RESUMEN
Tobacco addiction is characterized by a lack of control over smoking and relapse after periods of abstinence. Smoking cessation leads to a dysphoric state that contributes to relapse to smoking. After the acute withdrawal phase, exposure to stressors increases the risk for relapse. Blockade of melanocortin 4 (MC4 ) receptors has anxiolytic and antidepressant-like effects in animal models. The aim of these studies was to investigate the role of MC4 receptors in the dysphoria associated with nicotine withdrawal and stress-induced reinstatement of nicotine seeking. To study stress-induced reinstatement, rats self-administered nicotine for 16 days and then nicotine seeking was extinguished by substituting saline for nicotine. Nicotine seeking was reinstated by intermittent footshock stress. The intracranial self-stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Elevations in the ICSS thresholds are indicative of a dysphoric state. The selective MC4 receptor antagonists HS014 and HS024 prevented stress-induced reinstatement of extinguished nicotine seeking. Drug doses that prevented stress-induced relapse did not affect responding for food pellets, which indicates that the drugs did not induce sedation or motor impairments. In the ICSS experiments, the nicotinic acetylcholine receptor antagonist mecamylamine elevated the ICSS thresholds of the nicotine-dependent rats. Pre-treatment with HS014 or HS024 did not prevent the elevations in ICSS thresholds. These studies indicate that MC4 receptors play a critical role in stress-induced reinstatement of nicotine seeking, but these receptors may not play a role in the dysphoria associated with acute nicotine withdrawal.
Asunto(s)
Comportamiento de Búsqueda de Drogas/fisiología , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Receptor de Melanocortina Tipo 4/metabolismo , Estrés Psicológico/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Tabaquismo/metabolismo , Animales , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Nicotina/efectos adversos , Agonistas Nicotínicos/efectos adversos , Péptidos Cíclicos/farmacología , Ratas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Recurrencia , Síndrome de Abstinencia a Sustancias/etiologíaRESUMEN
BACKGROUND: Cocaine and methamphetamine (METH) are two commonly abused drugs that have behavioral-stimulant properties. These stimulant effects are partially mediated by the dopaminergic system. Recent evidence has suggested that the histamine H(3) receptor (H(3)R) may modulate the release of dopamine induced by METH. The aim of the present study was to examine the role of H(3)R in the behavioral-stimulant effects of cocaine and METH in mice and monkeys. METHODS: Nonhabituated, experimentally naïve mice (n = 5-6) were pretreated with the H(3)R agonist imetit 30 min before METH or cocaine, and activity was measured for 90 min. The behavioral-stimulant effects of METH and cocaine were also studied in squirrel monkeys (n = 3) under a fixed-interval schedule of stimulus termination. Monkeys were pretreated with imetit 30 min before the peak behavioral-stimulant doses of METH or cocaine derived from individual subjects. RESULTS: Pretreatment with imetit did not affect basal activity in mice. Imetit significantly attenuated the behavioral-stimulant effects of METH, but not cocaine. In monkeys, no dose of imetit tested significantly altered the behavioral-stimulant effects of METH or cocaine. CONCLUSION: These results suggest a role of H(3)R in the behavioral-stimulant effects of METH, but not cocaine, in mice and no role in monkeys.
Asunto(s)
Conducta Animal/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Cocaína/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Imidazoles/farmacología , Metanfetamina/farmacología , Receptores Histamínicos H3/fisiología , Tiourea/análogos & derivados , Animales , Condicionamiento Operante/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Esquema de Refuerzo , Saimiri , Tiourea/farmacologíaRESUMEN
The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.
Asunto(s)
Ácidos Grasos/química , Melanocitos/efectos de los fármacos , Oligopéptidos/síntesis química , Receptores de Melanocortina/agonistas , Acilación , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ligandos , Melanocitos/citología , Melanocitos/enzimología , Ratones , Monofenol Monooxigenasa/metabolismo , Oligopéptidos/química , Oligopéptidos/farmacología , Isoformas de Proteínas/agonistas , Receptor de Melanocortina Tipo 1/agonistas , Receptor de Melanocortina Tipo 3/agonistas , Receptor de Melanocortina Tipo 4/agonistas , Receptores de Corticotropina/agonistas , Relación Estructura-ActividadRESUMEN
The melanocortin pathway is an important participant in skin pigmentation, steroidogenesis, obesity, energy homeostasis and exocrine gland function. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). The melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," and it has been well-documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library, based upon the template Ac-His-DPhe-Arg-Trp-NH(2), consisting of 26 members that have been modified at the DPhe(7) position (alpha-MSH numbering) and pharmacologically characterized for agonist and antagonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the identification of the tetrapeptide Ac-His-(pI)DPhe-Arg-Trp-NH(2) that is a full nanomolar agonist at the mMC1 and mMC5 receptors, a mMC3R partial agonist with potent antagonist activity (pA(2) = 7.25, K(i) = 56 nM) and, but unexpectedly, is a potent agonist at the mMC4R (EC(50) = 25 nM). This ligand possesses novel melanocortin receptor pharmacology, as compared to previously reported peptides, and is potentially useful for in vivo studies to differentiate MC3R vs MC4R physiological roles in animal models, such as primates, where "knockout" animals are not viable options. The DNal(2') substitution for DPhe resulted in a mMC3R partial agonist with antagonist activity (pA(2) = 6.5, K(i) = 295 nM) and a mMC4R (pA(2) = 7.8, K(i) = 17 nM) antagonist possessing 60- and 425-fold decreased potency, respectively, as compared with SHU9119 at these receptors. Examination of this DNal(2')-containing tetrapeptide at the F254S and F259S mutant mMC4Rs resulted in agonist activity of this mMC4R tetrapeptide antagonist, similar to that observed for the SHU9119 peptide, supporting our previously proposed hypothesis that the Phe 254 and 259 transmembrane six receptor residues are important for differentiating melanocortin sequence-based MC4R antagonists vs the agouti-related protein (AGRP) sequence-based antagonists.
Asunto(s)
Oligopéptidos/síntesis química , Fenilalanina/química , Receptores de Corticotropina/efectos de los fármacos , Receptores de Péptidos/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Oligopéptidos/química , Oligopéptidos/farmacología , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/agonistas , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Melanocortina , Receptores de Péptidos/agonistas , Receptores de Péptidos/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
The melanocortin pathway is an important participant in obesity and energy homeostasis. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). The melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp", and it has been well documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library based on the template Ac-His-DPhe-Arg-Trp-NH(2), consisting of 17 members that have been modified at the His(6) position (alpha-MSH numbering) and pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. These studies provide further experimental evidence that the His(6) position can determine MC4R versus MC3R agonist selectivity and that chemically nonreactive side chains may be substituted for the imidazole ring (generally needs to be side chain protected in synthetic schemes) in the design of MC4R-selective, small-molecule, non-peptide agonists. Specifically, the tetrapeptide containing the amino-2-naphthylcarboxylic acid (Anc) amino acid at the His position resulted in a potent agonist at the mMC4R (EC(50) = 21 nM), was a weak mMC3R micromolar antagonist (pA(2) = 5.6, K(i) = 2.5 microM), and possessed >4700-fold agonist selectivity for the MC4R versus the MC3R. Substitution of the His(6) amino acid in the tetrapeptide template by the Phe, Anc, 3-(2-thienyl)alanine (2Thi), and 3-(4-pyridinyl)alanine (4-Pal) resulted in equipotency or only up to a 7-fold decrease in potency, compared to the His(6)-containing tetrapeptide at the mMC4R, demonstrating that these amino acid side chains may be substituted for the imidazole in the design of MC4R-selective non-peptide molecules.
Asunto(s)
Histidina/química , Oligopéptidos/síntesis química , Fragmentos de Péptidos/síntesis química , Receptores de Corticotropina/agonistas , alfa-MSH/química , Animales , Línea Celular , Técnicas Químicas Combinatorias , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Oligopéptidos/química , Oligopéptidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/genética , Receptores de Melanocortina , Receptores de Péptidos/agonistas , Receptores de Péptidos/genética , Relación Estructura-ActividadRESUMEN
The melanocortin pathway is involved in the regulation of several physiological functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The endogenous melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," postulated to be important for melanocortin receptor molecular recognition and stimulation. Herein, we report a tetrapeptide library, based upon the template Ac-His-D-Phe-Arg-Trp-NH(2), consisting of 20 members that have been modified at the Trp(9) position (alpha-MSH numbering) and pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. Results from this study yielded compounds that ranged in pharmacological properties from equipotent to a loss of melanocortin receptor activity at up to 100 microM concentrations. Interestingly, modification of the Trp(9) in the tetrapeptide template at the MC1R resulted in only up to a 220-fold potency change, while at the MC4R and MC5R, up to a 9700-fold decrease in potency was observed, suggesting the MC1R is more tolerant of the modifications examined herein. The most notable results of this study include identification that the Trp(9) indole moiety in the tetrapeptide template is important for melanocortin-3 receptor agonist potency, and that this position can be used to design melanocortin ligands possessing receptor selectivity for the peripherally expressed MC1 and MC5 versus the centrally expressed MC3 and MC4 receptors. Specifically, the Ac-His-D-Phe-Arg-Tic-NH(2) and the Ac-His-D-Phe-Arg-Bip-NH(2) tetrapeptides possessed nanomolar MC1R and MC5R potency but micromolar MC3R and MC4R agonist potency. Additionally, these studies identified that substitution of the Trp amino acid with either Nal(2') or D-Nal(2') resulted in equipotent melanocortin receptor potency, suggesting that the chemically reactive Trp indole side chain may be replaced with the nonreactive Nal(2') moiety for the design of nonpeptide melanocortin receptor agonists.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/química , Oligopéptidos/síntesis química , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/agonistas , Triptófano/química , Sustitución de Aminoácidos , Animales , Ligandos , Ratones , Oligopéptidos/química , Oligopéptidos/farmacología , Receptores de Corticotropina/metabolismo , Receptores de Melanocortina , Relación Estructura-ActividadRESUMEN
Agouti-related protein (AGRP) is one of two known naturally occurring antagonists of G-protein coupled receptors. AGRP is synthesized in the brain and is an antagonist of the melanocortin-3 and -4 receptors (MC3R, MC4R). These three proteins are involved in the regulation of energy homeostasis and obesity in both mice and humans. The human AGRP protein is 132 amino acids and contains five disulfide bridges in the C-terminal domain. Previous reports of the NMR structures of hAGRP(87-132) and a truncated 34 amino acid form consisting of four disulfide bridges identified that AGRP contains an inhibitor cystine knot (ICK) structural fold, and that is the first mammalian example. Herein, we report a bicyclic hAGRP analogue that, when compared to hAGRP(87-132), possesses equal binding affinity but is 80-fold less potent at the mouse MC4R. Using NMR, computer assisted molecular modeling (CAMM), and cluster analysis, we have identified five structural families, two of which are highly populated, of this bicyclic hAGRP analogue. Computational docking experiments of this bicyclic hAGRP derivative, using a three-dimensional homology molecular model of the mouse MC4R, identified that three of the five structural families could be docked into the MC4R without problems from steric hindrance. Those three docked mMC4R-bicyclic hAGRP family structures were compared with putative hAGRP(87-132) ligand-receptor interactions previously reported (Wilczynski et al. J. Med. Chem. 2004, 47, 2194) in attempts to identify a "bioactive" conformation of the bicyclic hAGRP peptide and account for the 80-fold decreased ligand potency compared to hAGRP(87-132).
Asunto(s)
Péptidos Cíclicos/síntesis química , Proteínas/química , Receptores de Melanocortina/antagonistas & inhibidores , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , AMP Cíclico/biosíntesis , Motivos Nodales de Cisteina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Conformación Proteica , Ensayo de Unión RadioliganteRESUMEN
The agouti-related protein (AGRP) is an endogenous antagonist of the centrally expressed melanocortin receptors. The melanocortin-4 receptor (MC4R) is involved in energy homeostasis, food intake, sexual function, and obesity. The endogenous hAGRP protein is 132 amino acids in length, possesses five disulfide bridges at the C-terminus of the molecule, and is expressed in the hypothalamus of the brain. We have previously reported that a monocyclic hAGRP(103-122) peptide is an antagonist at the melanocortin receptors expressed in the brain. Stereochemical inversion from the endogenous l- to d-isomers of single or multiple amino acid modifications in this monocyclic truncated hAGRP sequence resulted in molecules that are converted from melanocortin receptor antagonists into melanocortin receptor agonists. The Asp-Pro-Ala-Ala-Thr-Ala-Tyr-cyclo[Cys-Arg-DPhe-DPhe-Asn-Ala-Phe-Cys]-Tyr-Ala-Arg-Lys-Leu peptide resulted in a 60 nM melanocortin-1 receptor agonist that is 100-fold selective versus the mMC4R, 1000-fold selective versus the mMC3R, and ca. 180-fold selective versus the mMC5R. In attempts to identify putative ligand-receptor interactions that may be participating in the agonist induced stimulation of the MC4R, selected ligands were docked into a homology molecular model of the mMC4R. These modeling studies have putatively identified hAGRP ligand DArg111-mMC4RAsn115 (TM3) and the hAGRP DPhe113-mMC4RPhe176 (TM4) interactions as important for agonist activity.
Asunto(s)
Proteínas/química , Receptor de Melanocortina Tipo 1/agonistas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptor de Melanocortina Tipo 4/agonistasRESUMEN
Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide template Tyr-c[Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that is equipotent to alpha-MSH at the mMC1, mMC3, and mMC5 receptors but is 30-fold more potent than alpha-MSH at the mMC4R. Additionally, these studies identified a new and novel >200-fold MC4R versus MC3R selective peptide Tyr-c[Asp-D-Phe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) template. Furthermore, when the His-DPhe-Arg-Trp sequence is used to replace the hAGRP Arg-Phe-Phe residues in the "mini"-AGRP (hAGRP87-120, C105A) template, a potent nanomolar agonist resulted at the mMC1R and MC3-5Rs.
Asunto(s)
Oligopéptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/síntesis química , Receptor de Melanocortina Tipo 4/metabolismo , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , AMP Cíclico/biosíntesis , Diseño de Fármacos , Humanos , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Receptor de Melanocortina Tipo 4/agonistas , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The melanocortin pathway is involved in the regulation of several physiological functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) located in the brain are implicated as participating in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). All the endogenous (POMC-derived) melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp." Herein, we report 12 tetrapeptides, based upon the template Ac-His(6)-DPhe(7)-Arg(8)-Trp(9)-NH(2) (alpha-MSH numbering) that have been modified at the Arg(8) position by neutral, basic, or acidic amino acid side chains. These peptides have been pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the observation that removal of the guanidinyl side chain moiety results in decreased melanocortin receptor potency, but that this Arg(8) side chain is not critical for melanocortin receptor agonist activity. Additionally, incorporation of the homoArg(8) residue results in 56-fold MC4R versus MC3R selectivity, and the Orn(8) residue results in 123-fold MC4R versus MC5R and 63-fold MC5R versus MC3R selectivity.
Asunto(s)
Arginina/química , Oligopéptidos/efectos de los fármacos , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/efectos de los fármacos , Animales , Línea Celular , Humanos , Ratones , Oligopéptidos/química , Receptores de Corticotropina/clasificación , Receptores de Melanocortina , Relación Estructura-ActividadRESUMEN
The agouti-related protein (AGRP) is an endogenous antagonist of the brain melanocortin receptors (MC3R and MC4R) and is believed to be involved in feeding behavior and energy homeostasis. Previous results identified that the human AGRP decapeptide Yc[CRFFNAFC]Y binds to the MC3R and MC4R and acts as an antagonist at the MC4R but not at the MC3R. We have synthesized the amidated version of this decapeptide as well as performed elongation studies at both the N-and C-terminus of the monocyclic hAGRP(109-118) peptide. This study was designed to identify monocyclic peptide fragments of the hAGRP(86-132) to determine the minimal active monocyclic sequence necessary for antagonism at the MC3R. For binding studies, radiolabeled 125I-NDP-MSH versus 125I-hAGRP(86-132) were utilized to determine if there were differences in the ability of the AGRP fragments prepared herein to competitively displace the 125I-NDP-MSH versus AGRP(86-132) radiolabel. The binding IC(50) values of radiolabeled hAGRP(86-132) versus NDP-MSH were identical within experimental error, supporting the hypothesis that AGRP and NDP-MSH interact with overlapping binding epitopes at the MC3R and MC4R. The most notable results include identification of the TAYc[CRFFNAFC]YAR-NH(2) (pA(2)=6.1, K(i)=790nM, mMC3R) and the Yc[CRFFNAFC]YARKL-NH(2) (pA(2)=6.2, K(i)=630nM, mMC3R) peptides as the minimal monocyclic AGRP-based fragments possessing antagonist pharmacology at the MC3R. Interestingly, extension of the AGRP(109-118) decapeptide at both the N- and C-terminus by two amino acids conferred detectable mMC3R antagonism, while retaining high nanomolar MC4R antagonist and micromolar MC1R agonist pharmacological properties. These data support the hypothesis that elongation of the hAGRP(109-118) decapeptide results in antagonism at the MC3R while retaining MC1R agonist activity and MC4R antagonist activity.
Asunto(s)
Fragmentos de Péptidos/farmacología , Receptor de Melanocortina Tipo 3/antagonistas & inhibidores , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Expresión Génica , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Receptor de Melanocortina Tipo 3/genética , Receptor de Melanocortina Tipo 3/metabolismoRESUMEN
Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Proteínas/farmacología , Receptores de Melanocortina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/química , Proteína Relacionada con Agouti , Aminoácidos/química , Línea Celular , AMP Cíclico/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Oligopéptidos/síntesis química , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Proteínas/química , Receptores de Melanocortina/antagonistas & inhibidores , Receptores de Melanocortina/metabolismo , Proteínas Recombinantes de Fusión/químicaRESUMEN
The melanocortin system is implicated in multiple physiological pathways including pigmentation, inflammation, erectile function, feeding behavior, energy homeostasis, weight homeostasis, and exocrine gland function, just to list a few. The endogenous agonists for the melanocortin receptors include the gene transcripts derived from the proopiomelanocortin gene and include the core tetrapeptide His-Phe-Arg-Trp sequence postulated to be important for melanocortin receptor selectivity and stimulation. Posttranslational processing of the proopiomelanocortin derived agonists results in the N-terminal acetylation and C-terminal amidation of alpha-melanocyte stimulation hormone (alpha-MSH). In this study we generated 25 N-terminally "capped" tetrapeptides containing the core sequence X-His-D-Phe-Arg-Trp-NH(2) and pharmacologically characterized them at the mouse melanocortin MC(1) receptor, melanocortin MC(3) receptor, melanocortin MC(4) receptor, and melanocortin MC(5) receptor. The N-terminal "capping" groups consisted of linear, cyclic, or aromatic moieties and all resulted in full agonist activity at the melanocortin receptors examined in this study. Increasing aliphatic chain length increased potency of the tetrapeptide derivatives, with the addition of octanoyl capping group resulting in 70- to 110-fold increased tetrapeptide potency at the melanocortin MC(1) receptor (EC(50)=0.4 nM), melanocortin MC(3) receptor (EC(50)=4.0 nM), and melanocortin MC(4) receptor (EC(50)=0.4 nM) while only enhancing potency at the melanocortin MC(5) receptor (EC(50)=0.8 nM) by 8-fold, compared to the tetrapeptide His-D-Phe-Arg-Trp-NH(2). This octanoyl derivative surprisingly resulted in a 14-fold greater potency than alpha-MSH (EC(50)=5.4 nM) at the mouse melanocortin MC(4) receptor implicated in feeding behavior and obesity. The 3,3,3-triphenylpropionyl derivative resulted in greater than 14 microM agonist potencies at the melanocortin MC(1) receptor, melanocortin MC(3) receptor, and melanocortin MC(4) receptor and possessed a 140 nM agonist EC(50) value at the melanocortin MC(5) receptor. This 3,3,3-triphenylpropionyl-His-D-Phe-Arg-Trp-NH(2) peptide is a 100-fold selective agonist for the melanocortin MC(5) receptor, versus the other melanocortin receptors studied herein, and is the first melanocortin MC(5) receptor selective tetrapeptide derivative reported to date with nanomolar potency.
Asunto(s)
Oligopéptidos/farmacología , Receptores de Corticotropina/agonistas , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Oligopéptidos/síntesis química , Oligopéptidos/química , Plásmidos/genética , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/genética , Receptores de Melanocortina , TransfecciónRESUMEN
Tobacco addiction is one of the leading causes of preventable death worldwide. Despite the negative health outcomes of tobacco use and a desire to quit, there is a low success rate of maintaining abstinence. Nicotine, the main psychoactive component of tobacco smoke, is mildly rewarding and maintains smoking behavior. Nicotine withdrawal induces somatic symptoms that may contribute to smoking behavior. However, it has been hypothesized that the negative affective signs are of greater motivational significance in contributing to relapse and continued tobacco use than the somatic symptoms of nicotine withdrawal (Markou and Koob (Eds.) Intracranial self-stimulation thresholds as a measure of reward, Vol. 2, Oxford University Press, New York, 1993; Koob et al. Semin Neurosci 5: 351-358, 1993). Intracranial self-stimulation (ICSS) has been established as a method to assess the bivalent properties of nicotine exposure and withdrawal from acute and chronic nicotine administration. Thus, ICSS provides a means to measure the negative affective aspects of nicotine withdrawal in animal models and may contribute to the understanding of the neurobiological bases of nicotine dependence and the development of effective treatment strategies to facilitate nicotine abstinence.
Asunto(s)
Estimulación Encefálica Profunda/métodos , Nicotina/administración & dosificación , Tabaquismo , Animales , Conducta Animal , Masculino , Modelos Animales , Nicotina/efectos adversos , Ratas , Autoadministración , Fumar , Síndrome de Abstinencia a Sustancias , Trastornos Relacionados con SustanciasRESUMEN
Extrasynaptic glutamate has been shown to regulate dopamine function in the mesocorticolimbic pathway, which plays an important role in the behavioral pharmacology of psychostimulants. Basal levels of glutamate are primarily regulated by the cystine-glutamate transporter and provide glutamatergic tone on extrasynaptic glutamate receptors. The present study examined the effects of a cystine-glutamate transporter enhancer on the neurochemical and behavioral effects of cocaine and amphetamine in nonhuman primates. It was hypothesized that augmenting extrasynaptic glutamate release with N-acetyl-L-cysteine (NAC), a cystine prodrug, would attenuate cocaine- or amphetamine-induced increases in extracellular dopamine and their corresponding behavioral-stimulant and reinforcing effects. In vivo microdialysis was used to evaluate cocaine-induced changes in extracellular dopamine (DA) in the caudate nucleus (n=3). NAC significantly attenuated cocaine-induced increases in dopamine but had inconsistent effects on amphetamine-induced increases in dopamine (n=4). Separate groups of subjects were either trained on a fixed-interval schedule of stimulus termination (n=6) or on a second-order schedule of self-administration (n=5) to characterize the behavioral-stimulant and reinforcing effects of psychostimulants, respectively. Systemic administration of NAC did not alter the behavioral-stimulant effects of either cocaine or amphetamine. Furthermore, cocaine self-administration and reinstatement of previously extinguished cocaine self-administration were not altered by pretreatment with NAC. Hence, drug interactions on caudate neurochemistry in vivo were not reflected in behavioral measures in squirrel monkeys. The present results in nonhuman primates do not support the use of NAC as a pharmacotherapy for cocaine abuse, although rodent and clinical studies suggest otherwise.
Asunto(s)
Acetilcisteína/farmacología , Sistema de Transporte de Aminoácidos y+/metabolismo , Cocaína/administración & dosificación , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Acetilcisteína/uso terapéutico , Sistema de Transporte de Aminoácidos y+/agonistas , Animales , Conducta Adictiva/tratamiento farmacológico , Conducta Adictiva/metabolismo , Cocaína/antagonistas & inhibidores , Masculino , Saimiri , AutoadministraciónRESUMEN
Smoking is one of the leading preventable causes of disease, disability, and death in the USA and leads to more than 400,000 preventable deaths per year. Nicotine is the major alkaloid present in tobacco smoke, and many of the negative effects of smoking are attributed to nicotine. Nicotine is not only the addictive component of tobacco smoke, but also highly associated with carcinogenesis and induces oxidative stress. Furthermore, the administration of nicotine via subcutaneous mini-osmotic pumps or by injection is an established method in preclinical studies for this area of research. Thus, preclinical research on the negative effects of tobacco smoke and tobacco addiction has focused primarily on the effects of nicotine. However, there are over 4,500 components found in tobacco smoke, many of which are highly toxic. Other components may also contribute to the addictive properties of tobacco smoke. Furthermore, the negative effects of tobacco smoke are not isolated to the smoker but can have negative effects to those exposed to the secondhand smoke (SHS) stream. SHS exposure is the third leading cause of preventable death. Approximately 38,000 deaths per year are attributed to SHS exposure in the USA. SHS exposure increases the risk of heart disease by approximately 30% and is associated with increased risk of stroke, cancer, type II diabetes, as well as pulmonary disease. Thus, methods of administering tobacco smoke in a controlled environment will further our understanding of tobacco addiction and the role tobacco smoke in other disease states. Moreover, combining smoke exposure with proteomics can lead to the discovery of biomarkers that can be potentially useful tools in screening, early diagnosis, prevention, and treatment of diseases caused by SHS.
Asunto(s)
Nicotina/administración & dosificación , Nicotina/efectos adversos , Contaminación por Humo de Tabaco/efectos adversos , Animales , Exposición a Riesgos Ambientales , Masculino , Modelos Animales , Estrés Oxidativo , Proteómica/métodos , Ratas , Ratas Sprague-Dawley , Humo/efectos adversos , Fumar , Espectrometría de Masas en Tándem/métodos , Tabaquismo/mortalidadRESUMEN
These studies investigated if pre-exposure to tobacco smoke affects the locomotor response to tobacco smoke, nicotine, and amphetamine in adult rats. The rats were habituated to an open field for 3-4 days and then exposed to tobacco smoke for 2h/day for 13-14 days. The effect of exposure to tobacco smoke on locomotor activity was investigated after 1, 7, and 14 days of smoke exposure and after one 2-hour exposure session that followed a 3-week off period. The effects of tobacco smoke on the locomotor responses to nicotine (0.04 and 0.4 mg/kg, base) and amphetamine (0.1 and 0.5mg/kg) were investigated on day 14, one day after the last smoke exposure session. The locomotor response to tobacco smoke was increased after 7 and 14 days of smoke exposure and after one exposure session after the 3-week off-period. The acute administration of the high dose of nicotine (0.4 mg/kg) led to a brief period of hypoactivity that was followed by a period of hyperactivity. Pre-exposure to tobacco smoke attenuated the nicotine-induced hypoactivity and potentiated the nicotine-induced hyperactivity. The low dose of nicotine (0.04 mg/kg) did not affect locomotor activity in the control rats but increased the total distance traveled in the tobacco smoke exposed rats. Exposure to tobacco smoke did not affect the locomotor response to amphetamine. These findings indicate that exposure to tobacco smoke leads to tolerance to the depressant effects of nicotine and potentiates the stimulant effects of nicotine and tobacco smoke.
Asunto(s)
Anfetamina/administración & dosificación , Actividad Motora/efectos de los fármacos , Nicotiana , Nicotina/administración & dosificación , Fumar , Factores de Edad , Animales , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Sinergismo Farmacológico , Exposición por Inhalación/efectos adversos , Masculino , Actividad Motora/fisiología , Ratas , Ratas Wistar , Fumar/efectos adversosRESUMEN
Brain disorders and environmental factors can affect neurogenesis and gliogenesis in the hippocampus. These studies investigated the effects of chronic exposure to tobacco smoke on progenitor cell proliferation and the survival and phenotype of new cells in the dentate gyrus of adolescent rats. The rats were exposed to tobacco smoke for 4h/day for 14 days. To investigate cell proliferation, the exogenous marker 5-bromo-2'-deoxyuridine (BrdU, 200mg/kg, ip) was administered 2h into the 4-h smoke exposure session on day 14. The rats were sacrificed 2-4h after the administration of BrdU. To investigate cell survival, the same dose of BrdU was administered 24h before the start of the 14-day smoke exposure period. These rats were sacrificed 24h after the last smoke exposure session. Tobacco smoke exposure decreased both the number of dividing progenitor cells (-19%) and the number of surviving new cells (-20%), labeled with BrdU in the dentate gyrus. The decrease in cell proliferation was not associated with an increase in apoptotic cell death, as shown by TUNEL analysis. Colocalization studies indicated that exposure to tobacco smoke decreased the number of new immature neurons (BrdU/DCX-positive) and transition neurons (BrdU/DCX/NeuN-positive) and increased the number of new glial cells (BrdU/GFAP-positive). These findings demonstrate that exposure to tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats. These effects may play a role in the increased risk for depression and cognitive impairment in adolescent smokers.
Asunto(s)
Giro Dentado/citología , Giro Dentado/fisiología , Inhibición Neural/fisiología , Neurogénesis/fisiología , Neuroglía/citología , Neuroglía/fisiología , Fumar/efectos adversos , Factores de Edad , Animales , Proliferación Celular/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Proteína Doblecortina , Masculino , Inhibición Neural/efectos de los fármacos , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/fisiología , Neurogénesis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Ratas , Ratas Wistar , Fumar/patologíaRESUMEN
The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC(3) in the regulation of food intake, JRH322-18 a mixed MC(3) partial agonist/antagonist and MC(4) agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC(4)) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC(4)KO mice significantly reduced cumulative food intake 24h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC(3) and MC(4) knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC(3) and MC(4) knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC(3) and MC(4) knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC(3) plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Receptor de Melanocortina Tipo 3/metabolismo , Secuencia de Aminoácidos , Animales , Ingestión de Alimentos/genética , Técnicas de Inactivación de Genes , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Receptor de Melanocortina Tipo 3/agonistas , Receptor de Melanocortina Tipo 3/antagonistas & inhibidores , Receptor de Melanocortina Tipo 3/genética , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , Saciedad , alfa-MSH/análogos & derivados , alfa-MSH/química , alfa-MSH/farmacologíaRESUMEN
Epidemiological studies indicate that parental smoking increases the risk for smoking in children. However, the underlying mechanisms by which parental smoking increases the risk for smoking are not known. The aim of these studies was to investigate if preadolescent tobacco smoke exposure, postnatal days 21-35, affects the rewarding effects of nicotine and nicotine withdrawal in adult rats. The rewarding effects of nicotine were investigated with the conditioned place preference procedure. Nicotine withdrawal was investigated with the conditioned place aversion procedure and intracranial self-stimulation (ICSS). Elevations in brain reward thresholds in the ICSS paradigm reflect a dysphoric state. Plasma nicotine and cotinine levels in the preadolescent rats immediately after smoke exposure were 188 ng/ml and 716 ng/ml, respectively. Preadolescent tobacco smoke exposure led to the development of nicotine dependence as indicated by an increased number of mecamylamine-precipitated somatic withdrawal signs in the preadolescent tobacco smoke exposed rats compared to the control rats. Nicotine induced a similar place preference in adult rats that had been exposed to tobacco smoke or air during preadolescence. Furthermore, mecamylamine induced place aversion in nicotine dependent rats but there was no effect of preadolescent tobacco smoke exposure. Finally, preadolescent tobacco smoke exposure did not affect the elevations in brain reward thresholds associated with precipitated or spontaneous nicotine withdrawal. These studies indicate that passive exposure to tobacco smoke during preadolescence leads to the development of nicotine dependence but preadolescent tobacco smoke exposure does not seem to affect the rewarding effects of nicotine or nicotine withdrawal in adulthood.