Inhibition of GAP junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hypophosphorylated

Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n - 6 and n - 3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 μM arachidonic acid (20: 4 n - 6) resulted in a rapid and transient loss of communication competence. The response to 20μM 20: 4 (n - 6) was prolonged (>210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20: 4 (n - 6). The fatty acids, 18: 3 (n - 6), 20: 5 (n - 3), 22: 6 (n - 3) and the 15-hydroxy- and the 15-hydroperoxy-derivatives of 20: 4 (n - 6) were also powerful inhibitors of GJC, while 23: 4 (n - 6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20: 0, and the methyl ester of 20: 4 (n - 6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20: 4 (n - 6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20: 4 (n - 6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20: 4 (n - 6) was antagonized by preincubating WB cells with either nordihydroguai-aretic acid or (±)-isoproterenol and isobutylmethyl-xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20: 4 (n - 6). As expected, other inhibitors of gap Junetional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancment in the phosphorylation of C×43 protein. The data indicate that inhibition of GJC by 20: 4 (n - 6) has a novel mechanism which does not require phosphorylation of Cx43 but may require its metabolism to eicosanoids. In addition the inhibitory effect of 20: 4 (n - 6) can be modulated by an increase in intracellular cAMP concentration which has been reported to enhance cell-cell communication. The data also argue against a non-specific detergent action of the fatty acids.Charles S. T. Hii, Antonio Ferrante, Simon Schmidt, Deborah A. Rathjen, Brenton S. Robinson, Alf Poulo and Andrew W. Mur