INHIBITION BY FATTY-ACIDS OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE ACTIVITY IN BRUSH-BORDER MEMBRANES ISOLATED FROM HUMAN PLACENTAL VESICLES

1 The inhibitory effects of arachidonic acid (AA) and a number of stru cturally related fatty acids on cyclic AMP-dependent protein kinase ac tivity have been investigated in brush border membranes (BBM) prepared from human placental vesicles. 2 BBM vesicles were characterized by e lectron microscopy and displayed enrichment of the appropriate marker enzymes, alkaline phosphatase and gamma-glutamyltranspeptidase; BBM we re prepared by vesicle lysis in hypotonic medium. 3 Cyclic AMP-depende nt protein kinase (PKA) activity was measured in BBM. At 1 mu M, cycli c AMP stimulated a 4.2 +/- 0.06 fold increase over basal levels of [P- 32]-phosphate incorporation into the synthetic substrate kemptide and this effect was abolished by a selective PKA inhibitor. By use of syne rgistic pairs of site-selective cyclic AMP analogues, the kinase was i dentified as the type II enzyme. 4 Cyclic AMP-stimulated PKA activity was inhibited by 10 mu M AA and this effect was significantly enhanced by nordihydroguaiaretic acid (NDGA)+indomethacin (Indo), inhibitors o f the lipoxygenase and cyclo-oxygenase pathways of AA metabolism respe ctively. 5 Oleic acid, elaidic acid, but not caprylic or palmitic acid s, also significantly inhibited PKA activity and this effect was again enhanced by NDGA+Indo. While arachidonyl alcohol alone was not inhibi tory, in the presence of the metabolic inhibitors a significant reduct ion in stimulated activity was observed. 6 The commercially available PKA type II holoenzyme (activated by cyclic AMP), but not the free cat alytic subunit, was inhibitable by AA, oleic or elaidic acids. 7 These results suggest that PKA localized to the brush border membrane of hu man placental vesicles is inhibited by fatty acids which may compete w ith cyclic AMP for binding to the kinase regulatory subunit. The repor ted inhibition by fatty acids of cyclic AMP-dependent Cl- secretion in epithelial cells may therefore be due in part to negative regulation of a Cl- channel-associated PKA.