Melatonin synergizes with oxytocin to enhance contractility of human myometrial smooth muscle cells

Abstract

Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night.

Objective: The purpose of this study was to determine the effects of MEL on contractility and the contractile machinery in telomerase-immortalized human myometrial cells.

Design: To ascertain the effect of MEL on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodomelatonin +/- oxytocin (OT). The effects of iodomelatonin on gap junctions were also investigated. Additionally, expression levels of the type 2 MEL receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor.

Results: MEL was found to synergistically enhance OT-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched nonlaboring pregnant women. MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner.

Conclusions: MEL synergizes with OT to promote myometrial cell contractions and to facilitate gap junction activity in vitro. Such a synergy in vivo would promote coordinated and forceful contractions of the late term pregnant uterus necessary for parturition.

Figure 1

Functional MEL receptors in the human myometrium. A, Western blot for the MT2R in hTERT cells, uteroleioma cells stably transfected with MT1R, MT2R, or neither (neg). B and C, MT2R immunoreactivity in myometrial punches from pregnant nonlaboring patients (B) and patients in active labor (C). Numbers in B and C represent individual donor samples. D, Results from panels B and C in histogram form. *, P < 0.05 by χ² statistic. E, MT2R mRNA expression in tissues from the same laboring (IL) and nonlaboring (NIL) patients as in B and C. *, P < 0.05 relative to NIL.

Figure 2

Effects of I-MEL on OT-induced contractility. A, Contractility of hTERT myometrial cells treated with OT (dark bars) or cotreated with 1 nm I-MEL (light bars). *, P < 0.05 relative to control values; **, P < 0.05 relative to all columns marked with single asterisk. B, Effects of treatment with 10 μm of the PKC inhibitor C1, or 10 nm of the MT2R-specific antagonist 4P-PDOT, on I-MEL induced contractility. *, P < 0.05 relative to control. C, Effect of 4P-PDOT pretreatment on the contractility of samples cotreated with I-MEL and OT. **, P < 0.05 relative to OT-treated and I-MEL/OT/4P-PDOT-treated samples. *, P < 0.05 significantly elevated over controls. D, Effects of treatment with 1 nm of I-MEL (M) and/or OT on the phosphorylation of the myosin light chain regulatory subunit and the effect of cotreatment with 10 nm 4P-PDOT (PD) or 10 μm C1. Un, Untreated control cells; S, size markers.

Figure 3

Effects of I-MEL on the expression of the gap junction protein, Cx43. A, Effects of treatment with 1 nm I-MEL (filled bars) or control vehicle (open bars) on Cx43 mRNA levels in hTERT cells collected at 0, 4, and 8 h (mean ± sem; n = 9). B, Effects of treatment with I-MEL on Cx43 protein levels. C, Effect of cotreatment with 4P-PDOT on Cx43 mRNA levels.

Figure 4

Effects of MEL on gap junction communication in hTERT cells. The top row shows the cells under bright field, whereas the remaining figures were photographed under fluorescent light to demonstrate lucifer yellow dye spread within 10 min after scrape loading. Treatments include 1 nm I-MEL, or I-MEL after pretreatment of the cells with 10 nm 4P-PDOT or 10 μm C1. The bottom two images at higher magnification reveal lucifer yellow dye spread in greater detail.

Figure 5

Proposed model for the synergy between MEL and OT on nocturnal myometrial contractility in the laboring pregnant uterus. MEL acts synergistically with OT to increase PLC activity and associated signaling mechanisms, thereby enhancing myometrial contractility and gap junction-associated intercellular communication. DAG, Diacylglycerol.