Gap junctional intercellular communication: Methodologies for measurement and application to potential inhibitors of communication

As an adaptation to multicellular life, organisms have evolved aqueous channels which allow passive transport of small molecules between contiguous cells. These channels are called gap junctions. Junctional communication exercises homeostatic control on cellular physiology and takes part in regulating cell growth and differentiation. In adult organisms the blockage of junctional communication is implicated in the process of carcinogenesis. In embryo's, blockage results in abnormal development. In this investigation metabolic cooperation was used to investigate junctional communication. A modification of an existing method was developed whereby 6-thioguanine (6-tg) resistant and sensitive strains of V79 cells were plated in a 1:1 ratio in multiwell plates. The co-cultures were treated with 6-tg. Metabolism of 6-tg in the sensitive cells and subsequent transfer to resistant cells led to inhibition of DNA synthesis in both cell types as assayed by 3H-thymidine incorporation. DNA synthesis in segregated resistant cells is not affected by 6-tg because these cells cannot metabolize 6-tg due to an enzyme deficiency. The inhibition of DNA synthesis in resistant cells in co-culture demonstrates junctional communication. As a positive control junctional communication in the co-culture was inhibited by exposure to a known communication inhibitor, the phorbol ester TPA. Using this method the effects of compounds on junctional communication were investigated. Calcium ionophore A23187 and DDT did not inhibit junctional communication whereas paracetamol appeared to inhibit junctional communication. To elucidate the mechanism of action of paracetamol BHA, aspirin and indomethacin were tested without showing an effect on junctional communication. Nordihydroguairetic acid also appeared to inhibit communication similarly to paracetamol. Using dye transfer as a comparative measure of cell communication it was found that these compounds did not inhibit junctional communication but prevented the incorporation of the 6-tg metabolite into the resistant cells. Consequently hydroxyurea, a known inhibitor of DNA synthesis was tested and shown to mimic the effects of these compounds. The implications of these findings to junctional research are discussed