Enhanced contractile response to thrombin in the pregnant rat myometrium

Abstract

Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the first 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-surface fluorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT - PCR analysis). Thrombin (0.01 - 3 u ml(-1)) caused dose-dependent increase in [Ca(2+)](i) and tension in the rat myometrium, and this effect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the effects of thrombin. In Ca(2+)-free PSS, thrombin induced no increase in [Ca(2+)](i) and tension in the pregnant myometrium. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the thrombin-induced elevations of [Ca(2+)](i) and tension, and their effects were additive. 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. 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.

Figure 1

The contractile effects of thrombin on the rat myometrium. (a) and (b) Representative recordings showing the effects of thrombin (1 u ml⁻¹) on the tension of the non-pregnant rat myometrium (a), and on the [Ca²⁺]_i (upper trace) and tension (lower trace) on the day 18 pregnant rat myometrium (b) in the normal PSS. (c) The concentration-response curves of thrombin-induced contraction in the pregnant and non-pregnant myometrium. The contractile response to thrombin was quantitatively evaluated in two ways; the first peak level of tension development and the area under the tension trace for the initial 10 min. The developed tension was expressed as a percentage, assigning the values in normal and 40 nM K⁺ PSS to be 0% and 100%, respectively. Data are the mean±s.e.mean (n=7).

Figure 2

Gestational change of the contractile response to thrombin in the rat myometrium. (a) Comparison of the contractile response to thrombin based on the day of pregnancy. Contractile response was evaluated by the area under the tension trace, assuming those obtained with 40 mM K⁺ to be 100%. The data are the mean±s.e.mean (n=3–7). ^*P<0.05 as compared with the non-pregnant rats.

Figure 3

The contractile effects of thrombin receptor activating peptide on the rat myometrium. (a) and (b) Representative traces showing the effects of PAR1-AP (10 μM) on the tension of the non-pregnant rat myometrium (a) and on the [Ca²⁺]_i (upper trace) and tension (lower trace) of the pregnant rat myometrium (b) in the normal PSS. (c) The concentration-response curves of PAR1-AP-induced contraction. The tension development was evaluated by the area under the tension trace for the initial 10 min, assigning the values obtained with 40 mM K⁺ PSS to be 100%. The data are the mean±s.e.mean (n=5–7).

Figure 4

The effect of the serine protease inhibitor on the myometrial contractions induced by thrombin and PAR1-AP. (a) Representative trace showing the inhibitory effect of the serine protease inhibitor, 4-aminidophenylmethane-sulphonyl fluoride (p-APMSF, 10 μM) on the 1 u ml⁻¹ thrombin-induced contraction of the pregnant rat myometrium. After observing the effect of p-APMSF-treated thrombin and stimulation once with 40 mM K⁺, the contracting effect of the untreated thrombin was examined. (b) Representative trace showing the effect of 10 μM p-APMSF on the 30 μM PAR1-AP-induced contraction of the pregnant rat myometrium. After observing the effect of p-APMSF-treated PAR1-AP and stimulation once with 40 mM K⁺, the contracting effect of the untreated PAR1-AP was examined.

Figure 5

Mobilization of Ca²⁺ in the pregnant rat myometrium during the thrombin-induced contraction. (a) (b) (c) and (d) Representative traces of the thrombin-induced changes in [Ca²⁺]_i (upper trace) and tension (lower trace) in the pregnant rat myometrium observed in the Ca²⁺-free PSS containing 0.1 mM EGTA (a), and in the normal PSS containing 3 mM Ni²⁺ (b), 10 μM diltiazem (c), and 30 μM SK-F96365 (d) in the normal PSS. (e) Summary of the inhibitory effects of diltiazem and SK-F96365 on the thrombin-induced increase in [Ca²⁺]_i and tension. Control; the 1 u ml⁻¹ thrombin-induced [Ca²⁺]_i elevation and tension development in the normal PSS with no inhibitors. The bottom and top levels of columns for [Ca²⁺]_i indicate the values obtained just before the initiation of contraction by thrombin and at the peak contractile response, respectively. The developed tension was evaluated by the area under the tension trace. The [Ca²⁺]_i and developed tension were expressed as a percentage, assigning the values obtained with 40 mM K⁺ PSS to be 100%. The data are the mean±s.e.mean (n=5–8). ^*P<0.05, ^**P<0.01 as compared with the control.

Figure 6

Expression of PAR1, PAR2 and PAR4 mRNA in the rat myometrium during the pregnancy. (a) Representative photographs showing the expression of PAR1, PAR2, PAR4 and β-actin mRNA in the myometrium of non-pregnant (lane 1), pregnant day 11 (lane 2), pregnant day 18 (lane 3), pregnant day 22 (lane 4) and postpartum rats (lane 5), as well as in the rat placenta (lane 6). Lane M, HincII digest of ΦX174 as the DNA size marker. Lane B, PCR product obtained with no addition of RT products. The predicted size of the products is indicated on the side of the photographs. (b) The changes in the expression of β-actin and PAR1 in the rat myometrium during pregnancy. The expression level was expressed as the value relative to that seen in the non-pregnant myometrium. The data are the mean±s.e.mean (n=5–6). ^*P<0.05 as compared with the non-pregnant rats.