Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation.

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10 h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

Fluorescent ligand for human progesterone receptor imaging in live cells.

We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression.

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.

Growth hormone-releasing hormone antagonists inhibit growth of human ovarian cancer.

Epithelial ovarian carcinoma is the leading cause of cancer-related deaths among women with gynecologic malignancies. Antagonists of the growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of various cancers through endocrine, autocrine, and paracrine mechanisms. In this study, we have investigated the effects of GHRH antagonists (GHRHa) in ES-2 human clear cell ovarian cancer and in UCI-107 human serous ovarian cancer in vitro and in vivo. We evaluated the expression of mRNA for GHRH receptor, the binding to GHRH receptors, in specimens of ES-2 ovarian cancer. We evaluated also the in vitro effects of GHRHa on ES-2 cells and the in vivo effect of 2 different GHRHa on ES-2 and UCI-107 tumors. Nude mice bearing xenografts on ES-2 and UCI-107 ovarian cancer were treated with JMR-132 and MZ-J-7-118, respectively. Tumor growth was compared to control. ES-2 cells expressed mRNA for the functional splice variant SV1 of the GHRH receptor. JMR-132 inhibited cell proliferation in vitro by 42% and 18% at 10 and 1 µM concentration, respectively. Specific high affinity receptors for GHRH were detected in ES-2 cancer samples. In vivo daily subcutaneous injections of GHRHa significantly reduced tumor growth compared to a control group in both animal models. Our results indicate that GHRHa such as JMR-132 and MZ-J-7-118 can inhibit the growth of human ovarian cancer. The efficacy of GHRHa in ovarian cancer should be assessed in clinical trials.

Discovery of PF-184563, a potent and selective V1a antagonist for the treatment of dysmenorrhoea. The influence of compound flexibility on microsomal stability.

The V1a receptor has emerged as an attractive target for a range of indications including Raynaud's disease and dysmenorrhoea. As part of an effort to discover a new class of orally active V1a antagonist, we optimised a highly lipophilic, metabolically unstable lead into a range of potent, selective and metabolically stable V1a antagonists. In this communication, we demonstrate the series-dependent effect of limiting the number of rotatable bonds in order to decrease Cytochrome P450-mediated metabolism. This effort culminated in the discovery of PF-184563, a novel, selective V1a antagonist with excellent in vitro and in vivo properties.

Central and peripheral anti-inflammatory effects of maprotiline on carrageenan-induced paw edema in rats.

OBJECTIVE: To explore the site of action of maprotiline, as an atypical antidepressant, on carrageenan-induced paw edema. SUBJECTS: Male Wistar rats were used. METHODS: Firstly, the anti-inflammatory effect of systemic maprotiline (12.5, 25 and 50 mg kg(-1)) was assessed using a paw edema model. Secondly, different doses of maprotiline were administrated intracerebroventricularly, intrathecally and locally before carrageenan challenge. Finally, we tried to reverse the anti-inflammatory effect of maprotiline by propranolol (10 mg kg(-1)), prazosin (4 mg kg(-1)), yohimbine (10 mg kg(-1)), naloxone (4 mg kg(-1)) and mifepristone (5 mg kg(-1)). RESULTS: Systemic, intracerebroventricular and subplantar application of maprotiline significantly inhibited peripheral edema, but intrathecal maprotiline did not alter the degree of paw swelling. The applied antagonists failed to change the anti-inflammatory activity of maprotiline. CONCLUSION: These results demonstrate that maprotiline has a potent anti-inflammatory effect and this effect is linked to the peripheral and supraspinal actions of the drug.

Individual behavioral and neuronal phenotypes for arginine vasotocin mediated courtship and aggression in a territorial teleost.

The neuropeptide arginine vasotocin (AVT) is well known to modulate both aggression and affiliation, yet few studies relate individual behavioral state to a quantitative assessment of AVT distribution in the brain. Here, using a wild population of beaugregory damselfish, Stegastes leucostictus, we assess: (1) the effect of AVT on courtship, and (2) with reference to our previous study on AVT modulation of aggression in this species, the relationship between AVT-like immunoreactive (ir) fiber distribution in the forebrain's preoptic area and individual courtship and aggression levels. Exogenous AVT did not affect courtship, yet Manning compound, an arginine vasopressin (AVP) V1a receptor antagonist, significantly lowered but did not eradicate courtship. Consistent with AVT's known facilitation of aggression in this species, the density of AVT-ir fibers in the preoptic area was significantly negatively correlated to aggression. Our findings match similar behavioral and immunoreactive patterns of neuropeptide secretion in other taxa. Unlike aggression, preoptic AVT-ir fiber density was not significantly correlated to individual courtship levels. The results suggest a differential involvement of preoptic AVT neurons and/or their receptors in supporting the expression of aggression and courtship.

TSH/IGF1 receptor crosstalk: Mechanism and clinical implications.

Increasing evidence of interdependence between G protein-coupled receptors and receptor tyrosine kinase signaling pathways has prompted reevaluation of crosstalk between these receptors in disease and therapy. Investigations into thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF1) receptor crosstalk, and its application to the clinic have in particular shown recent progress. In this review, we summarize current insights into the mechanism of TSH/IGF1 receptor crosstalk. We discuss evidence that crosstalk is one of the underlying causes of TSHR-based disease and the feasibility of using combinations of TSH receptor and IGF1 receptor antagonists to increase the therapeutic index for the treatment of Graves' hyperthyroidism and Graves' ophthalmopathy.

Methylprednisolone inhibits IFN-gamma and IL-17 expression and production by cells infiltrating central nervous system in experimental autoimmune encephalomyelitis.

BACKGROUND: Glucocorticoids have been shown to be effective in the treatment of autoimmune diseases of the CNS such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms and the site of glucocorticoids' actions are still not completely defined. The aim of this study was to investigate the in vivo effect of the synthetic glucocorticoid methylprednisolone (MP) on the expression and production of proinflammatory cytokines interferon (IFN)-gamma and interleukin (IL)-17 by cells infiltrating CNS tissue. METHODS: Experimental autoimmune encephalomyelitis was induced in Dark Agouti (DA) rats by immunization with rat spinal cord homogenate mixed with adjuvants. Commencing on the day when the first EAE signs appeared, DA rats were injected daily for 3 days with MP and/or RU486, an antagonist of glucocorticoid receptor. Cytokine production and gene expression in CNS-infiltrating cells and lymph node cells were measured using ELISA and real time PCR, respectively. RESULTS: Treatment of rats with MP ameliorated EAE, and the animals recovered without relapses. Further, MP inhibited IFN-gamma and IL-17 expression and production in cells isolated from the CNS of DA rats with EAE after the last injection of MP. The observed effect of MP in vivo treatment was not mediated through depletion of CD4+ T cells among CNS infiltrating cells, or through induction of their apoptosis within the CNS. Finally, the glucocorticoid receptor-antagonist RU486 prevented the inhibitory effect of MP on IFN-gamma and IL-17 production both in vitro and in vivo, thus indicating that the observed effects of MP were mediated through glucocorticoid receptor-dependent mechanisms. CONCLUSION: Taken together, these results demonstrate that amelioration of EAE by exogenous glucocorticoids might be, at least partly, ascribed to the limitation of effector cell functions in the target tissue.

Metabolic activation of mifepristone [RU486; 17beta-hydroxy-11beta-(4-dimethylaminophenyl)-17alpha-(1-propynyl)-estra-4,9-dien-3-one] by mammalian cytochromes P450 and the mechanism-based inactivation of human CYP2B6.

Mifepristone [RU486; 17beta-hydroxy-11beta-(4-dimethylaminophenyl)-17alpha-(1-propynyl)-estra-4,9-dien-3-one] inactivates CYP2B6 in the reconstituted system in a mechanism-based manner. The loss of 7-ethoxy-4-(trifluoromethyl)-coumarin deethylation activity of CYP2B6 is concentration- and time-dependent. The inactivation requires NADPH and is irreversible. The concentration of inactivator required to give the half-maximal rate of inactivation is 2.8 microM, and the maximal rate constant for inactivation at a saturating concentration of the inactivator is 0.07 min(-1). Incubation of CYP2B6 with 20 microM RU486 for 15 min resulted in 61% loss of catalytic activity, 60% loss of the reduced cytochrome P450 (P450)-CO complex, and a 40% loss of native heme. The partition ratio is approximately 5, and the stoichiometry of binding is approximately 0.6 mol RU486/mol P450 inactivated. SDS-polyacrylamide gel electrophoresis and high-pressure liquid chromatography analysis showed that [(3)H]RU486 was irreversibly bound to CYP2B6 apoprotein. RU486 is metabolized to form three major metabolites and bioactivated to give reactive intermediates by purified P450s in the reconstituted system. After incubation of RU486 with the purified P450s and liver microsomes from rats and humans in the presence of glutathione (GSH) and NADPH, GSH conjugates with MH(+) ions at m/z 769, 753, and 751 were detected by liquid chromatography-tandem mass spectrometry. Two GSH conjugates with MH(+) ions at m/z 753 are formed from the reaction of GSH with RU486. The adducts are formed after addition of an activated oxygen to the carbon-carbon triple bond of the propynyl moiety. This suggests that oxirene intermediates may be involved in the mechanism of inactivation. It seems that the potential for drug-drug interactions of RU486 may not be limited only to CYP3A4 and should also be evaluated for drugs metabolized primarily by CYP2B6, such as bupropion and efavirenz.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist.

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

Sequence and characterization of a coactivator for the steroid hormone receptor superfamily.

A yeast two-hybrid system was used to identify a protein that interacts with and enhances the human progesterone receptor (hPR) transcriptional activity without altering the basal activity of the promoter. Because the protein stimulated transactivation of all the steroid receptors tested, it has been termed steroid receptor coactivator-1 (SRC-1). Coexpression of SRC-1 reversed the ability of the estrogen receptor to squelch activation by hPR. Also, the amino terminal truncated form of SRC-1 acted as a dominant-negative repressor. Together, these results indicate that SRC-1 encodes a coactivator that is required for full transcriptional activity of the steroid receptor superfamily.

Evolutionary origin and divergence of GnIH and its homologous peptides.

Probing undiscovered hypothalamic neuropeptides that play important roles in the regulation of pituitary function in vertebrates is essential for the progress of neuroendocrinology. In 2000, we discovered a novel hypothalamic dodecapeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). GnIH acts on the pituitary and gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus via a novel G protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotropin release and synthesis. Similar findings were observed in other avian species. Thus, GnIH is a key factor controlling avian reproduction. To give our findings a broader perspective, we also found GnIH homologous peptides in the hypothalamus of other vertebrates, such as mammals, reptiles, amphibians and teleosts. GnIH and its homologs share a common C-terminal LPXRFamide (X=L or Q) motif. A mammalian GnIH homolog also inhibited gonadotropin release in mammals like the GnIH action in birds. In contrast to higher vertebrates, hypophysiotropic activities of GnIH homologs were different in lower vertebrates. To clarify the evolutionary origin of GnIH and its homologs, we further sought to identify novel LPXRFamide peptides from the brain of sea lamprey and hagfish, two extant groups of the oldest lineage of vertebrates, Agnatha. In these agnathans, LPXRFamide peptide and its cDNA were identified only from the brain of hagfish. Based on these findings over the past decade, this paper summarizes the evolutionary origin and divergence of GnIH and its homologous peptides.

G protein-coupled receptors of the hypothalamic-pituitary-gonadal axis: a case for Gnrh, LH, FSH, and GPR54 receptor ligands.

The hypothalamic-pituitary-gonadal (HPG) axis, important in reproduction and sex hormone-dependent diseases, is regulated by a number of G protein-coupled receptors. The recently "deorphanized" GPR54 receptor activated by the peptide metastin is thought to be the key regulator of the axis, mainly by releasing gonadotropin-releasing hormone (GnRH) from the hypothalamus. The latter decapeptide, through the activation of the GnRH receptor in the anterior pituitary, causes the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which subsequently activate their respective receptors on the gonadotrope cells. In this review we will discuss the small molecule agonists and antagonists that are currently being developed to intervene with the action of these four receptors. For GnRH receptors, 14 different chemical classes of non-peptidic antagonists have been reported, while for the LH receptor three classes of agonists have been described. Both agonists and antagonists have been introduced for the FSH receptor. Recently, the first non-peptidic agonist for GPR54 was reported.

Sex differences in cell proliferation and glucocorticoid responsiveness in the zebra finch brain.

Neural proliferation is a conserved property of the adult vertebrate brain. In mammals, stress reduces hippocampal neuronal proliferation and the effect is stronger in males than in females. We tested the effects of glucocorticoids on ventricular zone cell proliferation in adult zebra finches where neurons are produced that migrate to and incorporate within the neural circuits controlling song learning and performance. Adult male zebra finches sing and have an enlarged song circuitry; females do not sing and the song circuit is poorly developed. Freshly prepared slices from adult males and females containing the lateral ventricles were incubated with the mitotic marker BrdU with or without steroid treatments. BrdU-labeled cells were revealed immunocytochemically and all labeled cells within the ventricular zone were counted. We identified significantly higher rates of proliferation along the ventricular zone of males than in females. Moreover, acute administration of corticosterone significantly reduced proliferation in males with no effects in females. This effect in males was replicated by RU-486, which appears to act as an agonist of the glucocorticoid receptor in the songbird brain. The corticosterone effect was reversed by Thiram, which disrupts corticosterone action on the glucocorticoid receptor. Sex differences in proliferation and responses to stress hormones may contribute to the sexually dimorphic and seasonal growth of the neural song system of songbirds.

Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay.

The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.

Opposite effects of oxytocin on proliferation of osteosarcoma cell lines.

Oxytocin stimulates proliferation of human osteoblast-like (hOB) cells and human osteosarcoma cells (SaOS-2). In contrast, oxytocin has also been shown to inhibit proliferation of other cell lines such as breast cancer cells. The aim of the present study was to investigate the effects of different concentrations of oxytocin on cell proliferation in osteosarcoma cell lines of different stages of differentiation: SaOS-2, TE-85, and UMR-106. For this purpose cells were incubated with oxytocin (1-1000 pmol/l). Cell proliferation was measured by [(3)H]thymidine incorporation and a commercially available kit (EZ4U). Incubation with oxytocin during 24 h increased proliferation of SaOS-2 cells significantly (100 pmol/l: p<0.01). In contrast, 24 h of incubation with oxytocin decreased proliferation of TE-85 (100 pmol/l: p<0.01) and UMR-106 cells significantly (100 pmol/l: p<0.01). The effects of oxytocin in SaOS-2 and TE-85, but not in UMR-106 cells, were abolished when the cells were incubated with both oxytocin and an oxytocin antagonist (1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin). Instead incubation with the oxytocin antagonist alone decreased proliferation of UMR-106 cells significantly (p<0.001). Thus oxytocin has the capacity to both stimulate and inhibit cell proliferation of osteosarcoma cells. This effect might be dependent on the stage of differentiation of the cancer cells.

Inhibitory effect of somatostatin on prostaglandin E2 synthesis by primary neonatal rat glial cells.

Glial inflammation plays an integral role in the development of neurodegenerative disease. Although somatostatin is known to be a local anti-inflammatory factor in the periphery, evidence of a similar function in the brain is scarce. The aim of the present study was to investigate the effect of somatostatin on prostaglandin E(2) synthesis in primary neonatal rat glial cells. The data shows that high concentrations of somatostatin (10(-5)-10(-4)) significantly increased prostaglandin synthesis. By contrast, when used at physiologically relevant concentrations (10(-9)-10(-7) M), somatostatin and somatostatin receptor agonists decreased prostaglandin E(2) synthesis in non-stimulated glial cells as well as in lipopolysaccharide-induced prostaglandin synthesis. The inhibitory effect of somatostatin in lipopolysaccharide-treated cells could be mimicked by protein kinase A inhibitor and was prevented by forskolin. These observations suggest the presence of a novel neuro-immune feedback pathway through which somatostatin inhibits glial prostaglandin synthesis, and thus may prove to play a role in brain inflammation. This action of somatostatin may have a therapeutic potential in pathological conditions of the brain, where an inflammatory response is involved.

Involvement of amino acids flanking Glu7.32 of the gonadotropin-releasing hormone receptor in the selectivity of antagonists.

The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.