Type of Document Dissertation Author Sharkey, James Thomas Author's Email Address email@example.com URN etd-11052009-201851 Title Melatonin Regulation of the Oxytocin System in the Pregnant Human Uterus Degree Doctor of Philosophy Department Biomedical Sciences, Department of Advisory Committee
Advisor Name Title James M. Olcese Committee Chair Akash Gunjan Committee Member Branko Stefanovic Committee Member Choogon Lee Committee Member Hank W. Bass University Representative Keywords
Date of Defense 2009-11-04 Availability unrestricted AbstractThe mechanisms underlying the gestational and circadian timing of parturition in humans are not fully understood. Studies of the timing of initiation of spontaneous labor show a peak between 2400 and 0500. This peak in labor onset coincides with peak serum melatonin levels in humans. Melatonin, N-acetyl-5-methoxytryptamine, is the molecular messenger of circadian night. A monoamine hormone produced by the pineal gland, melatonin, is released into the blood directly in a circadian manner controlled by input from suprachiasmatic nuclei (SCN). Peak levels occur several hours after darkness and its release is inhibited by light via photic input transmitted from the eye via the retino-hypothalamic tract to the SCN. Once in the circulation melatonin can act on numerous tissues via its receptors or via antioxidant mechanisms inferred by its indole ring. Our laboratory recently characterized the expression of the melatonin receptors in the human myometrium and showed that the expression of these receptors is suppressed until late pregnancy. In an effort to understand better the significance of melatonin in the human myometrium, we explored the mechanisms through which this hormone influences the expression of the oxytocin receptor in vitro. The stable melatonin analog iodo-melatonin (I-MEL) was presented to cultured telomerase-immortalized smooth muscle myometrial cells of the human telomerase reverse transcriptase line under physiological doses and durations. Pharmacological inhibitors of melatonin binding (4P-PDOT), gene transcription (actinomycin), phospholipase C (U73122), and protein kinase C (C1) signaling were used to define the mechanism of melatonin action. Our results reveal that melatonin significantly reduces oxytocin receptor mRNA levels primarily via the melatonin type 2 receptor, MT2R. We assayed OTR mRNA levels over 24 hours after treatment with the transcriptional inhibitor, Actinomycin, with and without cotreatment melatonin. Our data suggest the melatonin-dependent decrease in oxytocin receptor transcripts involves reduction of the OTR mRNA accumulation rate rather than enhanced rates of transcript degradation. Melatonin effects were abolished by pre-treating the cells with the phospholipase C inhibitor U73122 or the protein kinase C inhibitor C1. Melatonin, like oxytocin, can negatively regulate oxytocin receptor transcription in human myometrial cells via modulation of protein kinase C signaling.
Due to the similarities between the melatonin and oxytocin signaling pathways including reduction of OTR mRNA levels, we next sought to determine the effects of melatonin on contractility and the contractile machinery in telomerase-immortalized human myometrial cells. To ascertain the effect of melatonin on myometrial contractility in cell cultures, we performed gel retraction assays with cells exposed to I-MEL, oxytocin and the pharmacological inhibitors 4P-PDOT, U73122, C1 and combinations of ligands and inhibitors. I-MEL was found to synergistically enhance oxytocin-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. The effects of I-MEL on gap junctions were also investigated as gap junction proteins have been shown to be upregulated by melatonin in other tissues and have also been shown to be important for coordination of contractions in the laboring uterus. I-MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that I-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. Additionally, expression levels of the type 2 melatonin receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched non-laboring pregnant women.
To ascertain the signaling pathway of melatonin and leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to I-MEL with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on IP3/DAG/ Protein Kinase C (PKC) signaling were also investigated as these signaling molecules were implicated by our previous pharmacology experiments. I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity and its effects on contractility were insensitive to Rho kinase inhibition. In order to examine another possible method of contractile sensitization, we assayed for caldesmon phosphorylation and upstream Erk1/2 activation. I-MEL did increase phosphorylation of Erk1/2 and caldesmon, which was inhibited by the MEK inhibitor, PD98059 or the PKC inhibitor, C1. These findings lead us to surmise that melatonin sensitizes myometrial cells to subsequent pro-contractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway resulting in specific activation of PKCα and Erk1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and crossbridging.
This research revealed a new role for melatonin in reproductive physiology, sensitizing myometrial cells to a subsequent pro-contractile oxytocin signal. This function would help explain the increased nocturnal uterine contractility and increased incidence of parturition observed in late term human pregnancy. Synergistic actions of melatonin on oxytocin-induced contractility may be of clinical relevance in that it could provide a means to lower the oxytocin dose used in the induction of labor and thus reducing the contraindications associated with oxytocin induction of labor.
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