Discovery of the molecular mechanisms subserving human uterine quiescence during pregnancy and their dysregulation in spontaneous preterm labor is the objective of this proposal. We will test the hypothesis that failure of preterm human myometrium to relax to nitric oxide-(NO) is the result of dysregulated S-nitrosation of specific smooth muscle contractile proteins. Our long-term goal is to find new effective tocolytics to treat women who enter labor too soon. Preterm labor leads to preterm delivery, a global problem accounting for 75% of fetal morbidity and mortality. No drugs reliably prevent labor in patients who enter labor preterm, thereby allowing their pregnancies to go to term. Therapeutic approaches to manage spontaneous preterm labor (SPTL) are employed without clear evidence of benefit for acute or maintenance tocolysis. NO-mediated relaxation of myometrium is cGMP-independent. Preterm myometrium fails to relax to NO. Discovering the mechanism of action of S-nitrosated contractile proteins can suggest new therapeutic targets to manage SPTL. We propose that gestational quiescence until term results from regulated post-translational S- nitrosation of myosin light chain kinase (MLCK), the regulatory light chain (MYL9) and profilin-1 (PFN1). Addition of NO relaxes term, but not preterm laboring tissues as a result of S-nitrosation differences that alter the function of these CAPs in SPTL. Discovering the effect of regulated S-nitrosations on the mechanism of contractile protein action in term tissues, term tissues from patients in labor and in SPTL (with controls for gestational timing, tocolytic and antenatal steroid use, infection and gestational length) will establish whether or not NO is an endogenous relaxation signal. Comparison of this S-NO fingerprint with that measured following relaxation of the tissue by NO addition in each pregnancy state is novel because SPTL is not simply early labor, will likely be influenced by infection and/or gestational length and because NO- induced relaxation of spontaneous and oxytocin-induced contractions of preterm myometrium is blunted. S-nitrosation differences between labor and SPTL point to altered quiescence mechanisms. Gestational length comparisons in the guinea pig will establish a model in which to investigate S-nitroso regulation of CAP proteins. We describe innovative experiments employing pregnant guinea pigs and tissues from pregnant women and in vitro functional assays designed to reveal the mechanisms underlying the failure of preterm tissues to relax to NO. Completion of this research will suggest therapeutic strategies for the treatment of SPTL such as the S-nitrosoglutathione reductase that regulates S- nitroso protein levels and is known to provide therapeutic benefit in asthma and for which an inhibitor is in development.
The research planned in this application will employ tissues from pregnant women and a guinea pig model of preterm labor. Relaxation to nitric oxide is dysfunctional in human preterm labor. We will examine the role of S-nitrosation of contraction/relaxation associated proteins by the endogenous nitric oxide donor GSNO. Regulation of CAP protein function by S-nitrosation is unknown. By describing the nature of S-nitrosation differences in CAP proteins in preterm laboring myometrium, we will discover new therapeutic targets for the prevention of spontaneous preterm labor.
|Barnett, Scott D; Buxton, Iain L O (2017) The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy. Crit Rev Biochem Mol Biol 52:340-354|