Search

Intrauterine or intraperitoneal administration of lipopolysaccharide (LPS) into normal mice at midgestation induces preterm delivery (PTD) within 24 h through a mechanism dependent on Toll-like receptor signaling and expression of inflammatory cytokines. The exact participants in the cellular network involved in PTD are not known. Although the activities of innate immune cells are thought to be important, the extent to which this process depends on T and B cells has yet to be examined. Mice deficient in T and B cells due to genetic deficiency in the recombination activating gene 1 (Rag1−/−) were given LPS intraperitoneally on Day 15 of gestation and found to be susceptible to LPS-induced PTD. This was found to involve many of the inflammatory mediators reported as important in normal mice. Moreover, at a low dose (3 μg), pregnant Rag1−/−mice were found to be more susceptible to PTD than a cohort of normal mice on the same genetic background. This increased susceptibility was partially reversed by transfer, on Day 10 of gestation, of whole lymphocytes or purified CD4+T cells. Transfer of purified CD4+T cells to Rag1−/−mice resulted in a uterine draining node population of FOXP3+cells, suggesting that these cells may contribute to resistance to LPS-induced PTD. Overall, the data suggest that, although T and B lymphocytes are not critical positive regulators of LPS-induced PTD, CD4+T cells play a protective and regulatory role, and thus could be a target for preventive or therapeutic manipulation.

Objective: Phospholipid scramblases (PLSCRs), a family of novel membrane proteins, facilitate the translocation of aminophospholipids from the inner to the exterior leaf of the cell membrane. Four isoforms of PLSCR (PLSCR1—4) have been reported in mouse and human. Tlie studies described in this report sought to characterize the uterine expression of the PLSCR isoforms in the near-term pregnant rat. Methods: Uterine tissue was obtained from timed-pregnant Sprague-Dawley rats. Total RNA was isolated, treated with DNase, and used in qualitative and quantitative reverse transcriptase—polymerase chain reaction (RT-PCR) studies utilizing PLCSR isoform PCR primers. A rat spleen cDNA bacteriophage library was used as a template for PCR-based sequencing to determine the cDNA and translated amino acid sequences for the PLSCR3 and PLSCR4 homologs expressed in rats. The 5' and 3' untranslated regions were obtained using 5' and 3' Rapid Amplification ofcDNA Ends (RACE) techniques. Results: RT-PCR studies confirmed expression of PLSCR3 and PLSCR4 in the endometrial and myometrial layers of the pregnant rat uterus; in contrast, PLSCR1 and PLSCR2 were not found in uterine tissues. Tlte cDNA sequence for the rat PLSCR3 homolog was found to be 1642 nucleotides, having 92% identity with mouse and 80% with human PLSCR3. The rat PLSCR4 homolog has a cDNA sequence of 1879 nucleotides, having an 89% identity with mouse and 12% identity with human PLSCR4 homologs. Conclusion: The intrauterine expression of PLSCR3 and PLSCR4 provides a dynamic mechanism by which aminophospholipid translocation can be regulated, thereby modulating the activity of various membrane proteins that are involved in inflammation and coagulation-related events.

Objective: Fg12and thrombin potentially play roles during inflammation-induced preterm delivery. These studies sought to demonstrate functional prothrombinase enzyme activity in rat uterus, consistent with the expression of Fg12in this tissue. Methods: Myometrial and other tissue obtained from non-pregnant and timed-pregnant rats was homogenized in Trisbuffered saline. Prothrombinase activity was determined based on the kinetic metabolism of a chromogenic thrombin substrate. Homogenates were incubated with prothrombin followed by the addition of the thrombin substrate. Thrombin activity was determined by comparing tissue activity to a standard curve generated using 0.01 to 0.04 units of active thrombin. Western blot studies were also performed to confirm tissue prothrombinase activity in myometrial homogenates. Prothrombin was incubated with tissue homogenates; the reactions were then terminated with sodium dodecyl sulfate (SDS) loading buffer. Results: Prothrombinase activity in myometrial tissue was observed to be 0.047 to 0.011 U thrombin/10 min/µg protein. Heat denaturation and calcium removal eliminated prothrombinase activity, whereas the addition of Factor V enhanced activity. The Western blots confirmed the presence of prothrombin, the anticipated prethrombin fragments, and thrombin. Consistent with the enzyme studies, the thrombin band formed upon incubation with myometrial homogenates from pregnant and nonpregnant rats. In contrast, the thrombin band was not apparent with removal of calcium, heat denaturation and treatment with serine protease inhibitors. Conclusions: In summary, these studies have confirmed functionally active prothrombinase activity in rat myometrium, supporting the hypothesis that Fg12is expressed in this tissue.

Objective:Phospholipid scramblases (PLSCRs), a family of novel membrane proteins, facilitate the translocation of aminophospholipids from the inner to the exterior leaf of the cell membrane. Four isoforms of PLSCR (PLSCR1-4) have been reported in mouse and human. The studies described in this report sought to characterize the uterine expression of the PLSCR isoforms in the near-term pregnant rat.Methods:Uterine tissue was obtained from timed-pregnant Sprague-Dawley rats. Total RNA was isolated, treated with DNase, and used in qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies utilizing PLCSR isoform PCR primers. A rat spleen cDNA bacteriophage library was used as a template for PCR-based sequencing to determine the cDNA and translated amino acid sequences for the PLSCR3 and PLSCR4 homologs expressed in rats. The 5′ and 3′ untranslated regions were obtained using 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) techniques.Results:RT-PCR studies confirmed expression of PLSCR3 and PLSCR4 in the endometrial and myometrial layers of the pregnant rat uterus; in contrast, PLSCR1 and PLSCR2 were not found in uterine tissues. The cDNA sequence for the rat PLSCR3 homolog was found to be 1642 nucleotides, having 92% identity with mouse and 80% with human PLSCR3. The rat PLSCR4 homolog has a cDNA sequence of 1879 nucleotides, having an 89% identity with mouse and 72% identity with human PLSCR4 homologs.Conclusion:The intrauterine expression of PLSCR3 and PLSCR4 provides a dynamic mechanism by which aminophospholipid translocation can be regulated, thereby modulating the activity of various membrane proteins that are involved in inflammation and coagulation-related events.

Objective:Fgl2 and thrombin potentially play roles during inflammation-induced preterm delivery. These studies sought to demonstrate functional prothrombinase enzyme activity in rat uterus, consistent with the expression of Fgl2 in this tissue.Methods:Myometrial and other tissue obtained from non-pregnant and timed-pregnant rats was homogenized in Tris-buffered saline. Prothrombinase activity was determined based on the kinetic metabolism of a chromogenic thrombin substrate. Homogenates were incubated with prothrombin followed by the addition of the thrombin substrate. Thrombin activity was determined by comparing tissue activity to a standard curve generated using 0.01 to 0.04 units of active thrombin. Western blot studies were also performed to confirm tissue prothrombinase activity in myometrial homogenates. Prothrombin was incubated with tissue homogenates; the reactions were then terminated with sodium dodecyl sulfate (SDS) loading buffer.Results:Prothrombinase activity in myometrial tissue was observed to be 0.047 to 0.077 U thrombin/10 min/μg protein. Heat denaturation and calcium removal eliminated prothrombinase activity, whereas the addition of Factor V enhanced activity. The Western blots confirmed the presence of prothrombin, the anticipated prethrombin fragments, and thrombin. Consistent with the enzyme studies, the thrombin band formed upon incubation with myometrial homogenates from pregnant and nonpregnant rats. In contrast, the thrombin band was not apparent with removal of calcium, heat denaturation and treatment with serine protease inhibitors.Conclusions:In summary, these studies have confirmed functionaly active prothrombinase activity in rat myometrium, supporting the hypothesis that Fgl2 is expressed in this tissue.

Objective:Enhanced tyrosine phosphorylation of phosopholipase C-γ1 (PLCγ1) is associated with increased spontaneous contractile activity. PLCγ1 phosphorylation is regulated by cellular protein tyrosine kinases and tyrosine phosphatases (PTPs). The studies in this report were undertaken to characterize the expression of two PTPs known to bind to PLCγ1: Src-homology phosphatase type-1 (SHP-1) and type-2 (SHP-2).Methods:Uterine and other tissues were obtained from non-pregnant (estrus) and pregnant (gestational day 12 through day 1 postpartum) Sprague-Dawley rats. PTP activity in myometrial homogenates was determined using an in vitro fluorometric PTP assay with and without bpV(phen) (a nonselective PTP inhibitor), or PTP-Inhibitor 1 (PTP-I1, a SHP selective inhibitor). Western blots were performed using polyclonal antibodies to SHP-1 and SHP-2. Immunoprecipitation studies were performed to demonstrate an association between PLCγ1 and the SHP proteins.Results:The in vitro PTP assays demonstrated comparable enzyme activity in myometrium from estrus and pregnant animals. BpV(phen) produced a 93% reduction in PTP activity (P <.05); similarly, PTP-11 produced an 86% reduction in enzyme activity (P <.05). Western blots confirmed robust expression of both SHP-1 and SHP-2 protein in rat uterus. SHP-1 expression decreased significantly at the end of gestation; in contrast, SHP-2 levels remained stable. Immunoprecipitation studies confirmed an association between the SHP proteins and PLCγ1.Conclusion:These studies have demonstrated that SHP-1 and SHP-2 are expressed in rat myometrium and appear to be responsible for the PTP activity in this tissue, thereby providing a molecular mechanism for the modulation of PLCγ1 phosphotyrosine levels in the rat uterus.

Objective: Enhanced tyrosine phosphorylation of phospholipase C-? 1 (PLCy 1) is associated with increased spontaneous contractile activity, PLCy 1 phosphorylation is regulated by cellular protein tyrosine kinases and tyrosine phosphatases (PTPs). The studies in this report were undertaken to characterize the expression of two PTPs known to bind to PLCyl: Src-homology phosphatase type-1 (SHP-1) and type-2 (SHP-2). Methods: Uterine and other tissues were obtained from non-pregnant (estrus) and pregnant (gestational day 12 through day 1 postpartum) Sprague-Datvley rats. PTP activity in myometrial homogenates was determined using an in vitro fluorometric PTP assay with and without bpV(phen) (a nonselective PTP inhibitor), or PTP-Inhibitor 1 (PTP-I1, a SHP selective inhibitor). Western blots were performed using polyclonal antibodies to SHP-1 and SHP-2. Immunoprecipitation studies were performe d to demonstrate an association between PLCyl and the SHP proteins. Results: The in vitro PTP assays demonstrated comparable enzyme activity in myometrium from estrus and pregnant animals. BpV(phen) produced a 93% reduction in PTP activity (P &lt;. 05); similarly, PTP-II produced an 86% reduction in enzyme activity (P &lt;. 05). Western blots confirmed robust expression of both SHP-1 and SHP-2 protein in rat uterus. SHP-1 expression decreased significantly at the end of gestation; in contrast, SHP-2 levels remained stable. Immunoprecipitation studies confirmed an association between the SHP proteins and PLCyl. Conclusion: These studies have demonstrated that SHP-1 and SHP-2 are expressed in rat myometrium and appear to be responsible for the PTP activity in this tissue, thereby providing a molecular mechanism for the modulation of PLCyl phosphotyrosine levels in the rat uterus.

Objective: Three protease-activated receptor (PAR1, 3, and 4) isoforms have been shown to be responsible for the cellular effects of thrombin; another PAR isoform (PAR2) is responsible for the cellular effects of trypsin. The present studies sought to test the hypothesis that one (or more) of these PAR isoforms is expressed in myometrial tissue, thereby accounting for the uterotonic effects of these novel agonists. Methods: The rat PAR3 and 4 isoforms were cloned from a rat spleen cDNA library. PAR isoform mRNA expression was determined by using reverse-transcriptase polymerase chain reactions (PCR) in Sprague-Dawley rats. Confirmation of the identity of the amplified mRNA was done by sequence analysis. Relative quantification of the PAR1 and PAR2 isoforms was performed using a real-time quantitative reverse transcriptase PCR (RT-PCR) technique. PAR protein expression was confirmed by Western blots using polyclonal antibodies. Results: The rat PAR3 and 4 homologues showed significant sequence homology to the mouse and human amino acid and nucleotide sequences. The RT-PCR studies confirmed PAR1-4 expression in myometrium from rats in estrus. PAR3 was expressed in uterus, spleen, kidney, liver, lung, brain, and heart. PAR4 was expressed in uterus, spleen, and lung. Messenger RNAfor the PAR1 and 2 isoforms was expressed during the second half of gestation in myometrium from timed-pregnant rats. In contrast, mRNA for the PAR3 and 4 isoforms was not detected in gestational myometrium. PAR protein expression appeared to match tissue mRNA expression patterns. Conclusion: These RT-PCR studies confirmed ubiquitous expression of the PAR1 and PAR2 isoforms in myometrium and other rat tissues; in contrast, the PAR3 and PAR4 isoforms are expressed in a tissue-specific and gestationally related pattern.

Preterm birth is a leading cause of neonatal morbidity and mortality. Despite a growing body of evidence correlating inflammation with preterm birth, the signal transduction pathways responsible for the emptying of the uterus in the setting of intrauterine inflammation has not been elucidated. We now report a unique, reproducible mouse model of localized intrauterine inflammation. This model results in 100% preterm delivery with no maternal mortality. Using our model, we also show that platelet-activating factor is a crucial mediator of both inflammation-induced preterm birth and fetal demise. Using C3H/HeJ mice, we demonstrate that toll-like receptor-4 (TLR-4) plays a role in lipopolysaccharide-induced preterm birth but not in inflammation-induced fetal death. Immunohistochemistry studies demonstrate the presence of the platelet-activating factor receptor in both endometrial glands and smooth muscle in uterine tissues. Molecular studies demonstrate the differential expression of platelet-activating factor receptor and TLR-4 in uterine and cervical tissue throughout gestation. Quantitative polymerase chain reaction revealed an up-regulation of TLR-4 in the fundal region of the uterus in response to intrauterine inflammation. The use of this model will increase our understanding of the significant clinical problem of inflammation-induced preterm birth and will elucidate signal transduction pathways involved in an inflammatory state.

Objective:To sequence and characterize the expression of the prothrombinase Fgl-2 in the Sprague-Dawley rat.Methods:Reverse-transcriptase polymerase chain reaction was performed on RNA from spontaneoulsy cycling adult pregnant Sprague-Dawley rats by using specific Fgl-w primers. The resulting amplicon was also used to screen a rat spleen bacteriophage library and to probe a Northern blot of various tissues. The rat Fgl-2 amino acid sequence was compared with the known sequences in mouse and human.Results:Fgl-w specific amplicon bands were observed in the rat brain, kidney, liver, ovary, spleen, and gestational day 22 and postpartum uterus. The rat Fgl-2 cDNA and amino acid sequence were found to be homologous with those of human (86% and 74%, respectively) and mouse (91% and 87%, respectively). Northern blotting demonstrated two different-sized transcripts (1.3 and 3.4 kb), and expression was observed in the cevix, heart, liver, ovary, and nongestational and gestational day 22 myometrium.Conclusion:Thrombin is classically generated from the clevage of the proenzyme prothrombin by activated factors V and X. In tissues thrombin appears to be generated by a novel prothrombinase Fgl-2 (fibrinogen-like protein) whose activity is stimulated by proinflammatory mediators. Fgl-2 provides the mechanistic coupling between proinflammatory cytokines and generation of active thrombin independent of the coagulation cascade. Our studies confirmed the expression of Fgl-2 mRNA in several rat tissues, including the pregnant uterus, where it could play a key role in the initiation of parturition especially in response to local or systemic infection.

Objective: To sequence and characterize the expression of the prothrombinase Fgl-2 in the Sprague-Dawley rat. Methods: Reverse-transcriptase polymerase chain reaction was performed on RNA from spontaneoulsy cycling adult pregnant Sprague-Dawley rats by using specific Fgl-w primers. The resulting amplicon was also used to screen a rat spleen bacteriophage library and to probe a Northern blot of various tissues. The rat Fgl-2 amino acid sequence was compared with the known sequences in mouse and human. Results: Fgl-w specific amplicon bands were observed in the rat brain, kidney, liver, ovary, spleen, and gestational day 22 and postpartum uterus. The rat Fgl-2 cDNA and amino acid sequence were found to be homologous with those of human (86% and 74%, respectively) and mouse (91% and 87%, respectively). Northern blotting demonstrated two different-sized transcripts (1.3 and 3.4 kb), and expression was observed in the cevix, heart, liver, ovary, and nongestational and gestational day 22 myometrium. Conclusion: Thrombin is classically generated from the clevage of the proenzyme prothrombin by activated factors V and X. In tissues thrombin appears to be generated by a novel prothrombinase Fgl-2 (fibrinogen-like protein) whose activity is stimulated by proinflammatory mediators. Fgl-2 provides the mechanistic coupling between proinflammatory cytokines and generation of active thrombin independent of the coagulation cascade. Our studies confirmed the expression of Fgl-2 mRNA in several rat tissues, including the pregnant uterus, where it could play a key role in the initiation of parturition especially in response to local or systemic infection.

Objective:Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus.Methods:Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptaase—polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin).Results:The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase—polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment within heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin.Conclusion:These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.

Objective: Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Methods: Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptaase—polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). Results: The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase—polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment within heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. Conclusion: These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.

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 pretreatd with indomethacia or vehicle (ethanol), which were then stimulated with thrombin. Forthe in vivo contraction studies, rats were pretreated with intraperitoneal injections of indomethacia 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 traducers, were computer digized, normalized for spontaneous activity, and statistically analyzed. Prostaglandin (PG) F2αwas 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 PGF2αconcentration in vitro; indomethacin (1 μM) pretreatment completely inhibited that increase in PGF2αproduction. Despite the suppression of PGF2αproduction, 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 andthrombin 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.

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 pretreatd with indomethacia or vehicle (ethanol), which were then stimulated with thrombin. Forthe in vivo contraction studies, rats were pretreated with intraperitoneal injections of indomethacia 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 traducers, were computer digized, normalized for spontaneous activity, and statistically analyzed. Prostaglandin (PG) F2awas 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 PGF2aconcentration in vitro; indomethacin (1 µM) pretreatment completely inhibited that increase in PGF2aproduction. Despite the suppression of PGF2aproduction, 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 andthrombin 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.

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 PP1αand PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1αin 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 μM; 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 PP1αwas expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1αwas 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.

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 vitromyometrial 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.

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 Vectastian ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were peformed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation.Results:These studies confirmed significant expression of the PKC-α, -β2, -δ, -η, and -ζ isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-β1, -γ, -ε, and -θ isoforms was observed. Expression of the PKC-α, -β2, and -η isoforms decreased modestly during the latter days of gestation; in contrast, PKC-δ and -ζ remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-α, -β2, -δ, -η, and -ζ 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.

These studies sought to test the hypothesis that tyrosine kinase-stimulated phasic myometrical 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 vivo 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 F2α (two classic uterotonic agonists). Cytosolic calcium studies were performed using Fura-2-loaded myometrial strips. During these studies, pervanadate was observed to produce cytosolid calcium oscillations and phasic contractions in myometrical tissue comparable to those produced in response to oxytocin and prostaglandin F2α. The pervandate-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 myometrical activity. Further confirming the important and shared role for the PI-signaling pathway during pervanadate-stimulated myometrical contractions, no significant additive effects were observed when classic uterotonic agonists such as oxytocin or prostaglandin F2α were combined with pervanadate.

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 vitrocontraction 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.

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 Vectastian ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were peformed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation. Results: These studies confirmed significant expression of the PKC-a, -ß2, -d, -?, and -? isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-ß1, -?, -e, and -? isoforms was observed. Expression of the PKC-a, -ß2, and -? isoforms decreased modestly during the latter days of gestation; in contrast, PKC-d and -? remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-a, -ß2, -d, -?, and -? 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.

These studies sought to test the hypothesis that tyrosine kinase-stimulated phasic myometrical 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 vivo 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 F2a (two classic uterotonic agonists). Cytosolic calcium studies were performed using Fura-2-loaded myometrial strips. During these studies, pervanadate was observed to produce cytosolid calcium oscillations and phasic contractions in myometrical tissue comparable to those produced in response to oxytocin and prostaglandin F2a. The pervandate-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 myometrical activity. Further confirming the important and shared role for the PI-signaling pathway during pervanadate-stimulated myometrical contractions, no significant additive effects were observed when classic uterotonic agonists such as oxytocin or prostaglandin F2a were combined with pervanadate.

Nine cases of pregnancy complicated by diabetes and prior renal transplantation are reviewed. Maternal and fetal death occurred in a patient with foot and leg ulcers associated with preexisting peripheral vascular disease. Pregnancy-induced hypertension occurred in six cases. Spontaneous weight-bearing fractures occurred in two patients. No episodes of renal allograft rejection occurred. Evidence of fetal compromise was present in six cases. All fetuses were delivered by cesarean section prior to term, with live births occurring from 31 1/2 to 36 weeks' gestation. A single case of hypospadias was the only congenital defect. Prepregnancy screening for complications of diabetes and renal transplantation is advised and euglycemia should be achieved before and during pregnancy. Advanced diabetic vascular disease puts these gestations at significant risk.(JAMA 1986;255:911-915)

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 heterotrimetric G proteins, results in stimulation of the phospholipase C-β (PCL-β) 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-γ (PCL-γ) 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 receptive 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 of 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 intermitten calcium transients that result in the development and maintenance of phasic myometrial contractions.

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-pergnant Sprangue-Dawley rats. Western blot studies were performed using polycclonal 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 of these G-protein α 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 α subunits in rat myometrical 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.

OBJECTIVES:Oxytocin-stimulated uterine contractions are associated with repetitive cycles of elevated cytosolic calcium, ie, cytosolic calcium oscillations. The studies in this report were per formed to test the hypothesis that phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the myometrial intracellular oscillator.METHODS:In vitro isometric contraction studies were performed using longitudinal strips of myometrium from nonpregnant, adult Sprague-Dawley rats. Cumulative dose-response studies were performed using oxytocin and aluminum fluoride with and without 2-nitro-4-carboxyphenyl- N,N-diphenylcarbamate (NCDC), an inhibitor of PI-PLC.RESULTS:Stimulation of G-proteins coupled to PI-PLC with aluminum fluoride resulted in a significant increase in phasic myometrial contractions comparable to those produced by oxytocin. Inhibition of PI-PLC with NCDC resulted in significant suppression of oxytocin- and aluminum fluoride-stimulated myometrial contractions. In contrast, doses that suppressed agonist-stimulated contractions had only a minimal effect on KCl-stimulated tonic myometrial contractions.CONCLUSIONS:These studies provide significant support for the novel hypothesis that PI-PLC is an important component of the agonist-stimulated cytosolic calcium oscillator that generates phasic myometrial contractions.(J Soc Gynecol Invest 1994;1:49-54)

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 heterotrimetric G proteins, results in stimulation of the phospholipase C-ß (PCL-ß) 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-? (PCL-?) 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 receptive 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 of 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 intermitten calcium transients that result in the development and maintenance of phasic myometrial contractions.

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 of these G-protein a 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 a subunits in rat myometrial tissue. Immunohistochemistry confirmed that Gq was expressed at high levels in the myometrial layer of the pregnant and nonpregnant uterus. Tliese 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, (J Soc Gynecol Invest 1997;4:209-217) Copyright © 1997 by the Society for Gynecologic Investigation.

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 teirodotoxin-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. Tire effects of nifedipine (1.0–5 µmol/L), a calcium channel blocker, were compared to tetrodotoxin (0.01–1 µmol/L), a sodium channel blocker. Results: Oxytocin-stimulated simultaneous cytosolic calcium oscillations and phasic contractions were completely inhibited by 1 µmol/L nifedipine; in contrast, 1 µmol/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.

One of the roles previously reported for protein kinase C (PKC) is modulation of the activity of the phosphatidylinositol signaling pathway. Studies were performed to test the hypothesis that activation of PKC results in inhibition of agonist-stimulated phasic myometrial contractions: contractions that appear to be mediated by phosphatidylinositol signaling mechanisms comparable to those producing cytosolic calcium oscillations. In vitro isometric contraction studies were performed using myometrium from adult Sprague-Dawley rats. Oxytocin and aluminium fluoride (a G-protein activator) produced comparable increases in phasic contractile activity. Phorbol 12,13-dibutyrate (PDB) significantly suppressed agonist-stimulated phasic myometrial contractions; in contrast, phorbol 13,20-diacetate (PDA), an inactive phorbol ester, had no significant effect on myometrial contractions. Prolonged exposure of myometrial tissue to PDB failed to down-regulate myometrial PKC and had no consistent effect on spontaneous and oxytocin-stimulated phasic contractions. These studies have provided support for a role for PKC as an intracellular regulator of the phosphatidylinositol signaling pathway, which itself appears to be part of the myometrial calcium oscillator that results in agonist-stimulated phasic myometrial contractions.

Objectives: Oxytocin-stimulated uterine contractions are associated with repetitive cycles of elevated cytosolic calcium, ie, cytosolic calcium oscillations. The studies in this report were performed to test the hypothesis that phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the myometrial intracellular oscillator. Methods: In vitro isometric contraction studies were performed using longitudinal strips of myometrium from nonpregnant, adult Sprague-Dawley rats. Cumllative dose-response studies were performed using oxytocin and aluminum fluoride with and without 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), an inhibitor of PI-PLC. Results: Stimulation of G-proteins coupled to PI-PLC with aluminum fluoride resulted in a significant increase in phasic myomttrial contractions comparable to those produced by oxytocin. Inhibition of PI-PLC with NCDC resulted in significant suppression of oxytocin- and aluminum fluoride-stimulated myomttrial contractions. In contrast, doses that suppressed agonist-stimulated contractions had only a minimal effect on KC—stimulated tonic myomttrial contractions. Conclusions: These studies provide significant support for the novel hypothesis that PI-PLC is an important component of the agonist-stimulated cytosolic calcium oscillator that generates phasic myomttrial contractions. (J Soc Gynecol Invest 1994;1:49–54)

Catecholamines are important in the modulation of smooth muscle contractile activity; this study was undertaken to evaluate adrenoceptor stimulation of intracellular inositol-phosphate production in a genital tract smooth muscle myocyte. DDT1MF-2 smooth muscle myocytes, derived from a hamster ductus deferens leiomyosarcoma, were loaded with 3H-inositol, incubated in 10 mM LiC1, then stimulated with adrenergic agonists with and without antagonists. Subsequently, the inositol phosphates were isolated by anion-exchange chromatography. In the presence of norepinephrine (NE), inositol trisphosphate (IP3) was produced by 30 s and peaked at 2 min; inositol I -phosphate was also apparent by 30 s, and continued to increase over 15 min. Clonidine (an alpha-2 agonist), isoproterenol, and NE in the presence of phentolamine or prazosin (an alpha-2 antagonist) failed to increase IP3. In contrast, NE in the presence of yohimbine (an alpha-2 antagonist) or propranolol stimulated IP3production to levels comparable to that stimulated by NE alone. These studies provide evidence that inositol phosphate production is involved in alpha-1 adrenergic signal transduction in DDT1MF-2 myocyte.

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.

Ultrasound-facilitated intrauterine transfusion was performed on 35 fetuses. Eleven fetuses were hydropic and less than 26weeks’ gestation at the time of the first intrauterine transfusion (JUT). Only two (18°/c) neonates survived. Twelve fetuses were not hydropic and less than 26weeks’ gestation at time of IUT. Six (50%) neonates survived. Corrected neonatal survival rates for three hydropic and nine nonhydropic fetuses transfused after 26weeks were 100% and 78%, respectively. The presence of ascites documented by ultrasound is an adequate indication for an IUT and permitted earlier detection of sicker fetuses; however, as a therapeutic aid, ultrasound neither diminished the fetal morbidity and mortality associated with the procedure nor completely eliminated the need for radiography to confirm proper intra-abdominal localization of the transfusion tubing.

Experimental data from fetal human and animal research suggest that the fetal sympathoadrenal system, composed of the adrenal medulla, sympathetic neurons, and extra-adrenal chromaffin tissue functions from early fetal life to maintain fetal homeostasis. The extra-adrenal chromaffin tissue undergoes maturation at 9 to 11 weeks of gestation, whereas the adrenal medulla and sympathetic nervous system mature later in fetal life. The fetal catecholamine response to hypoxia, mediated predominantly by norepinephrine, is an important component of the fetal cardiovascular response to hypoxia, i.e., through α-receptor stimulation, fetal cardiac output redistribution occurs. Fetal catecholamine secretion in response to substrate availability, through α- and β-receptor stimulation, provide a mechanism by which the fetus can utilize its own substrate stores. Pulmonary β-receptor stimulation by catecholamines has been demonstrated to increase lecithin synthesis, increase surfactant secretion, and decrease lung fluid production near term. β-Receptor stimulation has also been demonstrated to have trophic effects on the development of thermogenic brown adipose tissue. Although the exact stimulus for the initiation of parturition in the primate is unknown, fetal catecholamines, through direct myometrial α-adrenergic or dopaminergic receptor stimulation and/or through the stimulation of prostaglandin production, have the potential of facilitating the onset of parturition.

Objective:These studies sought to test the hypothess 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 parital 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 μmol/L), a calcium channel blocker, were compared to tetrodotoxin (0.01-1 μmol/L), a sodium channel blocker.Results:Oxytocin-stimulted simultaneous cytosolic calcium oscillations and phasic contractions were completely inhibited by 1 μmol/L nifedipine; in contrast, 1 μmol/L tetrodotoxin had no effect on the oxytocin-stimulated 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 tetrodotoxinsensitive sodium channels does not appear to play a comparably important role.

During the 3-year period from January, 1976 through December 1978, 1,497 repeat cesarean sections were performed at one hospital. To ensure that the correct gestational age had been established prior to electively timed repeat cesarean section, a protocol was recommended which emphasized menstrual history, early bimanual pelvic examination, and mid-pregnancy ultrasonography. Amniocentesis for confirmation of pulmonary maturity was not performed routinely, but if the above-mentioned criteria were not met, predelivery amniocentesis was recommended. Among the 23 infants who weighed over 1,800 grams and developed the respiratory distress syndrome (RDS), there were four who had been delivered by electively timed repeat cesarean section. In two of these four, both the birth weight and the pediatric assessment confirmed that they were 38 and 40 weeks' gestation, as anticipated. The other two infants appeared to be premature both by birth weight and pediatric assessment. If the overall experience is considered, the incidence of iatrogenic prematurity with RDS was 0.13% (two cases), and the incidence of potentially preventable RDS was 0.27% (four cases). None of the infants experienced serious complications of the RDS, and none died. In the entire group of repeat cesarean sections, the frequency with which amniocentesis was performed to confirm pulmonary maturity was estimated to be 12.7%.

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.

To determine the efficacy of perioperative cefoxitin in preventing infections after primary cesarean section, a randomized placebo-controlled, double-blind clinical trial was performed. Among 266 participants, those who received three perioperative 2 gm doses of cefoxitin (138) had significantly fewer serious infections (19.5% vs. 4.3%), fewer urinary tract infections (10.7% vs. 4.4%), less standard febrile morbidity (9.4% vs. 3.6%), and fewer courses of antibiotics postoperatively (23.4% vs. 11.6%). There was no reduction in the length of hospitalization. Use of perioperative cefoxitin was not accompanied by more side effects. Three doses of cefoxitin beginning as soon as the umbilical cord is clamped are safe and efficacious in preventing infection after primary cesarean section.

Previous reports have confirmed that steroid hormones modulate the expression of adrenergic receptors on the surface of smooth muscle myocytes. The present study was undertaken to evaluate the mechanism by which testosterone modulates alpha-1 adrenergic receptor expression in the DDT1 MF-2 transformed smooth muscle cell. Utilizing 3H-prazosin radioligand binding studies, alpha-1 adrenergic receptors were noted to increase more than 2 fold in response to incubation with 10-8 M testosterone for 96 hours. Dihydrotestosterone similarly stimulated a significant increase in alpha-1 receptors; whereas, estradiol and hydrocortisone appeared to suppress the expression of this receptor in DDT myocytes. The testosterone effect was dose related with a maximal response observed in response to 10 -7M testosterone at both 48 and 96 hours. Kinetic experiments utilizing 10-8 M testosterone demonstrated a peak effect on alpha-1 receptor expression at 96 hours, and maintenance of the effect for at least 168 hours (7 days). The testosterone effect was completely prevented at both 48 and 96 hours by inhibition of transcription with actinomycin-D, or inhibition of translation with cycloheximide. Consistent with the receptor binding studies, RNA blotting studies have demonstrated maximal alpha-1 receptor mRNA levels at 48–96 hours of testosterone stimulation. In conclusion, these in vitro experiments have confirmed that physiologic concentrations of testosterone stimulate the increased expression of alpha-1 receptors in the DDT1 MF-2 myocyte after a delay of 48–96 hours; and that this effect appears to be mediated by transcription, translation, and synthesis of new proteins in these genital tract myocytes.

Summary: The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8 M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.

We studied the effect of diabetic nephropathy on the course of pregnancy, perinatal outcome, and infant development and determined the influence of pregnancy on maternal hypertension and renal function. Maternal proteinuria usually increased during pregnancy (>3 gm/24 hours in 69%), and hypertension was present by the third trimester in 73%. The degree of proteinuria correlated with diastolic pressure and creatinine clearance. After pregnancy pregnancy, proteinuria declined in 65% of the mothers, hypertension was absent in 43.5%, and the expected rate of fall in creatinine clearance was not accelerated. Among 35 patients, abortion occurred spontaneously or was performed electively in 25.7%, and 71% of the remainder underwent delivery before 37 weeks. Birth weight was related to maternal blood pressure and creatinine clearance. Neonatal morbidity was common, but the perinatal survival rate was 89%. Infants seen at follow-up without congenital anomalies had normal development at 8 to 36 months of age. We concluded that perinatal outcome has significantly improved for diabetic women with nephropathy.

Previous investigation has demonstrated biologically significant concentrations of catecholamines in amniotic fluid, which increase with gestation. The half life, metabolic clearance rate, and metabolic fate of these hormones in the amniotic compartment are yet to be established. This study was undertaken to demonstrate the ability of human chorioamnion to metabolize dopamine in vitro. Incubation experiments demonstrated that 3H-dopamine is rapidly metabolized to dihydroxyphenylacetic acid, 3-methoxy,4-hydroxyphenylacetic acid, and 3-methoxy,4-hydroxyphenylethanol—all products of monoamine oxidase. No significant 3-methoxytyramine, a catechol-o-methyltransferase product, was observed. Incubation experiments with pargyline, a monoamine oxidase inhibitor, resulted in significant reduction in 3H-dopamine metabolism. Catecholamines and their interaction with prostaglandin synthesis have been theorized to be a fetal signal for the initiation of parturition. The ability of chorioamnion to metabolize catecholamines could, therefore, provide another control mechanism by which fetal catecholamines are modulated.

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.