Korean J Physiol Pharmacol.  2008 Aug;12(4):137-142. 10.4196/kjpp.2008.12.4.137.

Relaxation Effect of Synthetic Ceramide Analogues in Cat Esophageal Smooth Muscle Cells

• Lee DW
• Park SY
• Ryu JS
• Kim SH
• Im CU
• Choi SH
• Lee SE
• Ko SK
• Sohn UD

• KMID: 1838350
• DOI: http://doi.org/10.4196/kjpp.2008.12.4.137

Abstract

Ceramide has emerged as a novel second messenger for intracellular signalling. It is produced from sphingomyelin and is involved in the control of cell differntiation, proliferation, and apoptosis. C2- ceramide, short chain ceramide, plays a role in mediating contraction of cat esophageal smooth muscle cells. We examined the effect of synthesized ceramide analogues on the C2-ceramide and ACh-induced contraction in esophageal smooth muscle cells isolated with collagenase. CY3523, CY3525, or CY3723 inhibited C2-ceramide induced contraction, in a time dependent manne. Each analogue also inhibited the contraction in concentration dependent manners. CY 3523, CY 3525, and CY 3723 had no effect to the contraction induced by PMA. The inhibition with CY3523, CY3525 and CY3723 on the C2- ceramide induced contraction was recovered by PMA. These analogues decreased the density of MAPK bands (p44/42 or p38) in the western blot. These results suggest that ceramide analogues can inhibit C2-ceramide induced contraction via PKC and MAPK dependent pathway.

Keyword

Mitogen-activated protein kinase; Smooth muscle; Protein kinase C; C2-ceramide

MeSH Terms

Animals
Apoptosis
Blotting, Western
Cats
Collagenases
Contracts
Muscle, Smooth
Myocytes, Smooth Muscle
Negotiating
Protein Kinase C
Relaxation
Second Messenger Systems
Sphingosine
Collagenases
Protein Kinase C
Sphingosine

Figure

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  Fig. 1. CY3523, CY3525, CY3723 inhibited C2 ceramide induced contraction in a time-dependent manner. Isolated cells were incubated in ceramide amalogues (10−5 M) for indicated times. Data are means±S.E. of 4 experiments (ANOVA, p<0.001).

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  Fig. 2. Dose-dependent contractile inhibition response of smooth muscle cells from feline esophagus to CY3523 (A), CY3525 (B), or CY3723 (C). Isolated smooth muscle cells were incubated in CY3523 for 5min for indicated dose, then stimulated for 30 s with ACh (10−9 M) and C2 ceramide (10−7 M). Data are means±S.E. of 4 experiments (ANOVA p < 0.001).

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  Fig. 3. Effect of CY3523, CY3525, and CY3723 on PMA induced contraction. Isolated smooth muscle cells were treated with CY3523, CY3525 and CY3723 (10−5 M, incubated 5 min). No inhibitory effect was shown with phorbol 12 myristate 13-acetate (PMA), PKC activator.

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  Fig. 4. Effect of CY3523, CY3525, and CY3723 on anisomycin induced contraction. Anisomycin (p38 MAPK activator) produced contraction. CY3523, CY3525 and CY3723 had a little contractile inhibition of anisomycin induced contraction.

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  Fig. 5. The effect of phorbol 12 myristate 13-acetate (PMA) on CY3523, CY3525, CY3723. Inhibition of C2-ceramide induced contraction by ceramide anaogues was recovered with PMA (100 nM, PKC activator) treatment. Data are means±S.E. of 4 experiments (student t-test, p<0.01).

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  Fig. 6. The effects on the analogues of MAPK activation. (A) Phosphorylated p44/42 MAPK was detected with anti-phospho p44/42 MAPK antibody. In lower panel, the each level on the control displayed as the ratio of phospho- p44/42 divided by total p44/42 MAPK density (n=3). (B) p38 MAPK was inhibited by synthetic ceramide analogues. Phosphorylated p38 MAPK was detected with anti-phospho p38 MAPK antibody. In lower panel, the each level on the control displayed as the ratio of phospho-p38 density divided by total p38 MAPK density (n=3).

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