The effects of retinoic acid on mmp-2 production, proliferation and ultrastructural morphology in rat uterus.

AIMS: Retinoic acid (RA) has a vital importance in order to ensure continuity and morphology in many tissues. Matrix metalloproteinases (MMPs) have significant roles in proliferation, the formation of cancers, and metastasis. In this study the effects of RA on MMP-2 production in cells of rat uterus were investigated. METHODS: Twenty-four adult Spraque Dawley rats were divided into two groups, the experimental group was treated with 40mg/kg/day 13-cis RA for 5days by gavage. Uterine tissue sections were treated with BrdU and MMP-2 antibodies, evaluated using light microscopy. Tissues were fixed with 2.5% glutaraldehyde and evaluated using transmission electron miroscopy. RESULTS: MMP-2 immunoreactivity decreased in the stromal cells compared with the control group and no staining of MMP-2 was observed in glandular epithelium in the experimental group. BrDU labeling of cells showed significant decrease in RA-treated group versus control group cells. Based on the electron microscopy evaluation, the surface epithelial cells of the experimental group showed vacuolization, and an accumulation of lipofuscin bodies was also observed in the gland epithelium. Cells involving autophagic vacuoles contained excess lipid granules in the entire uterus layers especially localized at the border of the endometrium and myometrium. CONCLUSION: RA had negative effects on cell proliferation and cell morphology and inhibited MMP-2 expression.

The inhibitory influence of calix[4]Arene of C-90 on the activity of Ca2+,Mg2+-ATPases in plasma membrane and sarcoplasmic reticulum in myometrium cells.

Our study on the plasma membrane vesicles and myometrium cells treated with 0.1% digitonin showed that inhibitory effect of calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulphonylimino)-methylamino- 25,26,27,28-tetrapropoxy-calix[4]arene) on the plasma membrane Ca2+,Mg2+-ATPase was more significant than on the Ca2+,Mg2+-ATPase in sarcoplasmic reticulum (the inhibition coefficient I0.5 values were 20.2 ± 0.5 µM and 57.0 ± 1.4 µM for the plasma membrane Ca2+,Mg2+-ATPase and Ca2+,Mg2+-ATPase in sarcoplasmic reticulum, respectively (n = 5)). Inhibition kinetics of calix[4]arene C-90 effect on the Ca2+,Mg2+- ATPase activities in plasma membrane and sarcoplasmic reticulum were studied. This substance inhibited both pumps as complete noncompetitive inhibitor. Calix[4]arene C-90 caused the increase of intracellular Ca2+ concentration and decrease of hydrodynamic diameter in smooth muscle cells similar to the action of uterotonic drug oxytocin.

Calix[4]arene C-90 and its analogs activate ATPase of the myometrium myosin subfragment-1.

Numerous female reproductive abnormalities are consequences of disorders in uterus smooth muscle (myometrium) contractile function. In this work, we described activators of ATPase, which could be used for development of effective treatments for correcting this dysfunction. Myosin ATPase localized in the catalytic domain of myosin subfragment-1 transforms a chemical energy deposited in macroergic bonds of ATP into mechanical movement. It was shown that сalix[4]arene C-90 and its structural analogs functionalized at the upper rim of macrocycle with four or at least two N-phenylsulfonуltrifluoroacetamidine groups, are able to activate ATP hydrolysis catalyzed by myometrium myosin subfragment-1. It was shown with the method of computer modeling that N-phenylsulfonуltrifluoroacetamidine groups of calix[4]arene C-90 interact with responsible for binding, coordination and the hydrolysis of ATP amino acid residues of myosin subfragment-1. The results can be used for further research aimed at using calix[4]arene C-90 and its analogs as pharmacological compounds that can effectively normalize myometrium contractile hypofunction.

Activation of the MAPK/ERK Cell-Signaling Pathway in Uterine Smooth Muscle Cells of Women With Adenomyosis.

We investigated whether the myometrium might be intrinsically different in women with adenomyosis. We studied whether the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs) and phosphoinositide 3-kinase/mammalian target of rapamycin/AKT (PI3K/mTOR/AKT) cell-signaling pathways, implicated in the pathogenesis of endometriosis, might also be activated in uterine smooth muscle cells (uSMCs) of women with adenomyosis and measured the production of reactive oxygen species (ROS), proinflammatory mediators that modulate cell proliferation and have been shown to activate the MAPK/ERK pathway in endometriosis. The uSMC cultures were derived from myometrium biopsies obtained during hysterectomy or myomectomy in women with adenomyosis and controls with leiomyoma. Proliferation of uSMCs and in vitro activation of the MAPK/ERK cell-signaling pathway were increased in women with adenomyosis compared to controls. The activation of the PI3K/mTOR/AKT pathway was not significant. The ROS production and ROS detoxification pathways were not different between uSMCs of women with adenomyosis and controls suggesting an ROS-independent activation of the MAPK/ERK pathway. Our results also provide evidence that protein kinase inhibitors and the rapanalogue temsirolimus can control proliferation of uSMCs in vitro suggesting an implication of the MAPK/ERK and the PI3K/mTOR/AKT pathways in proliferation of uSMCs in women with adenomyosis and leiomyomas.

17ß-Estradiol Induces Overproliferation in Adenomyotic Human Uterine Smooth Muscle Cells of the Junctional Zone Through Hyperactivation of the Estrogen Receptor-Enhanced RhoA/ROCK Signaling Pathway.

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. Recent models favor abnormal thickening of the junctional zone (JZ) may be the causative factor in the development of ADS. RhoA, a small guanosine triphosphatase which controls multiple cellular processes, is involved in the control of cell proliferation. Here we demonstrate that treatment of human uterine smooth muscle cells (SMCs) of the JZ with 17ß-estradiol (E2) increased expression of RhoA and its downstream effectors (-associated coiled coil containing protein kinase [ROCK] 1 and ROCK2). Compared with non-ADS cells, RhoA, ROCK1, and ROCK2 were overexpressed and hyperactivated in ADS cells. These effects were suppressed in the presence of ICI 182,780, supporting an estrogen receptor (ER)-dependent mechanism. Hyperactivation of ER-enhanced RhoA/ROCK signaling was associated with overproliferation in ADS human uterine SMCs of the JZ. Moreover, E2-induced overproliferation was accompanied by downregulation of cyclin-dependent kinases inhibitors (CKIs; p21(Waf1/Cip1) and p27(Kip1)) and upregulation of cyclin-dependent kinases (CDKs) and cyclins (cyclin D1, cyclin E1, CDK2, CDK4, and CDK6).

[Extracting and study of biochemical properties of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium].

The method of extraction and purification of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium has been elaborated. Kinetic characteristics of T-kinase from non-malignant and tumor tissue of women myometrium have been studied. It has been shown, that malignization of myometrium is accompanied by a decrease in affinity of thiamine pyrophosphokinase from tumor to thiamine and by an increase in sensitivity of the enzyme from tumor to thiochrome.

The androgen receptor mediates antiapoptotic function in myometrial cells.

During pregnancy, myometrial phenotype is programmed into three characteristic stages referred to as the early proliferative, the midterm hypertrophic, and the late contractile stage. Increased myometrial growth in the early and midterm of pregnancy involves a complex process of cell proliferation, antiapoptosis and differentiation. We have previously demonstrated that the androgen receptor (AR) is required for myometrial cell proliferation by modulating IGF-1 signaling during early pregnancy. Here, we report that AR also exerts its antiapoptotic function in human myometrial cells. Enhanced AR expression protects, whereas AR silencing sensitizes myometrial cells to both intrinsic and extrinsic apoptotic stimuli. AR agonist inhibits, whereas AR antagonist induces myometrial cells to undergo apoptotic cell death. Gene microarray analysis confirms that the central functions of AR in myometrial cells are to regulate cell cycling and apoptosis through three major gene groups involving the epidermal growth factor (EGF) signaling, RNA splicing and DNA repair processes. AR mediates its antiapoptotic function through two distinct pathways. In the receptor-dependent pathway, AR is required for the expression of several protein factors within the EGF signaling pathway. Through the PI3K/Akt pathway, AR enhances the expression of the antiapoptotic protein Mcl-1. In the ligand-dependent pathway, AR agonist triggers the activation of Src kinase, which in turn phosphorylates STAT3 to increase Mcl-1 expression. We conclude from these results that the AR signaling exerts antiapoptotic function in myometrial cells, further supporting its key role in programming of myometrial phenotype.

Nicotinamide adenine dinucleotide phosphate oxidase is differentially regulated in normal myometrium versus leiomyoma.

Uterine fibroids are the most common benign tumor in women. The goal of this study was to investigate whether nicotinamide adenine dinucleotide phosphate oxidase (NOX), a major source of superoxide and subsequent oxidative stress, was differentially regulated in myometrium versus leiomyoma. Expression levels of NOXs1-5, dual oxidase (DUOX), DUOX2, NOX organizer (NOXO) 1, NOX activator 1, p47(phox), p67(phox), and p22(phox) were determined in cells treated with hypoxia by real-time reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry in tissues. Expression of NOX4 increased in fibroid compared to myometrial tissues and cells. The NOX2, DUOX1, and p67(phox) were higher while p22(phox) was lower in fibroid than that in myometrial cells. Hypoxia increased NOX4, DUOX1, and NOXO1 and decreased p22(phox) in myometrial and reduced DUOX1 in fibroid cells. The NOX1, NOX3, NOX5, and DUOX2 were undetectable. Fibroid cells are characterized by a unique NOX profile, which promotes a severe prooxidant state that may be responsible for their development. Targeting these subunits may be beneficial for future therapeutic interventions.

Increased progesterone receptor A expression in labouring human myometrium is associated with decreased promoter occupancy by the histone demethylase JARID1A.

Progesterone regulates female reproductive function predominantly through two nuclear progesterone receptors (PRs), PR-A and PR-B. During human parturition myometrial PR expression is altered to favour PR-A, which activates pro-labour genes. We have previously identified histone H3 lysine 4 trimethylation (H3K4me3) as an activator of myometrial PR-A expression at labour. To further elucidate the mechanisms regulating PR isoform expression in the human uterus at labour, we have (i) determined the methylation profile of the cytosine-guanine dinucleotides (CpG) island in the promoter region of the PR gene and (ii) identified the histone-modifying enzymes that target the H3K4me3 mark at the PR promoters in term and preterm human myometrial tissues obtained before and after labour onset. Bisulphite sequencing showed that despite overall low levels of PR CpG island methylation, there was a significant decrease in methylated CpGs with labour in both preterm (P < 0.05) and term (P < 0.01) groups downstream of the PR-B transcription start site. This methylation change was not associated with altered PR-B expression, but may contribute to the increase in PR-A expression with labour. Chromatin immunoprecipitation revealed that the histone methyltransferase, SET and MYND domain-containing protein 3 (SMYD3), bound to the PR gene at significantly higher levels at the PR-A promoter compared with the PR-B promoter (P < 0.010), with no labour-associated changes observed. The H3K4 demethylase, Jumonji AT-rich interactive domain 1A (JARID1A), also bound to the PR-A, but not to the PR-B promoter prior to term labour, and decreased significantly at the onset of labour (P = 0.014), providing a mechanism for the previously reported increase in H3K4me3 level and PR-A expression with labour. Our studies suggest that epigenetic changes mediated by JARID1A, SMYD3 and DNA methylation may be responsible, at least in part, for the functional progesterone withdrawal that precipitates human labour.

[Kinetics of inhibitory effect of calix[4]arene C-90 on activity of transporting plasma membrane Ca2+, Mg2+-ATPase of smooth muscle cells].

In experiments on the suspension of myometrium cell plasma membrane, processed by 0.1% digitonin, the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(threeftor)methyl(phenilsulphonilimino)-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene) on the activity of Ca2+,Mg2+-ATPase was investigated. The authors also examined the influence of calix[4]arene in different concentration on affinity of enzyme (Ca2,Mg2+-ATPase) for the ATP and ions of Mg and Ca, and its influence on cooperative effect and maximum velocity of ATP hydrolysis. It is shown that calix[4]arene does not influence the affinity of Ca2+,Mg2+-ATPase for the ATP, which means that these two compounds have different binding centers. Also calix[4]arene has no influence on affinity and cooperative effect of Ca ions, if it is used in concentration lower than 50 µM. Calix[4]arene slightly increases coefficient of Ca2+,Mg2+-ATPase activation by magnesium chloride. In all three cases, where ATP, Mg and Ca ions are used to test the impact of calix[4]arene, maximum velocity of ATP hydrolysis significantly decreases. All these results clarify that calix[4]arene implements its inhibitory action through mechanism of uncompetitive inhibition of Ca2+,Mg2+-ATPase activity.

[Protective effect of tiacalix[4]arene-tetrasulphonate on heavy metal inhibition of myometrium myosin subfragment-1 ATP-hydrolase activity].

Heavy metals have a negative effect on the contractility of uterine smooth muscles (myometrium), these effects can lead to various pathologies of a women reproductive system. To overcome these effects the methods for correcting the myometrium contractile activity are to be developed. Catalyzed by myosin ATPase ATP hydrolysis is the most important reaction in the molecular mechanism of myometrium contraction. We have found an inhibitory effect of 0.03-0.3 mM Ni2+, Pb2+ and Cd2+ on enzymatic hydrolysis of ATP by myosin subfragment-1 obtained from swine uterine smooth muscles. We have demonstrated that 100 µM thiacalix[4]arene-tetrasulphonate (C-798) recovered to the control level of ATPase activity of myosin subfragment-1 in the presence of heavy metal cations. One of the most probable mechanisms of C-798 corrective activity is based on its ability to chelate heavy metals, thus cations Pb, Cd and Ni can be removed from the incubation medium. Computer simulation has demonstrated that the protective effect of C-798 may also be the result of weakening the interaction of heavy metal ions with amino acid residues of the myosin molecule near the active site of ATP hydrolase. The obtained results can be used for further research aimed at assessing the prospects of thiacalix[4]arene-tetrasulfonate as pharmacological compounds.

[Kinetic properties of calixarene C-90 action on the myometrial plasma membrane Ca2+,Mg2+-Atpase activity and on the Ca2+ concentration in unexcited cells of the myometrium].

Plasma membrane Ca2+,Mg2+-ATPase is an important element of general myometrium tonus control mechanism, which also makes a contribution to muscle tension relaxation after its contraction. Expiriments were done on the myometrial cell plasma membrane suspension, which was treated with 0.1% digitonin solution. The authors have investigated the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(trifluor) methyl(phenylsulphonylimino)-methylamino-25,26,27,28-tetra propoxi-calix[4]arene) on the Ca2+,Mg2+-ATPase activity (the magnitude of 10.5 was 20.2 +/- 0.5 mkM). The inhibitory action of calix[4]arene C-90 on the activity of Ca2+, Mg2+-ATPase is explained as cooperative action of four trifluormethyl(phenylsulfonylimino)methylamino groups that are spatially oriented on the calix[4]-arene base rather than with the action of tetra-phenol macrocycle or separate pharmacophore sulphonilamidin groups. Considering established kinetic pattern of calix[4]arene C-90 inhibitory action on the plasma membrane Ca2+,Mg2+-ATPase activity, stationary kinetical model of basal calcium concentration control in unexcited uterus myocytes was developed. It is assumed that obtained results may be promising for creation of new generation ("supramolecular") pharmacological agent - uterus basal tonus stimulator - on the base of calix[4] arene C-90.

Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells.

STUDY QUESTION: Can biologically active vitamin D3 [1,25(OH)2D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells? SUMMARY ANSWER: 1,25(OH)2D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells. WHAT IS KNOWN ALREADY: Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)2D3 can regulate the expression and activities of MMPs in human uterine fibroid cells. STUDY DESIGN, SIZE, DURATION: Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)2D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay. MAIN RESULTS AND THE ROLE OF CHANCE: 1-1000 nM 1,25(OH)2D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)2D3. 1,25(OH)2D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)2D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)2D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)2D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable. LIMITATIONS, REASONS FOR CAUTION: This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals an important biological function of 1,25(OH)2D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)2D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.

[The calix[4]arene C-107 is highly effective supramolecular inhibitor of the Na+,K(+)-ATPase of plasma membranes].

The inhibition of the Na+,K(+)-ATPase activity of the myometrium cell plasma membranes with calixarene C-107 (5,17-diamino(2-pyridyl) methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) was investigated. It has been shown that calixarene C-107 reduced the Na+,K(+)-ATPase activity more efficiently than ouabain did, while it did not practically influence the "basal" Mg(2+)-ATPase activity of the same membrane. The magnitude of the cofficient of inhibition I0.5 was 33 +/- 4 nM, Hill coefficient was 0.38 +/- 0.06. The model calixarene C-150--the calixarene "scaffold" (26,28-dihydroxy-25,27-dipropoxycalix[4]arene), and the model compound M-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid)--a fragment of the calixarene C-107, had practically no influence on the enzymatic activity of Na+,K(+)-ATPase and Mg(2+)-ATPase. We carried out the computer simulation of interaction of calixarenes C-107 and the mentioned model compound with ligand binding sites of the Na+,K(+)-ATPase of plasma membrane and structure foundation of their intermolecular interaction was found out. The participation of hydrogen, hydrophobic, electrostatic and pi-pi (stacking) interaction between calixarene and enzyme aminoacid residues, some of which are located near the active center of Na+,K(+)-ATPase, was discussed.

Serum lactate dehydrogenase profile as a retrospective indicator of uterine preparedness for labor: a prospective, observational study.

BACKGROUND: Lactate dehydrogenase (LDH) isoenzymes are required for adenosine triphosphate production, with each of five different isoenzymes having varying proficiencies in anaerobic versus aerobic environments. With advancing pregnancy, the isoenzyme profile in uterine muscle shifts toward a more anaerobic profile, speculatively to facilitate uterine efficiency during periods of low oxygen that accompany labor contractions. Profile shifting may even occur throughout labor. Maternal serum LDH levels between 24-48 hours following delivery predominantly originate from uterine muscle, reflecting the enzymatic state of the myometrium during labor. Our purpose was to describe serum LDH isoenzymes 24-30 hours post-delivery to determine if cervical dilation rates following labor admission were associated with a particular LDH profile. We also compared differences in post-delivery LDH isoenzyme profiles between women admitted in pre-active versus established active labor. METHODS: Low-risk, nulliparous women with spontaneous labor onset were sampled (n = 91). Maternal serum LDH was measured at labor admission and 24-30 hours post-vaginal delivery. Rates of cervical dilation during the first four hours after admission were also measured. Spearman's rho coefficients were used for association testing and t tests evaluated for group and paired-sample differences. RESULTS: More efficient dilation following admission was associated with decreased LDH1 (p = 0.029) and increased LDH3 and LDH4 (p = 0.017 and p = 0.017, respectively) in the post-delivery period. Women admitted in established active labor had higher relative serum levels of LDH3 (t = 2.373; p = 0.023) and LDH4 (t = 2.268; p = 0.029) and lower levels of LDH1 (t = 2.073; p = 0.045) and LDH5 (t = 2.041; p = 0.048) when compared to women admitted in pre-active labor.Despite having similar dilatations at admission (3.4 ± 0.5 and 3.7 ± 0.6 cm, respectively), women admitted in pre-active labor had longer in-hospital labor durations (12.1 ± 4.3 vs. 5.3 ± 1.4 hours; p < 0.001) and were more likely to receive oxytocin augmentation (95.5% vs. 34.8%; p < 0.001). CONCLUSIONS: More efficient cervical dilation following labor admission is associated with a more anaerobic maternal serum LDH profile in the post-delivery period. Since LDH profile shifting may occur throughout labor, watchful patience rather than intervention in earlier labor may allow LDH shifting within the uterus to more fully manifest. This may improve uterine efficiency during labor and decrease rates of oxytocin augmentation, thereby improving birth safety.

Immunohistochemical localization of selected pro-inflammatory factors in uterine myomas and myometrium in women of various ages.

Uterine myomas represent one of the most frequently manifested benign tumors in women. They originate from smooth muscle cells of myometrium or its blood vessels. Many studies suggest that inflammation and pro-inflammatory factors may play a role in the carcinogenesis with an involvement of the transcription factor NF-kappaB which activity can be controlled by various environmental factors, including many cytokines. The aim of the study was to investigate the expression of NF-B, interleukin-1ß (IL-1ß), tumor necrosis factor a (TNF-α), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in myometrium and uterine myomas of women of various age. The expression of NF-kappaB, selected cytokines and enzymes was estimated in women of reproductive or perimenopausal age by semiquantitative immunohistochemistry. The expression of the examined proteins was higher in myomas than in control myometrium and was dependent on the size of myomas and the age of women. However, the expression of the cytoplasmic NF-kappaB observed in uterine myomas was independent on the size of myomas and no significant differences were observed in the number of stained nuclei between control and myoma groups. Thus, the expression of proinflammatory factors in myomas was not accompanied by the nuclear activation of NF-kappaB p65. The results of our study indicate that the examined factors may be involved in the pathogenesis of benign tumors and not only malignant diseases.

Increased constrictor tone induced by ouabain treatment in rats.

Ouabain (Oua)-induced hypertension in rodents provides a model to study cardiovascular changes associated with human hypertension. We examined vascular function in rats after a long-term treatment with Oua. Systolic blood pressure was measured by tail-cuff plethysmography in male Sprague-Dawley rats treated with Oua (≈ 25 µg/d) or placebo for 8 weeks. Blood pressure increased in Oua-treated animals, reaching 30% above baseline systolic blood pressure after 7 weeks. At the end of treatment, vascular responses were studied in mesenteric resistance arteries (MRAs) by wire myography. Contraction to potassium chloride in intact and denuded arteries showed greater sensitivity in Oua-treated animals. Contraction to phenylephrine and relaxation to acetylcholine were similar between groups with a lower response to sodium nitroprusside in Oua-treated arteries. Sensitivity to endothelin-1 was higher in Oua-treated arteries. Na⁺-K⁺ ATPase activity was decreased in MRAs from Oua-treated animals, whereas protein expression of the Na⁺-K⁺ ATPase α2 isoform was increased in heart and unchanged in mesenteric artery. Preincubation with indomethacin (10⁻5 M) or Nω-nitro-L-arginine methyl ester (10⁻4 M) abolished the differences in potassium chloride response and Na⁺-K⁺ ATPase activity. Changes in MRAs are consistent with enhanced vascular smooth muscle cell reactivity, a contributor to the increased vascular tone observed in this model of hypertension.

Role of serine-threonine phosphoprotein phosphatases in smooth muscle contractility.

The degree of phosphorylation of myosin light chain 20 (MLC20) is a major determinant of force generation in smooth muscle. Myosin phosphatases (MPs) contain protein phosphatase (PP) 1 as catalytic subunits and are the major enzymes that dephosphorylate MLC20. MP regulatory targeting subunit 1 (MYPT1), the main regulatory subunit of MP in all smooth muscles, is a key convergence point of contractile and relaxatory pathways. Combinations of regulatory mechanisms, including isoform splicing, multiple phosphorylation sites, and scaffolding proteins, modulate MYPT1 activity with tissue and agonist specificities to affect contraction and relaxation. Other members of the PP1 family that do not target myosin, as well as PP2A and PP2B, dephosphorylate a range of proteins that affect smooth muscle contraction. This review discusses the role of phosphatases in smooth muscle contractility with a focus on MYPT1 in uterine smooth muscle. Myometrium shares characteristics of vascular and other visceral smooth muscles yet, during healthy pregnancy, undergoes hypertrophy, hyperplasia, quiescence, and labor as physiological processes. Myometrium presents an accessible model for the study of normal and pathological smooth muscle function, and a better understanding of myometrial physiology may allow the development of novel therapeutics for the many disorders of myometrial physiology from preterm labor to dysmenorrhea.

Expression of endothelial nitric oxide synthase in the uterus of patients with leiomyoma or adenomyosis.

AIM: To confirm the difference in the expression of endothelial nitric oxide synthase in the normal endometrium and myometrium of women who have leiomyoma or adenomyosis compared with controls, and its correlation with the pathogenesis of menorrhagia or dysmenorrhea in patients with uterine leiomyoma. METHODS: Fifty-one hysterectomized patients were divided into three groups: (i) patients with leiomyoma (n=24); (ii) those with adenomyosis (n = 19); and (iii) the control group (n=8). The expression of endothelial nitric oxide synthase was confirmed on immunohistochemistry and analyzed using an evaluation nomogram. RESULTS: The expression of endothelial nitric oxide synthase was significantly higher in the leiomyoma group and the adenomyosis group as compared with the control group. In the subgroup analysis of leiomyoma depending on symptoms (menorrhagia or dysmenorrhea or both), the expression of endothelial nitric oxide synthase was significantly higher in the symptomatic subgroup than the asymptomatic subgroup (endometrium P=0.0029, myometrium P=0.0276). CONCLUSIONS: Based on the findings that the expression of endothelial nitric oxide synthase was significantly higher in the uterus with leiomyoma or adenomyosis, it can therefore be inferred that nitric oxide might have a pathological effect on the uterus with the above diseases. In particular, it is also presumed that endothelial nitric oxide synthase is closely associated with menorrhagia and dysmenorrhea.

Human prolyl hydroxylase expression in uterine leiomyoma during the menstrual cycle.

BACKGROUND: To investigate the role of prolyl hydroxylase (PH), a key enzyme of collagen synthesis, in human uterine leiomyoma, PH expression was determined in the normal uterine myometrium and the leiomyoma tissues during the menstrual cycle. METHODS: The tissues were obtained from 40 regularly cycling women (aged 29 to 53 yr) who were undergoing abdominal hysterectomy for symptomatic uterine leiomyoma. Immunohistochemistry for human PH with specific monoclonal antibody was used for analysis. RESULTS: Immunohistochemical staining for PH revealed intense staining of leiomyoma cells in the uterine leiomyoma throughout the menstrual cycle, as compared with the adjacent normal myometrium. In the secretory phase, weak or no immunostaining for PH was detected in the normal myometrial tissues. CONCLUSIONS: These results suggest that increased expression of PH might play an role in the physiology of uterine leiomyoma during the menstrual cycle.