title: Investigating the role of hydrogen sulfide in the myometrium
Creators: name: Robinson, Hayley
Publisher: name: University of Liverpool Repository
Year: 2013

description: Preterm births are increasing worldwide; currently 7 % of UK births are preterm. Prematurity is the principal cause of neonatal mortality and a major cause of paediatric morbidity. Uterine contractility before term leads to pre-term labour. Therefore to reduce pre-term delivery, new pathways and drugs that inhibit uterine contractility are of interest. The gasotransmitter H2S has been shown to inhibit myometrial contractility without much mechanistic insight and thus is of potential interest. Hydrogen sulfide (H2S) is produced in vivo from L-cysteine, by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). At least two enzymes degrade H2S; thiosulfate sulphur transferase (TST) and ethylmalonic encephalopathy 1 (Ethe1). Thus H2S will be regulated within cells. NaHS, which releases a rapid bolus of H2S, reduces myometrial contractility. However it is not clear if an H2S-generating system is present throughout gestation or if more physiological modes of H2S production can affect contractility. Previous studies used NaHS as a H2S producer, which is toxic and releases H2S as a non-physiological bolus and thus alternative H2S donors suitable for drug development are sought. A new H2S generating compound, GYY4137, developed to slowly release H2S which better reflects physiological conditions, appears to be such a drug. The aims of this work were to: (1) characterise the non-pregnant human myometrial tissue to determine the inherent spontaneous activity, to monitor whether the contractions were stable enough to assess H2S effects when compared to the term human myometrium. In addition, to monitoring changes in contractility in response to age, menopausal state and whether the women have endometriosis, (2) examine throughout gestation, the effects of GYY4137 on rat myometrial contractility, (3) investigate the differences in response to GYY4137 in non- pregnant versus pregnant human myometrium (4) compare GYY4137 effects to those of NaHS and L-cysteine in rat and human tissues, (5) elucidate the mechanism of H2S effects, and (6) determine the myometrial expression of enzymes governing tissue H2S levels. Non-pregnant human myometrium gave rise to stable spontaneous contractions. The older women become the lower the amplitude and area under the curve of contractions. High K+ depolarisations were also diminished with advanced age. As women reach post menopause contractions are found to decline when compared to pre-menopausal women. Women with endometriosis showed decreased amplitude with increased frequency of contractions when compared to their fertile, healthy counterparts. This finding suggested a potential involvement of altered myometrial activity in women suffering this condition. NaHS, L-cysteine and GYY produce uterine relaxation in a dose-dependent manner using rat and human tissues. NaHS and GYY4137 effects increased throughout gestation using rat myometrial tissue, possibly due to changes in H2S removal rates. TST, a H2S breakdown enzyme was not detectable in different gestation rat as well as in non- pregnant and term pregnant human myometrial tissue, implying no involvement.. Labouring rat myometrium however was not affected by either H2S producer. These data suggest that H2S contributes to uterine quiescence until labour onset. Term human myometrial contractions, both spontaneous and oxytocin-stimulated produced similar inhibitory responses to H2S producers NaHS and GYY4137. Non-pregnant spontaneous human contractions were unaffected on application of H2S producers. GYY4137 and L-cysteine decreased Ca transients, suggesting it affects L-type Ca channels, perhaps via sulfhydration of residues. These data were further supported upon use of BayK 8644 a calcium channel opener showing reduced effects of L-cysteine. KATP channels were also shown to be involved in the mechanism of H2S in the myometrium as use of KATP channel blocker glibenclamide caused reduced effects of the H2S producer GYY4137. These data suggest that H2S is an attractive target for therapeutic manipulation of human myometrial contractility and drugs such as GYY4137 will be effective. Both CBS and CSE are present in all the tissues tested in this thesis. It was also demonstrated that these enzymes were down regulated at term perhaps showing a role in preparing the myometrium for the onset of labour as the enzymes have been shown to further decline in labouring tissues. In conclusion, the work I have undertaken in this thesis strengthens the evidence of a physiologically important role for H2S in the myometrium and suggests it targets ion channels to affect calcium signalling and thus contractions.