Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia

Molecular Human Reproduction vol.5 no.11 pp. 1048–1054, 1999 Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia S.Zervou1, L.D.Klentzeris2 and R.W.Old1,3 1Cell and Molecular Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, and 2Cardiff Assisted Reproduction Unit, University Hospital of Wales, Heath Park, Cardiff CF4 4XW, UK 3To whom correspondence should be addressed Menorrhagia (excessive menstrual bleeding) is a common clinical problem of unknown aetiology. The freeradical and vasodilator nitric oxide (NO) relaxes the myometrial smooth muscle and is a strong candidate for the cause of excessive blood loss in menorrhagic patients. The aim of this study was to measure NO production in women with and without menorrhagia to detect nitric oxide synthase (NOS) isoforms in uterine cells and to investigate any steroid effects on myometrial NOS expression. We showed for the first time that menorrhagic endometrium produces significantly higher amounts of NOx (the sum of NO2– and NO3–) than control endometrium (P < 0.01). Inducible NOS (iNOS) protein was detected by immunoblotting in endometrial and myometrial tissue extracts. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) experiments revealed an induction of myometrial smooth muscle endothelial NOS (eNOS) expression by progesterone and 17β-oestradiol, while myometrial iNOS expression was unaffected by steroid hormones. These results are consistent with the hypothesis that NO plays a role in excessive menstrual bleeding and provide the first evidence on steroid regulation of eNOS in the human non-pregnant uterus. Key words: 17-oestradiol/menorrhagia/nitric oxide/progesterone Introduction Menorrhagia, defined as excessive menstrual blood loss (Hallberg et al., 1966), remains a challenge at the therapeutic and research level. In the UK, 31% of reproductive age women suffer from menorrhagia and 80–90% of these women receive unnecessary and ineffective treatment (Rees, 1991). More than 80% of menorrhagic women do not have an organic disease, such as uterine tumours (polyps, fibroids, carcinomas), infection or pregnancy. Recent work has shown that blood clotting disorders were responsible for a proportion of menorrhagia cases, but not for the majority of them (Kadir et al., 1998). Much attention has been focused on the uterus itself, and it seems that locally derived factors and vascular changes control the amount of blood loss during menstruation. The correlation between uterine artery pulsatility index and amount of menstrual blood loss, suggests that vascular factors may be involved in the pathogenesis of menorrhagia (Hurskainen et al., 1999). Previous studies have also shown increased concentrations of prostaglandins F2α and E2 in menstrual fluid of women with menorrhagia (Rees et al., 1984b), and increased prostaglandin E2 receptors in myometrium, correlated with menstrual blood loss (Cameron et al., 1988). The free radical gas nitric oxide (NO) is produced after catalytic oxidation of L-arginine by the enzyme nitric oxide synthase (NOS). NOS occurs in three distinct isoforms: two constitutive, Ca21-dependent, isoforms endothelial NOS (eNOS) and neuronal NOS (nNOS), and the Ca21-independent inducible NOS (iNOS) (Moncada et al., 1991). nNOS (NOS 1048 I) was first identified in neuronal tissues and is expressed constitutively, like eNOS (NOS III) which was first identified as a constitutive enzyme in vascular endothelial cells. iNOS (NOS II), first described in macrophages, can be induced by cytokines or bacterial lipopolysaccharides (LPS) (Fosterman et al., 1991). NO action has been associated with platelet aggregation, relaxation of smooth muscles in the gastrointestinal and respiratory tract, neurotransmission and immunoactivation. Such regulatory functions of NO are mediated via cyclic guanosine monophosphate (cGMP) (Moncada et al., 1991). NO production has been assayed in the pregnant rat uterus, by measuring its oxidation products nitrite (NO2–) and nitrate (NO3–), and an L-arginine-NO-cGMP pathway was found to exist throughout pregnancy (Yallampalli and Garfield, 1993; Yallampalli et al., 1993; Jaing et al., 1996). In the rat and human myometrium, an L-arginine-NO system has an important role in inhibiting uterine contractility and possibly, maintaining pregnancy (Izumi et al., 1993). However, NOS expression decreases at term, when myometrial contractions lead to labour (Weiner et al., 1994b; Buhimschi et al., 1996; Dong et al., 1996; Bansal et al., 1997; Riemer et al., 1997; Norman et al., 1999). Various studies have suggested a role of NO in regulation of menstrual blood loss (Norman, 1996; Norman and Cameron, 1996; Telfer et al., 1997), and more recent work showed that NOS activity is increased during menstruation, suggesting a role of NO in menstrual blood release and in endometrial apoptosis (Tschugguel et al., 1999). As well as affecting vasodilation by relaxing the vascular smooth muscle, uterine-derived NO appears to act as a © European Society of Human Reproduction and Embryology NO in the uterus of women with menorrhagia suppressor of smooth muscle contractility, by maintaining uterine quiescence during pregnancy and relaxing the nonpregnant human myometrium (Cameron and Campbell, 1998). Ca21-dependent isoforms eNOS and nNOS were previously detected in human pregnant and non-pregnant endometrium and myometrium, while iNOS was detected in the pregnant and non-pregnant endometrium and pregnant myometrium (Bansal et al., 1997; Telfer et al., 1997; Norman et al., 1999). In view of the suggested roles of NO in pregnancy and menstruation, it is of interest to study eNOS and iNOS expression. eNOS is a known vasodilator of myometrial vasculature and neighbouring smooth muscle cells (Tschugguel et al., 1997) and iNOS was reported to have a steroid-mediated action in the pregnant rat uterus (Ali et al., 1997). Data on the steroid regulation of NOS isoforms in the human non-pregnant uterus is still unclear. Ca21-dependent NOS isoforms were reported to be induced by oestrogens in pregnant guinea pigs (Weiner et al., 1994a) and in non-pregnant sheep (Figueroa and Massmann, 1995). A progesterone-, but not 17β-oestradiol-mediated regulation of vascular adaptations by NO was reported during pregnancy in rats (Buhimschi et al., 1995). Progesterone was also suggested to be the steroid hormone with the major steroid action on vascular tension during pregnancy (Liao et al., 1996). Inducible NOS was found to play a role in initiation of labour and cervical ripening in rats and progesterone was found to differentially regulate iNOS in the rat cervix and uterus (Ali et al., 1997). Recently, a synergistic role of NO and progesterone during the establishment of pregnancy in the rat was demonstrated (Chwalisz et al., 1999). However, other workers (Telfer et al., 1997) observed neither a role of exogenous progestagens (...truncated)