Enhanced contractile response to thrombin in the pregnant rat myometrium

Abstract

1 Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the ®rst time, that thrombin induced myometrial contraction. To determine the mechanism of thrombin-induced myometrial contraction, we simultaneously measured intracellular Ca 2+ concentration ([Ca 2+ ] i ) and tension of fura-PE3-loaded rat myometrium using front-surfacē uorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT ± PCR analysis). 2 Thrombin (0.01 ± 3 u ml 71 ) caused dose-dependent increase in [Ca 2+ ] i and tension in the rat myometrium, and this eect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the eects of thrombin. 3 In Ca 2+ -free PSS, thrombin induced no increase in [Ca 2+ ] i and tension in the pregnant myometrium. Both diltiazem (10 mM) and SK-F 96365 (10 mM) signi®cantly inhibited the thrombininduced elevations of [Ca 2+ ] i and tension, and their eects were additive. 4 RT ± PCR analysis revealed an approximately 10 fold increase in the level of thrombin receptor mRNA in the pregnant myometrium compared to that obtained in the non-pregnant myometrium. 5 In conclusion, the contractile response to thrombin was greatly enhanced in the pregnant myometrium, mainly due to the up-regulation of thrombin receptor. We propose that initiation of a post-parturitional myometrial contraction is one of the most important physiological roles of thrombin receptor.

Enhancement of trypsin‐induced contraction by in vivo treatment with 17β‐estradiol and progesterone in rat myometrium

“…The thermal cycle profile used for the amplification of PAR1, PAR2, PAR3 and PAR4 fragments was composed of the initial denaturation at 94°C, for 2 min and the following 40‐cycle amplification step consisting of 15 s denaturation at 94°C, 15 s annealing at 52°C and 30 s extension at 68°C. The expression of β ‐actin was determined as a control as described previously ( Shintani et al ., 2000a ). The RT–PCR products were separated on 3% agarose gel containing 0.05 μ g ml −1 ethidium bromide.…”

“…We have previously reported that thrombin and trypsin induced an enhanced myometrial contraction in the pregnant rat myometrium ( Shintani et al ., 2000a ; 2001 ). Among the known four members of PARs, PAR1 was the only receptor expressed in the rat myometrium, and it was found to be upregulated in the pregnant rat myometrium ( Shintani et al ., 2000a ; 2001 ). The upregulation of PAR1 was thus considered to contribute to the enhancement of the contractile response to thrombin in the pregnant rat ( Shintani et al ., 2000a ).…”

“…Among the known four members of PARs, PAR1 was the only receptor expressed in the rat myometrium, and it was found to be upregulated in the pregnant rat myometrium ( Shintani et al ., 2000a ; 2001 ). The upregulation of PAR1 was thus considered to contribute to the enhancement of the contractile response to thrombin in the pregnant rat ( Shintani et al ., 2000a ). On the other hand, we obtained evidence that PAR1 did not mediate the contractile response to trypsin in the rat myometrium.…”

“…In the present study, we tested the hypothesis that sex hormones are responsible for the upregulation of the receptors for thrombin and trypsin and the enhancement of the contractile responses to thrombin and trypsin. For this purpose, we treated nonpregnant rats with 17 β ‐estradiol and progesterone in vivo , and then examined the contractile response to thrombin, trypsin, PAR1‐activating peptide (PAR1‐AP), PAR2‐AP and PAR4‐AP in the isolated myometrium as described previously ( Shintani et al ., 2000a ; 2001 ). We also examined the expression of the four known members of PARs by reverse transcription–polymerase chain reaction (RT–PCR).…”

Changes in amniotic fluid concentration of thrombin–antithrombin III complexes in patients with preterm labor: Evidence of an increased thrombin generation

“…The uterotonic effect of thrombin may be exerted by its receptor, PAR-1, which mediates many of the effects of thrombin on platelet activation[153,154], proinflammatory cytokine secretion[17,155,156], local tissue remodeling after injury[157,158], fetal blood vessel development and stabilization[159–161], as well as uterine contractions[162–164]. Indeed, in a study on stem cells gene expression, PAR-2 and PAR-1 were among the top 25 up-regulated genes in stem cells of amniotic fluid origin[165], suggesting that thrombin can exert its intracellular and proinflammatory effect via local mechanisms through its receptor.…”