C(2)-ceramide-induced circular smooth muscle cell contraction involves PKC-epsilon and p44/p42 MAPK activation in cat oesophagus. Mitogen-activated protein kinase.
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.
Shin
Chang Yell
CY
Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul, South Korea.
Lee
Yul Pyo
YP
Lee
Tai Sang
TS
Song
Hyun Ju
HJ
Sohn
Uy Dong
UD
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002415:Cats; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004947:Esophagus / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D000906:Antibodies; D007527:Isoenzymes; C064769:N-acetylsphingosine; D010455:Peptides; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013110:Sphingosine