The comparative actions of uterine inhibiting drugs

Currently there is not an in vitro or in vivo method of quantitating the potency of uterine inhibiting drugs. Electric field stimulation (EFS) of pregnant and parturient rabbit myometrial strips has been effectively used to assay oxytocic potency of prostaglandins and it is shown in this study to be useful in assaying potency of beta-sympathomimetic compounds. Decreasing electric field stimulation (DEFS) provides three measureable endpoints: tension, excitability, and threshold voltage. Three compounds were tested for these parameters: isoproterenol, ritodrine, and isoxsuprine. It is also revealed that so-called uterine inhibition by these compounds represents the creation of a chaotic fibrillating muscle which is unable to respond in a synchronous way to produce a contraction of maximal tension.     

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.     

Effect of fluoxetine on contractile activity of pregnant rat uterine rings

OBJECTIVE: We sought to compare the effects of fluoxetine, imipramine, and nortriptyline on spontaneous and serotonin-activated contractile activity of the uterine rings from midterm and term pregnant rats. STUDY DESIGN: Uterine rings from timed-pregnant Sprague-Dawley rats on day 14 (midgestation) and day 22 (term gestation) were used for isometric tension recording. Responses to cumulative concentrations of fluoxetine, imipramine, nortriptyline, and serotonin in the absence and presence of the monoamine reuptake inhibitors were studied. RESULTS: Neither of the monoamine reuptake inhibitors significantly influenced spontaneous contractile activity, whereas the concentration-dependent increase in activity induced by serotonin was inhibited in rings from both midterm and term pregnant rats. CONCLUSIONS: The reported increase in preterm delivery in women receiving fluoxetine during the third trimester cannot be explained by a direct effect on uterine contractility.     

The secretory immune system in the uterus of the pregnant rat: production of secretory component by uterine tissues

Secretory component (SC) was measured in amniotic fluid, fetal serum, and maternal serum and compared with SC production during in vitro culture of uterine tissue segments from pregnant rats. The concentrations of SC in amniotic fluid did not change between days 14 and 20 of pregnancy. Similarly, there was no change in maternal or fetal serum during pregnancy, although, the levels of SC in sera were consistently higher than those in amniotic fluid. When uterine segments were incubated in vitro, release of SC was greater in the absence of cycloheximide than in the presence of cycloheximide at all stages of pregnancy. In contrast to SC values in amniotic fluid, however, SC production by uterine tissue changed markedly during pregnancy. SC levels were low during early pregnancy (day 7 post coitus) and increased to levels found in non-pregnant diestrous rats just prior to parturition (day 20). The findings suggest that the endocrine balance during pregnancy may play a central role in regulation of the uterus immune system. The pattern of SC release may reflect a need both to ensure protection of the fetus from the IgA immune system in early pregnancy and to prevent maternal infection during parturition by reactivation of this system.     

The effect of aminophylline on uterine smooth muscle contractility and prostaglandin production in the pregnant rat uterus in vitro

The effect of aminophylline on contractility and prostaglandin production was investigated in a 20-day pregnant rat uterus preparation in vitro. A significant decrease was observed in frequency (24.4% of control, p less than 0.05) and contractile force (13.8% of control, p less than 0.001) in rat uterine segments treated with aminophylline. Treated segments produced more prostaglandin E2 (139.6% of control, p less than 0.001) and less prostaglandin F2 alpha (73.8% of control, p less than 0.001) during the 45-minute observation period. No significant difference in thromboxane B2 or 6-ketoprostaglandin F1 alpha production was noted. This is the first observation of prostaglandin levels in rat uterine segments treated with aminophylline. It could not be concluded from this study whether the observed changes in prostaglandin production are directly related to the relaxant effect of aminophylline.     

Macrophage density in the pregnant rat uterine cervix is modulated by mast cell degranulation

The uterine cervix at term undergoes histomorphological changes that resemble an inflammatory process. The aim of this study was to better characterize these changes, describing the temporal and spatial pattern of macrophages and mast cells (MC) distribution in the uterine cervix and assessing whether both cells exert a coordinated action on angiogenesis. Macrophages and MC were identified by immunohistochemistry in cervical tissue from cycling, pregnant and postpartum rats. In order to inhibit MC degranulation, pregnant rats were injected with disodium cromoglycate. The expression of vascular endothelial growth factor (VEGF) by macrophages was also evaluated. Results showed that macrophage density increased towards parturition and declined at postpartum, whereas MC density showed an inverse pattern. Interestingly, disodium cromoglycate-treated rats showed an increased number of macrophages. VEGF expression in macrophages was detected neither in control nor in treated animals; however, a coordinated action between MC and macrophages on angiogenesis could not be excluded. The present study provides a detailed mapping of macrophage and MC densities and distribution in the rat uterine cervix. Moreover, an association between macrophages and MC along pregnancy is shown, and evidence that macrophage density in the rat cervix is modulated by MC degranulation is presented.     

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.     

Endovascular trophoblast invasion and associated structural changes in uterine spiral arteries of the pregnant rat

The involvement of endovascular trophoblast in fibrinoid deposition, replacement of the endothelium and vascular smooth muscle breakdown is studied in spiral arteries of the mesometrial triangle from day 15 to day 21 of rat pregnancy, by examining arterial cross sections after staining for cytokeratin, PAS, CD31 and alpha-actin. From day 15 to day 18 of pregnancy, fibrinoid deposition underneath the endovascular trophoblast increases gradually, whereas the amount of endovascular trophoblast in invaded arteries remains constant. CD31 staining is significantly reduced in sub-ET (= underlying the endovascular trophoblast) as compared to extra-ET (= outside the endovascular trophoblast) and no-ET (= non-invaded arterial sections) at each time-point of pregnancy examined (P < 0.005 and P < 0.0005 at each day of pregnancy), whereas alpha-actin staining is reduced both in sub-ET and in extra-ET as compared to no-ET. During pregnancy, CD31 staining in sub-ET initially declines, but increases significantly on day 21 (P < 0.001 versus d20) suggesting re-endothelialization of the vascular wall. In conclusion, changes in spiral arteries of pregnant rats reveal striking similarities with physiological changes seen in human pregnancy, thus emphasizing the usefulness of this species as an experimental model for studying normal and complicated pregnancies in humans.     

Effects of recombinant human relaxin on pregnant rat uterine artery and myometrium in vitro

OBJECTIVE: The purpose of this study was to evaluate the effects of recombinant human relaxin on the uterine artery and myometrial contractility in pregnant rats. STUDY DESIGN: Uterine artery and myometrial rings from mid and term pregnant rats were used. Relaxin effect was studied on phenylephrine-induced contraction in the presence or absence of nitric oxide synthase inhibitor, N omega-nitro-l-arginine methyl ester, soluble guanylate cyclase inhibitor, 1H-oxadiazolo-quinoxaline-1-one, or adenylate cyclase inhibitor, SQ-22,536. The myometrial inhibitory effect of relaxin was studied on spontaneous and oxytocin- or protein kinase C activator-induced contractions. RESULTS: Uterine artery relaxation by relaxin was greater at mid pregnancy compared with term. Relaxin effect was decreased by SQ-22,536, 1H-oxadiazolo-quinoxaline-1-one and N omega-nitro-l-arginine methyl ester at mid pregnancy. Relaxin inhibited spontaneous contractions at mid pregnancy but not at term. Relaxin had no effect on oxytocin- or indolactam-V-induced contractions. CONCLUSION: Relaxin effect is mediated by nitric oxide, soluble guanylate cyclase, and adenylate cyclase in mid pregnant uterine artery. Relaxin inhibits spontaneous uterine activity at mid pregnancy. Relaxin effect decreased at term gestation in both tissues.     

Role of nucleotide cyclases in the inhibition of pregnant rat uterine contractions by the openers of potassium channels

OBJECTIVE: Our objective was to study the involvement of adenylate and guanylate cylases in spontaneous uterine contractions and inhibition induced by the opening of potassium channels. STUDY DESIGN: Uterine rings from rats at mid and term gestation and from rats at term gestation in labor were suspended in organ chambers for isometric tension recording. Concentration-response relationships to an opener of adenosine triphosphate-dependent potassium channels, levcromakalim, or to an opener of calcium-dependent potassium channels, NS 1619, were studied in the absence and presence of inhibitors of adenylate cyclase (MDL 12330 A, 2 x 10(-5) mol/L; SQ 22536, 10(-4) mol/L) or guanylate cyclase (LY 83583,3 x 10(-6) mol/L). RESULTS: MDL 12330 A and SQ 22536 accentuated contractions in rings from rats at mid gestation but not at term gestation or at term gestation in labor. LY 83583 inhibited contractions in the rings from all 3 groups. Levcromakalim was equally effective in inhibiting contractions of rings from all 3 groups. MDL 12330 A, but not SQ 22536, decreased sensitivity and maximal inhibition induced by levcromakalim (term gestation greater than mid gestation greater than term gestation in labor). LY 83583 decreased the sensitivity to and maximal inhibition induced by levcromakalim in rings from pregnant rats at mid gestation. NS 1619 attenuated contraction of rings from rats at mid gestation and, to a lesser extent, at term gestation but accentuated contractions in rings from animals at term gestation in labor. MDL 12330 A, but not SQ 22536 or LY 83583, attenuated the changes induced by NS 1619 in rings from all 3 groups. CONCLUSIONS: (1) The influence of nucleotide cyclases on basal uterine contractility depends on gestational age. (2) The inhibition of uterine contractions that results from the opening of calcium-dependent potassium channels depends on adenylate cyclase, whereas that of adenosine triphosphate-dependent potassium channels depends on both adenylate and guanylate cyclases. 3. Activation of adenosine triphosphate-dependent potassium channels is more efficient than activation of calcium-dependent potassium channels. 4. The inhibition induced by calcium-dependent potassium channel openers, but not adenosine triphosphate-dependent potassium channel openers, decreases as pregnancy progresses, and at delivery the activation of spontaneous contractile activity is evident.     

Chemotactic factor in the pregnant rabbit uterine cervix.

OBJECTIVE: Neutrophil accumulation is one of the characteristic changes observed in uterine cervical stroma at term pregnancy, but chemotactic activity in the tissue is obscure. Our study examined the existence and production of chemotactic factor in the rabbit uterine cervix. STUDY DESIGN: Uterine cervical explants of rabbits at term pregnancy and nonpregnant rabbits were cultured with and without interleukin-1 alpha. Rat neutrophilic chemotaxis in culture media was evaluated with a Boyden chamber. RESULTS: Tissue extract from the pregnant rabbit uterine cervix at term pregnancy contained more chemoattractive activity than the nonpregnant cervix. Production of chemoattractant from cultured rabbit cervical explants at term pregnancy was also higher than that from nonpregnant explants. The addition of interleukin-1 alpha to the culture system promoted its production. This chemoattractant was characterized as a true chemotactic factor and heat-stable and trypsin-sensitive protein with an apparent relative molecular mass of 16,200. So far, these properties are very similar to those of the interleukin-8 family. Rabbit uterine cervical fibroblast is characterized as a chemotactic factor-producing cell in the rabbit uterine cervix. CONCLUSION: These results indicate that interleukin-8-like chemotactic factor participates in the cervical ripening at term pregnancy and that the production of this factor is controlled effectively by interleukin-1.     

The uterine lumen of the pregnant guinea-pig contains a large macrophage population

Cellular constituents of the uterine lumen were investigated. Fourteen pregnant sows of 40 + days' gestation were anaesthetized and naturally occurring peritoneal fluid was collected. A uterine horn was delivered and 0.25 ml Gey's solution injected into the uterine lumen to collect free cells. The fluid was aspirated into a syringe and the cells extracted, counted and prepared for phagocytosis experiments and microscopy. The cells were stained with alpha-naphthyl-acetate-esterase (ANAE) to determine the fraction that was non-specific esterase-positive, a feature of mononuclear phagocytes. Differential cells counts were also made. Both uterine and peritoneal compartments yielded large numbers of cells (greater than 10(6)/ml). Peritoneal fluid cells were 47 +/- 6 per cent (SD) macrophages and 49 +/- 6 per cent eosinophils (the remainder being 'other' cells); 47 +/- 6 per cent also stained positively for ANAE. Uterine cells were 78 +/- 12 per cent macrophages, the remainder being mostly lymphocytes (18 +/- 11 per cent); 85 +/- 13 per cent stained positively with ANAE. Electron microscopy of the uterine cells confirmed that most had morphology consistent with being mononuclear phagocytes. Uterine and peritoneal cells phagocytized carbon particles and yeast cells when incubated at 37 degrees C. The origin and role of this macrophage population is unknown but uterine lumenal macrophages may be present to remove antigen-antibody complexes thus facilitating uptake of maternally derived IgG by the fetal yolk sac.