Factors associated with premature ovarian failure, early menopause and earlier onset of menopause in Japanese women.

OBJECTIVE: The purpose of this study is to clarify the median age at natural menopause and the proportions of women with premature ovarian failure (POF) and early menopause (EM) by using Kaplan-Meier cumulative estimates and differences in reproductive and lifestyle factors associated with POF, EM and median age at menopause in a large population of Japanese women. SUBJECTS AND METHODS: This study is a cross-sectional analysis of the Japan Nurses' Health Study (JNHS). We analyzed data for 24,152 pre- and postmenopausal women who were 40 years or older at the JNHS baseline survey. RESULTS: The overall estimated median age at natural menopause was 52.1 years, and the proportions of women with POF and EM were 0.28% and 1.67%, respectively. Older generation, cigarette smoking, low body mass index, regular menstruation cycles at 18-22 years of age, nulliparity and unilateral oophorectomy were associated with earlier onset of natural menopause. Only unilateral oophorectomy was associated with increased risk of POF, and nulliparity and unilateral oophorectomy were associated with increased risk of EM. CONCLUSION: Unilateral oophorectomy is a common factor associated with earlier onset of menopause, EM and POF, although other reproductive and lifestyle factors are not associated with POF or EM.

Effects of oral estradiol and levonorgestrel on cardiovascular risk markers in postmenopausal women.

PURPOSE: This study aimed at investigating changes in postmenopausal women's cardiovascular risk markers induced by hormone therapy regimens of low (1.0 mg) or ultra-low (0.5 mg) doses of micronized estradiol (mE(2)) and levonorgestrel (LNG). METHODS: Three randomized placebo-controlled trials were reanalyzed with regard to changes in cardiovascular risk markers, such as serum lipids, lipoproteins, and coagulation parameters. Trial 1 (n = 210) was an 8-week study comparing the effects of 1.0 or 0.5 mg of unopposed mE(2) on menopausal symptoms. Trial 2 (n = 194) was a 24-week study comparing the effects of 1.0 mg of mE(2) combined with 10, 20, or 40 µg of LNG on endometrial safety. Trial 3 (n = 195) was a 52-week study comparing the effects of 1.0 or 0.5 mg mE(2) + 40 µg LNG on bone metabolism. RESULTS: 1.0 mg of unopposed mE(2) reduced low-density lipoprotein cholesterol (LDL-C) after as little as 8 weeks. 1.0 mg mE(2) for 24 weeks lowered the serum levels of total cholesterol (TC) and LDL-C, whereas the addition of LNG caused decreases in the levels of high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) in a dose-dependent fashion. 1.0 or 0.5 mg mE(2) + 40 µg LNG for 52 weeks also lowered the levels of TC, HDL-C, LDL-C, and TG. Both regimens slightly lowered antithrombin and Protein C activities within normal limits. CONCLUSIONS: Hormone therapy using 1.0 or 0.5 mg of mE(2) and LNG lowers the serum levels of TC, HDL-C, LDL-C, and TG without significantly affecting coagulation/fibrinolysis parameters.

A natural S-equol supplement alleviates hot flushes and other menopausal symptoms in equol nonproducing postmenopausal Japanese women.

OBJECTIVE: The objective of this clinical trial was to examine the efficacy of a supplement containing natural S-(-)equol, a daidzein metabolite, in reducing menopausal symptoms. METHODS: In this multicenter, double-blind placebo-controlled trial, 160 equol nonproducing, postmenopausal Japanese women who experienced at least 1 hot flush/day were randomly assigned to consume 10 mg/day S-(-)equol (n=77 women) or placebo (n=83 women) for 12 weeks. Participants completed a standardized menopausal symptom checklist and rated five common menopause symptoms by a visual analog scale at baseline, week 12, and week 18 (6-week postintervention). Physical, blood, and urine examinations were conducted. One hundred twenty-six women completed the study. RESULTS: At baseline, daily hot flush frequency was 2.9±2.1 for the S-(-)equol group and 3.2±2.4 for the placebo group. After the 12-week intervention, the S-(-)equol group had a greater decrease from baseline in hot flush frequency compared with the placebo group (-1.9±1.8/day, -58.7%, vs. -1.0±2.0/day, -34.5%, p=0.009). The severity of hot flushes and neck or shoulder muscle stiffness significantly decreased in the S-(-)equol group compared with the placebo group. No changes in clinical parameters or serious adverse effects were reported. CONCLUSIONS: This is the first trial to show beneficial effects of a 10-mg natural S-(-)equol supplement consumed daily for 12 weeks on major menopausal symptoms, specifically, hot flushes and neck or shoulder muscle stiffness, in postmenopausal Japanese women. This supplement offers a promising alternative for management of menopausal symptoms.

Association of endometriosis-related infertility with age at menopause.

OBJECTIVE: The influence of past history of infertility, which is closely associated with reproductive factors, on menopause has not been clarified. The purpose of this study was to clarify the association of reproductive factors, including past history of infertility, with menopause in Japanese women. STUDY DESIGN: This study was a cross-sectional analysis of the Japan Nurses' Health Study (JNHS) baseline survey. The JNHS is a nationwide prospective cohort study on the health of Japanese women, and the baseline survey was conducted between 2001 and 2007. MAIN OUTCOME MEASURE: We analyzed data for 24,153 pre- and postmenopausal women who were enrolled in the JNHS at the ages 40-59 years. The main outcome measure was menopause. RESULTS: Reproductive factors influencing the onset of natural menopause were past history of infertility [relative risk (RR) (age-adjusted odds ratio and 95% confidence interval): 1.28 (1.10-1.48)], past history of endometriosis: 1.32 (1.07-1.64), age at menarche: 0.919 (0.886-0.953) and number of pregnancies: 0.943 (0.911-0.976). Endometriosis as the cause of infertility was significantly associated with earlier onset of menopause (3.43 [2.17-5.44]) after adjustment for age. The strong association of past history of infertility related to endometriosis was still significant after multivariate adjustment. CONCLUSION: Menopause in women who have a past history of infertility, especially in those who have suffered from endometriosis, is significantly earlier than that in women without such a history.

Relationship between estrogen, vasomotor symptoms, and heart rate variability in climacteric women.

The present study investigated resting cardiac autonomic function in Japanese climacteric women using heart rate variability (HRV) power spectral analysis to evaluate the relationship between HRV indices and estrogen, as well as the ability of each HRV index to predict vasomotor symptoms. Forty-five peri- and postmenopausal women completed a questionnaire about the presence of vasomotor symptoms (hot flashes, sweating). To analyze the relationship between HRV and hot flashes or sweating, we classified subjects into groups based on symptoms and combinations of symptoms: no hot flashes (H1), hot flashes (H2), non-sweating (S1), sweating (S2), neither hot flashes nor sweating (V1), either hot flashes or sweating (V2), and both hot flashes and sweating (V3). Values for total power and the low-frequency component of HRV were significantly lower in the H2 group than in H1 (p < 0.05); values for total power and the high-frequency and low-frequency components of HRV were significantly lower in group S2 than S1 (p < 0.05); and values for total power and the high-frequency and low-frequency component of HRV were significantly lower in groups V2 and V3 compared to V1 (p < 0.05). Clinical diagnosis of climacterium relies upon subjective complaints of patients. Our findings suggest that HRV indices may help to evaluate vasomotor symptoms.

Equol improves menopausal symptoms in Japanese women.

It has been well documented that the frequency of vasomotor menopausal symptoms, such as hot flashes and night sweats, of Japanese menopausal women is less than that of Western women. High intake of soy isoflavones in the traditional Japanese diet has been postulated as the possible explanation of the difference. Epidemiological studies have reported that the content of equol, which is a biologically active metabolite of the isoflavone, daidzein, is lower in the women who complain of severe vasomotor symptoms. To investigate the involvement of equol in the manifestation of menopausal symptoms, especially vasomotor symptoms, and the possible therapeutic role of a supplement containing equol (natural S-equol developed by Otsuka Pharmaceutical) on the menopausal symptoms of Japanese women, 3 randomized clinical trials were conducted. The studies indicated that a daily dose of 10 mg of natural S-equol improved menopausal symptoms. In the confirmation study, menopausal women who were equol nonproducers who consumed 10 mg/d of natural S-equol for 12 wk had significantly reduced severity and frequency of hot flashes as well as a significant reduction in the severity of neck or shoulder stiffness. The equol-ingesting group also showed trends of improvement in sweating and irritability and a significant improvement in the somatic category symptoms. Thus, it is concluded that the supplement containing natural S-equol, a novel soybean-derived functional component, has a promising role as an alternative remedy in the management of menopausal symptoms.

Long-term use of dienogest for the treatment of endometriosis.

AIM: To investigate the safety and efficacy of 52 weeks of dienogest treatment in patients with endometriosis. METHODS: One hundred and thirty-five patients with endometriosis received 2 mg of dienogest orally each day for 52 weeks. Adverse drug reactions and bone density were evaluated. Global improvement was assessed based on the changes in severity categories of five subjective symptoms during non-menstruation (lower abdominal pain, lumbago, dyschezia, dyspareunia, and pain on vaginal examination) and two objective findings (induration involving the pouch of Douglas and limited uterine mobility). RESULTS: The most common adverse drug reactions included metrorrhagia (71.9%), headaches (18.5%), and constipation (10.4%). No clinically significant changes were noted in the incidence or severity of reactions associated with the course of the treatment period (52 weeks). Changes from the baseline bone mineral density of the lumbar spine measured by dual-energy X-ray absorptiometry were -1.6 +/- 2.4% and -1.7 +/- 2.2% (mean +/- standard deviation) at 24 and 52 weeks, respectively, which were statistically significant decreases; however, there was no cumulative decrease. The proportions of patients assessed as marked or moderate improvement in terms of global improvement were 72.5% (95/131 cases) at 24 weeks and 90.6% (106/117 cases) at 52 weeks. CONCLUSION: The long-term effect of dienogest on bone mineral density was slight, whereas the efficacy increased cumulatively.

Dienogest is as effective as intranasal buserelin acetate for the relief of pain symptoms associated with endometriosis--a randomized, double-blind, multicenter, controlled trial.

OBJECTIVE: To compare the efficacy and safety of dienogest (DNG) with intranasal buserelin acetate (BA) in patients with endometriosis. DESIGN: Phase III, randomized, double-blind, multicenter, controlled trial. SETTING: Twenty-four study centers in Japan. PATIENT(S): Two hundred seventy-one patients with endometriosis. INTERVENTION(S): Dienogest (2 mg/day, orally) or BA (900 microg/day, intranasally) for 24 weeks. MAIN OUTCOME MEASURE(S): The pre- to posttreatment changes in the scores of five subjective symptoms during nonmenstruation (lower abdominal pain, lumbago, defecation pain, dyspareunia, and pain on internal examination) and two objective findings (induration in the pouch of Douglas and limited uterine mobility). RESULT(S): Dienogest reduced the scores of all symptoms and findings at the end of treatment, and the mean changes in the scores of all symptoms and findings except induration in the pouch of Douglas were comparable to those obtained with BA. Compared with BA, DNG was associated with irregular genital bleeding more frequently and with fewer hot flushes. The reduction in bone mineral density (BMD) during DNG treatment was significantly lower than that during BA treatment. CONCLUSION(S): DNG is as effective as intranasal BA in alleviating endometriosis, and causes less BMD loss.

Design of the Japan Nurses' Health Study: a prospective occupational cohort study of women's health in Japan.

The Japan Nurses' Health Study (JNHS) is a prospective occupational cohort study investigating the effects of lifestyle and healthcare practices on women's health. It was initiated in 2001, with a six-year entry period and a proposed ten-year follow-up. Participants comprise female registered nurses, licensed practical nurses, public health nurses, and midwives, aged 25 yr or over at the baseline survey. Participants were recruited in cooperation with the Japanese Nursing Association and the Japan Menopause Society. A self-administered baseline questionnaire was distributed, requesting demographic information, lifestyle factors, pharmaceutical drug use, physical condition, reproductive health, and disease history. A total of 49,914 women from all 47 prefectures in Japan responded to the baseline survey. Among them, approximately 18,000 agreed to be followed-up, and returned signed informed-consent sheets, together with their completed baseline questionnaires. Changes in lifestyle, healthcare, incidence of disease, and health outcomes over time will now be studied. The cohort receives annual JNHS newsletters and biennial follow-up questionnaires by mail.

Prevalence of diseases and statistical power of the Japan Nurses' Health Study.

The Japan Nurses' Health Study (JNHS) is a long-term, large-scale cohort study investigating the effects of various lifestyle factors and healthcare habits on the health of Japanese women. Based on currently limited statistical data regarding the incidence of disease among Japanese women, our initial sample size was tentatively set at 50,000 during the design phase. The actual number of women who agreed to participate in follow-up surveys was approximately 18,000. Taking into account the actual sample size and new information on disease frequency obtained during the baseline component, we established the prevalence of past diagnoses of target diseases, predicted their incidence, and calculated the statistical power for JNHS follow-up surveys. For all diseases except ovarian cancer, the prevalence of a past diagnosis increased markedly with age, and incidence rates could be predicted based on the degree of increase in prevalence between two adjacent 5-yr age groups. The predicted incidence rate for uterine myoma, hypercholesterolemia, and hypertension was > or =3.0 (per 1,000 women, per year), while the rate of thyroid disease, hepatitis, gallstone disease, and benign breast tumor was predicted to be > or =1.0. For these diseases, the statistical power to detect risk factors with a relative risk of 1.5 or more within ten years, was 70% or higher.

Quality of life in gynecological inpatients undergoing surgery.

This study investigates the changes in the quality of life (QOL) of gynecological patients undergoing surgery, and the relationship between these changes and clinical/demographic factors. Ninety patients were examined on three occasions using the Japanese version of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-core30 (EORTC QLQ-C30). Although the global QOL and physical function decreased before discharge, the emotional function was lowest before surgery. There was no difference between the benign and malignancy groups in most QOL subscales. With regard to the relationship between global QOL and physical function before discharge and other demographic/clinical factors, multiple regression analysis suggested that these were explained by employment status, benign/malignancy, region of tumor, chemotherapy, postoperative complication, and psychological symptoms during hospitalization.

Testosterone inhibits matrix metalloproteinase-1 production in human endometrial stromal cells in vitro.

Androgen receptor (AR) is reported to be expressed in human uterine endometrium, but not much information is available on the role of androgens in human endometrium. The purpose of this study is to investigate the role of androgens in the regulation of matrix metalloproteinase (MMP)-1, which is one of the important MMPs for menstruation and embryo implantation in human endometrium. Human endometrial stromal cells (HESCs) were obtained from human endometrium by enzymatic dissociation method. Purified HESCs were incubated with 17beta-estradiol (E2), testosterone, or E2 + testosterone. Progestins (natural progesterone or medroxyprogesterone acetate) or vehicle (dimethyl sulfoxide) were also added to the media instead of testosterone. Furthermore, hydroxyflutamide (FLU),a specific AR antagonist, was also supplemented to cultured media. The amounts of MMP-1 in cultured media and in HESC lysates were examined by ELISA measurements and western blotting analysis respectively. The expression of ARmRNA in HESCs RNA was analyzed by RT-PCR. Testosterone significantly inhibited MMP-1 in both cultured media and cell lysates in a dose-dependent manner. Progestins also inhibited MMP-1. Furthermore, FLU completely recovered the decrease of MMP-1 induced by testosterone. ARmRNA was detected in all HESCs RNA. The present study demonstrated that the secretion and production of MMP-1 in HESCs in vitro were inhibited by testosterone through androgen receptors in a manner similar to that seen for progesterone. These findings indicate that androgen may play an important role in morphological and functional changes of human endometrium.

Emotional state and coping style among gynecologic patients undergoing surgery.

The aim of the present study was to investigate changes in emotional state and the relationship between emotional state and demographic/clinical factors and coping style among gynecologic patients undergoing surgery. Using the Japanese version of the Profile of Mood States (POMS), 90 patients (benign disease: 32, malignancy: 58) were examined on three occasions: before surgery, before discharge, and 3 months after discharge. They were also examined using the Coping Inventory for Stressful Situations (CISS) on one occasion before discharge. The scores for the subscales depression, anger, and confusion were the highest after discharge while those for anxiety were the highest before surgery. The average scores of the POMS subscales for all subjects were within the normal range. With regard to the relationship between these emotional states and other factors, multiple regressions showed that the principal determinants of anxiety before surgery were religious belief, psychological symptoms during hospitalization and emotion-oriented (E) coping style; further, it was found that depression after discharge could be explained by chemotherapy, duration of hospitalization, and E coping style. The principal determinants of anger after discharge and vigor before surgery were length of education and E coping style, and severity of disease, chemotherapy, E coping style and task-oriented coping style, respectively. Those of post-discharge fatigue and confusion were length of education, psychological symptoms, and E coping style. In summary it is suggested that the following should be taken into account in patients undergoing gynecologic surgery: anxiety before surgery, depression, anger, and confusion after surgery, including coping styles.

Dysembryoplastic neuroepithelial tumor in pregnancy.

BACKGROUND: Dysembryoplastic neuroepithelial tumor is a rare, low-grade brain tumor that is characterized by intractable, partial seizures of juvenile onset. CASE: A 36-year-old pregnant woman with dichorionic, diamniotic twins temporarily lost consciousness at 27 weeks of gestation. Magnetic resonance imaging evaluation showed a 3-cm multicystic nodule in her right temporal lobe that was diagnosed, together with her history of recurrent anxiety attacks, as dysembryoplastic neuroepithelial tumor. The lesion did not enlarge during pregnancy, and healthy male infants were delivered by cesarean at 36 weeks. The tumor was successfully removed 2 months postpartum, and the patient has experienced no further seizures. CONCLUSION: Dysembryoplastic neuroepithelial tumor was conservatively managed during pregnancy without neurosurgical intervention.

Body fat and blood lipids in postmenopausal women are related to resting autonomic nervous system activity.

The present study investigated the activity of the autonomic nervous system (ANS), a major influence in normal physiological function, and its association with unfavorable postmenopausal states in body composition, lipid and/or glucose metabolism, or cardiovascular profiles. Body composition, blood pressure, and blood profiles of lipid and glucose of 175 postmenopausal women were measured. Resting ANS activity was assessed by heart rate variability (HRV) power spectral analysis. To scrutinize the influence of ANS activity levels on postmenopausal obesity-related factors, we divided the subjects into a low group ( < 220 ms(2)) and a high group ( > 220 ms(2)), based on the total power of HRV. Low-frequency (P < 0.01) and high-frequency power (P < 0.01) were both significantly lower in the low group. No significant difference was found in age, age at menopause, or years after menopause between the two groups. In contrast, body mass index (P < 0.05), percentages of body fat (P < 0.01), and systolic (P < 0.01) and diastolic (P < 0.01) blood pressure were significantly greater in the low group. As to blood lipid profiles, triglycerides (P < 0.05), total cholesterol (P < 0.05), and low-density lipoprotein cholesterol (P < 0.05) were significantly higher in the low group. Our findings indicate that reduced sympatho-vagal activity is associated with higher postmenopausal body fat content, blood pressure, and blood lipid concentrations. This study further implies that such autonomic depression could be a crucial risk factor in undermining the health and, ultimately, the quality of life, of postmenopausal women.

Transient ADC change precedes persistent neuronal death in hypoxic-ischemic model in immature rats.

A brief ischemia causes delayed neuronal death (DND) in some areas vulnerable to ischemia. Additionally, it causes a transient reduction in the apparent diffusion coefficients (ADCs) obtained from diffusion-weighted magnetic resonance imaging (DWI), which is a powerful tool to detect ischemic changes in the brain at a very early stage. The present study examined long-term histopathological changes in the hippocampal neurons up to 30 days after a very mild hypoxic-ischemic (HI) insult in immature rats. Three-week-old male rats were subjected to 15- and 30-min HI insults (15-min HI and 30-min HI) and serial DWI was performed. Only animals whose ADC reduction pattern was transient were examined histopathologically. ADCs decreased significantly during the insult, and the ADC values of 30-min HI group were significantly lower than those of 15-min HI group. Ischemic neuronal changes were observed up to 30 days after the insult in 30-min HI group, although ADCs in the chronic stage were within the normal range. In addition, neuron density in 30-min HI group was significantly lower in the chronic stage (on days 14 and 30) than in 15-min HI group. A very mild hypoxia-ischemia followed by a transient ADC reduction causes persistent neuronal death, which can be predicted by measuring ADCs during the acute insult.

Involvement of arginase in regulating myometrial contractions during gestation in the rat.

This study was designed to investigate the role of arginase in regulating myometrial contractions during gestation in the rat. Arginase activity in the myometrium was significantly decreased during the 7th-21st day of gestation, with the lowest value on the 14th day. However, the enzyme activity became significantly higher at term gestation (22nd day) than that in the non-pregnant myometrium. Arginase I protein was undetectable in the non-pregnant myometrium, at 7th and 14th day of gestation and after delivery. A slight positive signal for arginase I was detectable at 21st day of gestation. However, the protein was clearly up-regulated at term gestation (22nd day), although arginase II protein was down-regulated during gestation, with the lowest value on the 14th day. Gestational changes in arginase activity negatively correlated with those in cyclic GMP production, whereas the changes positively correlated with those in endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) contents. Myometrial arginase activity was inhibited by N(G)-hydroxy-L-arginine as an intermediate of NO production from L-arginine in a concentration-dependent manner. Both basal and stimulated guanylyl cyclase activities were enhanced at mid- and reduced at term gestation and after delivery, thereby partly increasing cyclic GMP production at mid- and partly decreasing the nucleotide production at term gestation and after delivery. These results suggest that the decreased arginase activity at mid-gestation possibly results from the down-regulation of arginase II protein. Whereas, the enhanced overall arginase activity at term gestation seems to be because of the induced functional arginase I in concert with the attenuated arginase II expression. The enhanced arginase activity at term gestation would be implicated in increasing myometrial contractions mediated by the increased ET-1. The increased peptide production at term gestation is possibly because of the reduced cyclic GMP production resulting from enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity.

Estrogen replacement effectively improves the accelerated intimal hyperplasia following balloon injury of carotid artery in the ovariectomized rats.

Present experiments were designed to investigate the effects of ovariectomy (OVX) and estrogen replacement (ER) on neointimal formation after balloon injury of the rat carotid artery. Young adult female rats were divided into 3 groups of sham operation (control), ovariectomy, and ovariectomy plus estrogen replacement. Estrogen replacement was initiated by implanting a sustained release pellet containing water-soluble 17beta-estradiol 1 week after the ovariectomy. Carotid arteries were harvested 2 weeks after the balloon injury for determinations. The balloon injury caused intimal hyperplasia, which was accompanied by the impaired endothelium-dependent relaxation and cyclic GMP production, and accumulation of asymmetric dimethylarginine (ADMA) as an endogenous NOS inhibitor. Bilateral ovariectomy accelerated the intimal hyperplasia. The acceleration was accompanied by the enhanced impairment of NO production, attenuated reendothelialization, and enhanced accumulation of ADMA. The estrogen replacement improved the accelerated intimal hyperplasia with concomitant improvement of the impaired NO production and accumulated asymmetric dimethylarginine, and facilitated reendothelialization. These results suggests that the enhanced impairment of NO production, which possibly results from the accumulated asymmetric dimethylarginine and lack of reendothelialization, may contribute to the acceleration of intimal hyperplasia by ovariectomy and that estrogen replacement effectively improves the intimal hyperplasia by restoring the impaired NO production through reducing endogenous NOS inhibitor and facilitating reendothelialization.

Involvement of GATA-4/-5 transcription factors in ovarian carcinogenesis.

To clarify the role of GATA transcription factors in ovarian carcinogenesis, we analyzed the expression and methylation states of GATA-4/-5/-6 in eight human ovarian cancer cell lines. GATA-4/-5 were methylated in three and two cell lines without their expression, respectively. Methylation of GATA-4/-5 was also detected in nine and five of 15 primary ovarian cancers, respectively. GATA-6 was not methylated in any cases. We transiently over-expressed GATA-5 in the JHOC-5 cell line using an adenovirus system, resulting in that apoptosis was induced and apoptosis-related genes, such as Apaf-1, were up-regulated. These data suggest that GATA-4/-5 may be involved in ovarian carcinogenesis.

Mediators involved in decreasing peripheral vascular resistance with carbachol in the rat hind limb perfusion model.

We examined the involvement of nitric oxide (NO) and/or endothelium-derived hyperpolarizing factor (EDHF) in decreasing peripheral vascular resistance in the rat hind limb perfusion model and analyzed the identity of EDHF in this model. The potency of carbachol (CCh) to produce relaxation was quantitatively similar to sodium nitroprusside (SNP). CCh-induced relaxation was abolished after endothelial denudation, but resistant to nitroarginine and indomethacin. The relaxation was inhibited by tetraethylammonium, ouabain, charybdotoxin plus apamin, and under depolarization. SNP-induced relaxation was accompanied by increased cGMP production, which was inhibited by ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-l-one). Although CCh produced a similar extent of relaxation to SNP, the cGMP level was 24 times lower than that with SNP. Low KCl produced a definite relaxation, which was inhibited by ouabain, but independent of NO, prostacyclin, and endothelium. 1-EBIO (1-ethyl-2-benzimidazolinone) as an activator of IK(Ca) channel also produced a concentration-dependent relaxation, which was inhibited by charybdotoxin, ouabain, and depolarization, but independent of NO and prostacyclin. Clotrimazole and 17-octadecynoic acid as inhibitors of P(450) monooxygenase inhibited the CCh-induced relaxation. Meanwhile, catalase at a concentration sufficient to inhibit H(2)O(2)-induced relaxation did not exert definite inhibition of the CCh-induced relaxation. These results suggest that CCh produces an endothelium-dependent, EDHF-dependent, and NO-cGMP-independent relaxation and that K(+) and metabolite(s) of P(450) monooxygenase possibly play an important role for this relaxation.