Intermediate filaments in smooth muscle from pregnant and non-pregnant human uterus.

The intermediate filament proteins desmin and vimentin from pregnant and non-pregnant uterine muscle and smooth-muscle cells in culture were analysed using SDS/PAGE. The desmin content in uterine muscle increases dramatically during pregnancy, whereas vimentin remains unchanged or changes very little. When muscle cells are kept in culture, a considerable increase in vimentin content is observed as compared with vimentin in freshly isolated non-pregnant uterine tissue. Our results strengthen the view that vimentin and desmin filaments have independent function and turnover, and point to a predominantly structural role for desmin filaments.     

The regulation of myosin phosphatase in pregnant human myometrium

Myometrial smooth muscle contractility is regulated predominantly through the reversible phosphorylation of MYLs (myosin light chains), catalysed by MYLK (MYL kinase) and MYLP (MYL phosphatase) activities. MYLK is activated by Ca2+-calmodulin, and most uterotonic agonists operate through myometrial receptors that increase [Ca2+]i (intracellular Ca2+ concentration). Moreover, there is substantial evidence for Ca2+-independent inhibition of MYLP in smooth muscle, leading to generation of increased MYL phosphorylation and force for a given [Ca2+]i, a phenomenon known as 'Ca2+-sensitization'. ROCK (Rho-associated kinase)-mediated phosphorylation and inhibition of MYLP has been proposed as a mechanism for Ca2+-sensitization in smooth muscle. However, it is unclear to date whether the mechanisms that sensitize the contractile machinery to Ca2+ are important in the myometrium, as they appear to be in vascular and respiratory smooth muscle. In the present paper, we discuss the signalling pathways regulating MYLP activity and the involvement of ROCK in myometrial contractility, and present recent data from our laboratory which support a role for Ca2+-sensitization in human myometrium.     

Preparation and P content of non-pregnant and pregnant human uterine myosin

Myosin content and phosphorus (P) concentration of myosin preparations were measured in non-pregnant and pregnant human myometrial tissue specimens. It was found that the amount of myosin gained from 1 g of minced myometrial tissue is 0.5 mg in the early follicular phase of the menstrual cycle, 0.6-0.7 mg in the late luteal phase, and 6-7 mg during pregnancy. Considering the different functional stages of the myosin sources and the performance characteristics of the methods, the estimated myosin content of non-pregnant myometrium is 1.0-1.5 mg, while 10-15 mg in pregnant myometrial tissue. A considerable amount of P is bound to the preparations. It is the smallest in the post-menstrual period and increases towards the end of the cycle. The largest amount of P is gained from fresh pregnant uterine samples. Analysis of the alkaline hydrolysate showed that the phosphate group was bound to amino acids, in the largest amount to arginine, less to histidine and the smallest amount to lysine and serine. As a function of the duration of storage, especially the P-Arg concentration was decreasing. The prolonged hydrolysis time decreases again the concentration of P-Arg with a consecutive increase of No. 1 and 2 P-containing peaks in the chromatographic profile of alkaline hydrolysate.     

TPA- and agonist-induced force development in myometrium from pregnant and non-pregnant rats

In myometrium from pregnant rats, 100 nM-TPA elevated resting tension and initially slightly enhanced the contraction induced by 138 mM-KCl. After 20 min this force development significantly declined. In saponin-treated skinned myometrial cells from pregnant rats, 100 nM-TPA enhanced the contraction induced by 0.3 microM-Ca2+, but reduced that induced by 1 microM-Ca2+. These findings suggest that the excitatory and inhibitory actions of TPA on the myometrium are probably due to its action on the contractile proteins. In myometrium from non-pregnant rats, TPA affected neither the resting tension, nor the amplitude of the evoked contractions, nor the Ca2+-induced contractions in skinned myometrium. While TPA only affected tension development in pregnant rats, both 1 mM-carbachol and 90 nM-oxytocin induced a tonic contraction in Ca-free solution independently of the hormonal status of the rats. The latter finding makes it unlikely that activation of protein kinase C is involved in the agonist-induced tonic force development in Ca-free solution.     

Regional differences in energy charge of the pregnant human uterus regardless of functional status in comparison with the non-pregnant uterus.

The energy status of the cell is mainly dependent on adenine nucleotides and can be expressed as energy charge (EC). EC is known to be kept at narrow limits near 0.90 under normal conditions in most cells. We recently reported remarkably low EC values in the human uterus under apparent steady-state conditions. The present paper is an extension of previous work. It shows that EC varies in different regions of the uterus in that the isthmic part in pregnant women displays a higher EC than the fundus of the uterus. There were no intergroup differences between non-pregnant and term pregnant women, nor between those who were in active normal labour, dysfunctional labour or those who were not in labour at all. On the other hand, EC in uterine muscle of post-menopausal women showed a significantly lower EC value. Human uterus seems to manage its metabolic requirements under different functional conditions in spite of low ATP and EC values. This suggests that ATP occurs in sufficient amounts to pertinent enzyme reactions, especially ATPases, which means Km values adapted for this unusually low ATP concentration.     

Oxytocin-induced phosphorylation of myosin light chain is mediated by extracellular calcium influx in pregnant rat myometrium.

Studies of oxytocin-induced phosphorylation of myosin light chain (MLC), resulting in myometrial contraction, suggest that extracellular Ca(2+) influx is involved in its signal transduction. To explore the possibility that intracellular Ca(2+) mobilization by oxytocin may also contribute to MLC phosphorylation, we investigated the relative contributions of these Ca(2+) sources to oxytocin signal transduction in myometrium of pregnant rat. In pregnant rat myometrium, oxytocin-induced Ca(2+) influx occurs via an L-type voltage-dependent Ca(2+) channel. Treatment with verapamil, an antagonist specific for these channels, significantly diminished MLC phosphorylation observed in response to oxytocin administration without affecting the release of Ca(2+) from intracellular Ca(2+) stores. Furthermore, oxytocin-induced MLC phosphorylation was not observed when extracellular Ca(2+) was not present. Our results clearly indicate that extracellular Ca(2+) influx, rather than release from Ca(2+) storage sites, is essential for oxytocin-induced MLC phosphorylation.     

Enzyme activities in human endometrium, myometrium and leiomyomas of the uterus.

Certain selected enzymes of carbohydrate metabolism were measured in samples of endometrium and myometrium from women who were premenopausal or postmenopausal. In addition, a number of samples of leiomyoma were obtained and assayed. Activities of several enzymes were higher in endometrium from premenopausal women compared to those in postmenopausal women; activities in myometrium were similar regardless of menopausal status. The activities of G6PD, ICD and GPI appeared to be lower in leiomyoma samples versus myometrial samples from premenopausal women; however, these differences were not apparent when enzyme activity was expressed per milligram protein.     

Localization of 3H-estradiol in the uterus of pregnant and non-pregnant armadillos

Summary The uptake and retention of radiolabeled estradiol by the uterus was examined in the armadillo. One pregnant and two non-pregnant armadillos were treated with 1.4 g/kg body weight of ³H-estradiol (E₂) by injection into the left ventricle, and one non-pregnant animal was injected with both the labeled hormone and 140 g/kg body weight of unlabeled E₂. One and a half hour after injection, the animals were sacrificed and the uteri were removed and processed for autoradiography. In the non-pregnant animals, nuclear localization was observed in the interstitial cells and glandular epithelium of the endometrium and the connective tissue cells and smooth muscle of the myometrium. Additionally, there was a gradation of uptake in the epithelial cells of the endometrium in that the glandular cells of the basal region were heavily labeled, while those cells in the sinusoidal, and luminal regions contained successively less label. The luminal cells were poorly labeled. In the pregnant female, the smooth muscle and glandular cells hypertrophied and their nuclei contained less label than was observed in the non-pregnant animals. The arteries of the myometrium were more easily distinguished in the pregnant animals and the nuclei of the endothelial cells and smooth muscle were more consistently labeled than those of the non-pregnant armadillos.     

Changes in the innervation and catecholamine concentrations in the myometrium of pregnant and non-pregnant sheep

The innervation of the myometrium in virgin, pregnant and post-partum ewes was examined using the SPG histofluorescence method and a high-performance liquid chromatographic catecholamine assay. In non-pregnant ewes, fluorescent axons were visible in all regions of the uterus. At 50 days of pregnancy the innervation was unchanged, but at 100 days axons were scarce over the whole uterus with the exception of the tubal extremities of the uterine horns. Noradrenaline concentrations were also significantly lower in late pregnancy, and significant variations occurred in different regions of the uterus, with the greatest concentrations present in the tubal extremities. At 13-16 weeks post partum, the density of innervation was variable, although the noradrenaline concentration was only slightly less than in virgin animals. Dopamine was also present in substantial quantities, but the mean concentrations remained unchanged during pregnancy.     

Purification and characterization of pregnant sheep myometrium myosin light chain kinase

Myosin light chain kinase (MLCK) has been purified from the myometrium of pregnant sheep. The Mr of the enzyme was determined from SDS-polyacrylamide gels to be 160,000. It requires Ca2+ and calmodulin for activation, and phosphorylates the 20,000-Da light chains of myosin at a rapid rate. The specific activity for the myosin light chains from turkey gizzards and rabbit uterine muscle are 7.7 and 5.4 mumol/min/mg, respectively. The Km for the former substrate is 40 microM and the Vmax of the reaction is 19 mumol/min/mg. Polyclonal antibodies raised against the enzyme cross-reacted with pregnant sheep myometrium (psm), turkey gizzard (tg), and chicken gizzard MLCK. Affinity purification of the antibodies on tg-MLCK Sepharose resulted in the preparation of two fractions of antibodies with different reactivity toward these proteins. Fraction A antibodies which did not bind to the affinity column cross-reacted only with psm-MLCK while Fraction B antibodies which bound to the column cross-reacted with all three proteins. Western blots of extracts of turkey gizzards, human myometrium, and various tissues from sheep showed cross-reactivity of both fractions of antibodies with a 160,000-Da protein in the extracts of sheep smooth muscles. Only Fraction B antibodies cross-reacted with a protein (130,000 Da) in turkey gizzards and human myometrium extracts. Prolonged tryptic digestion of psm-MLCK produced large fragments Mr approximately 60,000 which appears to be similar to that formed from tg-MLCK, and some smaller peptides. Fraction A antibodies cross-reacted with the small peptides while Fraction B antibodies cross-reacted with the large fragments but not vice versa. Further analysis of the tryptic peptides suggests that the epitopes of Fraction A antibodies are localized in a peptide which appears to be in the NH2-terminal region of the molecule.     

Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca²⁺. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0–2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca²⁺-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0–2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8–4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the V_max of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphrylation site B (S⁸²⁸).     

Complex effects of imatinib on spontaneous and oxytocin-induced contractions in human non-pregnant myometrium

Human myometrium includes two important cell populations involved in its contractility: smooth muscle fibers and interstitial cells. The pacemaking mechanism is not yet identified, but it is possible that myometrial smooth muscle cells contract in response to a signal generated by c-kit positive interstitial cells. The aim of this study was to investigate the effects of imatinib as a c-kit receptor antagonist on the spontaneous or oxytocin (OT) induced contractions of human non-pregnant myometrium in vitro. Myometrial strips were obtained from non-pregnant women (reproductive age) undergoing hysterectomy for benign indications. The strips were suspended in organ baths for recording of isometric tension. Imatinib effects were assessed on spontaneous contraction and after preexposure to OT.Direct exposure of myometrial strips to imatinib inhibits both amplitude and frequency of contractions (80-320 μM) in a dose dependent manner. Amplitude reverted back to 90% of the baseline amplitude by consequent addition of imatinib (until 480 μM). Total inhibition of myometrial contraction was obtained after addition of OT 60 nM. If myometrium was pre-exposed to OT (320 nM), imatinib 80-160 μm increased amplitude, while decreasing frequency. These data provide evidence that telocytes may be involved as modulators of the spontaneous contractions of the non-pregnant human uterus, via a tyrosine-kinase independent signaling pathway.     

Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin, and villin in rat and bovine exocrine glands

Actin, myosin, and the actin-associated proteins tropomyosin, alpha-actinin, vinculin, and villin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by means of immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. The presence of villin, which has so far been demonstrated only in intestinal and kidney brush border, was further confirmed by antibody staining of blotted electrophoresis gels of whole acinar cell extracts. Fluorescently labelled phalloidin, which reacts specifically with F-actin, gave similar staining, within the cell apex to that obtained with antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web, antibodies to myosin and tropomyosin gave only a weak immunostaining (confined to the lateral cell borders) whereas antibodies to actin and alpha-actinin led to a rather strong bead-like staining along the lateral and basal cell membrane most probably marking microfilament-associated desmosomes. Anti-villin immunofluorescence was confined to the apical terminal web. It is suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca(++)-dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins.     

Intracellular calcium of longitudinal muscles isolated from pregnant rat myometrium.

Longitudinal muscle cells were successfully isolated from pregnant rat myometrium (21 days of gestation) with more than a 95% survival rate. The approximate size of relaxed cells was 232.2 +/- 74 microns in length and 16.2 +/- 7.0 microns in width. Using the fluorescent indicator Fura-2, the concentration of intracellular free calcium ([Ca2+]i) in resting state cells was calculated to be 116 +/- 18.5 nM. The isolated cells responded well to K+, acetylcholine and oxytocin in terms of contraction as well as the increase in [Ca2+]i. The increase in [Ca2+]i induced by acetylcholine and K+ appeared to be mainly due to an influx of extracellular Ca2+. On the other hand, the oxytocin-induced increase in [Ca2+]i was mainly due to a release of Ca2+ from intracellular storage sites in the isolated cells. Isolated longitudinal muscle cells can serve as a useful tool in establishing the relationship between [Ca2+]i and regulation of the uterine contraction at the final stage of pregnancy.     

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.     

Expression of RND proteins in human myometrium

RHO GTPases are key regulators of the actin cytoskeleton and stress fiber formation. In the human uterus, activated RHOA forms a complex with RHO-associated protein kinase (ROCK) which inhibits myosin light chain phosphatase (PPP1R12A), causing a calcium-independent increase in myosin light chain phosphorylation and tension (Ca2+ sensitization). Recently discovered small GTP binding RND proteins can inhibit RHOA and ROCK interaction to reduce calcium sensitization. Very little is known about the expression of RND proteins in the human uterus. We tested the hypothesis that the uterine quiescence observed during gestation is mediated by an increase in RND protein expression inhibiting RHOA-ROCK-mediated PPP1R12A phosphorylation. Immunohistochemistry and immunoblotting were used to determine RHOA and RND protein expression and localization in nonpregnant, pregnant nonlaboring, and laboring patients at term and patients in spontaneous preterm labor. Changes in protein expression estimated by densitometry between different patient groups were measured. A significant increase of RND2 and RND3 protein expression was observed in pregnant relative to nonpregnant myometrium associated with a loss of PPP1R12A phosphorylation. RND transfected myometrial cells demonstrated a dramatic loss of stress fiber formation and a "rounding" phenotype. RND upregulation in pregnancy may inhibit RHOA-ROCK-mediated increase in calcium sensitization to facilitate the uterine quiescence observed during gestation.