Evaluation of RU28318 and RU40555 as Selective

Evaluation Of Ru28318 And Ru40555 As Selective-PDF Download

  • Date:24 Mar 2020
  • Views:31
  • Downloads:0
  • Pages:10
  • Size:596.92 KB

Share Pdf : Evaluation Of Ru28318 And Ru40555 As Selective

Download and Preview : Evaluation Of Ru28318 And Ru40555 As Selective

Report CopyRight/DMCA Form For : Evaluation Of Ru28318 And Ru40555 As Selective


214 Paul J Kim et al, determining these relative roles of MR and GR is in dies These functional studies were designed to con. vivo treatment with selective receptor antagonists rm in vivo the ability of these antagonists to block. Several different steroid analogues have been charac the effects of an agonist for MR or GR Furthermore. terized which appear to be effective in selectively these functional studies tested for possible agonist. antagonizing corticosterone effects mediated by MR effects of these putative antagonist compounds One. or GR However for each of these compounds the of the best characterized physiological roles of MR is. validation of their suitability for in vivo studies has mediation of sodium retention by the kidney and a. been limited complementary regulation of sodium appetite 27. In recent years a number of studies have used Adrenalectomy of rats results in increased sodium. RU28318 as a selective MR antagonist RU28318 intake which can be normalized by treatment with. has been reported to be superior to the more tra the MR selective agonist aldosterone 28 29. ditional anti mineralocorticoid spironolactone in Therefore we have tested the ability of RU28318 to. terms of having a higher in vivo anti mineralocorti functionally antagonize this MR mediated effect by. coid activity and a lower anti androgen activity 7 examining saline intake in adrenalectomized rats trea. However in vitro receptor binding studies suggest ted with aldosterone 2RU28318 For functional. that RU28318 may have considerable interaction with analysis of RU40555 we have examined the ability of. GR 8 9 RU40555 to antagonize dexamethasone GR selective. RU486 has been the most widely used compound agonist suppression of the HPA axis response to. for blocking GR however in many cases this com acute stress The suppression of the corticosterone. pound has been found to have agonist properties 10 acute stress response by dexamethasone has been well. 14 In addition for the last several years the distri established as a sensitive GR mediated effect 1. bution of RU486 in the U S A has been prohibited A limitation of these functional tests is that they. or severely restricted A potential alternative selective may not directly test the antagonist properties of. GR antagonist is RU40555 This compound has been these compounds within the brain There is evidence. described by its developer Roussel Uclaf for a central site of action for aldosterone modulation. Romainville France as having a similar receptor of sodium appetite 29 30 although the hippo. selectivity as RU486 af nity for both glucocorticoid campus is probably not involved 31 There is the. and progesterone receptors but a lower potency for possibility however that the normalization of saline. blocking glucocorticoid effects unpublished corre intake by a systemic dose of aldosterone is secondary. spondence with Roussel Uclaf to normalization of sodium retention within the kid. We report here studies that further evaluated the ney In addition the suppressive effect of dexametha. suitability of using RU28318 and RU40555 as selec sone on HPA axis activity has been repeatedly. tive MR and GR antagonists for in vivo studies in the demonstrated to depend primarily on dexamethasone. rat Both the dose and time response effects of these activation of GR located in the pituitary 32 34. drugs on available MR and GR receptor binding were Nevertheless by combining these functional tests. examined From these studies we can estimate the with evaluation of the ability of RU28318 and. proportion of MR and GR that are occupied in vivo RU40555 to occupy corticosteroid receptors within. by these compounds and thereby assess the extent to the brain and pituitary a better in vivo validation of. which these compounds selectively interact with MR the selective antagonistic properties of these com. and GR 15 16 For the receptor binding studies we pounds is provided than has previously been. have utilized the hippocampus and pituitary reported. Hippocampal tissue was selected as a representative. brain region The hippocampus is an ideal tissue for. available corticosteroid receptor measures because of MATERIALS AND METHODS. its high expression of both MR and GR In addition, there is considerable interest among researchers in Subjects. the relative roles of hippocampal MR and GR ex Experimental subjects were male Sprague Dawley. pression in regulating a number of hippocampal for rats 285 350 g at time of experimentation pur. mation related processes such as neuronal chased from Harlan Sprague Dawley Indianapolis. excitability 17 long term potentiation 18 primed IN Animals were housed in wire mesh hanging. burst potentiation 19 neurotransmitter receptor cages 3 rats per cage and were given food Purina. expression 20 23 neuronal atrophy 24 and neur laboratory rat chow and tap water ad lib The animal. onal survival 25 26 The pituitary was selected as a room was maintained on a 12 h light dark cycle. representative peripheral tissue that also has direct lights on at 7 00 a m All experimental procedures. relevance for corticosteroid negative feedback actions were conducted in the a m After arrival at the. on HPA axis activity University of Colorado at Boulder animal care facili. In addition to available receptor binding studies ties the animals were given a 2 week acclimation. we have tested these compounds in functional stu period before onset of experimentation All exper. Corticosteroid receptor antagonist validation 215, iments were approved by the University of Colorado 3H dexamethasone 10 nM nal concentration 2. Institutional Animal Care and Use Committee non radioactive competitors 3H Dexamethasone. was used because of its high af nity for both MR and. Corticosteroid agonists and antagonists GR in vitro 16 35 Available GR binding was deter. The MR antagonist used for these studies was mined from the amount of total 3H dexamethasone. RU28318 7 17alpha 17 hydroxy 3oxo 7 propl binding that was displaced by the selective GR ligand. pregn xene 21 carboxylic acid potassium The GR RU28362 0 5 mM 9 Available MR binding was. antagonist used for these studies was RU40555 17 determined by the amount of residual 3H dexa. beta hydroxy 11 beta 4 methyl 1 methylethyl a methasone speci c binding Nonspeci c binding was. minophenyl 17alpha prop 1 ynyl estra 4 9 diene 3 de ned as the amount of 3H dexamethasone bind. one Both antagonists were donated by Roussel ing that was not displaced by an excess of dexametha. Uclaf Romainville France Aldosterone and dexa sone 10 mM Non speci c binding was less than. methasone were used as selective MR and GR ago 10 of total binding Bound 3H dexamethasone. nists respectively Although dexamethasone binds was separated from unbound steroid by gel ltration. MR with a high af nity in an in vitro receptor binding 100 ml per incubate in triplicate in 1 ml Sephadex. assay 16 35 in vivo dexamethasone is a selective LH 20 Pharmacia columns The resulting eluate. agonist for GR 36 Aldosterone and dexamethasone was mixed with scintillation cocktail and the radioac. were purchased from Sigma St Louis MO tivity was measured using a scintillation counter. Propylene glycol was used as the vehicle for all antag Protein concentrations for each cytosolic sample were. onists and agonists For receptor binding studies determined using a Bradford assay 37 bovine serum. 1 2 4 3 H dexamethasone S A 50 Ci mmol was albumin was used for the protein standard The nal. obtained from Amersham Arlington Heights IL protein levels in each incubation tube ranged from. For corticosterone radioimmunoassay 1 2 6 7 0 5 1 5 mg ml. H N corticosterone S A 80 Ci mmol was,obtained from New England Nuclear Boston MA. Experiment 1 dose and time response study of the effect. of RU28318 on available MR and GR binding in the,ESTIMATES OF IN VIVO RECEPTOR hippocampus.
OCCUPATION BY ANTAGONISTS The in vivo selectivity of a range of doses of. Available corticosteroid receptor binding RU28318 for MR was tested For the dose response. comparison adrenalectomized rats n 3 4 were, Several important features of the receptor binding. injected s c 1 h before death with either vehicle pro. assay indicates that the available receptor binding. pylene glycol 1 ml kg or one of three doses of, data may provide an estimate of in vivo corticosteroid. RU28318 10 25 or 50 mg kg An additional group,receptor occupation by RU28318 and RU40555 see. of adrenalectomized rats n 3 was injected with the. DISCUSSION For all of the receptor binding,highest dose of RU28318 50 mg kg and were killed. measures the rats were adrenalectomized 24 h prior. 2 h after injection The 2 h time point was included. to the day of experimentation to prevent interference. in order to provide some indication as to whether the. from endogenous corticosterone On the day of ex, effects of the antagonists would be sustained for at.
perimentation rats were injected with RU28318 or,least 2 h after injection. RU40555 see below for doses and were then killed, by rapid decapitation 1 or 2 h later Immediately fol. lowing decapitation the hippocampus and pituitary, were rapidly removed and stored at 708C Trunk Experiment 2 dose and time response study of the effect. blood was also collected for determination of plasma of RU40555 on available MR and GR binding in the. corticosterone None of the plasma samples from hippocampus. adrenalectomized rats had plasma corticosterone Following the same design as experiment 1 the in. levels above detection limits of our assay 0 5 mg vivo selectivity of a range of doses of RU40555 for. 100 ml for a 20 ml sample GR was tested For the dose response comparison. The available cytosolic MR and GR binding was adrenalectomized rats n 3 4 were injected s c 1 h. measured using a radioligand receptor binding before death with either vehicle propylene glycol. assay 16 Tissues from individual animals were hom 3 ml kg or one of three doses of RU40555 10 20. ogenized in a buffer solution comprised of 10 mM or 30 mg kg An additional group of adrenalectom. Tris 1 mM EDTA 10 glycerin 20 mM molybdic ized rats n 3 was injected with the highest dose of. acid and 5 mM dithiothreitol at a pH of 7 4 at 48C RU40555 30 mg kg and was killed 2 h after injec. The homogenate was centrifuged 100 000g 30 min tion Due to the poor solubility of RU40555 all. and 150 ml of the supernatant fraction cytosol was injection solutions were adjusted to an injection. incubated for 18 24 h in the presence of 200 ml of volume of 3 ml kg. 216 Paul J Kim et al, Experiment 3 comparison of the effects of RU28318 and was injected 60 min prior to the onset of restraint. RU40555 on available MR and GR binding in the hip stress Thus there were 4 combinations of drug treat. pocampus and the pituitary ment 1 vehicle vehicle 2 RU40555 vehicle. This experiment compared the effect of antagonist 3 vehicle dexamethasone and 4. treatment on available MR and GR binding in both RU40555 dexamethasone n 4 These manipula. the hippocampus and the pituitary n 5 6 Vehicle tions were performed during the rst third of the light. propylene glycol 3 ml kg s c RU28318 50 mg kg period Restraint consisted of placing rats in a. s c or RU40555 30 mg kg s c were injected in Plexiglas tube 23 5 cm in length and 7 cm in diam. adrenalectomized rats Rats were then killed 60 min eter that inhibited forward backward and lateral. later movement Serial blood samples approximately, 100 ml sample were collected from the tip of the tail.
of each rat at 0 30 60 90 and 120 min after the, FUNCTIONAL STUDIES onset of stress Plasma corticosterone was measured. by radioimmunoassay Plasma samples were diluted, Experiment 4 mineralocorticoid receptor dependent func 1 50 in 0 01 M PBS and corticosteroid binding glo. tional bioassay bulins were heat inactivated by incubating the diluted. A functional validation of the ability of RU28318 samples for 1 h at 708C Samples and corticosterone. to antagonize an MR dependent effect in vivo was standards 25 2000 pg per tube were incubated. conducted using the two water bottle drinking test overnight with antiserum B21 42 Endocrine. in adrenalectomized rats This test relies on the Sciences Tarzana CA and 3H corticosterone. voluntary increased saline intake observed in rats after 20 000 cpm tube Antibody bound steroid was sep. adrenalectomy Low dose aldosterone replacement is arated from free steroid with dextran coated activated. known to reverse the increased saline intake 28 29 charcoal The antibody bound steroid was then. Subjects were individually housed and were given the mixed with scintillation cocktail and the radioactivity. choice of two drinking bottles lled with either tap was counted using a scintillation counter Assay sensi. water or 0 9 saline The relative daily liquid intakes tivity was 0 5 mg 100 ml for an assay volume of 20 ml. were monitored by measuring the weight of the two of plasma All samples were analyzed within the same. water bottles over a 24 h period The change in assay The intra assay coef cient of variation was. weight of the water bottles was then converted into 9 5 for a sample containing approximately 5 mg. the volume consumed 1 g 1 ml All subjects 100 ml and 4 4 for a sample containing approxi. except for the Sham rats were adrenalectomized mately 20 mg 100 ml n 4. 1 week prior to the onset of treatment The subjects. were then injected with antagonist RU28318 50 mg Data analysis and statistics. laboratory rat chow and tap water ad lib The animal room was maintained on a 12 h light dark cycle lights on at 7 00 a m All experimental procedures were conducted in the a m After arrival at the University of Colorado at Boulder animal care facili ties the animals were given a 2 week acclimation period before onset of experimentation

Related Books