Chemical structure of Tg.

<p>Chemical structure of Tg.</p

Tg dose- and time-dependently induced contractions of rat uterine strips and MLC20 phosphorylation in rat myometrial cells.

<p>(<b>A</b>) Representative recording of rat myometrial contractions induced by cumulative doses of Tg. Muscle tension was recorded isometrically with a tension transducer connected to a polygraph system. The solution for each strip was first changed to 40 mM K⁺ for 10 min to ensure contractile viability and to determine maximum contraction. (<b>B–E</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from rat myometrial cells treated with cumulative doses of Tg (B) or 400 nM Tg for different period of time (D). Quantitative analyses of the pMLC20-to-MLC20 ratio (C, E). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses. Data are expressed as means ± SEM.</p

PLC-IP3 signaling pathway is involved in Tg-induced myometrial contractions through the regulation of [Ca²⁺]_i.

<p>(<b>A</b>) Inhibitory effects of cumulative doses of 2-APB and NCDC on contractile responses after stimulation with Tg in rat myometrial strips (n = 3). Contractions were measured as the area under the curve (AUC) and expressed as a percentage of the response to 5 µM Tg. (<b>B–E</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of 2-APB (2-APB inhibitor) (B) or U73122 (PLCβ inhibitor) (D). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (C, E). Data are expressed as means ± SEM. *** <i>P</i><0.001 compared to control.</p

RhoA/ROK signaling pathway mediates Tg-induced myometrial contractions via regulating MLC20 phosphorylation.

<p>(A) Inhibitory effects of cumulative doses of Y27632 on contractile responses after stimulation with Tg in rat myometrial strips (n = 3). The control strips were added with vehicle at the same time after stimulation with Tg (5 µM). (B) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of Y27632.</p

Role of extracellular as well as intracellular calcium in Tg-induced uterine contractions and MLC20 phosphorylation.

<p>(<b>A</b>) Representative recordings of the cumulative dose responses on myometrial strips induced by Tg or PGF-2α in calcium-free solution. After the stimulators were increased to the maximal effective dose, calcium was added to a final concentration of 1.8 mmol/L. (<b>B</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of extracellular Ca²⁺. (<b>C</b>) Inhibitory effects of cumulative doses of nitrendipine on contractile responses after stimulation with Tg or PGF-2α in rat myometrial strips (n = 3). Contractions were measured as the area under the curve (AUC) and expressed as a percentage of the response to 5 µM Tg or 450 nM PGF-2α. (<b>D–E</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of thapsigargin (2.5 µM) (D). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (E). (<b>F</b>) Inhibitory effects of cumulative doses of Rethenium red on contractile responses after stimulation with Tg in rat myometrial strips (n = 3). Contractions were measured as the area under the curve (AUC) and expressed as a percentage of the response to 5 µM Tg. Data are expressed as means ± SEM. *** <i>P</i><0.001 and ** <i>P</i><0.01 compared to control.</p

Involvment of [Ca²⁺]_i increase and CaM activity in Tg-induced uterine contractions and MLC20 phosphorylation.

<p>(A) Confocal fluorescence images of myometrial smooth muscle cells loaded with Fluo-4 AM before and after 2.5 µM Tg stimulation. The fluorescence images were observed at the excitation wavelength of 510 nm and 12 s after Tg stimulation. (B) Time course of the change of the fluorescence in a Fluo-4 AM-loaded myometrial smooth muscle cells in response to Tg (2.5 µM). The arrow indicates the time at which Tg was added. (<b>C</b>) Inhibitory effects of cumulative doses of W-7 on contractile responses after stimulation with Tg in rat myometrial strips (n = 3). Contractions were measured as the area under the curve (AUC) and expressed as a percentage of the response to 5 µM Tg. (<b>D–E</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of W-7 (500 nM), a common antagonist of calmodulin (D). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (E). Data are expressed as means ± SEM. ** <i>P</i><0.01 compared to control.</p

MLCK is involved in Tg-induced MLC20 phosphorylation and uterine contractions.

<p>(<b>A</b>) Representative recording of the inhibitory effects of cumulative doses of MLCK inhibitor ML-7 on rat myometrial contractions induced by 5 µM Tg, while the control strips were added with vehicle at the same time after stimulation with Tg. (<b>B–C</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of ML-7 (500 nM), a specific inhibitor of MLCK (B). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (C). Data are expressed as means ± SEM. ** <i>P</i><0.01 compared to control.</p