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.

1 The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface¯uorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT ± PCR). 2 In non-pregnant rats, 10 mM trypsin developed a force of up to 30.5+5.1% of that obtained during the 40 mM K + -depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca 2+ concentration and tension obtained with 3 mM trypsin was 143.2+6.0% and 63.2+7.9%, respectively. The depletion of the extracellular Ca 2+ abolished the trypsin-induced contraction.3 Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4 RT ± PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5 Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the prestimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6 In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

1 Using fura-2¯uorometry of [Ca 2+ ] i in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca 2+ ] i elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was signi®cantly (P50.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP speci®cally attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher anity PAR1-AP (haPAR1-AP) (Ala-pFArg-Cha-HArg-Tyr-NH 2 ) speci®cally attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was signi®cantly (P50.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

1 Using fura-PE3¯uorimetry and a-toxin permeabilization, the characteristics of the contractile responses to neurokinin A (NKA) were determined in the pregnant rat myometrium. 2 NKA induced contractions in rat myometrium in a concentration-dependent manner. There were no signi®cant dierences in the maximum contractions and EC 50 values between the pregnant and non-pregnant myometrium, however, the contraction of only the former was greatly enhanced in the presence of phosphoramidon (PPAD), an endopeptidase inhibitor. -sensitizing eect by NKA was also observed in the a-toxin permeabilized myometrium. 6 These results indicated that in the pregnant rat myometrium: (1) the responsiveness to NKA increased, although it was masked by the increase in the endopeptidase activity; (2) NKA induced contractions of the myometrium by increasing both [Ca 2+ ] i and the myo®lament Ca 2+ sensitivity and (3) The NKA-induced [Ca 2+ ] i elevation was partly due to the intracellular Ca 2+ release and mainly due to the Ca 2+ in¯ux, which was thought to be through both voltage dependent calcium channels and non-speci®cation channels.

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