Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Abstract

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.

Keywords: myometrium; oxytocin; oxytocin receptor; pharmacological chaperones; pregnancy.

Figure 1

SR49059 and L371,257 increase cell surface abundance of variant and wild-type oxytocin receptor in HEK293T cells.A, effects of compounds on V281M OXTR cell surface abundance in HEK293T cells plotted relative to cell surface OXTR abundance in cells transfected with WT OXTR and treated with vehicle. ∗p < 0.05 compared with vehicle-treated cells by one-way ANOVA with Dunnett’s multiple comparisons test. B, effects of compounds on WT OXTR cell surface abundance relative to that in vehicle-treated cells. ∗∗p < 0.0055 compared with vehicle by one-sample t test (α corrected for multiple comparisons by Bonferroni method). Data shown are mean and standard error from N = 3 independent trials. OXTR, oxytocin receptor.

Figure 2

SR49059 and L371,257 rescue trafficking and functional defects of V281M OXTR.A–C, subcellular localization of OXTR (green) and (A) ER marker PD1 (magenta) or (B) Golgi marker Golgin-97 (magenta) in HEK293 cells stably transfected with WT OXTR-GFP and V281M OXTR-GFP. Results shown are representative from three independent trials. C, quantitation of colocalization by Pearson’s r. Each point represents an individual image. ∗∗∗∗p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D, oxytocin-induced IP1 production in HEK293T cells transfected with WT or V281M OXTR and treated with vehicle or SR49059. Data are shown as mean and standard error from N = 5 independent trials. ∗∗∗∗p < 0.0001 compared with V281M + vehicle by sum-of-squares F test. OXTR, oxytocin receptor.

Figure 3

SR49059 and L371,257 increase OXTR trafficking and oxytocin response in immortalized human myometrial cells.A, effect of compounds on cell surface localization of OXTR in hTERT-HM cells. Data shown are mean and standard error from N = 3 independent trials. ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA with Dunnett’s multiple comparisons test. B, change in surface OXTR on hTERT-HM cells after incubation with SR49059 or L371,257 for the indicated time points. Error bars show standard error for N = 5 (SR49059) and N = 3 (L371,257). C, change in surface OXTR after 12-h incubation with SR49059, L371,257, cycloheximide, and brefeldin A as indicated. Bars with “a” are statistically different from vehicle-treated cells at p < 0.001 (one-sample t test). ∗p < 0.05 by one-way ANOVA with Šidák’s multiple comparisons test; ns, not significant. D, oxytocin-induced IP1 production in hTERT-HM cells treated with SR49059 or L371,257. Data are shown as mean and standard error from N = 5 independent trials. ∗∗∗∗p < 0.0001 compared with vehicle-treated cells by sum-of-squares F test. OXTR, oxytocin receptor.

Figure 4

SR49059 and L371,257 increase oxytocin-induced IP1 production in primary human myometrial cells.A–E, oxytocin-induced IP1 production in primary human myometrial samples from five term nonlaboring samples (TNL 1–5). Error bars show standard error from N = 4 technical replicates. p-values from comparison of curve E_max (sum-of-squares F test). F, E_max from N = 5 primary cell samples. p values from Friedman ANOVA with Dunn’s multiple comparisons test.