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

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 PKCε 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-ε pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.

Lysophosphatidic acid induces integrin activation in vascular smooth muscle and alters arteriolar myogenic vasoconstriction

“…LPA is a small phospholipid with a wide array of biological functions such as, cell proliferation and survival, platelet aggregation, cell migration, and SMC contraction (Cheng et al, 2009 ; Cui, 2011 ; Gaaya et al, 2012 ; Nam et al, 2013 ). The biological effects of LPA are mediated through specific G-protein coupled cell surface receptors named LPAR1-6 (Choi et al, 2010 ; Yanagida and Ishii, 2011 ) and through intracellular receptors such as the peroxisome proliferator-activated receptor-γ (Cheng et al, 2009 ).…”

The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

“…Adult male cats weighing between 3 and 5 kg were anesthetized and esophageal smooth muscle tissue was prepared as previously described ( Biancaniet al, 1987 ; Murthy and Makhlouf, 1998 ; Nam et al, 2013 ). The tissue was digested overnight maintaining a temperature of 4°C with HEPES-buffered solution thatcontained 1 mg/ml papain, 1 mM dithiothreitol, 1 mg/ml BSA and 0.5 mg/ml collagenase (type F, Sigma).…”

“…Developed films from ECL were scanned and analyzed densitometrically using Scion Image. Phosphorylation of MLC 20 was calculated as the ratio of phosphorylated MLC 20 to total MLC 20 ( Cao et al, 2001 ; Ijzeret al, 2009 ; Nam et al, 2013 ). All measurements were done in the presence of adenosine A1 and A2 antagonists (1 mM DPCPX and 0.1 mM CGS-15943, respectively) ( Murthyet al, 1995 ).…”