Inhibition of volume-regulated anion channels in cultured endothelial cells by the anti-oestrogens clomiphene and nafoxidine. (1/8)

1. We have used the whole-cell patch clamp technique to study the effect of the partial anti-oestrogens clomiphene and nafoxidine, the pure anti-oestrogens ICI 182,780 and RU 58,668 and the oestrogen ss-estradiol, on the volume-regulated anion channel (VRAC) in cultured pulmonary artery endothelial (CPAE) cells. 2. In contrast to the pure anti-oestrogens and ss-estradiol, clomiphene and nafoxidine potently inhibited the volume-sensitive chloride current, I(Cl,swell), activated by challenging CPAE cells with a 25% hypotonic solution. For clomiphene, the estimated IC(50) and Hill coefficient were 1.03+/-0.14 microM and 1.40+/-0.21 respectively. In the case of nafoxidine, these values were 1.61+/-0.29 microM and 1.24+/-0.19. 3. The inhibition induced by the pure enantiomers of clomiphene, zuclomiphene and enclomiphene, was not different from that of the racemic mixture, indicating that the interaction between clomiphene and VRAC is not stereoselective. 4. Clomiphene and nafoxidine inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.98+/-0.17 and 1.66+/-0.21 microM respectively, concentrations similar to those for half-maximal block of VRAC. 5. In conclusion, the nonsteroidal partial anti-oestrogens nafoxidine and clomiphene are potent inhibitors of volume-regulated anion channels. The inhibition by clomiphene is not stereoselective and occurs at concentrations close to therapeutically relevant concentrations. Finally, both drugs inhibit the proliferation of endothelial cells.  (+info)

Enclomiphene does not alter the postpartum interval of suckled beef cows. (2/8)

The objective of this study was to determine whether an antiestrogen (enclomiphene) would shorten the interval to first estrus and conception in postpartum beef cows. Sixty postpartum Angus beef cows were stratified by age, body condition, and calving date and were randomly assigned to one of two treatment groups. Group 1 cows (n = 24) received three silastic implants, each containing 150 mg of enclomiphene, on d 20 postpartum. Implants were removed on d 30 postpartum. Group 2 cows (n = 28), received empty implants and served as controls. Cows were artificially inseminated at first detected estrus. Estrus detection and ovulation were further verified by increased serum progesterone. Concentrations and pulse frequencies of LH were determined from blood samples collected at 15-min intervals for 6 h on d 20, 25, 30, and 40 postpartum. Hypothalami and pituitaries were collected from four cows in each treatment group on d 30 postpartum and analyzed for concentrations of estradiol receptors. Concentrations of total and unoccupied hypothalamic and pituitary estradiol receptors were reduced by enclomiphene. Neither concentrations nor pulse frequencies of LH differed significantly between treatment groups on any of the 4 d. Days to first estrus did not differ (P greater than .05) between enclomiphene-treated (57 +/- 6; n = 24) and control (56 +/- 4; n = 28) cows. Days to conception did not differ between treated (81 +/- 9) and control (79 +/- 8) cows. The dose of enclomiphene used in this study reduced hypothalamic and pituitary estrogen receptors but did not alter secretion of LH or days to first estrus in the postpartum beef cow.  (+info)

CYP2D6 is primarily responsible for the metabolism of clomiphene. (3/8)

Clomiphene is a first line treatment for anovulation, a common cause of infertility. Response to clomiphene is variable and unpredictable. Tamoxifen is structurally related to clomiphene, and also shows considerable variation in response. CYP2D6 and CYP3A4 are major contributors to the metabolism of tamoxifen. The aim of the present work was to define the role of CYP2D6 and CYP3A4 in the in vitro metabolism of enclomiphene, regarded by some as the more active isomer of clomiphene. Enclomiphene (25 microM) was incubated with human liver microsomes (from 4 extensive (EM) and 1 poor metaboliser with respect to CYP2D6) and with microsomes from lymphoblastoid cells expressing CYP2D6. Microsomes from all the EM livers and recombinant CYP2D6 metabolised enclomiphene (the disappearance of drug ranged from 40-60%). No metabolism was detected in microsomes from the PM liver. Quinidine (1 microM) completely inhibited the metabolism of enclomiphene by all the EM livers and by recombinant CYP2D6 (p<0.001, one way ANOVA). Ketoconazole (2 microM) had no significant effect on enclomiphene metabolism in 3 out of the 4 EM livers. The extent of enclomiphene metabolism was correlated with the amount of CYP2D6 present (p<0.001, Pearson correlation test). The findings indicate that CYP2D6 is primarily responsible for the metabolism of enclomiphene.  (+info)

Effects of enclomiphene on estradiol-induced changes in LH secretion in ewes. (4/8)

Experiments were conducted to characterize the ability of the antiestrogen enclomiphene (ENC) to block the effects of estradiol on secretion of LH in ovariectomized ewes. To determine whether ENC could block an estradiol-induced LH surge, ewes (n = 4/group) were administered 10 to 250 mg ENC followed 30 min later by 25 micrograms estradiol. Ten or 25 mg ENC suppressed the estradiol-induced LH surge in one of four ewes, whereas 100- or 250-mg doses suppressed the LH surge in three and four of four ewes, respectively. In ewes that received a single treatment of 100 mg ENC plus 25 micrograms estradiol, serum concentrations of LH remained below 1 ng/ml for 3 wk. Compared with untreated ewes, the number of pituitary GnRH receptors was elevated (P less than .05) at 12 d and 28 d, but pituitary content of LH had decreased (P less than .05) by 28 d in ewes treated with 100 mg ENC. To determine whether ENC could block the inhibitory effects of estradiol on serum concentrations of LH, ewes received injections of .03, .1, 1 or 10 mg ENC every 4 d. Half the ewes treated with each dose also received estradiol implants. Injection of .03, .1 or 1 mg ENC alone did not affect serum concentrations of LH, whereas the 10-mg dose decreased serum concentrations of LH below 1 ng/ml by wk 1 of treatment. No dose prevented the inhibition of serum concentrations of LH caused by estradiol implants. In ovariectomized ewes, ENC was antagonistic to estradiol; it prevented the positive effects of estradiol required to induce an LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)  (+info)

Postpartum interval in beef cows shortened by enclomiphene. (5/8)

This study was designed to determine whether an anti-estrogen can block the negative effect of estrogens on luteinizing hormone (LH) release and therefore decrease the postpartum interval in suckled beef cows. In Exp. I, eight suckled postpartum beef cows were randomly assigned to treatment and control groups. Treatment cows received 1 g/d clomiphene citrate (im) from d 21 to 28 postpartum, while control cows were injected with saline. On d 28 postpartum, there was no difference (P greater than .05) in mean total and basal LH concentrations or LH pulse frequency between treatment and control cows. All control cows exhibited estrus on d 52 +/- 3; treatment cows exhibited estrus on d 134 +/- 12 (P less than .05). In Exp. II, 17 suckled cows were randomly assigned to three treatment groups: 1) control group (n = 6) receiving one empty implant, 2) 10-cm enclomiphene implant group (n = 5) and 3) 30-cm enclomiphene implant group (n = 6). The silastic implants were placed sc on d 20 and removed on d 29 postpartum. Mean total LH concentrations during d 24 to 29 postpartum in the 30-cm enclomiphene implant group were higher than the 10-cm implant (P less than .05) and control group (P less than .05). The postpartum period in the 30-cm enclomiphene group (45 +/- 6 d) was shorter than the 10-cm implant (94 +/- 24 d) and control (96 +/- 20 d) groups (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)  (+info)

Gap junction modulation in rat uterus. II. Effects of antiestrogens on myometrial and serosal cells. (6/8)

The ability of several triphenylethylene antiestrogens to affect the modulation of gap junctions in rat uterine myometrial and serosal cells was examined in animals 60 days following hypophysectomy. Five daily injections of enclomiphene, zuclomiphene, tamoxifen, nafoxidine, CI 628 or CI 680 (500 micrograms per injection) promote uterine luminal epithelial cell hypertrophy characteristic of exogenous estrogen administration. These same compounds, however, fail to induce myometrial cell or increase the number of serosal cell gap junctions, respectively, which is also characteristic of exogenous estrogen treatment. Pretreatment of animals with antiestrogens blocks the ability of estradiol benzoate (E2 B) to induce gap junction formation in myometrial cells when followed by combined injections of E2 B and antiestrogens (both 250 micrograms) administered daily for 5 days. Therefore, with respect to the parameter of myometrial cell gap junction stimulation, all of the antiestrogens examined act as pure estrogen antagonists. These same antiestrogen pretreatments only weakly antagonized the ability of E2 B to modulate serosal cell gap junction membrane. These studies indicate the presence of different mechanisms for the estrogenic modulation of gap junctional membrane in myometrial and serosal cells.  (+info)

The inhibition of the estrogen receptor's positive cooperative [3H]estradiol binding by the antagonist, clomiphene. (7/8)

The calf uterine estrogen receptor showed positive cooperativity of [3H]estradiol equilibrium binding; the Scatchard plot was convex and the Hill coefficient was 1.69 +/- 0.021 (n = 14). The effects of the estrogenic antagonists, zuclomiphene (cis-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate) and enclomiphene (trans-23-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate), on the positive cooperativity of [3H]estradiol binding were measured by titrating the receptor with a variable concentration of [3H]estradiol and antagonist while maintaining a constant excess in a specific ratio of the antagonist to the [3H]estradiol. With a 45- to 55-fold molar excess of zuclomiphene or an 820- to 900-fold molar excess of enclomiphene above the [3H]estradiol concentration, the receptor's positive cooperative [3H]estradiol binding was inhibited. A transition from a convex to a linear Scatchard plot and a decrease in the Hill coefficient from 1.69 to 1.10 +/- 0.02 (n = 6) were induced. The specifically bound [3H]estradiol was inhibited 43 to 50% by the zuclomiphene and enclomiphene. The addition of unlabeled estradiol in a 1- or 2.3-fold molar excess above that of the [3H]estradiol concentration produced a 50 to 75% competitive displacement of the specifically bound [3H]estradiol; nevertheless, the Scatchard plot remained convex and the Hill coefficient was 1.74 and 1.80, respectively. Thus, inhibition of the positive cooperativity of [3H]estradiol binding by the clomiphene isomers was not due to dilution of the specifically bound [3H]estradiol by the antagonist. These data demonstrate that there are two molecular mechanisms by which an estrogen antagonist interferes with the function of the receptor: as a competitor, thus blocking the estrogen receptor's binding site to an agonist, and second by inducing conformational changes that inhibit site:site interactions and receptor activation.  (+info)

Effects of enclomiphene and zuclomiphene on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of small and large ovine luteal cells. (8/8)

We examined the effects of enclomiphene and zuclomiphene, alone and in combination with oestradiol, on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of both large (granulosa-lutein) and small (theca-lutein) ovine luteal cells. Isolated large and small luteal cells derived from intact, enucleated ovine corpora lutea were incubated for 48-120 h with or without 22R-hydroxycholesterol or pregnenolone (2.5 microM) and a range of enclomiphene, zuclomiphene, and/or oestradiol concentrations (3-100 microM), both with and without ovine luteinizing hormone (100 ng/ml). Spent media were assayed in duplicate for progesterone content by radioimmunoassay. Enclomiphene, zuclomiphene, and oestradiol exhibited equivalent dose-dependent inhibitory effects on basal and gonadotrophin-stimulated small and large ovine luteal cell progesterone secretion under all substrate conditions. Both cell types became more sensitive to clomiphene inhibition with increasing time in culture. In combined treatments, the effects of oestradiol and either enclomiphene or zuclomiphene became additive in longer-term cultures and were never antagonistic. In this model system, (i) clomiphene, like oestradiol, appears to inhibit 3beta-hydroxysteroid dehydrogenase activity, (ii) both stereoisomers act as oestrogen agonists, (iii) neither demonstrates any anti-oestrogenic properties, and (iv) both large and small luteal cells become more sensitive to clomiphene inhibition with increasing duration of exposure.  (+info)