Thromboxane A2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity

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Abstract

Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP1, EP2, EP3, DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9α, 11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM–10 μM), a stable thromboxane A2 mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC50 value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4(Z)-6-(2-o-Chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1S-[1α,2α(Z),3α,4α]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [3H]SQ29548 binding site with similar Kd values (30 nM), but Bmax in the circular muscle (90.9±8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2±8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with pKi values in parentheses) was [1S-[1,2(Z),3(1E,3S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2S-[2α(Z),3β(1E,3R*)]]-5-heptenoic acid (CTA2, 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1S-[1α,2β(Z),3α(1E,3R*),5α]]-5-heptenoic acid (PTA2, 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1S-[1α,4α,5α(1E,3R*),6β(Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N-tert-butyl-N¢-[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 μM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP1, EP2 and EP3) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A2-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.

Introduction

Thromboxane A2 is a biologically potent arachidonic acid metabolite synthesized through cyclooxygenase and thromboxane A2 synthase pathways. Thromboxane A2 plays important physiological roles in the regulation of vascular and non-vascular smooth muscle tone, aggregation of platelets, cell proliferation and apoptosis. Vascular contraction and activation of platelets induced by thromboxane A2 are involved in the pathophysiology of myocardia and cerebral infarction Sjoberg and Steen, 1989, Mihara et al., 1989, Senchyna and Crankshaw, 1996, Rolin et al., 2001, Gao et al., 2000. These responses to thromboxane A2 are mediated by the thromboxane A2 receptor (TP), which belongs to a family of seven membrane-spanning receptors that transduce signals through the G protein Narumiya et al., 1999, Wise et al., 2002. Human, bovine and rat TP receptors have recently been cloned (Wise et al., 2002), and two isoforms (TPα and TPβ) have been identified in the human uterus according to the property of signal transduction pathways Miggin and Kinsella, 2002, Moore et al., 2002. TPβ receptor activation stimulates the phospholipase C-inositol 1,4,5-trisphosphate-Ca2+ pathway and RhoA-associated protein kinase. On the other hand, activation of the TPα receptor increases cytoplasmic cyclic AMP by stimulation of the Gs-adenylate cyclase-cyclic AMP pathway (Kinsella et al., 1997).

In the human uterus, 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), a stable and potent agonist of the TP receptor, has been shown to cause contraction of smooth muscle through activation of myogenic TP receptors, and specific binding sites of [125I] ([1S-[1 α,2 α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabi-cyclo[2.2.1]hept-2-yl]5-heptenoic acid) (I-BOP) have been found in the myometrial membrane Senior et al., 1992, Senchyna and Crankshaw, 1996. The results of a molecular biological study have also revealed that the TP receptor gene is expressed in human uterine smooth muscle (Swanson et al., 1992). It has been reported that the concentrations of thromboxane B2, a metabolite of thromboxane A2, increased during pregnancy in the human and rat decidua, placenta, chorion, amnion and myometrium, and reached peaks at the end of pregnancy Mitchell et al., 1978, Zamecnik et al., 1980. Immunohistochemical studies have shown that thromboxane A2 synthase is localized in human placenta, endometrial glands, uterine blood vessels and myometrial cells Wetzka et al., 1993, Wetzka et al., 1994, Swanson et al., 1992. These findings suggest that thromboxane A2 plays an important physiological role in regulation of contractility and vascular tone of the uterus during pregnancy and labor.

Isolated myometrial strips are useful for investigation of the characteristics of prostanoid receptors in the uterus, and remarkable species-related variations in mechanical responses (contraction, relaxation or a mixture of both responses) to prostanoids, including thromboxane A2, have been reported. These species-related variations in mechanical responses are caused by the difference in prostanoid receptor subtype populations in the uterus (Crankshaw, 2001). Functional and molecular biological studies have shown the presence of FP, TP, EP1, EP2, EP3, DP and IP receptors in the human uterus Senior et al., 1991, Senior et al., 1992, Senchyna and Crankshaw, 1996, EP1, EP3, FP, IP and TP receptors in the rat uterus Crankshaw and Gaspar, 1992, Goureau et al., 1992, FP, EP1,EP2, EP3 and TP receptors in the sheep uterus (Crankshaw and Gaspar, 1995) and EP1, EP3 and TP receptors in the guinea-pig uterus (Coleman et al., 1990). In our previous study, we characterized the subtypes of prostanoid receptors in the non-pregnant porcine uterus using naturally occurring prostaglandins and receptor selective-agonists. Contractile FP, EP1, EP3 receptors and relaxant DP, IP, EP2 receptors are present in the porcine uterus, and the distribution of these receptors has been shown to be smooth muscle layer-dependent (longitudinal and circular muscle layers) (Cao et al., 2002). The population of receptor subtypes in the porcine uterus appears to be very similar to that in the human uterus except for the TP receptor. However, the presence of the TP receptor in the porcine myometrium has not been clarified yet.

The aim of the present study was therefore to determine whether TP receptors are present in the porcine myometrium. For this purpose, mechanical responses of TP receptor agonists (U46619, 7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-6yl]-,[1S-[1α,4α,5α(1E,3R*),6β(Z)]]-5-heptenoic acid (U44069) and I-BOP) to uterine contractility and inhibitory effects of TP receptor antagonists (4(Z)-6-(2-o-chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-, [1S-[1α,2α(Z),3α,4α]]-]5-heptenoic acid (SQ29548)) on the response to U46619 were examined in isolated smooth muscles of the porcine uterus. The possible heterogeneous distribution of TP receptors in longitudinal and circular muscle layers was also examined by a radioligand ([3H]SQ29548) binding study. Moreover, in order to find out the role of endogenous thromboxane A2 in regulation of uterine contractility, changes in spontaneous contraction induced by indomethacin (a cyclooxygenase inhibitor) and N-tert-butyl-N¢-[(2-cyclohexylamino-5-nitrobenzene) sulfonyl]urea (BM-531, a TP receptor antagonist with thromboxane A2 synthase-inhibiting activity, Dogne et al., 2001) were also examined.

Section snippets

Tissue preparations

Fresh uteri, with the ovaries intact, from 120 sexually mature crossbred virgin gilts (about 6 months old) were provided by a local abattoir and were used in experiments on the day of slaughter. The pigs were judged to be in proestrus (about day 4 of in the 21-day estrus cycle of the pig) by gross examination of the follicle size (smaller than 2 mm in diameter) and by the appearance of the corpora lutea (McDonald, 1975). Uterine muscle segments (each about 15 mm in length) were isolated

Effect of U46619 on the mechanical activities of longitudinal and circular muscles isolated from the cornu

As shown in Fig. 1A, the isolated longitudinal muscle and circular muscle of the cornu contracted spontaneously in Krebs solution. The frequency of the contraction in the longitudinal muscle was significantly lower than that in the circular muscle as previously reported (longitudinal muscle, 4.1±0.4/10 min, n=18, circular muscle, 22.8±1.8/10 min, n=17, Kitazawa et al., 1998). U46619 (1 nM–1 μM) applied cumulatively to the organ bath stimulated the contractile activities of both smooth muscle

Discussion

We previously reported the muscle layer-dependent heterogeneous distribution of contractile (FP, EP1, EP3) and relaxant (IP, DP, EP2) prostanopid receptors in the non-pregnant porcine myometrium, but the pharmacological effects of thromboxane A2 in this tissue have not been reported (Cao et al., 2002). Therefore, the main objective of this study was to determine whether the porcine uterus expresses functional TP receptors and to elucidate the muscle layer- and region-related differences in TP

Acknowledgments

This work was partly supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (13660304).

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