C-2-ceramide-induced circular smooth muscle cell contraction involves PKC-epsilon and p44/p42 MAPK activation in cat oesophagus

Abstract

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-alphabetagamma, -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. (C) 2002 Elsevier Science Inc. All rights reserved.