The role of thrombin in preterm parturition.

OBJECTIVE: Previous reports from our laboratory have shown that thrombin is a potent uterotonic agonist; those studies have suggested a role for thrombin during parturition, especially with regard to intrauterine bleeding. Thrombin activation can be quantified in peripheral blood by measurement of thrombin-antithrombin III (TAT) complex levels. This study sought to determine whether thrombin activation, as measured by thrombin-antithrombin III levels, is associated with premature labor. STUDY DESIGN: Thrombin-antithrombin III levels were measured in patients and control subjects with preterm labor. Quantitative TAT levels were determined by use of an enzyme-linked immunoassay with a working range from 0 to 60 ng/mL. All patients were monitored for pregnancy outcome. Receiver operating curve analysis was performed to determine the optimal TAT cutoff values. Further statistical analyses with one-way ANOVA, the chi2 test, or the Fisher exact test were performed to determine statistical significance (P <.05). RESULTS: Patients admitted with preterm labor who were subsequently delivered within 3 weeks had significantly higher mean TAT levels (7.80 +/- 2.86 ng/mL; P <.05) than control subjects (5.77 +/- 1.43 mL) or patients with preterm labor who were not delivered within 3 weeks of presentation with preterm labor (5.57 +/- 1.69 ng/mL; P <.05). Given a diagnosis of preterm labor, a TAT level of 8.0 ng/mL had a positive predictive value of 80% for delivery within 3 weeks of enrollment. CONCLUSIONS: This study showed that TAT levels are elevated in patients with preterm labor who are destined to deliver before term. These results suggest that preterm labor resulting in premature delivery is associated with the activation of thrombin. Future studies will further elucidate the role of thrombin in preterm parturition and confirm whether tests for thrombin activation can accurately identify those patients destined for preterm delivery.

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-beta3, -gamma1, -gamma2, and -delta1 proteins in both the membrane and cytosolic fractions of rat myometrium; in contrast, only trace amounts of the phospholipase C-beta1 protein was observed in this tissue. The immunohistochemical studies demonstrated localization of the phospholipase C-beta3, -gamma1, -gamma2, -delta1 and to a lesser degree phospholipase C-beta1 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.

Thrombin-stimulated uterine contractions in the pregnant and nonpregnant rat.

OBJECTIVE: Thrombin generated during the active clotting of blood appears to be a potent uterotonic agonist; however, the mechanism underlying this effect on uterine smooth muscle is not well understood. We performed studies to confirm the uterotonic effects of thrombin and to determine whether prostaglandin production plays a role during the uterotonic effects of thrombin or clotting blood. METHODS: Uterine contraction studies were performed using adult nonpregnant and near-term pregnant rats. The in vitro isometric contraction studies used uterine strips pretreated with indomethacin or vehicle (ethanol), which were then stimulated with thrombin. For the in vivo contraction studies, rats were pretreated with intraperitoneal injections of indomethacin or vehicle (ethanol) then stimulated by intraluminal injection of fresh rat blood or thrombin into the uterus. The contraction data were acquired using isometric force transducers, were computer digitized, normalized for spontaneous activity, and statistically analyzed. Prostaglandin (PG) F2alpha was measured using an enzyme-linked immunoassay. RESULTS: The in vitro contraction studies demonstrated that both thrombin and actively clotting blood produce a significant increase in the frequency and intensity of uterine contractions. Thrombin stimulation was associated with a 54% increase in PGF2alpha concentration in vitro; indomethacin (1 microM) pretreatment completely inhibited that increase in PGF2alpha production. Despite the suppression of PGF2alpha production, pretreatment with indomethacin had no inhibitory effect on thrombin-stimulated contractile activity. In vivo contraction studies further confirmed that indomethacin (2 mg/kg) pretreatment had no effect on blood- or thrombin-stimulated contractile activity. CONCLUSIONS: We confirmed that thrombin and thrombin produced by actively clotting blood had a robust uterotonic effect in the rat and that prostaglandin production did not play a significant role in thrombin-stimulated contractions.

Constitutive androstane receptor expression in the rat cervix during gestation.

OBJECTIVE: To investigate expression of the constitutive androstane receptor (CAR) in the pregnant rat cervix. METHODS: Rat uterine tissue was obtained on gestational days 12, 16, 20, 21, and 22 (the day of parturition), and postpartum day 1. In addition, liver, lung, kidney, heart, and skeletal muscle tissue were obtained. Expression of the two known CAR isoforms was evaluated using reverse transcriptase-polymerase chain reaction. RESULTS: These studies confirmed CAR expression in the liver; however, CAR was not demonstrated in the myometrium or cervical tissue. CONCLUSIONS: The currently described CAR1 and CAR2 isoforms are not expressed in rat uterine tissue; therefore, they do not appear to participate in parturition in the pregnant rat.

Suppression of eukaryotic translation termination by selected RNAs.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

Effects of thrombin on myometrial contractions in vitro and in vivo.

OBJECTIVE: A previous report from our laboratory demonstrated that thrombin stimulates myometrial contractions by activating the phosphatidylinositol signaling pathway in a dose-dependent fashion. The studies described in this report sought to determine whether thrombin and blood stimulate myometrial contractions both in vivo and in vitro and whether these uterotonic effects could be suppressed or prevented with thrombin inhibitors. STUDY DESIGN: In vitro contraction studies were performed with proestrus and estrus rat myometrial tissue. In vivo contraction studies were performed with nonpregnant and timed-pregnant Sprague-Dawley rats. RESULTS: Pretreatment of thrombin with hirudin suppressed the uterotonic effects of thrombin in vitro. Fresh whole blood stimulated myometrial contractions in a dose-dependent fashion in vitro, and thrombin inhibitors decreased the myometrial response seen with blood alone. Thrombin increased the frequency, intensity, and tone of myometrial contractions in vivo in a dose-related manner. In pregnant animals increasing doses of whole blood increased the frequency and tone of myometrial contractions. In both pregnant and nonpregnant animals whole blood significantly stimulated myometrial contractions, whereas heparinization of the blood significantly suppressed this in vivo uterotonic effect. CONCLUSION: Thrombin is a potent uterotonic agent both in vitro and in vivo; furthermore, the uterotonic effects of blood appeared to be related to thrombin production during coagulation. These studies provide a possible mechanistic explanation for the observed increase in myometrial contractions in the presence of intrauterine bleeding and may also provide an insight into preterm labor associated with vaginal bleeding.

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 F(2)(alpha). 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.

Divergence in murine myometrium spontaneous and oxytocin-stimulated contractile responses to serine/threonine protein phosphatase-1 inhibition.

Reversible phosphorylation is essential in regulating uterine contractions. Identification, characterization, and functional understanding of myometrium protein phosphatase(s) are lacking. Okadaic acid (OA), which inhibits protein phosphatase-1 (PP1) and PP2A, has been shown to alter uterine contractions. Experiments were conducted to determine the 1) identity of the myometrial OA-sensitive PP, 2) influence of OA on spontaneous and oxytocin (OT)-stimulated myometrial contractions, and 3) expression of uterine PPs during sexual development. Western blot analysis indicated the presence of PP1(alpha) and PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1(alpha) in longitudinal and circular myometrial layers and endometrial epithelium. Conversely, PP2A was localized to the endometrial stroma. Cumulative addition of OA (n = 9; 10, 100, 250, 500, 1000 nM) did not significantly alter spontaneous contractions of mouse uterine horns in comparison to vehicle-treated controls (n = 9). By the end of the test period OA- and vehicle-treated uteri displayed a comparable decline in uterine contractions to 79.2% and 63.7%, respectively, of basal contractile activity. Pretreatment of uterine tissue with OA (1 microM; n = 7) significantly reduced contractile response to increasing concentrations of OT (8, 16, 32, 64 nM) in comparison to vehicle pretreatment (dimethyl sulfoxide; n = 7). At the end of the OT-administration period, contractile activity was 160.4% and 67.3% of basal contractile activity for vehicle (no OA) and OA-pretreated groups, respectively. During the early prepubertal period PP1(alpha) was expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1(alpha) was observed in both the longitudinal and circular myometrium. In summary, these studies have indicated 1) that PP1 is the primary myometrial OA-sensitive PP; 2) that inhibition of PP1 had no effect on spontaneous contractions, whereas it markedly inhibited OT-stimulated uterine contractions; and 3) that PP1 is differentially expressed in the circular and longitudinal myometrium in relation to sexual development.

The role of diacylglycerol as a modulator of oxytocin-stimulated phasic contractions in myometrium from pregnant and nonpregnant rats.

OBJECTIVE: The role of diacylglycerol in the phosphatidylinositol-signaling pathway is to activate protein kinase C. In the myometrium, protein kinase C activation leads to inhibition of phasic contractions. These studies are designed to determine why stimulation of the phosphatidylinositol-signaling pathway caused by oxytocin does not cause a paradoxical suppression of contractions through diacylglycerol production and protein kinase C activation. Specifically, these studies were performed to test the hypothesis that diacylglycerol catabolism is significant in myometrial tissue, thereby precluding its availability for the activation of protein kinase C. STUDY DESIGN: For these studies, uterine tissue was obtained from Sprague-Dawley rats both nonpregnant and with timed gestations. In vitro contraction studies were performed with cumulative additions of oxytocin (8-64 nmol/L) with and without R59022 (a diacylglycerol kinase inhibitor) or RHC80267 (a diacylglycerol lipase inhibitor). The contraction data were computer-digitalized, analyzed for total contractile activity, normalized for tissue cross-sectional area, and reported as the percentage of spontaneous activity. RESULTS: In myometrium from nonpregnant animals, inhibition of diacylglycerol lipase with RHC80267 had little effect on oxytocin-stimulated contractile activity, whereas inhibition of diacylglycerol kinase with R59022, although producing an increase in contractile frequency, markedly suppressed total oxytocin-stimulated contractile activity. In contrast, in myometrium from near-term pregnant animals both RHC80267 and R59022 produced marked suppression of oxytocin-stimulated contractile activity. CONCLUSIONS: These studies have demonstrated that prevention of diacylglycerol degradation, especially in response to inhibition of myometrial diacylglycerol kinase, results in the paradoxic oxytocin-mediated suppression of total myometrial contractile activity. These observations support the hypothesis that, when its catabolism is prevented, diacylglycerol produced in response to stimulation of the phosphatidylinositol-signaling pathway by oxytocin becomes available for protein kinase C activation, resulting in inhibition of myometrial contractile activity.

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.

The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4, 5-trisphosphate receptor modulator on myometrial contractions.

These studies were performed to evaluate the effect of 2-aminoethoxydiphenyl borate (2-APB), a novel membrane-permeable inositol 1,4,5-trisphosphate-receptor inhibitor on agonist-induced, spontaneous, and KCl-stimulated in vitro myometrial contractions. 2-APB significantly inhibited spontaneous myometrial contractions as well as phasic contractions induced by various uterotonic agonists. Confiriming its effects on intracellular calcium release, 2-APB inhibited phasic contractions in the absence of extracellular calcium. 2-APB had little effect on the tonic response to KCl stimulation, implicating its insignificant effect on voltage-gated calcium channels. The inhibitory effect of 2-APB on phasic contractions was completely reversed by washout. In summary, 2-APB effectively penetrated uterine tissue and significantly inhibited myometrial events previously shown to be mediated through activation of the PI-signaling pathway.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Cellular mechanisms underlying magnesium sulfate inhibition of phasic myometrial contractions.

These studies sought to test the hypothesis that magnesium inhibits extracellular calcium entry, thereby inhibiting intracellular calcium release and cytosolic calcium oscillations in myometrial smooth muscle. In vitro contraction studies were performed using oxytocin and other uterotonic agonists with and without the addition of magnesium in the presence and absence of extracellular calcium. Cytosolic calcium studies were performed using myometrial strips loaded with Fura 2. Oxytocin produced cytosolic calcium oscillations and simultaneous phasic contractions; both were inhibited by magnesium. The other uterotonic agonists tested also produced phasic contractions which were significantly inhibited by magnesium. The magnesium effect was reversible with washout and counteracted by Bay K 8644 (a calcium channel agonist). In the absence of extracellular calcium, intracellular calcium release in response to oxytocin was inhibited by magnesium. In summary, magnesium inhibited extracellular calcium entry, intracellular calcium release, cytosolic calcium oscillations, and phasic contractions of myometrial smooth muscle.

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-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 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-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 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.

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.

Identification of gestationally regulated genes in rat myometrium by use of messenger ribonucleic acid differential display.

OBJECTIVE: We hypothesized that the proteins contributing to myometrial changes during gestation could be identified indirectly by analyzing the changing pattern of messenger ribonucleic acid expression in the myometrium during pregnancy. STUDY DESIGN: Ribonucleic acid was extracted from myometrium of timed pregnant Sprague-Dawley rats on days 12, 16, 20, 21, and 22 of pregnancy and on day 1 post partum. The technique of messenger ribonucleic acid differential display, a simple and sensitive polymerase chain reaction-based method for rapidly identifying messenger ribonucleic acids whose levels increase or decrease, was performed with the nine different anchoring primers (oligodeoxythymidine11 VN: V = G, A, or C; N = G, A, or C) in combination with 24 different 10-base oligonucleotides of random sequence. The polymerase chain reaction products were separated by electrophoresis on a 5% polyacrylamide sequencing gel, and those whose levels changed were then cloned, sequenced, and compared with those in the GenBank database to determine whether they corresponded to a known sequence in the database or were novel. Semiquantitative reverse transcriptase-polymerase chain reaction was used to confirm differential expression of selected products. RESULTS: Messenger ribonucleic acid differential display revealed > 500 polymerase chain reaction products that were differentially expressed during gestation, 179 of which were cloned and sequenced. Of these, 157 were from messenger ribonucleic acids whose levels increased during gestation, and 22 were from transcripts that decreased. Eighty-seven (49%) were related to sequences in the GenBank database, of which 62 (35%) were from messenger ribonucleic acids encoding known proteins and 25 (14%) corresponded to known expressed sequence tags. The technique of semiquantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of messenger ribonucleic acids encoding beta-tropomyosin, type II phosphatidyl inositol-4-phosphate 5-kinase, and a novel myometrial messenger ribonucleic acid named RPU0901AC. CONCLUSION: Messenger ribonucleic acid differential display is a simple and sensitive method for rapidly identifying myometrial messenger ribonucleic acids that are differentially regulated during pregnancy. The identification of these differentially expressed messenger ribonucleic acids may lead to a better understanding of the molecular basis of normal and abnormal parturition.

Increase in serum leptin and uterine leptin receptor messenger RNA levels during pregnancy in rats.

Pregnancy is a physiological state associated with significant changes in appetite, thermogenesis, and lipid metabolism, functions which are regulated in part by a hormone, leptin, secreted by adipocytes. Leptin has also been shown to have a role in reproduction, promoting centrally-regulated maturation of the reproductive system and signaling the presence of adequate maternal energy stores for fertility. Here we demonstrate that serum leptin levels are modulated during normal rat pregnancy with a 1.8-fold increase during pregnancy followed by a decrease just before parturition. Leptin receptor mRNA levels in the uterus are also regulated with an increase about 2.7-fold during this same period, whereas there is no change in other tissues examined. The results suggest that leptin may play a role during pregnancy, perhaps regulating energy utilization.

Gq-protein alpha subunit expression and distribution in pregnant rat myometrial tissues.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.