AB281. SPR-08 Altered S-nitrosation of contractile proteins underlies dysfunctional quiescence in human preterm labor

Objective: Preterm labor is defined as labor prior to 37 weeks of gestation. There are no FDA approved drugs available to treat spontaneous preterm labor. Uterine smooth muscle is unique in that it relaxes in a cGMP-independent manner in the presence of nitric oxide (NO). However, NO-mediated relaxation in tissues from mothers who deliver preterm is blunted. S-nitrosation of cysteines via NO acts as an important mediator in a number of disease states. Our analysis of the myometrial S-nitrosoproteome has revealed that several smooth muscle contractile proteins are differentially S-nitrosated based upon the state of labor in women. An important regulator of protein S-nitrosation is the availability of S-nitrosoglutathione (GSNO), an endogenously expressed NO donor. The enzyme GSNO reductase (GSNOR) regulates GSNO levels in smooth muscle. We performed experiments to test the hypotheses that GSNOR activity is altered in the myometrium from women in preterm labor and that inhibition of GSNOR attenuates myometrial contraction and restores quiescence in spontaneous preterm labor.

Methods: Experiments were performed in the Myometrial Research Laboratory at UNR Med. Uterine biopsies were obtained with IRB approval and written informed consent from mothers undergoing Cesarean section and transported to the laboratory in physiological buffer for immediate processing. GSNOR expression was measured by western blot and normalized to GAPDH expression. GSNOR activity was measured by the decrease in absorbance at 340 nm via conversion of NADH to NAD⁺ by GSNOR in the presence of GSNO. Eight (8) nM N6022, a potent and specific inhibitor of GSNOR, was used to verify GSNOR specific activity. Human myometrial tissue was dissected and six uterine strips from each patient were mounted in tissue baths and tested for their ability to contract in response to KCl prior to addition of test agents.

Results: Human preterm myometrium fails to relax to NO addition whether in the presence or absence of oxytocin stimulation. GSNOR expression is increased in myometrial tissue from women in spontaneous preterm labor compared to term laboring controls. GSNOR enzymatic activity was also increased in protein lysates from myometrial tissue from patients undergoing spontaneous preterm labor than those in labor at term. A potent and selective inhibitor of GSNOR (N6022) decreases the peak force of contraction in guinea pig myometrium and restores sensitivity to NO.

Conclusions: GSNOR is more highly expressed in preterm laboring myometrium. Preterm laboring myometrium exhibits a blunted response to NO and the GSNOR inhibitor N6022 restores sensitivity to relaxation by NO. Our data suggests that increased GSNOR activity in spontaneous preterm labor contributes to a preterm contractile phenotype through the enzymatic degradation of endogenous GSNO.

Funding Source(s): NIH 1U54GM 104944

Keywords: Preterm labor; S-nitrosation; human; myometrium; in vitro contractility