Objective
Obesity is an increasing problem in obstetric practice. Apelin, secreted by adipocytes, is present in increased serum concentrations in an obese state. Our group has previously highlighted that the adipokines leptin and ghrelin have a potential role in metabolic modulation of uterine contractility in obese women. The aim of this study was to evaluate the effects of apelin on human uterine contractility in vitro.
Study Design
Biopsies of human myometrium were obtained at elective cesarean section. Myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of apelin in the concentration range of 1 nmol/L to 1 μmol/L. Control experiments were performed simultaneously.
Results
Apelin exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human myometrium. The mean maximal inhibition values were 36.8 ± 6.4% for spontaneous (n = 6; P = .002) and 30.4 ± 4.6% for oxytocin-induced contractions (n = 6; P < .0001).
Conclusion
Apelin inhibits human uterine contractility in vitro, raising the possibility that such metabolic modulation may play a physiological role in obese parturients.
The physical and metabolic sequelae of obesity are associated with a large number of pregnancy- and delivery-related complications, and it is evident that the prevalence and extent of these problems has increased recently. In recent years, much research interest has focused on a new range of secretory products from adipose tissue, collectively known as adipocytokines, many of which are now known to be closely linked to metabolic regulation of important systemic factors such as glucose tolerance, insulin resistance, cardiac and vascular function, and many other neuroendocrine mediated processes. There is also a limited knowledge, but growing awareness, of the potential roles of adipocytokines in normal and complicated pregnancies.
Apelin is one such adipocytokine, which is a novel bioactive peptide identified as the endogenous ligand of the orphan G protein-coupled receptor, APJ. The physiologically active apelin molecule is a 36–amino acid peptide. In humans it is secreted by many systems such as vascular smooth muscle, pituitary, and pancreatic tissues. Apelin has also been shown to be expressed in the placenta ; however it is primarily expressed by adipose tissue and serum levels are found to increase in the obese state. Serum apelin levels are also found to decrease in a fasting state and increase upon refeeding, suggesting that insulin may regulate apelin gene expression and secretion. Physiologically apelin has also been shown to be involved in the regulation of cardiovascular function and fluid homeostasis. Serum apelin levels have been shown to be decreased in the second trimester of pregnancy compared with nonpregnant controls; however, fetal levels have been shown to be markedly increased on day 1 and day 4 of life.
The possibility of altered metabolic modulation of uterine smooth muscle in association with obesity has been hypothesized over recent years. This hypothesis is due to reporting of the functional roles of secretory products of adipose tissue as well as the emerging concept that these secretory products play an important role in the pathophysiology of obesity related complications. Our group has previously shown that the adipokines, leptin and ghrelin, have exerted an effect on human uterine contractility in an in vitro model. These findings have highlighted the potential role of metabolic modulation of uterine contractility for women in obese body mass index (BMI) categories.
Apelin exerts an inotropic effect on the heart and simultaneously elicits vasodilatation in the peripheral circulation, and this role may be more pertinent in patients with type 2 diabetes mellitus. There are no data to our knowledge outlining the effects of apelin on nonvascular smooth muscle. The aim of this study was to evaluate the effects of apelin on in vitro uterine function in isolated preparations obtained during human pregnancy, in relation to spontaneous and agonist induced contractility.
Materials and Methods
Tissue collection
Biopsies of human myometrial tissue were obtained at elective cesarean section in the third trimester of pregnancy in the Department of Obstetrics and Gynaecology, University College Hospital, Galway, Ireland. Ethical committee approval for tissue collection was obtained from the Research Ethics Committee at University College Hospital Galway and recruitment of patients was by informed written consent.
Biopsies were excised from the upper portion of the lower segment of the uterus. Once collected, all tissue biopsies were placed in Krebs-Henseleit physiological salt solution (PSS) at pH 7.4 containing the following: 4.7 mmol/L potassium chloride, 118 mmol/L sodium chloride, 1.2 mmol/L magnesium sulphate, 1.2 mmol/L calcium chloride, 1.2 mmol/L potassium phosphate, 25 mmol/L sodium bicarbonate, and 11 mmol/L glucose (Sigma-Aldrich, Dublin, Ireland). Tissues were stored at 4°C and used within 12 hours of collection.
Tissue bath experiments
Longitudinal myometrial strips (measuring approximately 2 × 2 × 10 mm) were dissected free of uterine deciduas and serosa and mounted for isometric recording under 2 g of tension in organ baths as previously described. The tissue baths contained 10 mL of Krebs-Henseleit PSS maintained at 37°C (pH 7.4) and were gassed continuously with a mixture of 95% oxygen/5% carbon dioxide. Myometrial strips were allowed to equilibrate for a period of at least 1 hour, during which time the physiological salt solution was changed every 20 minutes. After equilibration, a 30 minute period was allowed to achieve spontaneous phasic contractions, or, alternatively, contractions were stimulated by bath exposure of the strips to oxytocin (0.5 nmol/L). Apelin was then added to the tissue bath in a cumulative manner at bath concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L and 1 μmol/L at 20 minute intervals.
Control experiments were performed simultaneously. Control strips were exposed to either PSS only (for spontaneous contractions) or PSS and 0.5 nmol/L oxytocin (for agonist induced contractions). In study strips and in controls, the integral of contractile performance was measured by calculating the area under the curve for a 20 minute period for each drug concentration as previously described. The measurement was expressed as a percentage of the integral obtained in the 20 minute period prior to any apelin addition using the PowerLab hardware unit and Chart version 4.0 software (AD Instruments, Hastings, UK).
The inhibitory effect of apelin was corrected for the reduction in the contractile activity observed in the control, and the effects of apelin were interpreted as the final additional relaxant effect. This value, when subtracted from 100% represents the mean maximum inhibition (MMI) of apelin on uterine contractility. The MMI provided therefore represents the net final inhibition resulting from exposure to apelin, ie, after subtraction of any alteration observed in control experiments.
Drugs and solutions
Apelin was purchased from Sigma-Aldrich. A stock solution was made in deionized water. Serial dilutions were made in deionized water on the day of experimentation. Fresh Krebs-Henseleit solution was made daily. A stock solution of oxytocin (1 mmol/L; Sigma-Aldrich) was prepared using deionized water. Serial dilutions were prepared in deionized water on the day of experimentation.
Statistical analysis
Comparisons of contractile effect, for each bath concentration of apelin, were performed using an analysis of variance followed by Tukey honestly significant difference post hoc testing to determine significant differences among data groups. A P < .05 was considered to be statistically significant. The statistical package SPSS for Windows version 14.0 (SPSS Inc, Chicago, IL) was used for these statistical calculations.
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