Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy

Nitric oxide (NO), derived from L-arginine by the action of nitric oxide synthase (NOS), is a mediator of many diverse biological activities, including vasodilation, neurotransmission and inhibition of platelet adhesion. A role for NO in the maintenance of rat and rabbit pregnancy is supported by a variety of studies. A recent study in women demonstrated that myometrial inducible NOS (iNOS) expression was greater in the early third trimester than either the late third trimester or in the non-pregnant condition, suggesting that increased iNOS expression is involved in the maintenance of human pregnancy. Constitutive NOS (cNOS) expression was not determined. The aim of this study was to compare constitutive NOS (both eNOS and bNOS) expression in the human non-pregnant uterus, preterm pregnant uterus (25-34 weeks gestation) and term pregnant uterus (>37 weeks gestation) using immunohistochemistry and Western blotting. Preterm pregnant samples were taken from women with a variety of pathologies necessitating early delivery. We found that eNOS and bNOS protein concentrations were greater in the preterm pregnant myometrium than non-pregnant myometrium. eNOS, but not bNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained preterm. We conclude that the constitutive isoforms of NOS are also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence.     

Control of intracellular Ca2+ activity in rat myometrium.

Four fractions enriched, respectively, in plasma membrane (PM), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria were isolated from estrogen-dominated rat myometrium. Ca2+ uptake by these fractions was studied in order to estimate the relative potential of the corresponding organelles for controlling intracellular Ca2+ activity. Ca2+ uptake properties of the PM, SER, and RER fractions were similar except that potentiation by oxalate was in the order RER greater than or equal SER greater than PM. However, studies with the ionophores X-537A and A23187 suggested that Ca2+ was transported into the lumen of membrane vesicles of all these fractions. Unlike that of skeletal muscle sarcoplasmic reticulum, Ca2+ uptake by the myometrial fractions was not supported by high-energy compounds other than ATP. Mitochondria took up much less Ca2+ at low, and much more Ca2+ at high, free Ca2+ concentrations than did the other fractions. The amount of Ca2+ taken up in 30 s from a 1 muM free Ca2+ solution in the presence of ATP was similar for all fractions. These results suggested that mitochondria may act as an important Ca2+ control system in rat myometrium when the intracellular Ca2+ concentration is near 1 muM or higher, whereas the PM, SER, and RER may be of major importance at Ca2+ levels of 0.3 muM or lower.     

Active pharmaceutical ingredients and mechanisms underlying phasic myometrial contractions stimulated with the saponin extract from Paris polyphylla Sm. var. yunnanensis used for abnormal uterine bleeding

BACKGROUND: Total steroidal saponins of Paris polyphylla Sm. var. yunnanensis (TSSP) have been widely used in China for the treatment of abnormal uterine bleeding (AUB). But until now, the main active constituents and the mechanisms underlying the pharmacological actions on uterine activity have not been described. METHODS: Total steroidal saponins were extracted with EtOH and purified by chromatography. In vitro isometric contraction studies were performed using myometrial strips from estrogen-primed or pregnant rats. Intracellular calcium was monitored under a confocal microscope using Fluo-3 AM-loaded myometrial cells. RESULTS: TSSP dose-dependently induced phasic myometrial contractions in vitro. Experiments with calcium channel blockers or kinase inhibitors demonstrated that the TSSP-stimulated myometrial contraction was mediated by an increase in [Ca(2+)](i) via influx of extracellular calcium and release of intracellular calcium. Through bioassay-guided separation, it was found that total spirostanol saponins exhibited contractile activity in myometrium and Pennogenin-3-O-alpha-L-arabinofuranosyl(1-->4)[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (PARG) was identified as the active ingredient of TSSP. Furthermore, the contractile response of rat myometrium to PARG was significantly enhanced with advancing pregnancy. CONCLUSIONS: These data provide evidence that myometrial contractility stimulated by TSSP results from [Ca(2+)](i) increase and supports the possibility that some spirostanol gylcosides may represent a new type of contractile agonist for the uterus.     

Luteinizing hormone/human chorionic gonadotropin receptors in myometrium and their role in uterine motility

The object of this paper is the existence of LH/hCG receptors in female uterine tissue. Specific and high-affinity binding sites for LH are present in the pig and rabbit uterus. Estradiol promoted the synthesis of LH receptors in porcine myometrium. Stimulation of LH receptors with hCG in estrogen-primed tissue had a quiescence effect on myometrial contractility in vitro. The physiological role of uterine LH receptors in maintenance of myometrial quiescence is discussed.     

Some aspects of calcium uptake by human myometrial mitochondria and microsomes relevant to relaxation

Calcium uptake by mitochondria and microsomes isolated from the human myometrum was studied at physiological Ca⁺⁺ concentrations. The initial rates as well as the maximum velocity of Ca uptake by mitochondria were 10–20 times higher than those by microsomes. The Ca⁺⁺ concentration for half-maximal transport in the mitochondria and microsomes was about 1 μM and 0.5 μM, respectively. The Ca uptake capacity of mitochondria measured after 20 min of uptake (1 μM Ca⁺⁺ in the medium) was 10–30 times higher than that of microsomes. The capacity but not the initial rates of Ca uptake by microsomes was increased in the presence of 5 mM oxalate. There was only minor differences in the Ca uptake kinetics of subcellular fractions isolated from the pregnant and non-pregnant myometria. The results of this study reinforce the argument for a domineering role of mitochondria in the relaxation of the human myometrium.     

败血症大鼠心肌线粒体钙转运能力的变化

为探讨心肌线粒体钙调节功能在休克大鼠心肌细胞钙超负荷发生中的作用。本文采用结扎大鼠盲肠加穿孔的腹膜炎败血症休克模型,观察到休克早期、晚期线粒体钙含量分别增加180%、330%,休克晚期线粒体钙转运能力明显降低(摄钙量减少34.6%,摄钙速率降低33%,P<0.01)。结果提示,心肌线粒体钙转运能力的降低可能是休克动物心肌钙超负荷,继而导致心功能障碍的重要原因之一。     

Effects of L-arginine and sodium nitroprusside on the spontaneous contractility of human non-pregnant uterus

BACKGROUND: The aim of this study was to investigate, in isolated human non-gravid myometrium, the involvement of the cyclic guanosine monophosphate (cGMP) pathway in nitric oxide (NO) induced relaxation. METHODS: Strips of human myometrium from hysterectomized women were suspended in organ baths for recording of isometric tension. Cumulative concentration-response curves for L-arginine and sodium nitroprusside were performed in the presence of methylene blue (10 micromol/l) or vehicle (control). The effect of increasing concentrations of 8-bromo-cGMP on uterine spontaneous contraction was also studied. RESULTS: L-arginine and sodium nitroprusside induced a concentration-dependent decrease in the amplitude of the myometrial spontaneous contractions. Pre-treatment with methylene blue enhanced the inhibitory effect of L-arginine and sodium nitroprusside on myometrial spontaneous contractions. In addition, 8-bromo-cGMP had no effect on spontaneous contractions in human myometrium. CONCLUSIONS: These data provide evidence that L-arginine and sodium nitroprusside inhibit the spontaneous contractions of the non-pregnant human uterus through a cGMP independent pathway.     

珠子参总皂甙、三七总皂甙及绞股兰总皂甙对心肌缺血再灌损伤的保护作用

采用Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流方法，以停灌３０分钟，再灌注２０分钟制备大鼠心肌缺血再灌损伤模型。三七总皂甙（ＰＮＳ３０ｍｇ／Ｌ）和绞股兰总皂甙（ＧＰ，３０ｍｇ／Ｌ和５０ｍｇ／Ｌ）显著降低不可逆室颤发生率，５０ｍｇ／Ｌ的ＧＰ显著抑制心肌ＬＤＨ释放，ＰＮＳ和ＧＰ显著提高缺血再灌心肌ＳＯＤ活性，ＧＰ明显降低其ＭＤＡ水平，ＰＮＳ明显降低心肌钙含量。在结扎家兔冠状动脉左室支２小时，再灌注３０分钟所造成的心肌缺血再灌损伤模型中，珠子参总皂甙ＰＪＳ，２５０ｍｇ／ｋｇ）和ＰＮＳ（２００ｍｇ／ｋｇ）轻度改善缺血再灌后的左室功能，显著降低再灌后血浆ＬＤＨ和ＣＰＫ水平，明显减轻心肌钙聚积。以上结果表明：ＰＪＳ，ＰＮＳ和ＧＰ对心肌缺血再灌损伤具有保护作用，ＰＪＳ和ＰＮＳ的作用机理可能与拮抗钙有关，ＧＰ的作用机理可能与抗脂质过氧化有关。     

Expression and modulation of Rho kinase in human pregnant myometrium

There is little information outlining the role of Rho kinase, RhoA, and calcium sensitization in regulation of human uterine contractility during pregnancy. The aims of this study were to investigate the expression of RhoA, and the Rho kinases ROCK I and ROCK II in human pregnant myometrium, to evaluate the effects of Rho kinase inhibition on pregnant human myometrial contractility in vitro, and to compare these effects with those of the calcium channel blocker nifedipine. RT-PCR using primers for RhoA, ROCK I and ROCK II was performed on mRNA isolated from human pregnant myometrium. Isometric recording was performed in isolated myometrial strips obtained at Caesarean section. The effects of the Rho kinase inhibitor Y-27632 (1 nmol/l to 10 mmol/l), and nifedipine (1 nmol/l to 10 mmol/l), on oxytocin (0.5 nmol/l) induced contractions were measured and compared. Expression of RhoA, ROCK I and ROCK II mRNA was identified in human pregnant myometrium (n = 3). Y-27632 exerted a potent relaxant effect on myometrial contractility with a pD(2) value (+/- SEM) of 7.63 +/- 0.38 (n = 6). The maximum net relaxant effect (+/- SEM) was 72.3 +/- 6.1% (n = 6). Corresponding values for nifedipine were 7.24 +/- 0.48 (n = 6; P = 0.469) and 93.40 +/- 3.1% (n = 6; P = 0.028). Rho A/Rho kinase-mediated calcium sensitization may play role in the physiology of human parturition, and pharmacological inhibition of this pathway may therefore provide a novel approach to tocolysis for pre-term labour.     

Increased calcium uptake by muscle mitochondria of cold-acclimated rats.

Skeletal muscle mitochondria of cold-acclimated rats have an altered morphology that is related to the occurrence of nonshivering thermogenesis. The transport of calcium by these mitochondria was studied in a search for an alteration in an energy-dissipating mechanism which might be related to the altered morphology and to the altered mode of thermogenesis in the cold-acclimated animal. The rates of calcium uptake, of calcium-stimulated respiration, and of state 4 respiration after calcium uptake were increased in the altered mitochondria. The capacity to accumulate calcium without phosphate was increased, whereas with phosphate all the calcium was removed from the medium and no difference in total uptake was seen. Spontaneous release of calcium was greater but sodium-induced release was unchanged. No effect of cyclic AMP or prostaglandin E1 on release of calcium was seen. The increase in rate of calcium uptake occurred gradually during the first 3-5 wk of acclimation to cold. The results are considered to give some support to the hypothesis that adaptive changes in the mitochondrial calcium transport cycle in skeletal muscle occur during acclimation to cold.     

Myometrial zonal differentiation and uterine junctional zone hyperplasia in the non-pregnant uterus

Human non-gravid myometrium differentiates in response to ovarian sex steroids into a subendometrial layer or junctional zone and an outer myometrial layer. Compared to the outer myometrial layer, the junctional zone myocytes are characterized by higher cellular density and lower cytoplasmic-nuclear ratio. These structural differences allow in-vivo visualization of the myometrial zonal anatomy by T2-weighted magnetic resonance (MR) imaging. The human myometrium is also functionally polarized. Video-vaginosonography studies have shown that propagated myometrial contractions in the non-pregnant uterus originate only from the junctional zone and that the frequency and orientation of these contraction waves are dependent on the phase of the menstrual cycle. The mechanisms underlying zonal myometrial differentiation are not known, but growing evidence suggests that ovarian hormone action may be mediated through cytokines and uterotonins locally released by the basal endometrial layer and endometrio-myometrial T-lymphocytes. Irregular thickening of the junctional zone due to inordinate proliferation of the inner myometrium, junctional zone hyperplasia, is a common MR finding in women suffering from menstrual dysfunction. Preliminary data suggest that junctional zone hyperplasia is further characterized by loss of normal inner myometrial function. Although irregular thickening of the junctional zone has been associated with diffuse uterine adenomyosis, the precise relationship between subendometrial smooth muscle proliferation and myometrial invasion by endometrial glands and stroma remains to be established.     

Characterization of calcium channel forming activity of Entamoeba histolytica in model biological membranes.

Entamoeba histolytica is an invasive enteric protozoan parasite whose cytolytic activity is associated with irreversible increases in target cell intracellular calcium. We studied the effect of homogenates of virulent E. histolytica on calcium permeability of bovine chromaffin granules and rat liver mitochondria, model membrane systems whose mechanisms of ion transport and permeability are well characterized. Treatment of chromaffin granules, with an extract of E. histolytica resulted in a dose-dependent increase in calcium uptake. These effects were similar to that seen with the calcium ionophore A23187 and indicate that the homogenate acts as a divalent cation ionophore. However, unlike what is observed with A23187, the Ca2+ uptake was greater in the presence of the permeable anion Cl- than in its absence. Also, in contrast to the calcium ionophore A23187, a homogenate of E. histolytica caused no calcium release from rat liver mitochondria but resulted in a dramatic increase in the rate of calcium accumulation. The amebic homogenate did not bind calcium in the absence of mitochondria. The increase in mitochondria calcium uptake occurred only in the presence of respiration and with normal state 3 and 4 oxygen consumption. Calcium accumulation occurred both in the presence and absence of 5 mM Pi. Substitution of NaCl or KCl for sucrose in the medium did not alter the enhanced mitochondria calcium uptake. Most data are consistent with the hypothesis that the E. histolytica homogenate is acting as a calcium ionophore transporting charged calcium electrophoretically across the chromaffin granules and the mitochondria inner membrane. This activity may contribute to the cytopathogenicity by E. histolytica.     

Regulation of Human Myometrial Contractility During Pregnancy and Labour: Are Calcium Homeostatic Pathways Important?

If we are to develop new strategies for the treatment and management of preterm and dysfunctional term labour, it is imperative that we improve current understanding of the control of human uterine activity. Despite many studies of animal pregnancy, there is a paucity of knowledge relating to the complex control of human myometrium during pregnancy. It is hypothesized that human myometrium is relatively quiescent during the majority of pregnancy and that as term approaches there is cascade of molecular events that prepare the uterus for labour. This review will consider the cellular mechanisms involved in the regulation of human myometrial activity and the modulation of these by hormonal and mechanical signals. In particular, the contribution of calcium homeostatic pathways to the control of human myometrial contractility during gestation will be discussed. Experimental Physiology (2001) 86.2, 247-254.     

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.     

Studies on calcium uptake by myometrial microsomes with particular reference to the dependence on inorganic phosphate and oxalate.

Ca uptake by microsomes isolated from non-pregnant rabbit myometrium was potentiated by both inorganic phosphate (Pi) and oxalate anions. Both Pi and oxalate had little effect on the initial rate of uptake but a pronounced effect on the capacity of Ca uptake measured after 20 min which was greater in the presence of oxalate than that of Pi (5 mM each). The presence or absence of sucrose in the uptake medium had a significant effect on oxalate-induced potentiation of Ca uptake but not on that potentiated by Pi or that measured in the absence of either potentiating anion. A part of Ca accumulated additionally under the influence of sucrose could be removed by washing microsomes with KCl. Another significant difference between the pontentiating effect of oxalate and Pi was observed when the pH of the incubation medium was varied. In the presence of oxalate the pH optimum was between 6.4--6.8, whereas that in its absence or in the presence of Pi the optimal pH was around 7.2. Reduction in pH from 7.2 to 6.8 along with the substitution of KCl by sucrose resulted in 3-fold increase in Ca uptake when oxalate was used as the potentiating anion. The results suggest that Ca is taken up by a different mechanism in the presence of oxalate than that in its absence or when oxalate anion is substituted with inorganic phosphate.     

Estrogen and the relaxant effect of intramural noradrenaline on calcium induced contractures in depolarized rat uterus

The influence of intramural noradrenaline on calcium induced contractures was studied in isolated preparations of rat uterus. The depolarized (127 mM KCl) myometrium of oophorectomized rats responded with contraction followed by a transient relaxation when exposed to 3 mM calcium. The threshold concentration of calcium, where the transient relaxation began to appear, was between 0.25 and 0.5 mM. Blockade of the beta-adrenoceptors with propranolol or noradrenaline depletion with reserpine completely removed the transient relaxation, indicating that the latter was due to release of intramural noradrenaline. Estrogen treatment abolished the relaxant effect of intramural noradrenaline, whereas progesterone was ineffective in this respect. Preparations from rats in natural estrus responded like estrogenized tissues, and diestrus preparations behaved as uteri of oophorectomized rats without estrogen treatment.     

Uterine myometrium as a cell patch as an alternative graft for transplantation to infarcted cardiac myocardium: a preliminary study

PURPOSE: Currently, only a small fraction of patients are able to receive reperfusion therapy for myocardial infarctions. We hypothesize that myometrial cell patch transplantation could be an alternative approach for the treatment of myocardial infarction. DESIGN: We performed a preliminary study to determine the feasibility of this novel therapeutic approach in a rabbit model. PROCEDURES: Six adult female New Zealand rabbits were used. Myocardial infarction was induced by left anterior descending artery ligation. A segment of uterus was removed via a laparotomy incision, and this uterine segment was transplanted as an autologous graft over the infarcted myocardium, which was then reinforced by greater omentum. Statistical methods and outcome measures: Hemodynamic measurements and histological studies. Main findings: All uterine myometrial patches survived in the test animals. Fluoroscopic hemodynamic measurements were made for ejection fractions at 8 weeks after the application of the uterine patch. Histological study demonstrated well-healed myometrial-myocardium junctions with minimum scar tissue. Angiogenesis occurred in the transplanted myometrium. Connexin 43 expression was demonstrated in the transplanted patches. CONCLUSION: Our noncontrolled preliminary rabbit experiments indicate that patches of uterine myometrium reinforced by greater omentum can be used as autologous transplant therapy for infracted myocardium. This is an innovative technique that could lead to future treatment for individuals who may suffer from an infarcted myocardium and may not be eligible for traditional reperfusion therapy.     

Characterization of frog muscle mitochondria.

Studies on oxidative phosphorylation revealed that, in frog skeletal muscle mitochondria (SKMM) from the thigh, the adenosine diphosphate/oxygen ratio (ADP/O) was 2.8 +/- 0.1 SE, and the respiratory control ratio was 9.5 +/- 0.9, with pyruvate/malate as the substrate. Oxygen uptake rate (Qo2) was 225 mumol O2 per minute per gram mitochondrial protein +/- 13; phosphorylation rate (ADP/O X Qo2 X 2) was 1,230 mumol ADP phosphorylation per minute per gram mitochondrial protein +/- 77; and the phosphorylation capacity (phosphorylation rate times tissue mitochondrial protein content) was 3.6 mumol ADP phosphorylated per gram wet weight of muscle +/- 0.2. Tissue mitochondrial protein content was determined by the measurement of nicotinamide adenine dinucleotide (NADH) oxidase activity. Electron microscopy (EM) revealed intact, isolated, energized twisted mitochondria of a condensed form. Frog sartorius muscle mitochondria gave similar oxidative phosphorylation parameters when investigated independently of the rest of the thigh. These values of SKMM respiration from the frog are similar to those values obtained from pigeon and rabbit heart and rat skeletal muscles. However, because of the low NADH-oxidase activity indicating reduced mitochondrial content (this was verified in low-magnification EM pictures), phosphorylation capacity was significantly reduced in frog skeletal muscle mitochondria.     

Circulating excitations and re-entry in the pregnant uterus

 The propagation of individual action potentials during spontaneous bursts of activity in isolated pregnant rat myometrium in the final stage of pregnancy was analyzed. Simultaneous recordings from 240 extracellular recording sites (inter-electrode distance 1 mm) made it possible to reconstruct, in spatial and temporal details, the conduction of the electrical impulse. On several occasions, the impulse was seen to be conducted in a circular fashion whereby the impulse repeatedly re-excited the myometrium. No evidence was found of circuits rotating around an area of depressed excitability or anatomical obstacle, suggesting that these circuits are similar to those proposed to occur in cardiac muscle by the ”leading circuit” model. Because of the anisotropic conduction properties of the myometrium, several circuits revolved in an ellipse with the long axis parallel to the longitudinal fiber direction, similar to functional re-entry in the anisotropic ventricle. Of a total of 46 bursts analyzed, myometrial re-entry occurred in 10 of the bursts. Furthermore, re-entries were found at day 17, day 19 and day 21 ( = term) stages of pregnancy suggesting that re-entry may occur throughout the final stage of gestation. In conclusion, functional re-entry, previously shown in the myocardium, may also occur in the pregnant myometrium. The presence of re-entry in the uterus could underlie the mechanism for uterine tachysystole, leading to dysfunctional labor. Received: 23 May 1996 / Received after revision: 19 September 1996 / Accepted: 1 October 1996