Phasic Myometrial Contractions Research Articles

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

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. 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. 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. 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.

The role of phospholipase Cγ1 tyrosine phosphorylation during phasic myometrial contractions

Phospholipase Cgamma1 (PLCgamma1) is expressed in myometrium and is activated by tyrosine phosphorylation. These studies sought to determine the association between PLCgamma1 tyrosine phosphorylation and spontaneous uterine contractions. In vitro contraction studies were performed with spontaneously contracting rat uterine strips along with strips that were treated with potassium bisperoxo(1,10 phenanthroline)oxovanadate (bpV(phen), a protein tyrosine phosphatase inhibitor. Additional studies were performed with phenylarsine oxide (a PLCgamma inhibitor) and other inhibitors. Western blots were performed to determine the phosphotyrosine PLCgamma1 levels. Spontaneous contractile activity and tyrosine phosphorylation of PLCgamma1 (but not PLCgamma2) were increased significantly in response to bpV(phen); in contrast, oxytocin and thrombin produced comparable contractile activity but did not alter phosphotyrosine-PLCgamma1. Phenylarsine oxide and neomycin significantly decrease bpV(phen)-stimulated contractions and PLCgamma1 tyrosine phosphorylation; other inhibitors only suppressed contractions. These studies support the hypothesis that spontaneous myometrial contractions are associated with tyrosine phosphorylation of PLCgamma1; both of which are further enhanced by the inhibition of protein tyrosine phosphatase activity.

Role of Src family kinases during tyrosine phosphorylation of phospholipase C-γ1 during phasic myometrial contractions

The intracellular signaling events during spontaneous myometrial contractions are yet to be defined. Previous reports have suggested that activation of PLC-γ1 in response to tyrosine phosphorylation plays an important role. These studies sought to characterize the Src kinase(s) involved in PLC-γ1 activation.

Effect of histamine on phasic and tonic contractions of isolated uterine tissue from pregnant women

Objective: To enhance our understanding of the uterotonic effect of histamine, we compared the effects of histamine on spontaneous phasic and tonic contractile activity of uterine strips from term pregnant nonlaboring women. Study Design: Longitudinal uterine strips were used from the lower uterine segment of term pregnant nonlaboring women undergoing elective cesarean section. The concentration-response relationship to histamine (10–8 to 10–4 mol/L) was determined in spontaneously contracting strips or in strips contracted tonically with a protein kinase C activator (–)–indolactam V in the presence of H1 receptor antagonist (S[+]-chlorpheniramine maleate), H2 receptor antagonist (cimetidine), or solvent. Results: Histamine increased spontaneous phasic myometrial contractions in a concentration-dependent manner. H1, but not H2, receptor antagonist significantly attenuated the response to histamine. Histamine significantly reduced tonic contractions of uterine strips induced by indolactam V. H1 histamine receptor antagonist abolished relaxation, whereas H2 histamine receptor antagonist had no effect. Conclusion: Histamine increases spontaneous, but inhibits tonic, contractions of uterine strips from term pregnant nonlaboring women. Both effects are mediated through activation of H1 receptors. (Am J Obstet Gynecol 2003;188:774-8.)

Protein expression of phospholipase C in pregnant and nonpregnant rat uterine tissue

Objective: The phosphatidylinositol signaling pathway appears to play a significant role in the intracellular events leading to agonist-stimulated phasic myometrial contractions. The studies described in this report were performed to characterize phospholipase C isoform expression at the protein level and to confirm histologic localization of these proteins within the myometrial smooth muscle layers of the uterus. Methods: For these studies, uterine tissue was obtained from timed- pregnant and spontaneously cycling adult female Sprague-Dawley rats. After isolation of myometrial cell membranes and cytosolic proteins, Western blots were performed by using phospholipase C isoform-specific antibodies. Tissue cross-sections of near-term pregnant rat uterus were used with the phospholipase C isoform-specific antibodies for immunohistochemical studies. Results: The Western blot studies confirmed expression of the phospholipase C-β3, -γ1, -γ2, and -δ1 proteins in both the membrane and cytosolic fractions of rat myometrium; in contrast, only trace amounts of the phospholipase C-β1 protein was observed in this tissue. The immunohistochemical studies demonstrated localization of the phospholipase C-β3, -γ1, -γ2, -δ1 and to a lesser degree phospholipase C-β1 isoforms within the longitudinal and circular smooth muscle layers of the near-term pregnant rat uterus. Conclusion: These studies have confirmed the simultaneous expression of several phospholipase C proteins within the smooth muscle cells of the pregnant and nonpregnant rat uterus, thereby providing support for the possible redundant role of these signal transduction enzymes during the generation of cytosolic calcium oscillations and phasic myometrial contractions. (Am J Obstet Gynecol 2001;185:1191-7.)

The mechanisms underlying the stimulatory effects of thrombin on myometrial smooth muscle

Objective: The mechanisms underlying the stimulation of uterine contractions in the presence of intrauterine hemorrhage have not been well defined. Thrombin, a blood coagulation factor, activates membrane receptors to result in the stimulation of the phosphatidylinositol signaling pathway and the mobilization of cytosolic calcium in platelets. Our studies sought to determine whether thrombin stimulates similar events in myometrial smooth muscle. Study Design: Cytosolic calcium imaging and in vitro contraction studies were performed with rat myometrial tissue. Results: At a concentration range of 1 to 100 U/mL thrombin produced phasic myometrial contractions, which were comparable in intensity to those produced by oxytocin and prostaglandin F2α. Thrombin-induced cytosolic calcium concentration oscillations were similar to those produced by oxytocin. Contractions stimulated by thrombin were significantly suppressed in response to inhibitors of the phosphatidylinositol signaling pathway. These studies also confirmed that membrane receptor–Gq protein coupling events play a more important role than tyrosine kinase–mediated events during thrombin stimulation of myometrial smooth muscle. Conclusion: Thrombin is a potent uterotonic agonist, and its effects in myometrium are mediated by intracellular signaling events comparable to those activated by classic uterotonic agents. The physiologic importance of thrombin appears to be related to its potential role in the stimulation of uterine contractions in the presence of intrauterine hemorrhage. (Am J Obstet Gynecol 2000;183:674-81.)

Tissue-specific protein kinase C isoform expression in rat uterine tissue.

Activation of the phosphatidylinositol signaling pathway plays a key role during the generation of agonist-stimulated phasic myometrial contractions. Protein kinase C (PKC), a component of this signaling pathway, has been previously shown to produce feedback inhibition of agonist-stimulated myometrial contractions. The studies described in this report were performed to survey the tissue-specific expression of several PKC isoforms in the rat uterus. Uterine tissue was obtained from timed pregnant and normally cycling adult female Sprague-Dawley rats. Immunohistochemical studies were performed using the Vectastain ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were performed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation. These studies confirmed significant expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-beta 1, -gamma, -epsilon, and -theta isoforms was observed. Expression of the PKC-alpha, -beta 2, and -eta isoforms decreased modestly during the latter days of gestation; in contrast, PKC-delta and -zeta remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in both circular and longitudinal smooth-muscle layers of the near-term pregnant rat uterus. In summary, these studies have confirmed significant levels of expression of several isoforms of PKC in estrus and near-term pregnant rat uterine tissue, which was most prominent in the smooth-muscle cells of the myometrium.

Tyrosine kinase-mediated activation of cytosolic calcium oscillations and phasic myometrial contractions.

These studies sought to test the hypothesis that tyrosine kinase-stimulated phasic myometrial contractions are mediated by activation of the phosphatidylinositol (PI)-signaling pathway and the generation of cytosolic calcium oscillations. For these studies, uterine tissue was obtained from adult female Sprague-Dawley white rats during the proestrus/estrus phase of the cycle. In vitro contraction studies were performed using pervanadate (a tyrosine phosphatase inhibitor) with and without inhibitors of the PI-signaling pathway, including 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (a phospholipase C inhibitor), thimerosal (an inositol-trisphosphate receptor/channel inhibitor), and Ruthenium red (a ryanodine receptor inhibitor), and with oxytocin or prostaglandin F2 alpha (two classic uterotonic agonists). Cytosolic calcium studies were performed using Fura-2-loaded myometrial strips. During these studies, pervanadate was observed to produce cytosolic calcium oscillations and phasic contractions in myometrial tissue comparable to those produced in response to oxytocin and prostaglandin F2 alpha. The pervanadate-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, the inositol-trisphosphate receptor, and the ryanodine receptor, thereby confirming the importance of the PI-signaling pathway during tyrosine kinase-associated myometrial activity. Further confirming the important and shared role for the PI-signaling pathway during pervanadate-stimulated myometrial contractions, no significant additive effects were observed when classic uterotonic agonists such as oxytocin or prostaglandin F2 alpha were combined with pervanadate.

Intracellular Signaling and Phasic Myometrial Contractions

Intracellular Signaling and Phasic Myometrial Contractions

Intracellular signaling and phasic myometrial contractions.

This article reviews recently reported observations regarding the intracellular signal transduction mechanisms involved in the generation of phasic contractions occurring in myometrial tissue. The presence of cell surface receptors for classic uterotonic agonists (including oxytocin, norepinephrine, vasopressin, acetylcholine, and prostaglandins [PGs]) has been well described; all are seven-membrane-spanning, G protein-coupled receptors. Occupancy of these receptors, coupled through members of the Gq and/or Gi families of heterotrimeric G proteins, results in stimulation of the phospholipase C-beta (PLC-beta) isoforms. Nonclassic uterotonic agonists, such as growth factors and cytokines, also activate the phosphatidylinositol (PI)-signaling pathway, in this case through tyrosine kinase receptor-mediated activation of the phospholipase C-gamma (PLC-gamma) isoforms. Several recent reports have demonstrated that activation of the PI-signaling pathway in uterine myocytes results in the development of cytosolic calcium oscillation-like phenomena. These cytosolic calcium oscillations appear to arise from repetitive cycles of emptying and refill of the endoplasmic reticulum calcium stores along with the influx of extracellular calcium. Calcium release from the endoplasmic reticulum calcium stores appears to be mediated by the inositol trisphosphate-sensitive and the ryanodine-sensitive receptor/channels; isoforms for both the these receptor/channels have been shown to be expressed in myometrial tissue. In summary, receptor-mediated activation of the PI-signaling pathway and the generation of cytosolic calcium oscillations appear to produce intermittent calcium transients that result in the development and maintenance of phasic myometrial contractions.

Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate–phenol–chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde–1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase–polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase–polymerase chain reaction studies have confirmed expression of the phospholipase C-β1a, phospholipase C-β3, phospholipase C-γ1, phospholipase C-γ2, and phospholipase C-δ1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-β3, phospholipase C-γ1, and phospholipase C-δ1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-β1a isoform was expressed at the highest level, the phospholipase C-β3, phospholipase C-γ1, and phospholipase C-γ2 isoforms at an intermediate level, and the phospholipase C-δ1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle. (Am J Obstet Gynecol 1998;178:848-54.)

Intracellular mechanisms underlying prostaglandin F2alpha-stimulated phasic myometrial contractions.

These studies sought to test the hypothesis that prostaglandin F2alpha (PGF2alpha)-stimulated phasic myometrial contractions are characterized by the activation of the phosphatidylinositol-signaling pathway resulting in the generation of cytosolic calcium oscillations. For the experiments described in this report rat myometrial tissue was used, after the tissue was loaded with fura 2, to perform cytosolic calcium imaging studies and to perform computer-digitalized in vitro isometric contraction studies. Consistent with the above hypothesis, the cytosolic calcium-imaging studies demonstrated PGF2alpha-stimulated cytosolic calcium oscillations occurring simultaneously with phasic contractions. The in vitro isometric contraction studies confirmed that previously reported inhibitors of the phosphatidylinositol-signaling pathway and cytosolic calcium oscillation mechanisms resulted in significant inhibition of PGF2alpha-stimulated phasic myometrial contractions. In summary, these studies have provided substantial support for the hypothesis that PGF2alpha-stimulated phasic myometrial contractions are generated by intracellular signaling mechanisms involving activation of the phosphatidylinositol-signaling pathway and the production of cytosolic calcium oscillation-like phenomena.

Gq-Protein Alpha Subunit Expression and Distribution in Pregnant Rat Myometrial Tissues

Multiple G-protein isoforms play an integral role in signal transduction; the Gq subtype of G-protein alpha subunits is involved in the activation of the phosphatidylinositol signaling pathway. The studies described herein evaluate the expression of Gq, along with Gs and Gi, in pregnant and nonpregnant rat myometrial tissues. Myometrium and other tissues were obtained from nonpregnant and timed-pregnant Sprague-Dawley rats. Western blot studies were performed using polyclonal G-protein isoform-specific antibodies. Immunohistochemical studies were performed using the same antibodies with specimens of myometrium, intestine, and skeletal muscle. The Western blot studies confirmed differential expression of all types of G-protein alpha subunit subtypes in rat myometrial tissues. In pregnant rat myometrium, the expression of Gq and Gs was sustained through day 22, whereas, Gi expression decreased on day 20 and remained low through the remainder of gestation. The immunohistochemical studies revealed significant staining for Gq and Gs in the myometrial layers of the pregnant and nonpregnant rat uterus; in contrast, immunostaining for Gi was minimal in nonpregnant myometrium, and even lower in myometrium from pregnant uteri. These studies have confirmed expression of the Gq, Gi, and Gs alpha subunits in rat myometrial tissue. Immunohistochemistry confirmed that Gq was expressed at high levels in the myometrial layer of the pregnant and nonpregnant uterus. These observations support the hypothesis that Gq expression is critically important for the transduction of hormone signals, such as those responsible for the generation of phasic myometrial contractions.

Effects of Sodium and Calcium Channel Blockade on Cytosolic Calcium Oscillations and Phasic Contractions of Myometrial Tissue

These studies sought to test the hypothesis that agonist-stimulated cytosolic calcium oscillations and phasic myometrial contractions are dependent on calcium influx through dihydropyridine-sensitive calcium channels, but not sodium influx through tetrodotoxin-sensitive sodium channels. Cytosolic calcium imaging studies and in vitro isometric contraction studies were performed using uterine tissue from proestrus/estrus Sprague-Dawley rats. The calcium imaging studies were performed after loading partial thickness strips of myometrium with Fura-2. For the in vitro isometric contraction studies, the contraction data were computer digitalized, analyzed for contraction area, and normalized for cross-section area. The effects of nifedipine (1.0-5 mumol/L), a calcium channel blocker, were compared to tetrodotoxin (0.01-1 mumol/L), a sodium channel blocker. Oxytocin-stimulated simultaneous cytosolic calcium oscillations and phasic contractions were completely inhibited by 1 mumol/L nifedipine; in contrast, 1 mumol/L tetrodotoxin had no effect on the oxytocin-stimulated calcium oscillations and contractions. Oxytocin, aluminum fluoride, potassium chloride, and ionomycin stimulated in vitro phasic myometrial contractions. Tetrodotoxin had no effect on these agonist-stimulated phasic contractions, whereas nifedipine produced a significant, dose-related inhibition of the phasic contractile activity. The studies described in this report support the hypothesis that the influx of extracellular calcium is an important component of the cellular mechanisms responsible for the cytosolic calcium oscillations occurring during phasic myometrial contractions. In contrast, sodium influx through tetrodotoxin-sensitive sodium channels does not appear to play a comparably important role.

(+)cis-Dioxolane Stimulation of Cytosolic Calcium Oscillations and Phasic Contractions of Myometrial Smooth Muscle

These studies sought to determine the intracellular mechanisms underlying muscarinic receptor stimulated phasic contractions of myometrial smooth muscle. Utilizing cytosolic calcium imaging studies with Fura-2 loaded muscle strips, along within vitroisometric contraction studies, we have demonstrated that phasic myometrial contractions stimulated in response to (+)cis-dioxolane, a muscarinic selective acetylcholine agonist, are mediated by activation of the M3 receptor subtype resulting in stimulation of the phosphatidylinositol signaling pathway and the generation of cytosolic calcium oscillations.

The Mechanisms Underlying Bay K 8644—Stimulated Phasic Myometrial Contractions

Phasic myometrial contractions appear to be produced by calcium transients resulting from the activation of the phosphatidylinositol-signaling pathway. Bay K 8644, an L-type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. These studies were performed to test the hypothesis that Bay K 8644-stimulated contractions were mediated through mechanisms involving phosphoinositide-specific phospholipase C activation and cytosolic calcium oscillation-like mechanisms. In vitro contraction studies and intracellular calcium imaging were performed on longitudinal strips of uterine tissue obtained from mature virgin Sprague-Dawley rats. Isometric contraction data were computer digitized, analyzed for contraction area, and normalized for cross-sectional area. Dose-response studies were performed using previously reported inhibitors of cytosolic calcium oscillation mechanisms. In addition, qualitative inositol-phosphate production studies were performed after prelabeling uterine tissue in vitro with 3H-inositol. Subsequently, the labeled inositol phosphates were separated and recovered using anion exchange chromatography. Bay K 8644 produced periodic calcium transients or oscillations along with a dose-related increase in contractile activity and a significant increase in inositol-phosphate production. In contrast, neomycin (an inhibitor of phospholipase C), adenine (an inhibitor of calcium-induced calcium release), nifedipine (an L-type calcium channel blocker), and EGTA (a calcium chelator) significantly inhibited Bay K 8644-stimulated contractile activity. These results are consistent with the hypothesis that Bay K 8644, through its facilitation of increased intracellular calcium, results in the activation of the phosphatidylinsitol-signaling pathway and cytosolic calcium oscillation-like phenomena, thereby resulting in the generation of phasic myometrial contractions.

The mechanisms underlying bay K 8644?stimulated phasic myometrial contractions

The mechanisms underlying bay K 8644?stimulated phasic myometrial contractions

The Effects of Ruthenium Red, an Inhibitor of Calcium-Induced Calcium Release, on Phasic Myometrial Contractions

Ruthenium red inhibits calcium-induced calcium release from the ryanodine-sensitive intracellular calcium stores; this study sought to evaluate the effects of ruthenium red on agonist-stimulated phasic myometrial contractions. Ruthenium red was found to significantly inhibitin vitroisometric contractions stimulated in response to oxytocin, prostaglandin F2α,aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium-induced calcium release from ryanodine-sensitive calcium stores is an important event during agonist-stimulated phasic myometrial contractions.

Cytosolic calcium oscillation-like phenomena occurring during acetylcholine-stimulated phasic myometrial contractions

Cytosolic calcium oscillation-like phenomena occurring during acetylcholine-stimulated phasic myometrial contractions

Ionomycin-stimulated phasic myometrial contractions.

Ionomycin, a calcium ionophore, facilitates the sustained entry of extracellular calcium; however, in myometrial tissue it stimulates phasic contractions. This study sought to define further this unanticipated effect of ionomycin and to begin to explore the possible mechanism(s) involved. Utilizing rat uterine strips, in vitro isometric contraction studies were performed to determine the effects of ionomycin with and without membrane-permeant inhibitors of cytosolic calcium oscillations. To determine the effects of ionomycin on phospholipase C, qualitative inositol phosphate production studies were performed. The in vitro contraction studies confirmed that ionomycin-stimulated phasic myometrial contractions were potentially dependent on stimulation of phospholipase C, calcium-induced calcium release, and additional calcium influx through dihydropyridine-sensitive membrane calcium channels. The inositol phosphate production studies confirmed that ionomycin stimulated phospholipase C in a dose-related fashion to levels comparable to oxytocin. In summary, these observations have confirmed the ability of ionomycin to generate dose-related phasic myometrial contractions through mechanisms potentially involving the phosphatidylinositol-signaling pathway.