90 s. These data are consistent with a direct interaction of heptanol with channel proteins or with their lipid environment.