Impact of tamsulosin and nifedipine on contractility of pregnant rat ureters in vitro.

OBJECTIVES: To evaluate the in vitro effect of tamsulosin and nifedipine on the contractility of pregnant rat ureters and to perform quantitative analysis of the pharmacological effects. Medical expulsive therapy (MET) is commonly used to treat urolithiasis. However, this treatment is seldom used in pregnant women since no studies support this practice. METHODS: This was an in vitro study on animal tissue derived from pregnant Sprague-Dawley rats. A total of 124 ureteral segments were mounted in an organ bath system and contractile response to methacholine (MCh) was assessed. Tamsulosin or nifedipine were added at cumulative concentrations (0.001-1 µM). The area under the curve (AUC) from isometric tension measurements was calculated. The effect of pharmacological agents and the respective controls were assessed by calculating the AUC for each 5-min interval. Statistical analyses were performed using the Mann-Whitney-Wilcoxon nonparametric test. RESULTS: Both drugs displayed statistically significant inhibitory activity at concentrations of 0.1 and 1 µM for tamsulosin and 1 µM for nifedipine when calculated as the AUC as compared to DMSO controls. CONCLUSION: Tamsulosin and nifedipine directly inhibit MCh-induced contractility of pregnant rat ureters. Further work is needed to determine the clinical efficacy of these medications for MET in pregnancy.

Phorbol 12,13-dibutyrate-induced protein kinase C activation triggers sustained contracture in human myometrium in vitro.

BACKGROUND: Although physiologic transition from rhythmic contractions to uterine retraction postpartum remains a poorly understood process, it has been shown that the latter is essential in the prevention of hemorrhage and its negative consequences. OBJECTIVE: To investigate the transition from oscillatory contractions to tonic contracture in human myometrium after delivery, a mechanism purported to facilitate postpartum hemostasis. Protein kinase C (PKC) plays a key regulatory role in human uterine contractions because it can prevent dephosphorylation of regulatory proteins and sensitize the contractile machinery to low Ca2+. Thus, activation of PKC by phorbol 12,13-dibutyrate (PDBu) may act as a strong uterotonic agent. STUDY DESIGN: Uterine biopsies were obtained from consenting women undergoing elective caesarian delivery at term without labor (N = 19). Isometric tension measurements were performed on uterine strips (n = 114). The amplitudes and area under the curve of phasic contractions and tonic responses were measured and compared. A total of 1 µM PDBu was added to the isolated organ baths, and maximal tension of the uterine contracture was determined in the absence and presence of either 1 µM of staurosporine, 100 nM nifedipine, or 10 µM cyclopiazonic acid to assess the role of PKC and calcium sensitivity on uterine contractility. RESULTS: On the addition of PDBu on either basal or oxytocin-induced activity, consistent contractures were obtained concomitant with complete inhibition of phasic contractions. After a 30-minute incubation period, the mean amplitude of the PDBu-induced tone represented 65.3% of the amplitude of spontaneous contraction. Staurosporine, a protein kinase inhibitor, induced a 91.9% inhibition of PDBu contractures, a process not affected by nifedipine or cyclopiazonic acid, thus indicating that this mechanism is largely Ca2+ independent. CONCLUSION: Pharmacologic activation of PKC leads to a significant contracture of the myometrium. Together, these data suggest that the up-regulation of PKC plays a physiologic role in the modulation of uterine contracture after delivery. A switch from phasic to strong tonic contractions potentially may facilitate postpartum hemostasis.

Antenatal montelukast treatment reduces uterine activity associated with inflammation in a pregnant rat model.

OBJECTIVE: The potency of acute montelukast treatment, a leukotriene receptor antagonist, has been demonstrated as tocolytic on in vitro myometrial contractility. This study assessed the ability of a 48h montelukast treatment to modify in vitro contractions under inflammatory conditions in a pregnant rat model. STUDY DESIGN: Pregnant Sprague-Dawley rats were injected intraperitoneally (gestational days 20-22) with lipopolysaccharides (LPS) 200µg/kg (4 treatments at 12h intervals) alone or combined with montelukast 10mg/kg/day or a saline solution for a 48h period. Uterine rings (n=72) were obtained by median laparotomy at day 22. Spontaneous contractile activities were compared using pharmacological compounds (oxytocin, nifedipine) along with assessment of contractile parameters. Myometrial subcellular fractions were also analyzed by Western blot to quantify oxytocin, cysteinyl leukotriene receptors and inflammation markers. RESULTS: In in vitro experiments, the area under the curve, the amplitude and the duration of phasic contractions were significantly reduced following 48h of LPS+montelukast treatment comparatively to the LPS group. Moreover, in this same group, oxytocin (10-9-10-7M) largely decreased uterine sensitivity (p=0.04). Following LPS and montelukast treatment, the tocolytic effectiveness of nifedipine (10-9-10-7M) was increased (p<0.01). Western blot analysis confirmed the presence of type 1 CysLT receptors in all treated groups. Hence, montelukast treatment restored TNF-α and COX-2 basal levels. CONCLUSION: Our results strongly suggest that montelukast treatment could facilitate a relative uterine quiescence by decreasing its sensitivity to uterotonic agent or by increasing tocolytic efficiency under proinflammatory conditions.

Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animal model.

OBJECTIVE: Abnormal uterine contraction patterns were recently demonstrated in uterine strips from pregnant women treated with Levothyroxine (T4). These abnormalities were correlated with an increased risk of C-section delivery and associated surgical complications. To date, no study has investigated whether uterine contractility is modified by hypothyroidism or T4 treatment. Herein, we analyze the physiological role of T4 on uterine contractions. STUDY DESIGN: Female non-pregnant Sprague-Dawley rats (N = 22) were used and divided into four groups: 1) control, 2) hypothyroidism, 3) hypothyroidism treated with low T4 doses (20 µg/kg/day) and 4) with high T4 doses (100 µg/kg/day). Hypothyroidism was induced by an iodine-deficient diet. Isometric tension measurements were performed in vitro on myometrium tissues in isolated organ baths. Contractile activity parameters were quantified (amplitude, duration, frequency and area under the curve) using pharmacological tools to assess their effect. RESULTS: Screening of thyroid function confirmed a hypothyroid state for all rats under iodine-free diet to which T4 was subsequently administered to counterbalance hypothyroidism. Results demonstrate that hypothyroidism significantly decreased contractile duration (-17%) and increased contractile frequency (+26%), while high doses of T4 increased duration (+200%) and decreased frequency (-51%). These results thus mimic the pattern of abnormal contractions previously observed in uterine tissue from T4-treated hypothyroid pregnant women. CONCLUSION: Our data suggest that changes in myometrial reactivity are induced by T4 treatment. Thus, in conjunction with our previous observations on human myometrial strips, management of hypothyroidism should be improved to reduce the rate of C-sections in this group of patients.

Leukotriene receptor antagonist as a novel tocolytic in an in vitro model of human uterine contractility.

OBJECTIVE: This study analyzed the ability of montelukast, a cysteinyl-leukotrienes receptor antagonist and anti-inflammatory agent, to produce a consistent tocolytic effect alone or in combination with nifedipine, a calcium (Ca(2+)) channel blocker currently used in clinical practice. STUDY DESIGN: Uterine biopsies were obtained from consenting women undergoing elective cesarean sections at term (n=20). Myometrial microsomal fractions were analyzed by immunoblotting to quantify relative cysteinyl leukotrienes receptor 1 (CysLTR1) levels. Isometric tension measurements were performed in vitro on human myometrial strips (n=120) in isolated organ baths in order to establish concentration-response curves to montelukast and to quantify changes in Ca(2+) sensitivity on ß-escin permeabilized tissues. RESULTS: Immunodetection analysis revealed the presence of CysLTR1 receptor in uterine tissues, fetal membranes and placenta. A significant increase in area under the curve (AUC) was quantified following the addition of leukotriene D4 (LTD4) (0.01-0.3 µM), an end-product of the lipoxygenase pathway. Conversely, addition of montelukast produced a significant tocolytic effect by decreasing the frequency and AUC (IC50=1 µM). Moreover, addition of montelukast also resulted in a reduced Ca(2+) sensitivity as compared to control tissues (EC50 values of 654 and 403 nM; p=0.02 at pCa 6), while an additive effect was observed in combination with 0.1 nM nifedipine (p=0.004). CONCLUSION: This original study demonstrates the potency of montelukast as a tocolytic agent in an in vitro human uterine model. Montelukast, in combination with nifedipine, could represent a therapeutic approach to reduce inflammation associated with prematurity while facilitating the inhibition of preterm labor.

Effect of cytochrome P-450 epoxygenase and hydroxylase metabolites on rat myometrium contractility in non-pregnancy, late pregnancy and late pregnancy under inflammatory conditions.

AIM: The aim of the present experimental study was to assess the tocolytic effect of eicosanoids on myometrium from non-pregnant and pregnant rats with or without an induced inflammatory condition. METHODS: Three hundred myometrial rings were obtained by median laparotomy from 50 Sprague-Dawley rats divided into three groups: (i) non-pregnant (n = 15); (ii) pregnant in absence (n = 20); or (iii) pregnant in presence (n = 15) of lipopolysaccharide treatment, timed at 22 days of pregnancy. Spontaneous contractile activities were compared by isometric tension measurements. The effects of epoxy- and hydroxyeicosanoids derived from arachidonic acid as well as specific enzyme inhibitors were assessed. Changes were expressed as percentage of basal activity by calculating the area under the curve as a function of drug concentration and compared to the effect of the vehicle. RESULTS: A decrease in contractile activity ranging 10-25% was observed upon addition of epoxy- and hydroxyeicosanoids. Increasing epoxyeicosanoid bioavailability by inhibiting their degradation induced a tocolytic effect in the non-pregnant group (20%) and in inflammation-induced condition (40%). There was a significant difference in reactivity between groups and pregnancy condition. Semiquantification of metabolic enzymes that produce (cytochrome P-450 epoxygenase) and degrade (soluble epoxide hydrolase) epoxyeicosanoids by western blot analysis revealed that these enzymes were mainly detected in the non-pregnant group. CONCLUSION: Eicosanoids can modify myometrial reactivity and their presence and effects are amplified in non-pregnant and in inflammation-induced condition. Our data suggest that in contrast to prostaglandins, epoxyeicosatrienoic acids are likely involved in the quiescence phase of parturition because they reduce the rhythmic contractile activity of uterine tissues in pregnant rats.

Effect of interleukin-6 receptor blockade on feto-maternal outcomes in a rat model of intrauterine inflammation.

AIM: To study the effect of blocking the inflammatory cascade with interleukin-6 receptor antibody (anti-IL-6R) on feto-maternal outcomes in a rat model. METHODS: Pregnant Sprague-Dawley rats (n = 38) were injected intraperitoneally (day 22) (control, anti-IL-6R 30 µg/kg, lipopolysaccharide [LPS] 250 µg/kg or 500 µg/kg alone or combined with anti-IL-6R) followed by preterm caesarian performed 12 h later. Resuscitated pups (n = 179) were given to surrogate mothers. Primary outcomes were maternal and pup mortality. RESULTS: Fifty percent of pregnant rats died after LPS 500 µg/kg + anti-IL-6R injection but none in other groups. Neonatal mortality at 24 h was 63% and 86% in LPS 500 µg/kg and LPS 500 µg/kg + anti-IL-6R groups, respectively (P < 0.05). Surviving pups in the latter group presented a severe growth deficit compared to the LPS 500 µg/kg group (P < 0.01) and showed no difference with controls for open field testing. Maternal cytokine analysis after LPS 500 µg/kg + anti-IL-6R injection showed a tendency for increased IL-1 production (P = 0.06). CONCLUSION: Paradoxically, the association of pregnancy, inflammation and anti-IL-6R increases the inflammatory effects of LPS.

Chronic levothyroxine and acute T3 treatments enhance the amplitude and time course of uterine contractions in human.

This study compares the functional consequences of levothyroxine (T4) treatment during pregnancy as well as the acute affects of triiodothyronine (T3) on spontaneous uterine contractile activities observed in vitro. Uterine biopsies were obtained from consenting women undergoing elective caesarean at term (n = 28). Spontaneous contractile activities from T4-treated pregnant women (n = 8) were compared with control patients (n = 20) by isometric tension measurements. Effects of acute T3 and T4 on control tissues were also monitored. Area under the curve, amplitude, time to peak, duration, and frequency were quantified. In uterine strips from women treated for hypothyroidism, phasic uterine contractions of larger amplitude (+77%) were observed, with a prolonged duration at 90% relaxation (+138%) and reduced frequency (-55%) compared with values of the control group. The addition of exogenous T3 in vitro on control strips induced a significant increase in the duration of the contractions and a significant decrease in frequency (P < 0.05), which partially mimics the results obtained in strips from T4-treated women. Significant modifications of contractile properties were observed in strips from pregnant women treated with levothyroxine, consistent with those observed with the addition of exogenous T3. Clinical practices of modern obstetrics should take into account the effect of thyroid hormones on uterine contractions' time course to ensure a tighter followup at the end of pregnancy to achieve safer delivery.

Inhibition of epoxy-eicosanoid degradation improves the tocolytic effects of indomethacin in the uterus from pregnant women.

The incidence of preterm birth is an increasing problem. Indomethacin, a non-specific cyclooxygenase inhibitor, has been largely used as tocolytic in the treatment of preterm labor. The aim of the present study was to assess a putative synergistic tocolytic effect between the inhibition of the production of prostanoids and stabilization of epoxides fatty acids, particularly arachidonate on spontaneous uterine contractile activity. The experimental work was performed on uterine biopsies from consenting women undergoing elective cesarean delivery at term. Isometric tension measurements were performed on fresh human myometrial strips. Contractile activities have been monitored upon individual and combined treatments of indomethacin, DDMS, an inhibitor of hydroxy-eicosanoids production and AUDA, an inhibitor of epoxy-eicosanoids degradation. Interestingly, a significant and consistent synergic effect was observed when indomethacin and AUDA were simultaneously added, raising the possibility of a combined clinical use of cyclooxygenase and sEH inhibitors in attempt to treat preterm labor.

Lipoxygenase and cyclooxygenase inhibitors reveal a complementary role of arachidonic acid derivatives in pregnant human myometrium.

OBJECTIVE: The aim of this study was to assess the involvement of lipoxygenase (LOX) metabolic pathways in uterine tissues from pregnant women as well as the combined inhibition of LOX and cyclooxygenase (COX) on contractile activity. STUDY DESIGN: Uterine biopsies were performed from consenting women undergoing elective caesarean sections at term (n = 24). Western blot analysis and isometric tension measurements were performed in vitro on fresh human myometrial strips. Concentration-response curves to arachidonic acid (AA) 861 and baicalein (5- and 12-LOX inhibitors, respectively) were performed. The combined effects of baicalein and indomethacin were also assessed. Contractile activities were quantified by calculating both amplitude and the area under the curve over 20 minute periods. RESULTS: 5- and 12-LOX were present in all tested tissues. Addition of AA861 or baicalein resulted in tocolytic effects (P < .05). Finally, the combined inhibition of both COX and 12-LOX pathways resulted in additive tocolytic effects. CONCLUSION: 5- and 12-LOX pathways modulate human myometrium contractility.

Why eicosanoids could represent a new class of tocolytics on uterine activity in pregnant women.

OBJECTIVE: The purpose of this study was to assess the effects of exogenous eicosanoids on spontaneous uterine contractile activity. STUDY DESIGN: Eight uterine biopsies were performed from women who were undergoing elective cesarean delivery. Tension measurements were performed in vitro on myometrial strips. Contractile activities were quantified by the calculation of the area under the curve. The effects of eicosanoids and specific enzyme inhibitors were assessed. Fractions from various uterine tissues were analyzed by Western blot. RESULTS: Data demonstrate the presence, in some tested tissues, of cytochrome P-450 epoxygenase and soluble epoxide hydrolase, which respectively produce and degrade epoxyeicosatrienoic acid regioisomers. Inhibition of soluble epoxide hydrolase with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid or omega-hydroxylase with N-methylsulfonyl-12,12-dibromododec-11-enamide resulted in a tocolytic effect; N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide, which is an epoxygenase inhibitor, had no effect. Exogenous epoxyeicosatrienoic acids displayed significant tocolytic effects on spontaneous contractile activities. CONCLUSION: Epoxy- and hydroxyeicosanoids represent new bioactive, arachidonic acid by-products with in vitro tocolytic activities. These findings suggest that cytochrome P-450 isozymes may represent relevant pharmacologic targets under physiopathologic conditions.