INHIBITION OF GAP JUNCTIONAL COMMUNICATION BY POLYUNSATURATED FATTY-ACIDS IN WB CELLS - EVIDENCE THAT CONNEXIN-43 IS NOT HYPERPHOSPHORYLATED

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 WE cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells, Studies wer e also conducted to investigate the possible mechanisms of action of t he fatty acids, Treatment of cells with 10 mu M arachidonic acid (20:4 n - 6) resulted in a rapid and transient loss of communication compet ence, The response to 20 mu M 20:4 (n - 6) was prolonged (>210 min) bu t was readily reversible by washing the cells with fatty acid-free bov ine serum albumin, Cells which had regained their communication compet ence 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 1 5-hydroperoxy-derivatives of 20:4 (n - 6) were also powerful inhibitor s of GJC, while 23:4 (n - 6) was a relatively weak inhibitor, The satu rated 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 pretre ating the cells with either phorbol-12-myristate-13-acetate (PMA) or i ndomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n - 6) in WE cells was neither dependent on PMA-responsive i sozymes of protein kinase C nor required the metabolism of the fatty a cids by cyclooxygenase. However, the effect of 20:4 (n - 6) was antago nized by preincubating WE cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethylxanthine, Western blot analys is of connexin 43 (Cx43), the major gap junctional protein expressed i n these cells, revealed no detectable changes to the electrophoretic m obility of Cx43 even after 60 min of incubation in the presence of 20: 4 (n - 6), As expected, other inhibitors of gap junctional permeabilit y including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enh ancment in the phosphorylation of Cx43 protein, The data indicate that inhibition of GJC by 20:4 (n - 6) has a novel mechanism which does no t require phosphorylation of Cx43 but may require its metabolism to ei cosanoids, In addition the inhibitory effect of 20:4 (n - 6) can be mo dulated by an increase in intracellular cAMP concentration which has b een reported to enhance cell-cell communication, The data also argue a gainst a non-specific detergent action of the fatty acids.