Korean J Physiol Pharmacol.  2013 Apr;17(2):139-147. 10.4196/kjpp.2013.17.2.139.

Signaling Pathway of Lysophosphatidic Acid-Induced Contraction in Feline Esophageal Smooth Muscle Cells

Affiliations
  • 1Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea. udsohn@cau.ac.kr

Abstract

Lysolipids such as LPA, S1P and SPC have diverse biological activities including cell proliferation, differentiation, and migration. We investigated signaling pathways of LPA-induced contraction in feline esophageal smooth muscle cells. We used freshly isolated smooth muscle cells and permeabilized cells from cat esophagus to measure the length of cells. Maximal contraction occurred at 10(-6) M and the response peaked at 30s. To identify LPA receptor subtypes in cells, western blot analysis was performed with antibodies to LPA receptor subtypes. LPA1 and LPA3 receptor were detected at 50 kDa and 44 kDa. LPA-induced contraction was almost completely blocked by LPA receptor (1/3) antagonist KI16425. Pertussis toxin (PTX) inhibited the contraction induced by LPA, suggesting that the contraction is mediated by a PTX-sensitive G protein. Phospholipase C (PLC) inhibitors U73122 and neomycin, and protein kinase C (PKC) inhibitor GF109203X also reduced the contraction. The PKC-mediated contraction may be isozyme-specific since only PKCepsilon antibody inhibited the contraction. MEK inhibitor PD98059 and JNK inhibitor SP600125 blocked the contraction. However, there is no synergistic effect of PKC and MAPK on the LPA-induced contraction. In addition, RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y27632 significantly, but not completely, reduced the contraction. The present study demonstrated that LPA-induced contraction seems to be mediated by LPA receptors (1/3), coupled to PTX-sensitive G protein, resulting in activation of PLC, PKC-epsilon pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.

Keyword

Contraction; Esophageal smooth muscle; LPA; RhoA/ROCK; Signaling

MeSH Terms

Amides
Animals
Anthracenes
Antibodies
Blotting, Western
Cats
Cell Proliferation
Contracts
Esophagus
Estrenes
Flavonoids
GTP-Binding Proteins
Indoles
Isoxazoles
Maleimides
Muscle, Smooth
Myocytes, Smooth Muscle
Neomycin
Pertussis Toxin
Propionates
Protein Kinase C
Pyridines
Pyrrolidinones
Receptors, Lysophosphatidic Acid
Type C Phospholipases
Amides
Anthracenes
Antibodies
Estrenes
Flavonoids
GTP-Binding Proteins
Indoles
Isoxazoles
Maleimides
Neomycin
Pertussis Toxin
Propionates
Protein Kinase C
Pyridines
Pyrrolidinones
Receptors, Lysophosphatidic Acid
Type C Phospholipases

Figure

  • Fig. 1 (A) Time-dependent contractile response of smooth muscle cells from feline esophagus to LPA (10-6 M). (B) Dose-dependent contractile response of smooth muscle cells from feline esophagus to LPA (30 s). Data are expressed as means±SEM of three independent experiments.

  • Fig. 2 (A) Identification of LPA receptor subtype in esophageal smooth muscle cell by western blot analysis. LPA1 and LPA3 receptors were detected at 50 kDa and 44 kDa. The blot also was probed with anti-GAPDH antibody as a loading control. (B) Effect of receptor antagonist on the contraction induced by LPA (10-6 M). The intact cells were preincubated with KI16425 (10-6 M), LPA receptor 1/3 antagonist, for 10 min. (C) Effect of PTX on the contraction induced by LPA (10-6 M). The intact cells were preincubated with PTX (400 ng/ml) for 60 min. Data are expressed as means±SEM of three independent experiments. **p<0.01 vs. control, two-tailed t-test.

  • Fig. 3 Effect of phospholipases on the contraction induced by LPA (10-6 M). The intact cells were preincubated with PLC inhibitors neomycin (10-6 M) or U73122 (10-6 M) for 10 min, PLD inhibitor ρCMB (10-5 M) for 10 min, or PLA2 inhibitor DEDA (10-5 M) for 1 min. Data are expressed as means±SEM of three independent experiments. **p<0.01 vs. control, two-tailed t-test.

  • Fig. 4 (A) Effect of protein kinase C inhibitor on the contraction induced by LPA (10-6 M). The intact cells were preincubated with PKC inhibitor GF109203X (3×10-6 M) for 10 min. (B) The permeabilized cells were preincubated with antibodies raised against PKC isozymes for 1 h. PKCε antibody reduced the contraction. Data are expressed as means±SEM of three independent experiments. **p<0.01 vs. control, two-tailed t-test.

  • Fig. 5 (A) Role of MAPK on LPA-induced smooth muscle cells contraction. The intact cells were pretreated with ERK1/2 inhibitor PD98059 (10-5 M), JNK inhibitor SP600125 (10-5 M), or p38 MAPK inhibitor SB202190 (10-5 M) for 30 min. (B) Synergistic effect of PKC and MAPK on LPA-induced contraction. The PKC inhibitor was co-treated with ERK1/2 or JNK inhibitor. Data are expressed as means±SEM of three independent experiments. **p<0.01 vs. control, two-tailed t-test.

  • Fig. 6 Effects of RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y27632 effect on the contraction induced by LPA (10-6 M). The permeabilized smooth muscle cells were preincubated with C3 exoenzyme for 30 min (10µg/ml), or Y27632 (10-5 M) for 10 min. Data are expressed as means±SEM of three independent experiments. **p<0.01 vs. control, two-tailed t-test.

  • Fig. 7 Expected intracellular signal pathways of LPA-induced contraction in esophageal smooth muscle cell. LPA-induced contraction seems to be mediated by LPA receptor (1/3), coupled to PTX-sensitive G protein, resulting in the activation of PLC, PKC-ε pathway, which subsequently mediates the activation of ERK and JNK. The data also suggest that RhoA/ROCK is involved in the LPA-induced contraction.


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