Small synthetic ligands of the cholecystokinin-B/gastrin receptor can mimic the function of endogenous peptide hormones.
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The gastric cholecystokinin-B/gastrin receptor (CCK-BR) is a key regulator of enterochromaffin-like cell function and proliferation. Over the last decade, a number of small non-peptide CCK-BR "antagonists" have been discovered. Here, we demonstrate that some of these non-peptide ligands in fact possess significant ability to activate the human CCK-BR, and are, therefore, more properly categorized as partial agonists. When tested in COS-7 cells transiently expressing the recombinant human CCK-BR, saturating concentrations of the small "peptoid" ligands PD 135,158 and PD 136,450 stimulated inositol phosphate formation to 23 and 43 percent, respectively, of the maximum response induced by a considerably larger endogenous peptide agonist, cholecystokinin octapeptide. In contrast, the benzodiazepine-derived CCK-BR ligand, YM022, acted as a "true" high-affinity antagonist of cholecystokinin-induced inositol phosphate formation (pA2 = 9.69). Consistent with recent findings in animal experiments, our data reveal that small synthetic ligands have the potential to function as either CCK-BR agonists or antagonists. These dual properties of synthetic molecules must be considered when evaluating candidate drugs for human disease. (+info)
High doses of gonadotrophin-releasing hormone antagonist in in-vitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles.
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The clinical application of gonadotrophin-releasing hormone (GnRH) antagonists instead of GnRH agonists, to prevent spontaneous premature luteinizing hormone surge during ovarian stimulation for assisted reproduction treatment has been advocated. A recent, double-blind, dose-finding study, including six dosages of the GnRH antagonist ganirelix, in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (FSH), has indicated that high doses of GnRH antagonist (1 or 2 mg once daily) are associated with a low implantation rate. This follow-up study reports on the pregnancy rate after replacement of cryopreserved embryos obtained in stimulation cycles of the above-mentioned trial. Ovarian stimulation was initiated on day 2 of the cycle, with daily injections of 150 IU recombinant FSH. Ganirelix (0.0625, 0.125, 0.25, 0.5, 1.0 or 2.0 mg) was administered once daily from stimulation day 6 onwards, up to and including the day of human chorionic gonadotrophin. Retrieved oocytes were fertilized by in-vitro fertilization (IVF) or intracytoplasmic sperm injection and a maximum of three fresh embryos was transferred. Excess embryos were frozen, and subsequently used in either natural or programmed cycles. Until June 1998, 11 ongoing pregnancies (12-16 weeks after embryo transfer) were achieved from 46 cycles in which embryos had been first frozen (23.9% per transfer). Six of these 11 patients had been treated with a high dose of ganirelix (1.0 or 2.0 mg) during the IVF cycles in which the embryos were obtained. In conclusion, our data suggest that high dosages of ganirelix do not adversely affect the potential of embryos to establish clinical pregnancy in freeze-thaw cycles. (+info)
Progesterone stimulation of human insulin-like growth factor-binding protein-5 gene transcription in human osteoblasts is mediated by a CACCC sequence in the proximal promoter.
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Insulin-like growth factor-binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates insulin-like growth factor mitogenic stimulation in osteoblast cell cultures. Progesterone (PG) increased IGFBP-5 expression in normal human osteoblasts and increased IGFBP-5 transcription in U2 human osteosarcoma cells. We developed a chloramphenicol acetyltransferase reporter construct containing the human IGFBP-5 proximal promoter sequence, which includes TATA and CAAT boxes, and five putative PG response element half-sites. 10(-8) M PG increased promoter activity of this construct in U2 cells co-transfected with a PG receptor isoform A (PR(A)) expression vector. Analysis of 5' deletion constructs indicates that PG transactivation of IGFBP-5 promoter activity does not require the PG response element half-sites but does require the region -162 to -124 containing two tandem CACCC box sequences. Mutation of the proximal CACCC box at -139 eliminated PG transactivation. Gel shift assays using a -162 to -124 DNA fragment, U2 cell nuclear extracts, and purified PR(A) protein indicate that nuclear factors bind to a CACCC sequence at -139 and that PR(A) alters the pattern of transcription factor interaction with the CACCC sequence. Using a luciferase reporter construct containing base pairs -252 to +24 of the IGFBP-5 promoter, we found that both PR(A) and PR(B) isoforms mediated PG stimulation of promoter activity. These results suggest that PG may stimulate IGFBP-5 gene transcription via a novel mechanism involving PR and CACCC-binding factors. (+info)
Duodenal loading with glucose induces fos expression in rat brain: selective blockade by devazepide.
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The role of CCK in mediating neuronal activity in the brain in response to dietary carbohydrate was measured by detecting Fos immunoreactivity in response to duodenal glucose load in rats after administration of the CCK-A receptor antagonist devazepide. In adult, male Sprague-Dawley rats, infusion for 30 min of 545 mg (2.18 kcal) dextrose through a duodenal cannula induced Fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), lateral division of the central nucleus of the amygdala (CeAL), and the external subnucleus of the lateral parabrachial nucleus (LPBE). Devazepide treatment (1 mg/kg) attenuated Fos expression in the NTS and AP by 81 and 78%, respectively, but not in the CeAL or LPBE. These results indicate that central neuronal activation is elicited by dietary glucose in the intestinal lumen and that activation of neurons in the NTS and AP is mediated by CCK-A receptors. (+info)
Regulation of calcitonin gene-related peptide receptors in the rat uterus during pregnancy and labor and by progesterone.
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Calcitonin gene-related peptide (CGRP) is a potent smooth muscle relaxant in a variety of tissues. We recently demonstrated that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine (125)I-CGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We found that (125)I-CGRP binding to membrane preparations from uteri was elevated during pregnancy and decreased during labor and postpartum. Changes in immunoreactive CGRP receptors were similar to the changes in (125)I-CGRP binding in these tissues, suggesting pregnancy-dependent regulation of CGRP receptor protein. CGRP receptors were elevated by Day 7 of gestation, and a precipitous decrease in these receptors occurred on Day 22 of gestation prior to the onset of labor. Both (125)I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells. Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17beta. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day 22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by (125)I-CGRP-binding, Western immunoblotting, and immunolocalization methods. These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased at term. These receptors are localized to the myometrial cells, and progesterone is required for maintaining CGRP receptors in the rat uterus. Thus, the inhibitory effects of CGRP on uterine contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy. (+info)
Regulation of the immunoglobulin G1 receptor: effect of prolactin on in vivo expression of the bovine mammary immunoglobulin G1 receptor.
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Induction of colostrogenesis in non-pregnant cows was used to evaluate the relationship between prolactin (PRL) and mammary immunoglobulin G1 (IgG1) receptor expression. Six of eleven non-pregnant, non-lactating Holstein cattle responded to a standard lactation induction protocol by development of elevated IgG1 concentrations in mammary secretions. In order to increase the diversity in PRL concentrations, two of the six cattle were treated with bromocriptine, and two others were treated with recombinant bovine PRL. Serum alpha-lactalbumin, serum PRL and mammary secretion IgG1 concentrations were measured throughout the experiment. Biopsies of mammary tissue were collected after induction of lactation, and after treatments to alter serum PRL. Immunohistochemistry was used to evaluate IgG1 receptor expression. Administration of recombinant bovine (rbPRL) was associated with increased lactogenic activity, decreased secretion IgG1 concentrations, and decreased IgG1 receptor expression. Decreased serum PRL, due to bromocriptine, was associated with decreased lactogenic activity and maintenance of IgG1 receptor expression. Results of this experiment are consistent with an effect of PRL in decreasing the expression of the bovine mammary IgG1 receptor at the onset of lactogenesis. (+info)
Dissociated glucocorticoids with anti-inflammatory potential repress interleukin-6 gene expression by a nuclear factor-kappaB-dependent mechanism.
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Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases. However, major side effects severely limit their therapeutic use. Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways. We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity. To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation. We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds. We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction. (+info)
Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms.
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BACKGROUND: In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. Recently, nongenomic effect of aldosterone (ALDO) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of ALDO on NHE and the intracellular signaling mechanisms for these effects have not fully been determined in VSMCs. METHODS: The effects of short- (3 hr) and long- (24 hr) term exposure to ALDO on NHE activity were examined in cultured VSMCs from rat thoracic aortae by using single-cell pHi measurement with the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after acid load. RESULTS: The NHE activity significantly increased after short- and long-term exposure of VSMCs to ALDO (10(-6) M). The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) had no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. The antagonists of the mineralocorticoid receptor (MR) (spironolactone) and of the glucocorticoid receptor (GR) (RU38486) caused no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation (24 hr pre-exposure to phobol 12-myristate 13-acetate, PMA) inhibited both the short- and long-term ALDO effects. Exposure of VSMCs to PMA for 3 hours mimicked the short-term effect of ALDO on NHE activity. ALDO significantly increased PKC activity in VSMCs. The short-term ALDO effect was inhibited by disruptors of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels. CONCLUSIONS: The short-term effect of ALDO on NHE activity is not mediated through either MR or GR, occurs independent of gene transcription and protein synthesis, and occurs through a mechanism involving the structural elements of cytoskeleton. The long-term effect of ALDO on NHE activity occurs through both MR and GR and requires gene transcription and protein synthesis. Both short- and long-term effects of ALDO are mediated through PKC activation. Therefore, ALDO activates NHE by nongenomic and genomic mechanisms in VSMCs. (+info)