Regulation Of Myometrial Contractility Research Articles

Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590.

VU590 was the first publicly disclosed, submicromolar-affinity (IC50 = 0.2 μM), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC50 ∼ 8 μM), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K+-dependent, suggesting the VU590 binding site is located within the pore. Mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild-type and binding-site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714 but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology.

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K+ channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K+ conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.

Phosphorylation of h1 Calponin by PKC epsilon may contribute to facilitate the contraction of uterine myometrium in mice during pregnancy and labor

BackgroundThe timely onset of powerful uterine contractions during parturition occurs through thick and thin filament interactions, similar to other smooth muscle tissues. Calponin is one of the thin filament proteins. Phosphorylation of calponin induced by PKC-epsilon can promote the contraction of vascular smooth muscle. While the mechanism by which calponin regulates the contraction of pregnant myometrium has rarely been explored. Here, we explore whether PKC-epsilon/h1 calponin pathway contribute to regulation of myometrial contractility and development of parturition.MethodsWe detected the expression of h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon in the different stages of mice during pregnancy and in labor by the method of western blot and recorded the contraction activity of myometrium strips at the 19th day during pregnancy with different treatments by the organ bath experiments.ResultsThe level of the four proteins including h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice. The ratios of phosphorylated h1 calponin/h1 calponin and phosphorylated PKC-epsilon/PKC-epsilon were reached the peak after the onset of labor in myometrium in the mice. After the treatment of more than 10(9-) mol/L Psi-RACK (PKC-epsilon activator), the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1 calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-epsilon. Meanwhile, the change of the ratio of phosphorylated h1 calponin/h1 calponin was consistent with that of contraction force of mice myometrium strips.ConclusionsThese data suggest that in mice myometrium, phosphorylation of h1 calponin induced by the PKC-epsilon might facilitate the contraction of uterine in labor and regulate pregnant myometrial contractility.

Labour pains: giving birth to new mechanisms for the regulation of myometrial contractility

Labour pains: giving birth to new mechanisms for the regulation of myometrial contractility

Treatment with an inhibitor of catechol-O-methyltransferase activity reduces preterm birth and impedes cervical resistance to stretch in pregnant rats

Catechol-O-methyltransferase (COMT) enzyme catalyzes the methylation of the 2- or 4-hydroxyestrogens to 2- or 4-methoxyestrogens. Both the hydroxyestrogens and methoxyestrogens have been shown to block or enhance the effects of estrogen respectively. Our objective was to investigate the potential role of COMT in parturition and cervical ripening using a rat model. Immunohistochemistry was conducted to detect and localize the COMT protein in rat uterine tissues during pregnancy. We measured the longitudinal changes in urinary 2-hydroxyestrogen before, during, and after pregnancy in rats. Animal studies were conducted to determine the effect of treatment with a selective COMT inhibitor on (1) mifepristone-induced preterm birth and (2) cervical resistance to stretch in pregnant rats. The intensity of staining for the COMT protein differed within the luminal epithelium, uterine gland epithelium, endometrium, and myometrium during pregnancy. Levels of staining for the COMT protein in rat myometrium were highest on day 1 and lowest on days 8 and 13, but high levels returned by days 16 and 19 of pregnancy. The levels of urinary 2-hydroxyestrogen gradually increased in the first 2 weeks of pregnancy, peaked from days 16 to 18 of pregnancy, and then gradually returned to pre-pregnancy levels after delivery. The percentage of pups retained in the uterus of pregnant rats treated with both mifepristone and COMT inhibitor (48 +/- 15%) was significantly higher (P < 0.05) when compared with the value of pregnant rats treated with mifepristone alone (12 +/- 4%). The resistance to stretch was significantly higher (P < 0.05) in cervical tissues from the pregnant rats treated with COMT inhibitor (0.28) when compared with cervical tissues taken from rats treated with vehicle control (0.18). Modulation of COMT activity may play a role in the regulation of myometrial contractility and cervical ripening during pregnancy.

Identification of a non-selective cation channel current in myometrial cells isolated from pregnant rats.

Non-selective cation channel (NSCC) currents were identified in myometrial smooth muscle cells isolated from pregnant rats (day 18-20) using the whole-cell patch clamp method. NSCC currents had a linear current/voltage relationship and were time independent. Reduction of extracellular Na(+) substantially decreased the amplitude of NSCC currents, indicating that the NSCC is permeable to Na(+). NSCC currents were blocked by La(3+) and Gd(3+) with K(d) values of 2.2 and 1.0 micro M, respectively. The relative permeability of various monovalent cations for NSCC was estimated by measurement of the reversal potential. The relative permeability for K(+):Cs(+):Na(+):Li(+) was 1.3:1:0.9:0.8. NSCC also had a small, but detectable, permeability for Ca(2+). Extracellular Mg(2+) inhibited myometrial NSCC currents concentration dependently with a K(d) of 0.28 mM. The observed Mg(2+) block may reasonably explain the inhibitory effect of magnesium on uterine contractions in the treatment of pre-term labor. Our results suggest that the NSCC plays a role in the regulation of myometrial contractility.

Basal and interleukin-1beta-stimulated prostaglandin production from cultured human myometrial cells: differential regulation by corticotropin-releasing hormone.

During pregnancy, placental CRH acts on human myometrium via specific receptors and might play a role in the regulation of myometrial contractility and hence human parturition. The myometrium is the site of production and target of several PGs, which can be activated by cytokines, especially during infection-induced preterm labor. We established primary human myometrial cell cultures that express functional CRH receptors (CRH-R1alpha, -R1beta, -Rlc, and -R2beta) to investigate the possible regulation of PG production by CRH. We studied the effect of CRH on the two major myometrial PGs, PGE2 and 6-keto PGF1alpha. Human CRH was able to partially inhibit basal, interleukin-1beta-stimulated, and oxytocin-stimulated PGE2 production (56 +/- 11%, 45 +/- 8%, and 58 +/- 6% inhibition, respectively). This effect was blocked by a specific CRH receptor antagonist in a concentration-dependent manner. Furthermore, CRH had no effect on 6-keto PGF1alpha production, indicating that the CRH inhibitory action does not involve suppression of cyclooxygenase, the enzyme responsible for the production of both PGE2 and 6-keto-PGF1alpha. These data further support the view that during pregnancy, CRH may promote myometrial quiescence and might play an important role in the regulation of human labor.

Level of postoperative analgesia is a critical factor in regulation of myometrial contractility after laparotomy in the pregnant baboon: Implications for human fetal surgery

Objectives: We used 2 dosage levels of postoperative opioid administration to determine whether the degree of postoperative analgesia after laparotomy during the last third of baboon pregnancy alters maternal pituitary-adrenal function, androgen secretion, and placental estrogen production. We also determined the relationship of estrogen production to surgery-induced increase in myometrial contraction activity. Study Design: After laparotomy under halothane general anesthesia at 0.75 gestation, 10 pregnant baboons were administered intra-arterially either a normal dose or a double dose of the opioid analgesic buprenorphine for 48 hours. Maternal plasma samples for steroid hormone and oxytocin analyses were obtained at circadian time 1000 hours and at circadian time 1800 hours, 4 hours before and 4 hours after the lights went off, respectively. Myometrial electromyographic contraction activity was quantified for the 6 hours from circadian time 1100 hours to circadian time 1700 hours. Results: Maternal plasma cortisol and dehydroepiandrosterone sulfate concentrations were lower in the dark period (at circadian time 1800 hours) than during daylight (at circadian time 1000 hours) in the double-dose group but not the normal dose group. In contrast, maternal plasma estradiol level was higher at circadian time 1800 hours than at circadian time 1000 hours in the normal dose group but not in the double-dose group. Maternal plasma estrogen level was higher during the hours of darkness in the normal dose group than in the double-dose group. Furthermore the number of myometrial contractions around the onset of darkness was greater in the normal dose group than in the double-dose group. Conclusions: The double dose of analgesia results in lower maternal nighttime plasma estradiol concentrations and significantly less nocturnal contraction activity. These observations further confirm an association between increased maternal plasma estrogen concentrations and increased myometrial contractility in the nonhuman primate. In addition, they suggest that adjustment of the level of postoperative analgesia may be of importance in preventing premature labor after chuman intrauterine fetal surgery. (Am J Obstet Gynecol 1999;180:1196-201.)

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.

Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP.

1. There is conflicting evidence in the literature concerning the role of cyclic GMP in the regulation of myometrial contractility and the importance of hormonal status on the uterine response to cyclic GMP-elevating agents. The objective of the present study was to investigate further the importance of cyclic GMP in the control of uterine contractility, by monitoring the effects of cyclic GMP-elevating agents on spontaneous contractions and cyclic GMP levels in myometrial strips from pregnant rats and from ovariectomized rats under the influence of oestrogen and/or progesterone. 2. Sodium nitroprusside (SNP) 5 mM, atrial natriuretic peptide (ANP) 100 nM, L-arginine 1 mM and 8-bromo-cyclic GMP 100 mM had no relaxant effect on the spontaneous contractions of myometria from pregnant rats or from ovariectomized rats under the influence of oestrogen or progesterone. 3. Tissue levels of cyclic GMP were significantly elevated by SNP in all treatment groups, including pregnant animals. For example, in ovariectomized, progesterone-treated rats, SNP raised cyclic GMP levels approximately 8 fold from a basal level of 2.9 +/- 0.4 pmol mg(-1) protein to 24.8 +/- 4.0 pmol mg(-1) protein. ANP increased cyclic GMP levels approximately 2 fold in all treatment groups, except in the pregnant animals. L-Arginine elevated cyclic GMP significantly only in ovariectomized, vehicle-treated myometria. 4. The activity of cyclic GMP-dependent protein kinase (PKG) was significantly increased (3 fold) in myometria exposed to SNP (5 mM). Thus, the inability of SNP to relax uterine preparations was not due to a failure of SNP-elevated cyclic GMP to activate PKG. 5. The more potent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), at a concentration of 100 microM was able to inhibit spontaneous contractions significantly in myometrial preparations from both non-ovariectomized and ovariectomized rats treated with oestrogen or progesterone. 6. Tissue levels of cyclic GMP were markedly increased by SNAP at concentrations of 10, 30 and 100 microM. At 100 microM, cyclic GMP levels increased from 1.9 +/- 0.2 pmol mg(-1) protein to 74.0 +/- 18.0 pmol mg(-1) protein. However, complete or partial blockade of SNAP-induced increases in cyclic GMP levels by the soluble guanylyl cyclase inhibitor, ODQ (25 microM), had no effect on the relaxant response to SNAP. Thus, the relaxant effect of SNAP in this tissue appears to be mediated via a mechanism independent of cyclic GMP. 7. Taken as a whole, the results of the present study indicate that cyclic GMP does not play an important role in the control of contractility in the rat uterus.

Control of human parturition

Parturition is a process that is composed of five separate and distinct physiological components but which lead from one to the next and are, therefore, interdependent. As such, the regulation of myometrial contractility should not be examined in isolation but as part of this continuum. The initiation of labor begins with the biochemical events that result in the rupture of the fetal membranes, effacement of the cervix, and the switch from contractures to contractions. Because we have defined being in labor as the point at which contractions no longer revert to contractures, we suggest that labor is superimposed upon pregnancy in humans and nonhuman primates. There is no withdrawal or retreat from pregnancy, and no evidence exists that the concentrations or actions of progesterone diminish at term. Rather, the target tissues of labor are activated to perform their physiological functions, and these functions are initiated by stimulators. The best candidate for achieving activation is maternal estrogen, derived from fetal DHEAS, but major gaps in our knowledge of this process still exist. Prostaglandins are the most likely candidates as the stimulators of labor initiation, but close inspection of their precise roles also demands that clearer definition of their synthesis and actions be acquired. Coordination of and communication between the physiological events of labor motivates us to examine nontraditional mediators such as cytokines for their potential roles in the regulation of these events at normal term.

Endothelin‐1 receptors in the human myometrium: evidence for different binding properties in post‐menopausal as compared to premenopausal and pregnant women

We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n = 12), myomatous premenopausal women (n = 14) and pregnant women (n = 14), using 125I-labelled endothelin-1. The mean (+/- SD) maximal receptor density (Bmax) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 +/- 196, 1116 +/- 201 and 490 +/- 145 pmol/g protein, respectively). Receptor affinity (Kd) did not differ significantly between these groups. Among the pregnant women, mean Bmax and Kd values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.

Contractile Effects of Endothelin 1 and Endothelin 3 on Myometrium and Small Intramyometrial Arteries of Pregnant Women at Term

This in vitro study was undertaken to investigate the effects of endothelin 1 (ET-1) and endothelin 3 (ET-3) on intramyometrial arteries and myometrial tissue of the uterus from pregnant women at term. Segments of small myometrial arteries (outer diameter 100-500 microns) obtained at cesarean section were isolated and mounted in tissue chambers. The effect of ET-1 and ET-3 on isometric tension was compared with the action of noradrenaline. In similar experiments on myometrial strips, the effect of ET-1 and ET-3 was studied in comparison with oxytocin. ET-1 and ET-3 both produced contractions of arteries and myometrial strips, ET-1 being the most potent. The response of ET-1 was more powerful than the effect of the reference compounds, i.e. noradrenaline with regard to myometrial arteries and oxytocin with regard to the myometrium. It is concluded that endothelins are powerful constrictors of myometrial arteries from pregnant women and possess strong oxytocic effects. In particular, ET-1 could be involved in the regulation of myometrial contractility and/or uteroplacental blood flow.

Uterine activity and hyperactivity Studies of hormonal effects in the regulation of myometrial contractility and uterine blood flow of non‐pregnant women with special regard to the mechanisms of dysmenorrhea

Uterine activity and hyperactivity Studies of hormonal effects in the regulation of myometrial contractility and uterine blood flow of non‐pregnant women with special regard to the mechanisms of dysmenorrhea

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.

Preparation and characterization of rabbit myometrial cells in primary culture: influence of estradiol and progesterone treatment.

Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits. Isolated cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC) at a plating density of 0.5 X 10(6) cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent cultures. The addition of increasing doses of 17 beta-estradiol (E2) (0.1 nM to 1 microM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.

Identification and characterization of receptors for oxytocin in the myometrium of the pregnant ewe.

Receptors for oxytocin were identified in a particulate fraction from the myometrium of the pregnant ewe. Specific binding of [3H]-oxytocin was rapid, reversible, and saturable. It showed an absolute requirement for Mg2+ and the selectivity among oxytocin analogues expected for the receptor. On fractionation of the myometrium by differential and discontinuous gradient centrifugation, [3H]-oxytocin binding showed a fractionation pattern which was similar to that for plasma membrane markers, but different from those for endoplasmic reticulum or mitochondrial markers. It is concluded that oxytocin receptors are located in the plasma membrane of the ewe myometrium. This subcellular fraction is a useful preparation for the study of receptor mechanisms and the regulation of myometrial contractility.