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

Abstract

We have previously reported that the contractile response to thrombin and trypsin was enhanced in the pregnant rat myometrium. We herein determined whether or not sex hormones contribute to this enhancement and the expression of protease-activated receptors (PARs). The nonpregnant rats received daily injections of either 17beta-estradiol or progesterone, and then the contractile response of the myometrium was examined ex vivo. Treatment with either 17beta-estradiol or progesterone had almost no significant enhancing effect on the high K(+)- or oxytocin-induced contraction. On the other hand, both 17beta-estradiol and progesterone dose-dependently enhanced the contractile response to trypsin. A maximal enhancement was obtained at 25 and 40 mg kg weight(-1) day(-1) for 17beta-estradiol and progesterone, respectively. The extent of the enhancement of the trypsin-induced contraction seen in the sex hormone-treated rats in the present study was comparable to that reported in the pregnant rats. However, the contractile response to thrombin and PAR1/PAR2-AP, SFLLRNP was not enhanced either by progesterone or 17beta-estradiol. PAR2-AP and PAR4-AP failed to induce contraction under any conditions. PAR1 mRNA was scarcely detected in the control myometrium by an RT-PCR analysis, while it slightly increased only in the progesterone-treated rats. Neither PAR2 nor PAR4 mRNA was detected. We thus conclude that the responsiveness to trypsin, but not thrombin, is controlled by sex hormones. A novel type of receptor, other than PAR1, PAR2 or PAR4, is suggested to mediate the trypsin-induced contraction as in the case of the pregnant rat myometrium.

Figure 1

Effect of in vivo treatment with 17β-estratiol and progesterone on the trypsin-induced contraction in the isolated rat myometrium. (a–c) Representative recordings showing the contractile responses to 3 μM trypsin in the myometrial strips isolated from rats treated with vehicle (control; a), 40 mg progesterone kg weight⁻¹ day⁻¹ (b) and 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ (c) for 8 days. (c) also shows the representative recordings of the changes in [Ca²⁺]_i induced by 3 μM trypsin. (d) The concentration–response curves of the trypsin-induced contraction in the control myometrium and the myometrium treated with 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ for 8 days. The contractile response was analyzed by the area under the tension trace for the initial 10 min of contraction. The developed tension was expressed as a percentage, while assigning the value obtained in normal PSS and that obtained with 40 mM K⁺ to be 0 and 100%, respectively. The data are the mean±s.e.m. (n=3–6).

Figure 2

The dose-dependent effect of 17β-estradiol and progesterone on the trypsin-induced contraction in the rat myometrium. The rats were in vivo treated with the indicated daily dosage of 17β-estradiol (a, c) and progesterone (b, d) for 8 days. The extent of the contractile responses of the isolated myometrium to 0.3 μM (a, b) and 3 μM trypsin (c, d) were evaluated by the area under the tension trace for the initial 10 min of contraction. The developed tension was expressed as a percentage, while assigning the value obtained with 40 mM K⁺ to be 100%. The data are the mean±s.e.m. (n=3–6). ^*P<0.05 vs the control.

Figure 3

The time-dependent effect of 17β-estradiol and progesterone on the trypsin-induced contraction in the rat myometrium. The rats were in vivo treated with 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ (a, c) and 40 mg progesterone kg weight⁻¹ day⁻¹ (b, d) for the indicated days. The extent of the contractile responses of the isolated myometrium to 0.3 μM (a, b) and 3 μM trypsin (c, d) were evaluated by the area under the tension trace for the initial 10 min of contraction. The developed tension was expressed as a percentage, while assigning the value obtained with 40 mM K⁺ to be 100%. The data are the mean±s.e.m. (n=3–6). ^*P<0.05 vs the control.

Figure 4

Effect of the in vivo treatment with 17β-estradiol and progesterone on the contractile response to thrombin, SFLLRNP and oxytocin in the rat myometrium. Representative traces of the changes in tension induced by 1 U ml⁻¹ thrombin (a–c), 10 μM SFLLRNP (d–f) and 100 nM oxytocin (g–i) in the myometrium isolated from the rats treated with vehicle (control; a, d, g), 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ (b, e, h) and 40 mg progesterone kg weight⁻¹ day⁻¹ (c, f, i) for 8 days.

Figure 5

The dose-dependent effect of 17β-estradiol (a) and progesterone (b) on the contractile response to oxytocin, thrombin and SFLLRNP in the rat myometrium. The developed tension induced by 100 nM oxytocin, 1 μM oxytocin, 1 U ml⁻¹ thrombin and 10 μM SFLLRNP were evaluated by the area under the tension trace for the initial 10 min of contraction and expressed as a percentage, while assigning the value obtained with 40 mM K⁺ PSS to be 100%. The data are the mean±s.e.m. (n=3–6). ^*P<0.05 vs the control.

Figure 6

Effect of TFLLR-NH₂ on the myometrial contraction. Representative traces showing the response to 50 μM TFLLR-NH₂ in the myometrial strips isolated from the control rats (a) and the rats treated with 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ for 8 days (b). Similar results were observed in three separate experiments.

Figure 7

Effect of the in vivo treatment with 17β-estradiol and progesterone on the Ca²⁺-induced contraction in the α-toxin-permeabilized rat myometrium. The concentration–response curves of the Ca²⁺-induced contraction in the absence (a) and presence (b) of GTPγS in the α-toxin-permeabilized myometrial strips obtained from the rat treated with vehicle, 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ and 40 mg progesterone kg weight⁻¹ day⁻¹ for 8 days. The contraction was induced by a stepwise increase in the Ca²⁺ concentrations. The developed tension was expressed as a percentage, assigning the values obtained with the relaxing solution and the maximal tension obtained with 10 μM Ca²⁺ to be 0 and 100%, respectively. The data are the mean±s.e.m. (n=3–4).

Figure 8

The contractile response to thrombin, PAR2-AP (SLIGRL) and PAR4-AP (GYPGKFC) in the myometrium isolated from the progesterone-treated rats. Representative traces showing the response to 1 U ml⁻¹ thrombin (a), 30 μM SLIGRL (b) and 30 μM GYPGKFC (c) in the myometrial strips isolated from the rats treated with 40 mg progesterone kg weight⁻¹ day⁻¹ for 8 days. The responsiveness to 3 μM trypsin was examined at the end of each protocol. Similar results were obtained in three independent experiments.

Figure 9

Bath-transfer experiment. The myometrium isolated from the rat treated with 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ for 8 days was contracted by 3 μM trypsin (a) or 100 nM oxytocin (c). A serine protease inhibitor, p-APMSF, was added to the bath at the concentration of 10 μM. After 10 min, the organ bath solution was transferred to the reporter tissue (b, d).

Figure 10

Effect of the in vivo treatment with 17β-estradiol and progesterone on the expression of PAR1, PAR2, PAR3 and PAR4 mRNA in the rat myometrium. Representative photographs showing the RT–PCR products of PAR1 (545 bp), PAR2 (596 bp), PAR3 (404 bp), PAR4 (336 bp) and β-actin (224 bp) mRNA in the myometrium obtained from the rats treated with vehicle (lane 1), 40 mgprogesterone kg weight⁻¹ day⁻¹ (lane 2), 200 μg 17β-estradiol kg weight⁻¹ day⁻¹ (lane 3) for 8 days, and in the liver (lane 4), placenta (lane 5) and lung (lane 6) obtained from the control rats. The photos are representative of three independent experiments.