C(2)-ceramide-induced circular smooth muscle cell contraction involves PKC-epsilon and p44/p42 MAPK activation in cat oesophagus. Mitogen-activated protein kinase.
C(2)-ceramide-induced circular smooth muscle cell contraction involves PKC-epsilon and p44/p42 MAPK activation in cat oesophagus. Mitogen-activated protein kinase.
复制标题
C(2)-神经酰胺诱导的圆形平滑肌细胞收缩涉及猫食道中的 PKC-epsilon 和 p44/p42 MAPK 激活。
作者：
C. Shin;Yul Pyo Lee;T. Lee;H. Song;U. Sohn
我们研究了C(2)-神经酰胺(C(2))引起猫食道环状平滑肌细胞收缩的机制。C(2)对酶消化法分离的平滑肌细胞产生收缩作用，在30 S时达到高峰，5min后维持在平台期。对C(2)的反应具有浓度依赖性。H-7或白屈菜红碱可抑制C(2)诱导的收缩，而二酰基甘油(DAG)激酶抑制剂R59949则不起作用，提示这种收缩是蛋白激酶C(PKC)途径依赖的。为了测试PKC介导的收缩是否具有同工酶特异性，我们检测了PKC同工酶抗体对收缩的影响。PKC-epsilon抗体抑制C(2)的收缩，但不能抑制PKC-βII或-Gamma的收缩，提示PKC-epsilon介导了C(2)的收缩。为了表征介导平滑肌细胞收缩的特定PKC同工酶，我们使用从PKC-(α)(β)(伽马)、-α、-β或-epsilon的假底物序列衍生的N-肉豆蔻酰化肽(MYR-PKC)作为抑制物。MYR-PKC-epsilon仅抑制收缩，且呈浓度依赖性，提示PKC-epsilon同工酶参与了收缩。为了研究哪些丝裂原活化蛋白激酶(MAPK)参与了C(2)诱导的收缩，使用了特定的MAPK抑制剂(MEK抑制剂PD98059和p38 MAPK抑制剂SB202190)。预先孵育的PD98059以浓度依赖的方式阻断C(2)引起的收缩。但SB202190对收缩无明显影响。与C(2)刺激的细胞相比，C(2)可增加磷酸化的p44/p42 MAPK抗体的条带强度，而预先孵育PD98059则可降低条带的强度。综上所述，C(2)对猫食道平滑肌细胞有收缩作用。这种收缩是由PKC-epsilon介导的，导致p44/p42 MAPK的激活。
We investigated the mechanism of C(2)-ceramide (C(2))-induced circular smooth muscle cell contraction in cat oesophagus. C(2) produced contraction of smooth muscle cells isolated by enzymatic digestion, peaked at 30 s and was sustained at a plateau at 5 min. The response to C(2) was concentration-dependent. H-7 or chelerythrine inhibited C(2)-induced contraction, while the diacylglycerol (DAG) kinase inhibitor, R59949, had no effect, suggesting that the contraction is protein kinase C (PKC) pathway-dependent. To test if PKC-mediated contraction may be isozyme-specific, we examined the effects of PKC isozymes antibodies on contraction. PKC-epsilon antibody inhibited the contraction by C(2) but not by PKC-betaII or -gamma, suggesting that PKC-epsilon mediates the contraction by C(2). To characterize the specific PKC isozymes that mediate contraction of the smooth muscle cells, we used, as an inhibitor, N-myristoylated peptides (myr-PKC) derived from the pseudosubstrate sequences of PKC-(alpha)(beta)(gamma), -alpha, -delta, or -epsilon. myr-PKC-epsilon only inhibited the contraction, which was concentration-dependent, suggesting that PKC-epsilon isozyme is involved in the contraction. To examine which mitogen-activated protein kinases (MAPKs) are involved in C(2)-induced contraction, specific MAPK inhibitors (MEK inhibitor, PD98059, and p38 MAPK inhibitor, SB202190) are used. Preincubation of PD98059 blocked the contraction induced by C(2) in a concentration-dependent manner. However, SB202190 had no effects on contraction. C(2) increased the intensity of the bands identified by phosphospecific p44/p42 MAPK antibody and preincubation of PD98059 decreased the intensity of bands as compared with C(2)-stimulated cells. In conclusion, C(2) produced the contraction of smooth muscle cells of cat oesophagus. The contraction is mediated by PKC-epsilon, resulting in the activation of p44/p42 MAPK.