Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells.
The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A2 (PLA2) activity in subsequently prepared MDCK-D1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA2 activity of cPLA2 immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA2 in MDCK-D1 cells. The BK-promoted increase of cPLA2 activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA2. However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKC alpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D1 cells (C. Godson, K.S. Bell, and P.A. Insel. [corrected] J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKC alpha-selective inhibitor GF109203X nor transfection of cells with PKC alpha antisense cDNA altered BK-mediated AA release. We conclude that PKC alpha is unlikely to play an important role in the regulation of cPLA2 by BK receptors in MDCK-D1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA2 activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D1 cells regulate cPLA2 via phosphorylation mediated by kinases other than ERK and PKC alpha.
Xing
M
M
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Tao
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L
Insel
P A
PA
D000818:Animals; D001920:Bradykinin / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology*; D004285:Dogs; D004789:Enzyme Activation; D007527:Isoenzymes / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D019950:Mitogen-Activated Protein Kinase 1; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D018002:Receptors, Bradykinin / Q000502:physiology
D007527:Isoenzymes; D018002:Receptors, Bradykinin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D010741:Phospholipases A; D054467:Phospholipases A2; D001920:Bradykinin