Despite demonstration of the independence of myometrial smooth muscle relaxation to accumulation of cyclic GMP following addition of nitric oxide donors, resistance to this notion or a lack of awareness hampers research into the development of new tocolytics. We present evidence underscoring the lack of effect of cyclic GMP accumulation following stimulation of the soluble guanylyl cyclase enzyme. Earlier evidence focused on examining nitric oxide-mediated uterine smooth muscle relaxation in the presence of soluble guanylyl cyclase inhibition. Here we describe the failure of the haem-independent soluble guanylyl cyclase agonist BAY58-2667 to relax human pregnancy myometrium. These data suggest that examining the action of cGMP-independent relaxation can reveal new tocolytic targets.
Approximately 15 million preterm births occur annually worldwide 1. Preterm infants that survive are at risk for learning disabilities, cerebral palsy, vision and hearing loss, respiratory and digestive problems 2. In 2012, more than 11% of US births were premature 3. Although the etiology of spontaneous preterm birth is likely to be complex, disparate medical, environmental, and genetic risk factors are thought to converge on effector pathways in the uterine myometrium to influence contractility and birth timing in women 4. Indeed, the definition of preterm labor as contractions of the uterus resulting in changes in the cervix that start before 37 weeks of completed gestation is likely to be inadequate to address the mechanism(s) of preterm labor.
Tocolytics used to prevent early spontaneous preterm labor (SPTL) are not FDA approved for this use and on average delay labor for only 48 hours, a window for antenatal steroid, but hardly a solution to the problem. Microbial infection might initiate SPTL in some cases, but antibiotic treatment doesn’t prevent preterm birth 5,6. Because no available medications can reliably interrupt established spontaneous preterm labor (SPTL) and allow an afflicted pregnancy to continue to term, it is clear that we lack the tools needed to address this problem. Indeed, development of the artificial womb7 can be viewed as emphasizing the severity of the problem of prematurity as well as our lack of a complete understanding of uterine quiescence 8.
If we are to advance our understanding of preterm birth in order to prevent it, we posit that understanding the biochemical mechanisms of relaxation of the uterus is paramount. This is because tools such as terbutaline used to relax airway smooth muscle, or nifedipine used to relax vascular smooth muscle are borrowed pharmacology. Myometrial relaxation signaling is unique.
Some years ago our laboratory observed the ability of nitric oxide (NO) to relax term human myometrium and addressed the mechanism by which this occurs 9. While treatment of pregnant myometrium with NO donors relaxed the tissue, blockade of cGMP accumulation failed to block the relaxation as predicted 10,11. These results were immediately controversial as they challenged the dogma 12 established following the classic smooth muscle experiments of Furchgott13.
Our work has not been aimed at proposing NO as a tocolytic. Indeed, patients who enter labor spontaneously preterm without infection have a blunted relaxation response 14 to NO-mediated relaxation (Figure 1) suggesting that the mechanism of NO action may be involved in the pathophysiology of preterm labor.
Figure 1: The biochemical distinction that relaxation of the smooth muscle of the myometrium to NO donors is cGMP-independent does not argue that cGMP has no role. Cyclic GMP-dependent phosphorylation can readily be measured 15 and the ability of cGMP to relax the muscle is agonist-specific16.
More over, the ability of NO to relax the uterine smooth muscle in a fashion independent of the action of cGMP 17 is not entirely unique 18. Nonetheless, this finding has been the subject of skepticism despite several lines of evidence suggesting that animal as well as human myometrium is distinct in its response to NO 9,19,20.
Here we offer new evidence on the failure of cGMP to relax myometrium and suggest that since NO relaxes the term myometrium but not preterm tissues, there is an opportunity to examine the actions of NO to find targets that may be disparately regulated in preterm tissues.
Human myometrium from a patient delivering at term by Cesarean section (C/S) was obtained with written informed consent. All research was reviewed and approved by the University of Nevada Biomedical Institutional Review Board (IRB) and the Renown Hospital IRB for the protection of human subjects. The myometrial biopsy was obtained from a mother (gestational age 30 wk, singleton pregnancy) without infection. The sample was taken from the superior aspect of the standard transverse incision.
All clinical data on the treatments and the pregnancy course prior to C/S was collected without patient identifiers. Exclusion criteria included age <18 years, any infection including vaginitis or suspected chorioamnionitis, use of oxytocin for labor induction, history of drug abuse, co-morbid diagnoses such as HIV infection or AIDS, hepatitis C infection, uncontrolled diabetes, and any use of steroids including topical use. A patient meeting our experimental criteria, was consented by after receiving permission from the surgeon. Tissue was transported to the laboratory immediately in cold physiological buffer containing (120 mM NaCl, 5.0 mM KCl, 0.58 mM KH2PO4, 0.58 mM Na2HPO4, 2.5 mM MgCl2, 20 mM α-D-glucose, 2.5 mM CaCl2, 25 mM Tris, and 5.0 mM NaHCO3), microdissected under magnification to isolate smooth muscle devoid of obvious blood vessels and hung in contractile baths at 37oC in continuously oxygenated Krebs buffer. Rat aorta was obtained under institutional approval in accordance with federal guidelines for the care and use of animals in research. Rat aorta was prepared as rings with endothelium in tact were handled in the same fashion as myometrium.
Tissue contractions were recorded digitally using DataTrax™ software (WPI, Sarasota, FL). BAY58-2667 was obtained from Tocris as the hydrochloride and prepared as either a 1 mM or 10 mM stockin DMSO (to minimize vehicle in the bath while achieving the desired final concentration).
In order to examine the role of cGMP in uterine smooth muscle relaxation in the absence of haem-dependent activation of soluble guanylyl cyclase by NO, we have examined the effect of direct stimulation of myometrium by the haem-independent agonist BAY58-2667 (BAY58, cinaciguat) on uterine relaxation. BAY58 fails to relax human term myometrium despite the use of concentrations from 0.1 to 10 mM (Fig. 2A). Treatment of rat aorta with BAY58 resulted in immediate relaxation consistent with the known actions of cGMP in vascular smooth muscle (Fig. 1B). The EC50 for activation of the soluble guanylyl cyclase is 6.4 nM and its effect to relax vascular smooth muscle is sub-nM 21. Experiments repeated with term myometrium from multiple donors confirmed the failure of BAY to relax the tissue (Fig. 1C).
Figure 2: Cyclic GMP-independent Uterine Smooth Muscle Relaxation. (A) Human pregnant myometrium was mounted in tissue baths at 37oC with continuous oxygenation and stimulated to contract in the presence of oxytocin (100 nM). Following 60 minutes of calibration, tissues were challenged in the presence or absence of BAY58-2667 in a cumulative fashion as indicated in the figure. No response was seen to addition of BAY58-2667. Addition of the NO-donor at the end of the experiment confirmed the ability of the tissue to relax (not shown). (B) Rat aorta was hung in tissue baths with Krebs buffer at 37oC with continuous oxygenation and allowed to equilibrate for 60 minutes in the absence of drug. Addition of phenylephrine (1 mM) produced immediate contractions that could be relaxed by addition of BAY58-2667 (10 mM). (C) The failure of pregnant human myometrium to relax to BAY58 was repeatable, quantified as area under the curve over 10 minutes and demonstrated the absence of any dose-dependent effect on tension (Fig 1C). Additional experiments performed in guinea pig myometrium revealed the failure of BAY58 to relax the tissue at micromolar concentrations (not shown).
Relaxation signaling in myometrium is distinct from that of vascular smooth muscle. The failure of cGMP accumulation to relax the tissue 9 is well documented 17. The NO donor cysteine-NO relaxes oxytocin stimulated term human myometrium 22 with an effective dose 50% of 1 uM. Preterm tissues prepared in the same manner fail to relax in the presence of CysNO 14. The failure of SPTL tissues to respond suggests that a fundamental difference in the mechanism of relaxation to NO may underlie the dysfunction. Despite the complete blockade of cGMP accumulation, term myometrium still relaxes to NO donor addition as demonstrated in guinea pig 19, cynomolgus monkey 20 and human 9.
Glyceryl trinitrate is an NO donor initially thought to be useful as a tocolytic 23 and is still being employed by some 24 despite a lack of clear evidence of efficacy 25. The failure of NO-donors to prove useful in the treatment of women in labor preterm is underscored by the use of BAY58 that fails to relax the myometrium despite its ability to relax vascular muscle (Fig 1). Stimulation of soluble guanylyl cyclase by BAY58 results in the formation of cGMP in a dose-dependent manner 26 unaffected by the presence of ODQ, the selective inhibitor of soluble guanylyl cyclase. BAY58 selectively activates the oxidized/haem-free soluble guanylyl cyclase enzyme by binding to the enzyme’s haem pocket, causing pronounced cGMP-dependent relaxation in responsive tissues such as vascular smooth muscle 27, but not myometrium.
The demonstration of cGMP-independence of the actions of NO and NO donors in myometrium has been criticized on the basis that smooth muscle relaxation to NO-mediated cGMP accumulation is dogma and thus blocking cGMP accumulation is expected to block relaxation. Despite evidence supporting a cGMP signaling exception in myometrial smooth muscle 17 and our finding that the agonist-specific effect of cGMP accumulation is dependent not on cGMP, but rather the type of guanylyl cyclase stimulated 16, acceptance of these data has not been incorporated into efforts to develop novel tocolytics. We think that there is an exciting opportunity to investigate the S-nitrosation signaling that underlies the ability of NO-donors to relax myometrium in a cGMP-independent fashion and that the failure of NO-donors to relax preterm human myometrium suggests that differences in S-nitrosation signaling between term and preterm tissues will reveal new targets that can be explored as tocolytics.
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