The influence of interleukin-1beta on oxytocin signalling in primary cells of human decidua.

OBJECTIVE: Oxytocin (OT) and its corresponding receptor (OTR), synthesized within the pregnant uterus, play a key role in the process of (preterm) labor as part of a paracrine system that regulates symmetrical contractility. In the setting of intrauterine infection, a major cause of preterm labour and birth, decidua serves as a major source of cytokine production. The present study evaluates the time-dependent effect [0-24 h] of the inflammatory cytokine Interleukin-1beta (IL-1beta) treatment on OT signalling and OT stimulated prostaglandin release in primary cultures of human decidua. STUDY DESIGN: Primary cultures of human decidua (n=6) were treated with IL-1beta [5 ng/ml] for 0-24h and or indomethacin [100 microM]--an inhibitor of the prostaglandin synthesis--for 0-24 h or for 24 h. OT peptide expression, OTR binding, Inositol triphosphate production (IP(3)), and arachidonic acid (AA) as well as prostaglandin (PGE(2)) release were measured. RESULTS: IL-1beta transiently reduced cytoplasmic OT peptide at 4-6 h of IL-1beta incubation, while its secretion into the media was increased after 6 h of stimulation. The later was completely blocked by indomethacin. A decrease in OTR mRNA expression and a loss of OTR binding were detected after 8 h and 16 h of IL-1beta treatment, respectively. IL-1beta also decreased IP(3) production and AA release, but significantly enhanced OT mediated PGE(2) production. This effect was completely suppressed by the cyclooxygenase-2 (COX-2) inhibitor NS-398. CONCLUSION: Our data suggest, that IL-1beta indirectly increases OT secretion in primary cultures of human decidua in a time dependent fashion through the production of prostaglandins through COX-2 and that this increase in OT peptide may secondarily down-regulate the OTR and its signalling cascade. These findings might explain the poor effectiveness of oxytocin receptor antagonists as tocolytic agents in the setting of intrauterine infection.

Effect of IL-1beta and IL-6 on oxytocin secretion in human uterine smooth muscle cells.

PROBLEM: Infection-mediated preterm labor results in the production of inflammatory cytokines, including interleukin-1beta (IL-1beta) and interleukin-6 (IL-6). Oxytocin (OT) plays a key role in the process of labor. This study investigates the effect of IL-1beta and IL-6 on intra-and extracellular OT in human smooth muscle cells and evaluates IL-1beta induced changes in IL-6 production. METHOD OF STUDY: Primary cultures of human myometrium, obtained from term pregnant women (n = 7) were incubated with either IL-1beta or IL-6 for 0-24 hr. Intra- and extracellular OT peptide concentrations were measured by radioimmunoassay and IL-6 mRNA and protein were evaluated by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Both, IL-1beta and IL-6 led to an increase in OT secretion, which was accompanied by a reduction of the intracellular OT peptide pool. IL-1beta significantly induced IL-6 mRNA expression and protein secretion, which did not further enhance IL-1beta induced OT secretion. CONCLUSIONS: The induction of OT secretion by proinflammatory cytokines in human myometrium in vitro, supports the concept of a thus regulated infection-triggered preterm labor process in vivo.

Interleukin-6 up-regulates the oxytocin receptor in cultured uterine smooth muscle cells.

PROBLEM: Intra-uterine infection results in the production of inflammatory cytokines, including interleukin-6 (IL-6). Increased oxytocin-receptor (OTR) concentrations are associated with the onset of preterm labor. We hypothesize that infection up-regulates OTR expression through IL-6-induced transcription factors. METHOD OF STUDY: Primary cultures of human myometrium were treated for various time periods or with different concentrations of IL-6 and OTR mRNA as well as OTR binding were measured by means of reverse transcription polymerase chain reaction and 125I-ornithine-vasotocin-binding assay. To study underlying mechanisms of OTR changes with IL-6 treated, cells were also incubated with genistein or H7 (tyrosine and serine phosphorylation inhibitors), respectively. RESULTS: OTR mRNA increased 2.5-fold after 4 hr of IL-6 treatment and OTR binding 1.4-fold after 8 hr of cytokine stimulation. The IL-6-induced increase in binding was blocked by genistein and H7. CONCLUSIONS: IL-6 up-regulates uterine OTR mRNA expression and binding capacity in cultured human myocytes most likely through tyrosine and serine phosphorylation pathways involving the nuclear factor STAT-3.

Oxytocin signaling in human myometrium is impaired by prolonged exposure to interleukin-1.

Intra-amniotic infection leads to preterm labor and is associated with the local release of inflammatory cytokines by fetal membranes, resulting in the production of uterotonic prostaglandins. Oxytocin, however, also plays a key role in the initiation of labor. Short-term exposure of myometrium to interleukin (IL)-1 enhances oxytocin signaling and contractility. With intrauterine infection, however, myometrium is exposed to inflammatory cytokines for prolonged periods. The present study was conducted to demonstrate that myometrial oxytocin signaling is significantly impaired following prolonged exposure to IL-1. Myometrial cells were treated with IL-1 for 24 h. Oxytocin-stimulated inositol trisphosphate (IP(3)) production was measured in tritiated myoinositol-loaded myometrial cells. Arachidonic acid (AA) release was measured in tritiated AA-loaded myometrial cells. Increases in intracellular calcium were measure with fluo-3. Prostaglandin (PG) F(2alpha) and 6-keto-PGF(1alpha) were measured by ELISA assay. Prolonged exposure of myometrial cells to IL-1 resulted in a significant reduction in oxytocin-mediated signaling as measured by IP(3) production and AA release, as well as a decrease in intracellular calcium. Prolonged exposure of myometrial cells to IL-1, however, resulted in enhanced PG release. Oxytocin may not contribute significantly to the labor-inducing action of IL-1 in the setting of preterm labor with prolonged infection.

Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression.

PROBLEM: Uterine infection occurs in as much as 20% of preterm labor and results in increased decidual cytokines. The objective of this study was to examine the effect of interleukin-1 (IL-1) and the cyclooxygenase-2 (COX-2) inhibitor, NS-398, on myometrial prostaglandin (PG) production and COX-2 expression. METHOD OF STUDY: Human uterine myocytes were stimulated with IL-1 (0-50 ng/mL) over 24 hr. PGE2, PGF2alpha, and 6-keto F1alpha were measured by enzyme-linked immunosorbent assay. Both COX-1 and COX-2 proteins and mRNA were measured by western and northern blot, respectively. RESULTS: IL-1 increased PG production beginning at 6 hr, COX-2 protein increased beginning at 4 hr and continued to increase at 24 hr. COX-2 mRNA increased at 2 hr and peaked at 4 hr. NS-398 blocked PG production but had no effect on COX-2 protein or mRNA. CONCLUSIONS: IL-1 increases PG production by myometrium by increased COX-2 expression. NS-398 completely blocks IL-1-induced PG production. With intrauterine infection, IL-1 may induce labor through the autocrine production of uterotonic PGs.

Alterations in ovine myometrial beta-adrenergic cascade do not mediate tachyphylaxis to ritodrine.

The purpose of this study was to determine in vivo the dose response relationship between beta-adrenergic receptor (BAR) agonist concentration and various elements of the BAR cascade: receptor density, hormone-stimulable adenylyl cyclase activity, and contraction inhibition. A previously described, chronically-catheterized ovine model was used. Ritodrine was infused continuously over 24 h in 22 mixed-breed sheep. Each animal received a single, constant infusion rate. Myometrial biopsies were obtained before and after the drug infusions. BAR density was determined using tritiated dihydroalprenolol. Adenylyl cyclase activity was determined using the Gilman competitive protein-binding assay. Intermittent oxytocin boluses were given into the maternal aorta and contractile response was determined. Infusion rates of 0.06-4.0 micrograms/kg/min yielded steady-state ritodrine serum concentrations of 5-168 ng/ml. No significant correlation was found between the ritodrine concentration and the magnitude of decrease in BAR density. Significant correlations existed, however, between the ritodrine concentration and both the magnitude of decrease in adenylyl cyclase activity and the loss of contraction inhibition. There was no correlation noted between the BAR cascade alterations and the loss of contraction inhibition. Despite significant reductions in receptor density (down regulation) and dose-related reductions in hormone-stimulable adenylyl cyclase activity (uncoupling), ritodrine at low concentrations was still able to inhibit oxytocin-induced contractions, i.e., tachyphylaxis did not occur. Tachyphylaxis appeared to correlate only with the serum ritodrine concentration. Hence, alterations in the BAR cascade do not necessarily equate with a loss of end-organ response (tachyphylaxis). Previous concepts based on in vitro studies about the interaction of the BAR agonist with its receptor, the subsequent generation of intracellular messengers, and the resultant end-organ response may not apply in the intact animal.

Comparison of pulsatile and continuous ritodrine administration: effects on uterine contractility and beta-adrenergic receptor cascade.

In this study we compare the uterine contractility and beta-adrenergic receptor effects of identical doses of ritodrine administered intermittently or continuously for 24 hours in pregnant sheep. Ritodrine was administered intravenously to five animals as a pulse, 16 micrograms/kg every 1.5 hours, whereas five other animals received ritodrine as a continuous infusion of 0.18 microgram/kg/min. Ritodrine plasma concentrations at steady state were comparable in both groups and averaged 18 ng/ml. Animals receiving ritodrine pulses demonstrated no alteration of myometrial beta-adrenergic receptors or adenylyl cyclase activity, and ritodrine inhibited oxytocin-induced contractility comparably at 4 and 24 hours. Animals receiving ritodrine continuously had a significant decrease in myometrial beta-adrenergic receptors and adenylyl cyclase activity, yet ritodrine inhibition of oxytocin-induced uterine contractility was sustained for 24 hours. Oxytocin receptors were not affected by ritodrine administration and were similar in both groups. At the dose studied, oxytocin-induced contractions are comparably inhibited by ritodrine for 24 hours whether the drug is given continuously or in a pulsatile fashion.

Pharmacokinetics and pharmacodynamics of ritodrine after intramuscular administration to pregnant women.

To define the pharmacokinetics and pharmacodynamics of ritodrine after intramuscular injection, we administered 5 or 10 mg ritodrine into the gluteus or deltoid muscles of 12 pregnant volunteers. Six women received 5 mg and six received 10 mg into each muscle group on different days. We withdrew blood samples before and 12 times in the 6 hours after ritodrine injection. Blood pressure and heart rate were recorded at each time. Ritodrine was measured by high-performance liquid chromatography. Peak ritodrine concentrations (mean +/- SD) after a single 5 mg injection in the deltoid or gluteus were 38 +/- 13 and 26 +/- 8 ng/ml, respectively. After a 10 mg dose in the deltoid or gluteus, peak concentrations were 59 +/- 30 and 47 +/- 22 ng/ml, respectively. Although higher, the peak plasma concentrations after injections into the deltoid were not significantly greater than those after injection into the gluteus. None of the pharmacokinetic parameters differed according to dose or injection site. The pharmacodynamic effects of ritodrine were unaffected by injection site, but ritodrine caused a dose-related increase in heart rate and systolic blood pressure and a dose-related decrease in diastolic blood pressure. After a 10 mg injection, the maximal changes in heart rate, systolic, and diastolic blood pressure were 22%, 10%, and 19%, respectively. However, mean blood pressure was not altered by either the 5 or 10 mg dose. These findings indicate that there are few differences in pharmacokinetic parameters between deltoid and gluteal injection of ritodrine. The single intramuscular injection of 5 or 10 mg ritodrine results in labor-inhibiting concentrations with clinically insignificant cardiovascular effects.

Pharmacokinetics of ritodrine administered intravenously: recommendations for changes in the current regimen.

We define the pharmacokinetics of ritodrine in 13 pregnant women who received the drug intravenously. With constant infusion of 50 micrograms/minute, steady state ritodrine concentrations reached 28 +/- 11 ng/ml (SD) with a range of 15 to 45 ng/ml. This wide variation is a result of differences in plasma clearance, which ranged from 1.0 to 3.3 L/min, mean 1.94 +/- 0.71 L/min. The apparent volume of distribution was 6.95 +/- 3.54 L/kg, indicating that ritodrine is extensively bound to extravascular tissue. When an infusion of ritodrine is stopped, plasma concentrations fall rapidly initially with a distribution half-life of 5.9 +/- 6.0 minutes. After the initial rapid fall, plasma concentrations decrease more slowly with a mean disposition half-life of 156 +/- 51 minutes. On the basis of the pharmacokinetic parameters defined, we recommend that the current infusion regimen for ritodrine be changed. The infusion rate of ritodrine should start at 50 micrograms/minute rather than 100 micrograms/minute. The maximal infusion rate of 350 micrograms/minute should be increased and once labor is inhibited, the infusion rate should be reduced.

Pharmacokinetics of orally administered ritodrine.

The oral dosing regimen for ritodrine was based in large part on kinetic data obtained in nonpregnant subjects. There are limited kinetic data after oral administration of ritodrine in pregnancy. The purpose of the present study was to compare ritodrine kinetics in pregnant and nonpregnant women, evaluate the effect of feeding on ritodrine absorption in pregnant women, and determine if the plasma concentration of ritodrine is proportional to the dose administered in nonpregnant women. Plasma concentrations after a single 20 mg dose of ritodrine were significantly greater in fasting nonpregnant women than in fasting pregnant women. The area under the concentration time curve was 1372 +/- 385 and 1001 +/- 257 ng/ml/min, respectively. In pregnant women ingesting 20 mg of ritodrine, plasma concentrations were not significantly different in the fed or fasted state; plasma concentrations peaked at 11 ng/ml and were less than 3 ng/ml within 4 hours. In nonpregnant subjects the concentration of ritodrine in plasma was proportional to the dose. After ingestion of 10, 20, or 30 mg of ritodrine, the area under the curve was 751 +/- 253, 1372 +/- 385, and 2148 +/- 571 ng/ml/min, respectively. These data indicate that ritodrine concentrations in pregnant women after a 20 mg oral dose are low. Increases in dosage will probably result in proportional increases in plasma concentration. The maximal dose of ritodrine recommended for prevention of recurrent preterm labor should be increased.

Dexamethasone effects on ritodrine-induced changes in myometrial contractility and beta-adrenergic receptor function.

We have previously demonstrated in pregnant sheep that ritodrine infusion for 24 hours reduces myometrial beta-adrenergic receptor density and isoproterenol-stimulated adenylate cyclase activity. These receptor-associated changes were accompanied by an increasing inability of ritodrine to inhibit uterine contractility induced by a bolus of oxytocin. In the present study, we evaluated whether these ritodrine-induced effects could be altered by dexamethasone. Ten pregnant sheep at gestational ages of 92 to 130 days received ritodrine 2 micrograms/kg/min for 24 hours. Five animals also received dexamethasone 10 mg intravascularly twice during the ritodrine infusion. Before and at 4 and 24 hours of ritodrine infusion, the animals were given an identical dose of oxytocin as a bolus, and the area under the uterine pressure-time curve was quantified. Myometrial biopsy specimens were obtained before and after ritodrine infusion. Dexamethasone treatment prevented ritodrine-induced reductions in beta-adrenergic receptor density and isoproterenol-stimulated adenylate cyclase activity. Despite these receptor-associated effects, dexamethasone did not prevent the loss of tocolytic efficacy associated with prolonged ritodrine infusion.

Myometrial desensitization after ritodrine infusion.

We have developed a model in pregnant sheep to investigate pharmacological agents used for suppression of preterm labor. This model allows repetitive determinations of uterine contractility and simultaneous measurements of myometrial receptor and postreceptor events. We used the model to study the beta-adrenergic agent ritodrine. We infused 11 pregnant sheep with ritodrine and 3 with physiological saline for 24 h. Oxytocin boluses were given before and at 5 and 22 h after onset of the infusion. Myometrial biopsies were obtained before and immediately after the infusion. After 22 h of ritodrine infusion, uterine contractility in response to the same oxytocin bolus was 50% greater than at 5 h (P less than 0.02). Myometrial membrane beta-adrenergic receptor density decreased 49% (P less than 0.005), and catecholamine-stimulated adenylate cyclase activity was reduced 70% (P less than 0.005). The model thus demonstrates that use of the beta-adrenergic agonist ritodrine in a clinically relevant manner results in tachyphylaxis of its effects on both physiological parameters and the receptor cascade system.

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.