Non-pregnant Rat Myometrium Research Articles

Myoinositol Causes Myometrial Contractions in Isolated Non-Pregnant Rat Myometrium

Myoinositol Causes Myometrial Contractions in Isolated Non-Pregnant Rat Myometrium

HSPB8 and the Cochaperone BAG3 Are Highly Expressed During the Synthetic Phase of Rat Myometrium Programming During Pregnancy1

The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.

The relaxation of non-pregnant rat myometrium by resveratrol with participation of the NO–cGMP pathway

Background Resveratrol (RSV) is a phytoalexin produced by grapevines. The benefit of resveratrol to health is widely reported. Resveratrol has been found to promote vascular relaxation but its mechanism of action is unclear. Data about an influence of RSV on the contractility of smooth muscles of the uterus are not available. It has been claimed that NO promotes uterine relaxation by the elevation of cyclic GMP. It is generally accepted that NO increases the intracellular cGMP concentration through activation of solubile guanylate cyclase (sGC). The aim of our study were to investigate the effects of RSV on the contractility of rat uterus and to investigate the involvement of the NO–cGMP pathway in the relaxant effect of RSV on spontaneous rhythmic contractions (SRC) and phasic contractions provoked by oxytocin.

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.

Decreased Expression of the Rat Myometrial Relaxin Receptor (RXFP1) in Late Pregnancy Is Partially Mediated by the Presence of the Conceptus1

The actions of relaxin are mediated by relaxin family peptide receptor 1 (Rxfp1). In pregnant mice, myometrial Rxfp1 expression decreases at term, coinciding with the highest level of circulating relaxin. This down-regulation in Rxfp1 in reproductive tissues has not been investigated in other pregnant animals, nor are the regulatory mechanisms known. In the present study, we examined Rxfp1 gene and protein expression in the nonpregnant, pregnant, and postpartum rat uterus. The potential effects of local conceptus-derived factors on Rxfp1 expression were then examined in unilaterally pregnant rats. Immunoreactive RXFP1 was predominantly detected in the circular smooth muscle layer in the myometrium and in the decidualized endometrium. Rxfp1 was expressed in the rat myometrium from early to midgestation at levels similar to those in nonpregnant rat myometrium, with a significant reduction in expression at both the transcriptional and translational levels during late gestation. In unilaterally pregnant rats, myometrial Rxfp1 was higher in the nongravid compared to the gravid uterine horn, demonstrating a local negative influence of the fetal-placental unit on Rxfp1 expression. In summary, the down-regulation in myometrial Rxfp1 expression at the end of gestation in the rat is partially mediated by the fetal-placental unit and is indicative of a functional withdrawal of relaxin. This may represent a novel mechanism for the activation of spontaneous uterine contractions at labor in this species.

The effects and selectivity of β-adrenoceptor agonists in rat myometrium and urinary bladder

Recent evidence supports a role for beta(3)-adrenoceptors in human non-pregnant and pregnant myometrium. The present study was designed to characterize the pharmacology of beta-adrenoceptors involved in the function of non-pregnant rat myometrium by comparison of the activity of several beta-adrenoceptor agonists and antagonists in isolated rat uterus and urinary bladder. Contractions of myometrial and detrusor strips were induced by adding 1 nM oxytocin and 15 mM KCl respectively. Cumulative concentration-response curves to the selective beta(3)-adrenoceptor agonists, CL 316243 and BRL 37344 and the selective beta(2)-adrenoceptor agonist ritodrine were obtained in the presence and absence of the selective beta(2)-adrenoceptor antagonist ICI 118551 and the non-selective beta-adrenoceptor antagonist bupranolol. Both BRL 37344 (pD(2)=6.79+/-0.09) and ritodrine (pD(2)=6.89+/-0.19) produced potent inhibition of oxytocin-induced contractions in myometrial strips; CL 316243 was inactive at concentrations up to 10 microM. Concentration effect curves to both BRL 37344 and ritodrine were shifted (10 to 30-fold) to the right in the presence of ICI 118551 (10 nM). BRL 37344 (pD(2)=8.51+/-0.21) and CL 316243 (pD(2)=8.61+/-0.24) produced potent inhibition of detrusor strips, while ritodrine was almost 100-fold less potent (pD(2)=5.83+/-0.17). The response to all agonists was significantly attenuated by pretreatment with bupranolol (10 microM), but only ritodrine was affected by ICI 118551 (1 microM). These results demonstrate that relaxation of rat myometrium is mediated by beta(2)-adrenoceptors while, consistent with previous reports, the beta(3)-subtype is primarily responsible for relaxation of rat detrusor.

Oxytocin Receptors Differentially Signal via Gq and Gi Proteins in Pregnant and Nonpregnant Rat Uterine Myocytes: Implications for Myometrial Contractility

Oxytocin (OT) receptors are important regulators of myometrial contractility. By using the activity of large conductance Ca2+-activated K+ (BKCa) channels as readout, we analyzed OT signaling in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium in detail. In nystatin-perforated whole-cell patches from NPM cells, which leave the intracellular integrity intact, OT transiently increased BKCa-mediated outward currents (Iout). This OT-evoked Iout was caused by the Ca2+ transients in response to the Gq/11-mediated activation of phospholipase C and was inhibited by activation of protein kinase A (PKA). In an open-access whole-cell patch (OAP), the OT-induced transient rise in Iout was disrupted whereas the regulation of BKCa by the cAMP/PKA cascade remained intact. OT counteracted the isoprenaline, i.e. the beta-adrenoceptor/Gs-mediated effect in NPM cells measured in OAP. In contrast, OT further enhanced the beta-adrenoceptor/Gs-mediated effect on BKCa activity in PM cells. All OT effects in the OAP were mediated by pertussis toxin-sensitive Gi proteins and PKA. By quantitative real-time PCR and overexpression of the recombinant protein, we demonstrate that an up-regulation of the Gbetagamma-stimulated adenylyl cyclase II during pregnancy is most likely responsible for this switch. By studying the OT-evoked Iout in nystatin-perforated whole-cell patches of PM cells, we further detected that the OT receptor/Gibetagamma-mediated coactivation of adenylyl cyclase II enhanced the beta-adrenoceptor/Gs-induced suppression of the OT-evoked Ca2+ transients and thus diminishes and self-limits OT-induced contractility. The differential regulation of the PKA-mediated suppression of OT-evoked Ca2+ transients and BKCa activity likely supports uterine quiescence during pregnancy.

PAR-2 activating peptide-induced stimulation of pregnant rat myometrium contractile activity partly involves the other membrane receptors

Objective To study if spontaneous contractions augmented by proteinase-activated receptor-2 (PAR-2)-activating peptide serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL) involve coactivation of membrane chemoceptors and are associated with expression of PAR-2 mRNA in non-pregnant and pregnant rat myometrium. Materials and methods Non-pregnant, mid-pregnant, and late pregnant rat uterine horn and small intestine segments were snap-frozen in liquid nitrogen to determine PAR-2 mRNA levels by real time polymerase chain reaction (PCR). Uterine rings were used for isometric tension recording. Effect of SLIGRL (0.1 mM) on spontaneous contractions before and after exposure to ibuprofen (cyclooxygenase inhibitor, 1.0 μM), SQ-29548 (thromboxane A 2 receptor inhibitor, 1.0 μM), ketotifen (histamine 1 receptor inhibitor, 10 μM), WEB-2170BS (platelet-activating factor (PAF) receptor inhibitor, 10 μM), atropine (muscarinic receptor inhibitor, 0.1 μM), or ketanserin (serotonin receptor inhibitor, 10 μM) were compared. Paired t-test and one-way ANOVA followed by Dunnett's or Newman–Keuls post hoc tests were used for statistical analysis when appropriate. Significance P < 0.05. Results The agents did not significantly affect time-associated decay in spontaneous contractile activity in any group of the tissues. Activation of spontaneous contractions induced by SLIGRL in non-pregnant rat myometrium did not involve coactivation of membrane chemoceptors, while in mid-pregnant rat myometrium coactivation of prostanoid, histamine, and serotonin receptors and in late pregnant rat myometrium coactivation of thromboxane receptors was noted. Expression of PAR-2 mRNA was similar in non-pregnant, mid-pregnant, and late pregnant rat myometrium. Conclusions Expression of PAR-2 in rat myometrium is not dependent on gestational age. Stimulation of PAR-2 is associated with production/release of cyclooxygenase pathway product(s) activating thromboxane/prostaglandin H2 receptors, partial involvement of histamine H1 receptors and serotonin receptors in midpregnancy and thromboxane A2/prostaglandin H2 receptors in late pregnancy.

Differences in the gestational pattern of mRNA expression of the Rnd family in rat and human myometria

Uterine myometrial contractility remains a poorly characterized area of research in reproductive physiology. Rnd1, a novel member of the GTP-binding Rho protein family, inhibits Ca 2+-sensitization by specifically interfering with a RhoA/Rho-activated kinases-dependent mechanism in smooth muscle. In addition to Rnd1, there are two other members, Rnd2 and Rnd3, in the Rnd family of Rho proteins. In the present comparative study of myometrial contractility in rats and humans, we found that all three Rnd mRNAs were expressed in nonpregnant rat myometrium and in nonpregnant human myometrial tissues. Although all three mRNA levels increased significantly after gestation in rat myometria, only Rnd1 expression was significantly greater after gestation in human samples. In the ovariectomized rat, administration of estrogen and/or progesterone increased the expression of all Rnd mRNAs. These results suggest that universal Rnd family up-regulation during pregnancy in rats may have an important role for negative-feedback control of uterine contraction during gestation by inhibiting RhoA-mediated increase in Ca 2+ sensitivity of contractile elements. Such increases in Rnd levels may be due to augmented levels of reproductive steroids in rats. Our data also point to gestational differences between rats and humans in Rnd isoform patterns.

Expression of capacitative calcium TrpC proteins in rat myometrium during pregnancy.

External Ca2+ entry into myometrial smooth-muscle cells is important to uterine contraction and hence to labor progression and parturition. Proteins of the transient receptor potential (Trp) channel family are putative capacitative Ca2+ entry channels that respond to contractant-generated signals and intracellular Ca2+ store depletion. Quantitative reverse transcription-polymerase chain reaction was used to examine the relative expression of TrpC mRNAs in rat myometrium and determine their expression pattern during pregnancy and labor. rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNAs, but not rTrpC3 mRNA, were expressed in nonpregnant rat myometrium. With the exception of rTrpC7, the resulting products were sequenced and found to be identical with published sequences; new rTrpC7 sequence exhibited >88% homology to mouse and human TrpC7 coding regions. Relative to beta-actin mRNA, rTrpC4 mRNA was expressed in the greatest abundance. rTrpC1, 5, and 6 mRNAs were expressed at lower levels, whereas rTrpC2 and 7 mRNAs were barely detectable. This relative expression pattern was also observed throughout the course of gestation. There were no major differences in expression of rTrpC1, 2, 4, or 7 mRNAs between Day 13 and Day 21 of gestation or labor. Rat TrpC5 and TrpC6 mRNA expression decreased in pregnancy but was not altered between Day 13 and Day 21 or in labor. Western blot analysis generally confirmed these observations with respect to protein expression. These data suggest that rTrpC4 may play a major role in regulated Ca2+ entry in myometrial cells and throughout pregnancy but do not rule out contributions from other Trp proteins.

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity.

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.

Desensitization of beta2-adrenoceptor function in non-pregnant rat myometrium is modulated by sex steroids.

The effects of in vivo treatment with estrogen and progesterone on isoproterenol-induced uterine relaxation and beta(2)-adrenoceptor (beta(2)AR) mRNA production in non-pregnant rat myometrium were investigated. Whether homologous myometrial desensitization of beta(2)AR function was dependent on or modulated by the two steroids was also examined. Estrogen treatment alone or in combination with progesterone reduced maximal relaxation (E(max)) of isolated uterine strips subsequently challenged with isoproterenol whereas progesterone alone had no effect on this parameter. The reduction was accompanied by an enhanced beta(2)AR mRNA concentration. The concentration of isoproterenol giving half-maximal relaxing response (EC(50)) increased following estrogen treatment and this effect was curbed by progesterone. Isoproterenol had no effect on beta(2)AR transcription irrespective of the steroid regimes employed. E(max) of isolated uterine strips was reduced following prolonged in vivo treatment with isoproterenol but the effect was found only when estrogen alone was administered concomitantly. Finally, in vivo treatment with isoproterenol increased EC(50) of uterine strips subsequently stimulated with isoproterenol in vitro. This effect was independent of steroid treatment. We conclude that homologous desensitization of beta(2)AR function in non-pregnant rat myometrium in terms of sensitivity (EC(50)) is independent of sex steroids but in terms of maximal response (E(max)) occurs only in the presence of estrogen. We speculate whether progesterone withdrawal in connection with the well-known estrogen dominance at rat parturition may strengthen the desensitization induced by beta(2)AR activation and thus contribute to the transformation of the uterus from a quiescent to a highly contractile organ.

The regulation by ovarian steroids of prostaglandin synthesis and prostaglandin-induced contractility in non-pregnant rat myometrium. Modulating effects of isoproterenol.

The objectives of the present study were to investigate the effects of the reproductive steroids oestradiol and progesterone on myometrial levels of cyclooxygenase-2 (COX-2) mRNA and PGF(2alpha) induced myometrial contractility and to study whether the effect of beta(2)-adrenoceptor stimulation by isoproterenol on the myometrium alters these parameters. Oestrogen treatment of ovariectomized rats increased myometrial COX-2 mRNA whereas PGF(2alpha) receptor (PGF(2alpha)-R) mRNA was unchanged following this treatment and maximal contractility (E(max)) of isolated uterine strips challenged with PGF(2alpha) was unaltered. Progesterone treatment alone decreased COX-2 mRNA in comparison with values obtained from oestrogen-treated animals, and in combination with oestrogen the enhancing effect of progesterone on COX-2 mRNA was curbed. EC(50) of uterine strips challenged with PGF(2alpha) increased following oestrogen treatment whereas this parameter was substantially decreased following progesterone treatment. When oestrogen was combined with isoproterenol infusion mRNA values of both COX-2 and PGF(2alpha)-R were reduced. Finally, when isoproterenol infusions were given in combination with both oestrogen and progesterone, PGF(2alpha)-R mRNA and E(max )were enhanced as compared with similar rats not having received isoproterenol. We conclude that oestrogen increases COX-2 mRNA production and subsequent prostaglandin synthesis in non-pregnant rat myometrium. We further conclude that in the oestrogen-dominated rat myometrium the relaxing effect of beta(2)-adrenoceptor stimulation involves attenuation of both prostaglandin synthesis and PGF(2alpha)-R expression. We finally conclude that in the presence of both oestrogen and progesterone this effect of beta(2)-adrenoceptor stimulation is restrained.

Up-regulation of oxytocin receptors in non-pregnant rat myometrium by isoproterenol: effects of steroids.

The objective of the present study was to further elucidate our previous observation that beta2-adrenoceptor activation induces oxytocin receptor (OTR) expression in rat myometrium. We wanted to investigate whether the mechanism behind this effect was under the influence of gonadal steroids. Ovariectomized non-pregnant rats were treated with estrogen, progesterone or a combination of both for 3 days. Some rats were concomitantly treated with isoproterenol. Estrogen treatment increased both OTR mRNA production and maximal binding of [3H]-oxytocin to isolated myometrial plasma membranes, but it did not affect contractility of isolated uterine strips challenged with oxytocin. When the estrogen regimen was combined with isoproterenol treatment, an augmented maximal contractile response (Emax) to oxytocin was observed although no further increase in OTR mRNA and binding was seen. Progesterone treatment did not in itself alter OTR mRNA, OTR binding or Emax. However, OTRs were induced at the level of gene expression when progesterone was supplemented with isoproterenol infusion. Finally, progesterone suppressed the effect of estrogen on OTR mRNA production and binding when the two compounds were administered together. However, when isoproterenol treatment was added this effect was abolished and Emax was enhanced more than that seen following treatment with estrogen alone. These data suggest that beta2-adrenoceptor activation represents an important regulator of OTR expression/function in estrogen- and progesterone-dominated rat myometrium.

Inhibitory Effects of Isradipine on Spontaneous and Oxytocin- and Carbachol-Stimulated Contractions of Rat Myometrium

Our aim was to investigate the effect of isradipine, a second-generation calcium channel blocker, on spontaneous and oxytocin- and carbachol-stimulated contractions of myometrium isolated from nonpregnant and pregnant rats. Amplitude, frequency, duration and integrated area of spontaneous and oxytocin- and carbachol-stimulated contractions of nonpregnant and pregnant rat myometrium were compared before and after the treatment with isradipine (10^–6 to 10^–4 mol/l). Isradipine inhibited contractions of myometrial strips isolated from nonpregnant and pregnant rats in a concentration-dependent manner, with a significant effect on the amplitude (10^–5 to 10^–4 mol/l) and integrated area (3 × 10^–6 to 10^–4 mol/l) of spontaneous and oxytocin- and carbachol-stimulated contractions. Isradipine induced similar concentration-dependent effects on the frequency and duration of spontaneous and carbachol-stimulated contractions at higher concentrations (3 × 10^–5 to 10^–4 mol/l) but had no effect on frequency and duration of oxytocin-stimulated contractions. Isradipine appears to be an effective relaxant in rat myometrium. This effect of isradipine may become therapeutically advantageous in clinical application for preterm labor.

Mechanisms of galanin-induced contraction in the rat myometrium.

A neuropeptide, galanin, regulates the reproductive process and directly induces myometrial contraction. The aim of this study was to determine the mechanism of galanin-induced myometrial contraction. For this purpose, we simultaneously measured intracellular Ca2+ concentration ([Ca2+]i) and tension using fura-PE3-fluorometry and the rat longitudinal myometrium. The effect of galanin on the Ca2+ sensitivity of the contractile apparatus was examined in beta-escin permeabilized strips. The expression of galanin and the galanin receptors mRNAs in the rat myometrium were determined by reverse transcription polymerase chain reaction (RT-PCR). Galanin (10-300 nM) induced phasic contraction with or without oscillation in the pregnant rat myometrium in a concentration-dependent manner. The maximal response was obtained at 100 nM. There was no significant difference either in the maximal responses or EC50 values for galanin-induced myometrial contractions among myometriums from non-pregnant and pregnant (day 4, day 11, day 20, day 22) rats. In the day 20 and 22 pregnant myometriums, assigning the levels of [Ca2+]i and tension at 40 mM K+-depolarization to be 100%, galanin increased the [Ca2+]i and tension to 126.9+/-2.9% and 116.3+/-2.7%, respectively. Diltiazem (10 microM) inhibited the galanin-induced elevation of [Ca2+]i and tension to 71.9+/-2.4% and 16.2+/-0.7%, respectively. Ni2+, by itself, decreased the basal [Ca2+]i to -50.2+/-3.9% without affecting resting tension. After Ni2+ treatment, galanin-induced increases in [Ca2+]i and tension were -19.6+/-3.4% and 0.9+/-0.1%, respectively. In myometrium treated with diltiazem, no oscillation in [Ca2+]i and tension was observed. In Ca2+-free solution with 0.1 mM EGTA, galanin increased [Ca2+]i from -40.2+/-2.7% to -18.0+/-2.6% and induced transient contraction (3.6+/-0.8%). In beta-escin permeabilized myometrium, galanin enhanced the contraction induced by 0.3 microM Ca2+ in the presence of GTP. In the presence of GDPbetaS (1 mM) instead of GTP, galanin failed to increase the Ca2+ sensitivity of the contractile apparatus. RT-PCR revealed that galanin mRNA was hardly expressed in the non-pregnant rat myometrium and increased to reach a maximal level at mid pregnancy (day 11), but decreased to the same level as in the non-pregnant myometrium at term (day 22). Type 2 galanin receptor (GALR2) mRNA was found to be expressed in the rat myometrium whereas type 1 galanin receptor (GALR1) mRNA expression was not detected. In conclusion, galanin induces contraction of the rat myometrium by increasing [Ca2+]i as well as by increasing Ca2+ sensitivity of the contractile apparatus. Galanin-induced increases in [Ca2+]i are caused by both intracellular Ca2+ release and Ca2+ influx from extracellular space. The responsiveness of the rat myometrium to galanin does not change during pregnancy. The galanin mRNA is expressed in the rat myometrium and it is upregulated during mid-pregnancy. Rat myometrium expresses GALR2 but not GALR1 mRNA. By changing mRNA expression in the myometrium during pregnancy, galanin may act as a paracrine or autocrine mediator in the regulation of myometrial contractility.

Beta2-adrenoceptor desensitization in non-pregnant estrogen-primed rat myometrium involves modulation of oxytocin receptor gene expression.

The nona-peptide oxytocin (OT) induces contraction of the myometrium by interaction with specific plasma membrane associated OT receptors (OTR), whereas stimulation of beta2-adrenoceptors (beta2AR) causes relaxation. Homologous desensitization of the myometrium to both hormones has been described. However, a possible interaction between the two systems has not been investigated. In the present study, long-term in vivo treatment of non-pregnant estrogen-primed rats with isoproterenol decreased maximal relaxation of isolated uterine strips challenged with isoproterenol. Increased EC50 values of similarly treated animals suggest that the coupling between receptor occupancy and contractile response was impaired. Since beta2AR mRNA levels were left unchanged, we conclude that the homologous desensitization to beta2 stimulation is not due to changes in beta2AR gene expression. OT infusion did not alter beta2AR mRNA levels or isoproterenol-induced relaxation of isolated uterine strips. Treatment with OT had no effect on the amount of myometrial OTR mRNA. We have previously found that OT down-regulates OTR in the non-pregnant rat myometrium, but this therefore does not appear to take place at the level of mRNA production. Isoproterenol treatment resulted in a three-fold increase in OTR mRNA. This was accompanied by a 91% rise in OTR binding and an augmented contractile response of isolated uterine strips to OT, suggesting that the increased production of mRNA reflects formation of active receptors. Neither OTR affinity nor EC50 of in vitro strips was affected by isoproterenol treatment. We conclude that stimulation of beta2AR causes heterologous up-regulation of OTR in the non-pregnant estrogen-primed rat myometrium.

Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate–phenol–chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde–1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase–polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase–polymerase chain reaction studies have confirmed expression of the phospholipase C-β1a, phospholipase C-β3, phospholipase C-γ1, phospholipase C-γ2, and phospholipase C-δ1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-β3, phospholipase C-γ1, and phospholipase C-δ1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-β1a isoform was expressed at the highest level, the phospholipase C-β3, phospholipase C-γ1, and phospholipase C-γ2 isoforms at an intermediate level, and the phospholipase C-δ1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle. (Am J Obstet Gynecol 1998;178:848-54.)

The role of protein kinase C as a modulator of oxytocin-stimulated phasic contractions in pregnant and nonpregnant rat myometrium

The role of protein kinase C as a modulator of oxytocin-stimulated phasic contractions in pregnant and nonpregnant rat myometrium

The contracture produced by sodium removal in the non-pregnant rat myometrium.

1. Mechanical responses to Na removal were investigated in the circular and longitudinal muscles of the non-pregnant rat myometrium at 35 degrees C. In both muscles, reduction of the external Na concentration to less than 20 mM produced an initial acceleration of phasic contractions and a sustained tonic contracture. No difference was found with different Na substitutes (Tris-hydroxymethyl aminomethane, choline, dimethyl diethanol ammonium). However, when Mg was substituted for Na, only the tonic contracture was produced without the phasic contractions. 2. Readmission of 5-10 mM-Na, after exposure to Na-free solution, relaxed the contracture produced by Na removal. The degree of relaxation was dependent on the Na concentration readmitted and on the period of pre-treatment with Na-free solution, being stronger with longer pre-treatment. 3. In the presence of Na, excess Ca failed to increase the muscle tone. In the absence of Na, the tension development was closely related to the external Ca concentration up to 20 mM. In the absence of both Ca and Na, some tension remained. Even after pre-treatment with Ca-free solution containing 0.1-0.5 mM EGTA, removal of Na caused some mechanical response. A similar small tension development was observed when Na removal was repeated during prolonged absence of external Ca for more than 3 h. 4. Verapamil (2 X 10(-4) M) markedly suppressed the response to Na removal, but it did not block it, either in the presence or in the absence of Ca. Ouabain (10(-3) M) in the presence of verapamil potentiated the early phasic component of the response to Na removal, but the tonic component was little affected or even slightly reduced. 5. The results indicate that there are three components in the mechanical response to Na removal: the phasic and tonic components, which are highly Ca-dependent, and the third small tonic component, which is independent of external Ca. Most of the phasic and tonic responses seem to be due to an increase in Ca permeability, but this may be secondary to membrane depolarization. A Na-Ca exchange mechanism is also considered to contribute to the transient phase of the response to Na removal and to Na readmission.