Xing M, Tao L, Insel P A
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Am J Physiol. 1997 Apr;272(4 Pt 1):C1380-7. doi: 10.1152/ajpcell.1997.272.4.C1380.
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.
缓激肽（BK）在犬肾Madin-Darby细胞（MDCK）及其他细胞类型中的作用，涉及通过尚未明确的机制生成花生四烯酸（AA）及其产物。我们发现，BK可促进AA释放，并使后续制备的MDCK-D1细胞裂解物中的磷脂酶A2（PLA2）活性增加，这两者均依赖Ca2+，并被85-kDa的胞质型PLA2（cPLA2）抑制剂花生四烯酰三氟甲基酮所抑制。此外，用BK处理细胞可导致从裂解物中免疫沉淀的cPLA2的PLA2活性增加。因此，BK受体通过MDCK-D1细胞中的cPLA2介导AA释放。用马铃薯酸性磷酸酶处理细胞裂解物可逆转BK促进的cPLA2活性增加，这表明磷酸化是cPLA2激活的基础。然而，细胞外信号调节激酶（ERK）似乎并不负责这种磷酸化，因为用BK处理细胞（与用肾上腺素和佛波酯获得的结果相反）既不会导致该激酶的酶激活，也不会导致其磷酸化（通过分子量变化判断）。虽然蛋白激酶C的α亚型（PKCα）负责佛波酯处理MDCK-D1细胞所促进的AA释放（C. Godson、K.S. Bell和P.A. Insel. [已校正] J. Biol. Chem. 268: 11946 - 11950, 1993），但用PKCα选择性抑制剂GF109203X处理细胞或用PKCα反义cDNA转染细胞，均未改变BK介导的AA释放。我们得出结论，PKCα不太可能在MDCK-D1细胞中BK受体对cPLA2的调节中发挥重要作用。另一方面，酪氨酸激酶抑制剂赫司汀A可抑制完整细胞中BK促进的AA释放以及细胞裂解物中cPLA2的激活，这表明酪氨酸激酶参与了BK受体对这种脂肪酶的调节。综上所述，这些数据表明MDCK-D1细胞中的BK受体通过ERK和PKCα以外的激酶介导的磷酸化来调节cPLA2。
