Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent.
Bradykinin (BK)-induced release of arachidonic acid (AA) from Madin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol 12-myristate 13-acetate (PMA) caused a synergistic increase in BK- and A-23187-induced release of AA but alone had no effect on this release. Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS) abolished the synergistic effects of PMA but did not affect AA release caused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMA resulted in a reduction of AA release induced by BK or A-23187 addition, which corresponded to a decrease in cytoplasmic phospholipase A2 (cPLA2) activity as measured in cell extracts. Although Western blotting revealed no differences in cPLA2 expression as a result of PMA treatment, phosphorylation of the enzyme, as assessed by phosphoserine content, was significantly reduced in PKC-depleted cells. These results imply that, with PKC downregulation, subsequent BK stimulation results in a Ca(2+)-dependent translocation of a less phosphorylated, less active form of cPLA2. Any stimulation of PKC by BK addition did not appear as a significant event in onset responses leading to AA release. On the other hand, inhibition of the mitogen-activated protein kinase (MAPK) cascade with the MAPK kinase inhibitor, PD-98059, significantly decreased BK-induced release of AA, a finding that, with our other results, points to the existence of a PKC-independent route for stimulation of MAPK and the propagation of onset responses.
Kennedy
C R
CR
Department of Biochemistry, Faculty of Medicine, University of Ottawa, Ontario, Canada.
Hébert
R L
RL
Do
M T
MT
Proulx
P R
PR
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010768:Phosphoserine; D016718:Arachidonic Acid; D000001:Calcimycin; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin