Ovarian function in ewes after treatment with mifepristone early during the oestrous cycle.
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The aim of this study was to determine whether endogenous progesterone regulates synthesis and secretion of luteal oxytocin. In Expt 1, mature ewes (n = 5 per group) were assigned randomly to control or mifepristone (RU486) treatment groups. Ewes were injected s.c. twice a day with vehicle or 10 mg RU486 on days 5-7 of the oestrous cycle (oestrus = day 0). On day 8, after an i.v. injection with prostaglandin F(2alpha) (250 microg cloprostenol), venous blood samples were collected at frequent intervals to determine plasma oxytocin concentrations. Plasma oxytocin concentrations of RU486-treated ewes were not significantly different from those of control ewes. In Expt 2, ewes were injected s.c. each day with vehicle or 175 mg RU486 on days 2-5 of the oestrous cycle followed by administration of prostaglandin F(2alpha) on day 6. Four of five RU486-treated ewes showed 'split-oestrus' (oestrous behaviour for 36 h and then again at 84-108 h after the onset of initial oestrus). There was no significant difference in mean plasma oxytocin or progesterone concentrations between treatment groups. The mean masses of mature corpora lutea from control and RU486-treated ewes on day 6 of the oestrous cycle did not differ significantly (394.8 +/- 28.8 versus 319.5 +/- 48.3 mg). RU486-treated ewes contained mature corpora lutea, new corpora lutea (two of four ewes) and preovulatory follicles (>or= 10 mm, two of four ewes). The average interoestrous interval for RU486-treated ewes was 9 days more than that for control animals (26.2 +/- 2.9 versus 17 +/- 0.5 days; P < 0.025). (+info)
Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).
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Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure. (+info)
Incadronate amplifies prostaglandin F2 alpha-induced vascular endothelial growth factor synthesis in osteoblasts. Enhancement of MAPK activity.
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We have previously reported that prostaglandin F2 alpha (PGF2 alpha) activates p44/p42 mitogen-activated protein kinase (MAPK) through protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of vascular endothelial growth factor (VEGF) synthesis induced by PGF2 alpha and the effect of incadronate on the VEGF synthesis in these cells. PGF2 alpha significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pm and 10 microm. Cycloheximide reduced the PGF2 alpha effect. PGF2 alpha increased the levels of mRNA for VEGF. Cloprostenol, a PGF2 alpha-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF2 alpha-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF2 alpha-induced VEGF synthesis. The VEGF synthesis induced by PGF2 alpha was significantly attenuated in the PKC down-regulated cells. PGF2 alpha elicited the translocation of PKC beta I from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF2 alpha. Farnesyltransferase inhibitor failed to affect the PGF2 alpha-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF2 alpha. NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF2 alpha-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF2 alpha or 12-O-tetradecanoylphorbol-13-acetate, a PKC activator. Incadronate significantly enhanced the cloprostenol-increased level of VEGF concentration in mouse plasma in vivo. These results strongly suggest that PGF2 alpha stimulates VEGF synthesis through the PKC-dependent activation of p44/p42 MAPK in osteoblasts and that the incadronate enhances the VEGF synthesis at the point between PKC and Raf-1. (+info)
Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.
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Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61. (+info)
Roles of tumor necrosis factor-alpha of the estrous cycle in cattle: an in vivo study.
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We have suggested in a previous in vitro study that tumor necrosis factor-alpha (TNFalpha) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFalpha on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2alpha [PGF2alpha] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFalpha actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2alpha (cloprostenol, 100 microg; n = 3) or saline with TNFalpha at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 microg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFalpha treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFalpha (0.1 and 1 microg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 +/- 0.9 and 18.0 +/- 0.7 days, respectively) compared with the control (22.3 +/- 0.3 days, P < 0.05). One microgram of TNFalpha increased the PGF2alpha (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFalpha (10, 25, 50 microg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFalpha cause luteolysis, whereas high concentrations of TNFalpha activate corpus luteum function and prolong the estrous cycle in cattle. (+info)
Influence of exogenous adrenocorticotropic hormone on estrous behavior in cattle.
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A two-trial experiment was conducted to determine the influence of ACTH on estrous behavior in cattle. In Trial 1, Holstein heifers (n = 20) received an injection of prostaglandin F2 alpha (PGF) during a synchronized diestrus and 30 h later were allotted randomly to receive (i.m.) either 1) 4 mL of gelatin (Veh) or 2) 320 units of ACTH in 4 mL of gelatin (ACTH). Eleven days after the PGF injection, all heifers were again injected with PGF, and they received either Veh or ACTH to complete a cross-over design. Treatment with ACTH decreased (P less than .05) the duration of estrus (12.0 +/- 1.9 vs 18.0 +/- 1.6 h for Veh) and increased (P less than .001) the interval to estrus after PGF injection (62.9 +/- 2.6 vs 43.7 +/- 2.2 h for Veh). Peak serum concentrations of progesterone (P4) and cortisol (C) were elevated (P less than .001) after ACTH compared with Veh. In Trial 2, ovariectomized Holstein cows (n = 12) were injected (i.m.) with .5 mg of estradiol benzoate (EB) and, 10 h later, were allotted randomly to receive (i.m.) either 1) 4 mL of gelatin (Veh) or 2) 320 units of ACTH in 4 mL of gelatin (ACTH). Seven days after the initial EB injection, all cows were again injected with .5 mg of EB and, 10 h later, received either Veh or ACTH to complete a cross-over design. Treatment with ACTH decreased (P less than .01) the proportion of cows in estrus (2/12 vs 11/12 for Veh) and increased (P less than .01) peak serum concentrations of P4 and C.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Effect of oxytocin infusion on secretion of progesterone and luteinizing hormone and the concentration of uterine oxytocin receptors during the periovulatory period in cloprostenol-treated ewes.
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Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.
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Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)