Objective
We sought to test the hypothesis that sildenafil citrate (SC) at low concentrations potentiates the tocolytic effects of nifedipine in vitro.
Study Design
Myometrial biopsies were obtained from 22 term nonlaboring women undergoing scheduled cesarean delivery. Tissue strips were suspended in organ chambers for isometric tension recording, and incubated for 30 minutes with either SC at 231 ng/mL or solvent. The effects of cumulative doses (10 –10 to 10 –5 mol/L) of nifedipine on spontaneous and oxytocin-induced uterine contractility were then determined. Areas under the contraction curve were compared using 1-way analysis of variance with Tukey post hoc test (significance: P < .05).
Results
Nifedipine significantly inhibited spontaneous and oxytocin-induced myometrial contractility. Preincubation with SC increased response to nifedipine and significantly potentiated its inhibitory effect at 10 –8 mol/L, without affecting oxytocin-induced contractile response.
Conclusion
At concentrations within a therapeutic window, SC increases myometrial sensitivity to nifedipine.
The incidence of preterm birth in the United States rose from 9.4% in 1981 to 12.8% in 2006, with approximately half of the >500,000 preterm births each year resulting from spontaneous preterm labor. The considerable efforts directed toward the prevention and treatment of preterm labor have had relatively little effect on neonatal morbidities, mortality, and long-term sequelae.
Despite considerable advances in our knowledge of myometrial physiology, our current understanding of the mechanisms responsible for preterm labor and delivery remains limited. Throughout pregnancy, the production of relaxing factors such as nitric oxide (NO) regulates uterine homeostasis. Soluble guanylyl cyclase represents one NO-specific target in the myometrium, generating 3’5′ guanine cyclic monophosphate (cGMP) from guanosine 5′ triphosphate. While the action of cGMP and cGMP-dependent protein kinase explains much of NO-induced relaxation in nonuterine smooth muscles, the role of cGMP in human uterine relaxation is more controversial. Recent new evidence has confirmed the importance of this biochemical pathway in the maintenance of uterine quiescence during pregnancy.
Sildenafil citrate (SC) is a selective phosphodiesterase type 5 (PDE5) inhibitor that prevents cGMP degradation and promotes smooth muscle relaxation. SC-mediated myometrial relaxation was demonstrated in an animal model of preterm labor as well as in human myometrial samples, suggesting its potential use as a new tocolytic agent. However, the concentrations of SC reported in these studies far exceeded (micromolar to millimolar range) the ones achieved when the medication is used to relax the pulmonary vasculature in patients with pulmonary arterial hypertension (PAH) (nanomolar range), indicating that the effect on uterine smooth muscle may not be solely mediated by PDE5. Moreover, since hypotension is one of the dose-limiting factors in the treatment of PAH, the higher levels required for direct myometrial relaxation will certainly be associated with significant cardiovascular side effects.
While the higher concentrations needed may preclude clinical applications of SC as a tocolytic, lower levels may sensitize the myometrium to other tocolytics while limiting untoward cardiovascular effects. The aim of this study was to determine the effect of low concentrations of SC on the tocolytic response to nifedipine in vitro.
Materials and Methods
The study was approved by our institutional review board. Patients undergoing scheduled cesarean section at term gestation (37-42 weeks) were approached for consent. Exclusion criteria were presence of >3 contractions per hour, rupture of membranes, placenta previa, and uterine abnormalities affecting contractility (eg, leiomyomas).
In consenting women, 2 × 2 × 4 cm biopsies were taken from the upper edge of the transverse incision in the lower uterine segment, placed in Hanks balanced salt solution (Gibco BRL Products, Rockville, MD), and prepared into 9 × 2 × 2 mm strips.
Tissue organ chamber
Myometrial strips were mounted vertically in 10-mL organ chambers containing Krebs–Henseleit solution and prepared for isometric tension recording using stainless steel hooks and surgical silk sutures. One end of the strip was attached to a fixed support at the bottom of the chamber, while the other end was connected to an isometric force transducer. The temperature in the organ bath was maintained at 37°C and the solution was continuously bubbled with 5% carbon dioxide in air (pH 7.4). Strips were equilibrated at the passive tension of 1 g in Krebs solution. Isometric tension was measured with Harvard isometric force transducers (Harvard Apparatus, South Natik, MA) connected to a computer. The data were acquired and analyzed using Windaq data acquisition (Dataq Instruments Inc, Akron, OH). The study protocol is summarized in Figure 1 . The specimens were equilibrated for 1-2 hours until homogeneous, regular, and rhythmic contractions had developed for at least 30 minutes (contraction stabilization). The bath solution was changed every 30 minutes. Tissues with poor spontaneous contractile activity were discarded with the assumption that they represented scar tissue or that the tissue was not viable at the time of the experiment. In the remaining tissue, oxytocin (10 –9 mol/L) was added to half of the preparations collected from each patient to generate a higher pattern of contractions while the other preparations remained with spontaneous contractions. Tissue samples were then incubated with either 231 ng/mL of SC or distilled water for 30 minutes. The SC concentration used was the same as the average serum concentration at steady state when SC is taken at doses clinically used to treat PAH. Cumulative concentrations of nifedipine (10 –10 to 10 –5 mol/L) were then added. At the end of the tocolytic dose response, high potassium chloride (KCl) concentration (60 mmol/L) was added in each channel to confirm tissue viability, and contractility was recorded for 30 minutes.
Data analysis
The area under the curve defining myometrial contractions (integral activity) over a 30-minute period was used as a measure of contractility. Baseline activity was defined as the integral activity over the 30 minutes following stabilization of myometrial contractions (for spontaneous contractions) or oxytocin administration (for oxytocin-induced contractions). To correct for time-induced decay in contractility, 30-minute study intervals were maintained constant throughout the experiments and were compared to the 30-minute time period defining baseline activity ( Figure 1 ).
The effects of nifedipine cumulative concentrations were expressed as percentage of the baseline activity, dividing the integral activity calculated after adding each dose of tocolytic agent by the baseline myometrial contractility. KCl-induced contractile response was expressed as a multiple of the baseline contractility, dividing the integral activity in the 30 minutes that followed KCl administration by the baseline myometrial contractility. Dose-response curves were then generated for nifedipine, and areas under the curve were calculated using software (GraphPad Prism, version 3.00 for Windows; GraphPad Software, San Diego, CA). One-way analysis of variance with Tukey post hoc test was used for statistical analysis (significance: P < .05). Data were expressed as mean ± SEM.
Drugs and solutions
SC (Viagra; Pfizer Inc, New York, NY), nifedipine, and oxytocin (Sigma, St Louis, MO) were used. Oxytocin was dissolved in distilled water and sonicated for 5-15 minutes to aid solubilization. Nifedipine was dissolved in dimethyl sulfoxide and SC in distilled water. Solutions were aliquoted and kept at –20°C until use.
Results
A total of 22 patients were included in the study. After discarding the samples with poor contractile activity (n = 65), 121 myometrial strips were analyzed, including not >1 strip from every patient in the different study groups. Table 1 summarizes the demographic characteristics and the indications for cesarean delivery of the study population.
