Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.

High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle.
We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy.
Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments.
No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.

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.