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 structurally related fatty acids on cyclic AMP‐dependent protein kinase activity have been investigated in brush border membranes (BBM) prepared from human placental vesicles. 2 BBM vesicles were characterized by electron microscopy a...
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Veröffentlicht in: | British journal of pharmacology 1994-02, Vol.111 (2), p.509-514 |
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Zusammenfassung: | 1
The inhibitory effects of arachidonic acid (AA) and a number of structurally related fatty acids on cyclic AMP‐dependent protein kinase activity have been investigated in brush border membranes (BBM) prepared from human placental vesicles.
2
BBM vesicles were characterized by electron microscopy and displayed enrichment of the appropriate marker enzymes, alkaline phosphatase and γ‐glutamyltranspeptidase; BBM were prepared by vesicle lysis in hypotonic medium.
3
Cyclic AMP‐dependent protein kinase (PKA) activity was measured in BBM. At 1 μm, cyclic AMP stimulated a 4.2 ± 0.06 fold increase over basal levels of [32P]‐phosphate incorporation into the synthetic substrate kemptide and this effect was abolished by a selective PKA inhibitor. By use of synergistic pairs of site‐selective cyclic AMP analogues, the kinase was identified as the type II enzyme.
4
Cyclic AMP‐stimulated PKA activity was inhibited by 10 μm AA and this effect was significantly enhanced by nordihydroguaiaretic acid (NDGA) + indomethacin (Indo), inhibitors of the lipoxygenase and cyclo‐oxygenase pathways of AA metabolism respectively.
5
Oleic acid, elaidic acid, but not caprylic or palmitic acids, also significantly inhibited PKA activity and this effect was again enhanced by NDGA + Indo. While arachidonyl alcohol alone was not inhibitory, in the presence of the metabolic inhibitors a significant reduction in stimulated activity was observed.
6
The commercially available PKA type II holoenzyme (activated by cyclic AMP), but not the free catalytic subunit, was inhibitable by AA, oleic or elaidic acids.
7
These results suggest that PKA localized to the brush border membrane of human placental vesicles is inhibited by fatty acids which may compete with cyclic AMP for binding to the kinase regulatory subunit. The reported 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. |
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ISSN: | 0007-1188 1476-5381 |
DOI: | 10.1111/j.1476-5381.1994.tb14766.x |