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.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

Application of electron spin resonance for evaluation of the level of free radicals in the myometrium in full-term pregnancy with normal labour and uterine inertia

In order to identify and quantify free radicals in the tissues of patients with normal physiological and pathological states of births, we developed a method to evaluate the amount of free radicals in myometrium of subplacental area and from body of uterus, using electron spin resonance spectroscopy. Analysis of the concentration of free radicals in the myometrium in full-term pregnancy with normal labour and during uterine inertia was studied. The activities of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase in samples of these tissues were tested too. Low free radical concentrations in these tissues were associated with disturbances in contractile activity of myometrium along with reduction of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase activity. There proved to be an association between the level of free radicals in the tissues and alteration in the physiological processes.     

Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.     

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN on mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.     

In vitro effects of L-arginine on spontaneous and homocysteine-induced contractility of pregnant canine uteri

The L-Arginine-Nitric Oxide Synthase-Nitric Oxide (L-Arg-NOS-NO) system exerts a pivotal role in the maintenance of uterine quiescence during pregnancy, whereas Homocysteine (Hcy) promotes uterine contractility. The aim of this study was to test the in vitro effects of L-Arg on spontaneous and Hcy-induced contractions of uteri excised from pregnant bitches. 104 strips cut from pregnant uteri were mounted in an organ bath. 40 out of 104 strips (16 from mid-gestation uteri and 24 from close to term uteri, respectively) were exposed to cumulative doses of L-Arg; 40 strips (16 from mid-gestation-uteri and 24 from close to term-uteri, respectively) were exposed to N-nitro-L-arginine methyl ester (L-NAME), a NOS antagonist; the remaining 24 strips (from close-to-term uteri) were first exposed to a single dose of Hcy and then to increasing doses of L-Arg. L-Arg showed no effects on spontaneous contractility both in mid-gestation- and close to term-uterine strips, whereas it promoted a relaxant effect on Hcy-induced contractility. On the contrary, L-NAME increased amplitude of contraction both in mid-gestation and close to term strips. These findings suggest that the L-Arg-NO system is present in the uterus of pregnant bitches and that Hcy is able to modulate its actions. Further investigation of this system may provide the basis of future obstetrical therapies in bitches.     

An opioid antagonist stimulates myometrial activity in early postpartum cows

The influence of the opioid antagonist naltrexone on myometrial activity of cows was tested during the early postpartum period, when uterine contractions are a highly sensitive indicator of oxytocin actions. Registering intrauterine pressure oscillations in five first calf heifers revealed that myometrial activity was enhanced after naltrexone was administered (0.02 mg/kg BW). However, plasma concentrations of oxytocin were not consistently elevated. In addition, naltrexone did not reliably potentiate the suckling-induced peak of oxytocin release in plasma. In all cases, naltrexone injections caused increases in the cows' respiratory rates and distinct behavioral changes during the first days postpartum, but not later. We conclude that: 1) In the early postpartum period, an inhibitory opioidergic control of myometrial activity may exist in cattle. 2) This control does not seem to be caused by a change in circulating oxytocin levels. 3) Opioids may exert an inhibitory influence on respiratory functions in cattle.     

Sensitivity of ovine myometrial tissue to various eicosanoids in vitro

The sensitivity of sheep myometrial tissue to prostaglandin F_2α (PGF_2α), PGE₂, the thromboxane analog U-44069, and leukotrienes C₄ (LTC₄) and LTD₄ was investigated in a superfusion system. Tissues were obtained from eight oophorectomized ewes, with or without pretreatment with estradiol-17β. After equilibration, spontaneous activity was abolished by adding indomethacin to the superfusion fluid. The dose needed to induce a contraction with a peak level of 50% of the median peak level of spontaneous contractions increased from PGE₂ to PGF_2α, U-44069, LTC₄, and LTD₄. The differences between the doses required were significant for all compounds, except between LTC₄ and LTD₄. Estradiol-17β pretreatment caused an increase in the required dose of PGF_2α. The results of this study do not support the hypothesis that leukotrienes are involved in the regulation of myometrial activity.     

Functional interactions between the oxytocin receptor and the β2-adrenergic receptor: implications for ERK1/2 activation in human myometrial cells

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled β₂-adrenergic receptor (β₂AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and β₂AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and β₂AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in β₂AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the β₂AR alone or co-transfected with the OTR. Using this approach, we found that β₂AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that β₂AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel β₂AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors.