RESUMEN
Fossil bones are often the only materials available for chronological reconstruction of important archeological sites. However, since bone is an open system for uranium, it cannot be dated directly and therefore it is necessary to develop models for the U uptake. Hence, a radial diffusion-adsorption (RDA) model is described. Unlike the classic diffusion-adsorption (D-A) model, RDA uses a cylindrical geometry to describe the U uptake in fossil bones. The model was applied across a transverse section of a tibia of an extinct megamammal Macrauchenia patachonica from the La Paz Local Fauna, Montevideo State, Uruguay. Measurements of spatial distribution of Na, K, Ca, and Mg were also performed by neutron activation analysis (NAA). Gamma-ray spectrometric U-series dating was applied to determine the age of the bone sample. From U concentration profile, it was possible to observe the occurrence of a relatively slow and continuous uranium uptake under constant conditions that had not yet reached equilibrium, since the uranium distribution is a âª-shaped closed-system. Predictions of the RDA model were obtained for a specific geochemical scenario, indicating that the effective diffusion coefficient D/R in this fossil bone is (2.4 ± 0.6)10(-12) cm(2)s(-1). Mean values of Na, K, Ca, and Mg contents along the radial line of the fossil tibia are consistent with the expected behavior for spatial distributions of these mineral elements across a modern bone section. This result indicates that the fossil tibia may have its mineral structure preserved.
Asunto(s)
Arqueología/métodos , Fósiles , Modelos Teóricos , Paleontología/métodos , Tibia/química , Uranio/química , Adsorción , Animales , Calcio/química , Difusión , Mamíferos , Metales Ligeros/química , Análisis de Activación de Neutrones , Espectrometría gamma , Uranio/análisisRESUMEN
Several observations highlight the importance of the carbohydrate moiety for the biological activity of antitumoural anthracyclines. Here is reported the synthesis, cytotoxicity and topoisomerase II-mediated DNA cleavage intensity of the new oligosaccharide anthracyclines 1--4 modified in the sugar residue. Evaluation of cytotoxic potency on different cell lines, resulted in quite similar values among the different analogues. On the other hand, topoisomerase II-mediated DNA breaks level was different for the various compounds, and was not related to cytotoxicity, thus supporting previous observations reported for some monosaccharide anthracyclines modified in the carbohydrate portion.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Oligosacáridos/farmacología , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Carbohidratos/química , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Oligosacáridos/síntesis química , Oligosacáridos/química , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in -1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug's efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.
Asunto(s)
Antineoplásicos/farmacología , Disacáridos/farmacología , Doxorrubicina/farmacología , Apoptosis , Ciclo Celular/efectos de los fármacos , Aductos de ADN/metabolismo , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Disacáridos/química , Doxorrubicina/análogos & derivados , Humanos , Fracciones Subcelulares , Células Tumorales CultivadasRESUMEN
We have studied the effect of zofenopril, a new angiotensin-converting enzyme inhibitor in preventing cardiac injury induced by chronic doxorubicin treatment in rats. Cardiac function was assessed by measuring changes in electrocardiogram (ECG) tracings, haemodynamics and cardiac responses in vivo to isoprenaline, 4 weeks after suspension of doxorubicin treatment, in vehicle-treated rats and in animals receiving zofenopril (15 mg/kg/os/day) alone, doxorubicin (1.5 mg/kg i.v. once a week for 5 weeks) or zofenopril+doxorubicin treatment. Doxorubicin induced a significant lengthening of the QalphaT interval, which was completely prevented by zofenopril treatment. The cardiac positive inotropic effect induced by i.v. isoprenaline was selectively depressed by doxorubicin (no changes in chronotropic responses) and this adverse effect of doxorubicin was also prevented in zofenopril+doxorubicin pretreated rats. Doxorubicin induced a significant increase in relative heart weight, which was likewise prevented in zofenopril+doxorubicin treated rats. In separate experiments, zofenopril did not interfere with the antitumor activity of doxorubicin (inhibition of tumor growth in nude mice xenografted with A2780 human tumor line). In conclusion, the oral administration of zofenopril is able to significantly ameliorate, up to 4 weeks after the end of doxorubicin administration, doxorubicin-induced cardiotoxicity without affecting the antitumor activity of this anthracycline.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antineoplásicos/antagonistas & inhibidores , Captopril/análogos & derivados , Captopril/farmacología , Cardiotónicos/farmacología , Doxorrubicina/antagonistas & inhibidores , Electrocardiografía/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Isoproterenol/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antineoplásicos/efectos adversos , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Captopril/uso terapéutico , Doxorrubicina/efectos adversos , Quimioterapia Combinada , Femenino , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica/fisiología , Masculino , Ratones , Ratones Desnudos , Tamaño de los Órganos/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytotoxic drugs commonly used in cancer therapy promote tumor cell death by inducing apoptosis, but the cell death pathway(s) is likely dependent on the mechanism of drug action. In the present study, we investigated the mechanisms of cell death induced by doxorubicin (DXR) and the novel disaccharide anthracycline MEN 10755, in a human ovarian cancer cell line (A2780). Exposure to either anthracycline induced the up-regulation of several genes known to promote cell cycle arrest and DNA repair (WAF1/p21, GADD45) or apoptosis (bax, Fas). Although the expression of Fas was increased, an antagonistic anti-Fas antibody ZB4 did not inhibit anthracycline-induced apoptosis, suggesting that the stimulation of the Fas receptor did not play a critical role in the induction of apoptosis in this cell line. We also observed that neither MEN 10755 nor DXR were able to induce apoptosis in A2780 cells deprived of the nucleus but retaining an intact mitochondrial function (cytoplasts) and that apoptosis induced by either anthracycline was inhibited by cycloheximide, indicating that it is an active process requiring new protein synthesis. Both the caspases inhibitors, ZVAD-fmk and DEVD-cho, inhibited at similar extent apoptosis induced by either DXR or MEN 10755, suggesting an involvement of caspase-3 in this response. We conclude that, in a tumor cell line of epithelial origin, the apoptosis following exposure to anthracyclines is an active process requiring protein synthesis and drug interaction with nuclear structures. The pathway was Fas-independent but likely involved bax and caspase-3 as effectors of the cascade culminating in apoptosis.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Ováricas/patología , Anexinas/metabolismo , Caspasa 3 , Caspasas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Doxorrubicina/farmacología , Femenino , Citometría de Flujo , Humanos , Mitocondrias/efectos de los fármacos , Ensayos de Protección de Nucleasas , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas , Receptor fas/biosíntesisRESUMEN
Anthracycline antibiotics play an important role in cancer chemotherapy. The need for an improvement of their therapeutic index has stimulated an ongoing search for anthracycline analogues with improved properties. Analogue development was originally limited by a lack of information on the cellular drug target, nevertheless almost 20 years ago the mechanism of action of doxorubicin and daunorubicin was revealed and DNA topoisomerase II was recognised to be their main cellular target. Several anthracyclines interfere with topoisomerase II functions by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to tyrosine residues of the enzyme. Investigations on the sequence specificity of doxorubicin in vitro and in nuclear chromatin of living cell have led to a molecular model of drug receptor on the topoisomerase II-DNA complex. Anthracyclines are likely placed at the interface between the DNA cleavage site and the active site of the enzyme, forming a DNA-drug-enzyme ternary complex. Moreover, a quite detailed structure-function relationship has been established for anthracyclines. First, drug intercalation is necessary but not sufficient for topoisomerase II poisoning; second, the removal of the 4-methoxy and 3'-amino substituents greatly increases the drug activity and third, the 3' substituent of the sugar moiety markedly influences the sequence selectivity of anthracycline-stimulated DNA cleavage. These relationships have been exploited during the last decade by several groups, including ours, in the search for new anthracycline drugs with lower side effects and higher activity against resistant cancer cells. This review will focus on areas of the anthracycline field including synthesis of new analogues, new strategies of synthesis and recent developments in the area of drug delivery.
Asunto(s)
Antibióticos Antineoplásicos , Animales , Antibióticos Antineoplásicos/síntesis química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Diseño de Fármacos , Humanos , Relación Estructura-ActividadRESUMEN
The effects of aging on the activation of the cytoplasmic tyrosine protein kinase p56(lck) have been investigated in PBL from adult and elderly subjects upon activation with mitogens or different co-stimuli. Results show that the amount and phosphorylation of p56(lck) are reduced in PBL from elderly as compared to adult subjects. This finding suggests that alterations in p56(lck) may contribute to the age-associated loss of some T cell functions, such as proliferation and IL-2 production, which are found decreased in PBL from old individuals. However, p56(lck) seems irrelevant to the production of IFN-gamma and IL-4 which were both found increased in the PBL from old subjects, as expected from the relative expansion of memory versus naive T cell subpopulations in aging.
Asunto(s)
Envejecimiento/metabolismo , Leucocitos Mononucleares/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , División Celular , Células Cultivadas , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Leucocitos Mononucleares/citología , Mitosis , FosforilaciónRESUMEN
Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.
Asunto(s)
Envejecimiento/metabolismo , Antígenos Nucleares , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/fisiología , División Celular , Extractos Celulares , Núcleo Celular/metabolismo , Citocinas/biosíntesis , ADN/metabolismo , Dimerización , Humanos , Autoantígeno Ku , Leucocitos Mononucleares , MitosisRESUMEN
We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice. Cytokine production was evaluated in culture supernatants by CTLL proliferation (IL-2), ELISA (IFN-gamma), CT4.S proliferation (IL-4) and in mRNA extracted from activated CD4+ cells by RT-PCR (IL-2, IFN-gamma and IL-4). Results show that the production of IL-2, as protein and mRNA, is profoundly depressed by aging, whereas that of IFN-gamma, as protein and mRNA, firstly declines and then increases with age. The production of IL-4, as protein, monotonically declines with aging whereas, as mRNA, firstly decreases and then increases above the level in young mice. Spleen cells in culture were also incubated with mitogens and with a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12 or IFN-gamma) at various concentrations. It was found that recombinant cytokines by and large enhance cytokine production when the level induced by mitogens only is low. This conclusion applies to IL-2 and IFN-gamma production as protein and mRNA. The addition of recombinant cytokines also increases the production of IL-4 at the protein level in spleen cells from old mice but, at the mRNA level, only in spleen cells from young mice. This finding suggests age-related changes in IL-4-specific mRNA transcription rate and post-transcriptional half-life as well as translation kinetics.
Asunto(s)
Envejecimiento/inmunología , Citocinas/biosíntesis , Citocinas/farmacología , Regulación del Desarrollo de la Expresión Génica , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Femenino , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Células TH1/inmunología , Células Th2/inmunología , Transcripción GenéticaRESUMEN
The binding characteristics of the novel radioligand [3H][beta-Ala8]neurokinin A-(4-10) were assessed in hamster urinary bladder membranes. This labelled compound bound in a reversible, highly specific and concentration-dependent manner to a single class of high affinity binding sites with a Kd of 1.8 +/- 0.2 nM and a Bmax of 139 +/- 21 fmol/mg of protein. Specific binding of [3H][beta-Ala8]neurokinin A-(4-10) was displaced only by NK2, but not by NK1 or NK3, tachykinin receptor agonists and antagonists. Neurokinin A, [beta-Ala8]neurokinin A-(4-10), L 659877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], MEN 10376 (H-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH2), MEN 10627 [cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta)] and SR 48968 [(S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2- (3,4-dichlorophenyl)butyl]benzamide] displaced the binding with Ki values of 0.4 +/- 0.1 nM, 1.9 +/- 0.36 nM, 3.05 +/- 0.1 nM, 7.9 +/- 0.4 microM, 0.36 +/- 0.02 nM and 2.5 +/- 0.9 nM, respectively. Functional data, obtained in isolated hamster urinary bladder strips with the newly developed tachykinin NK2 receptor antagonists (MEN 10627 and SR 48968), showed a good agreement with binding data. This novel radioligand could represent a new useful tool for the assessment of tachykinin NK2 receptor antagonists.
Asunto(s)
Neuroquinina A/análogos & derivados , Fragmentos de Péptidos/farmacología , Receptores de Neuroquinina-2/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Unión Competitiva/efectos de los fármacos , Cricetinae , Técnicas In Vitro , Cinética , Ligandos , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Mesocricetus , Contracción Muscular/efectos de los fármacos , Neuroquinina A/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-3/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the superimposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 A distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the pi stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed.
Asunto(s)
Aminoácidos , Indoles/síntesis química , Antagonistas del Receptor de Neuroquinina-1 , Oligopéptidos/síntesis química , Sustancia P/antagonistas & inhibidores , Dicroismo Circular , Indoles/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Oligopéptidos/química , Conformación Proteica , Relación Estructura-Actividad , Sustancia P/análogos & derivados , Sustancia P/síntesis químicaRESUMEN
We have used the [3H]resiniferatoxin (RTX) binding assay to characterize for the first time a vanilloid (capsaicin) receptor in tracheobronchial tissues of the guinea pig. Membranes obtained from the trachea and the main bronchi bound RTX with an affinity of 1 nM; the cooperativity index was close to unity, indicating noncooperative binding. Specific [3H]RTX binding was fully inhibited by capsaicin (Ki = 500 nM) and capsazepine (Ki = 100 nM), but it was not inhibited at all by the inactive RTX structural analog resiniferonol 9, 13, 14-orthophenylacetate (10 microM), confirming the specificity of the binding. Neither was RTX binding inhibited by the functional vanilloid antagonist ruthenium red (100 microM). The density of specific RTX binding sites was similar in the trachea (Bmax = 150 fmol/mg protein) and the bronchi (Bmax = 170 fmol/mg protein). In keeping with the marked resistance of hamsters to capsaicin actions, no specific RTX binding could be detected in the airways of this species. By contrast, we have been able to demonstrate specific RTX binding sites in human bronchi: the estimated affinity for RTX, 2 nM, was similar to that (7 nM) determined in guinea pig bronchi. We conclude that (1) the [3H]RTX binding assay affords a novel biochemical marker for vanilloid-sensitive nerves in the airways, and (2) this binding assay may be a useful tool to explore species-related differences in the expression and pharmacologic profile of vanilloid receptors in the airways.
Asunto(s)
Bronquios/química , Capsaicina/metabolismo , Diterpenos/metabolismo , Neurotoxinas/metabolismo , Receptores de Droga/análisis , Tráquea/química , Adulto , Animales , Sitios de Unión , Bronquios/inervación , Capsaicina/análogos & derivados , Capsaicina/farmacología , Femenino , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Receptores de Droga/efectos de los fármacos , Tráquea/inervación , TritioRESUMEN
The cyclic pseudopeptides MEN 10,548, MEN 10,581, MEN 10,619, MEN 10,677, MEN 10,777 and MEN 10,867 were studied at tachykinin neurokinin (NK)1, NK2 and NK3 receptors on several in vitro bioassays. All compounds were potent and competitive antagonists at tachykinin NK1 and NK2 receptors of the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, showing the highest affinity for the hamster NK2 receptor (e.g., MEN 10,677: pKB = 9.3). By contrast, none showed affinity for NK3 receptors of the rat portal vein, up to 3 microM. In the guinea-pig isolated bronchus, the pseudopeptide compounds competitively antagonized the NK2 receptor-selective agonist [beta Ala8]-NKA (4-10) with potencies comparable to those shown at the rabbit NK2 receptor. In addition, the pseudopeptides were from 3.5-fold (MEN 10,677) to 16-fold (MEN 10548) more potent antagonists against septide than against [Sar9]SP sulfone, two agonists reportedly selective for two distinct sites/subtypes of the NK1 receptor. In binding experiments at human IM9 cells, the pseudopeptide compounds displaced [3H]substance P from human NK1 receptor, showing similar affinities to those displayed at the NK1 receptor in the guinea-pig ileum or bronchus against substance P methylester or septide, as agonists, respectively. This new class of pseudopeptide antagonists, by showing a comparable and high affinity at both tachykinin NK1 and NK2 receptors, might be proposed for treatment/prevention of airway diseases in which endogenous tachykinins play a role by activation of both receptors.
Asunto(s)
Antagonistas del Receptor de Neuroquinina-1 , Péptidos Cíclicos/metabolismo , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-3/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Unión Competitiva , Cricetinae , Cobayas , Humanos , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Mesocricetus , Datos de Secuencia Molecular , Péptidos Cíclicos/química , Vena Porta/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Conejos , Ratas , Ratas Wistar , Tráquea/efectos de los fármacosRESUMEN
We describe the in vitro and in vivo pharmacological properties of MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta), the first example of a polycyclic peptide tachykinin NK2 receptor antagonist. MEN 10,627 is endowed with high affinity for NK2 receptor expressed in various species with pKB values ranging between 10.1 (hamster trachea) and 8.1 (rabbit pulmonary artery). The antagonism is of competitive type in both functional and radioligand binding assays. A 100- to 10,000-fold selectivity was found vs. NK1 or NK3 receptors expressed in various species. As an NK2 receptor antagonist, MEN 10,627 is 10- to 100-fold more potent than the monocyclic peptide antagonist L 659,877 or cyclo(Met-Gln-Trp-Phe-Gly-Leu). At the hamster NK2 receptor, MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist SR 48,968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide], whereas the converse is true for the rabbit NK2 receptor. Furthermore, MEN 10,627 is, up to micromolar concentrations, devoid of antagonist properties toward a wide range of transmitters of both peptide and nonpeptide nature. In urethane-anesthetized rats in vivo, MEN 10,627 (10-100 nmol/kg i.v.) produced long-lasting inhibition of contraction of the urinary bladder and duodenum produced by i.v. administration of the NK2 receptor agonist [beta Ala8]NKA(4-10), without affecting the responses produced by i.v. administration of the NK1 receptor agonist [Sar9]SP sulfone or acetylcholine. In anesthetized rats, both MEN 10,627 and SR 48,968 blocked urinary bladder contraction induced by the NK2 receptor agonist after intravenous, intranasal or intraduodenal administration. Equieffective doses of MEN 10,627 producing about 50% inhibition of the response to [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo, were 0.01, 0.03 and 3 mumol/kg after intravenous, intranasal and intraduodenal administration, respectively. The corresponding doses of SR 48,968 were 0.03, 0.1 and 1 mumol/kg, after intravenous, intranasal and intraduodenal administration, respectively. In conclusion, MEN 10,627 is a potent and selective NK2 receptor antagonist, endowed with high potency and long duration of action in vivo, which is not restricted to parenteral administration.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Péptidos Cíclicos/farmacología , Receptores de Neuroquinina-2/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Benzamidas/farmacología , Cricetinae , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Piperidinas/farmacología , Conejos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Neuroquinina-2/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/fisiologíaRESUMEN
In a human astrocytoma cell line U373 MG, the activation of the neurokinin 1 (NK1) receptor by substance P (SP) increase, in a concentration-related manner (1 nM to 10 microM), the basal release of interleukin-6 (IL-6) as assayed by an ELISA method, in cell supernatants after 18 h of incubation. Septide, a selective NK1 receptor agonist, is equipotent to SP in inducing the IL-6 release showing similar Emax (2644 +/- 285 and 2830 +/- 271 pg/ml) and EC50 (15.6 +/- 3.6 and 13.8 +/- 3.2 nM). However, in binding assays on intact cells, septide was an about 50-fold weaker displacer of the binding of [3H][Sar9,Met(O2)11]SP than SP (Ki's were 0.28 +/- 0.1 nM and 14.2 +/- 5.0 nM for SP and septide, respectively). NK2- and NK3-selective agonists (up to 1 microM) had no binding or functional effect. Highly selective non-peptide (CP96,345) or peptide (GR82,334) NK1 receptor antagonists were more effective in antagonizing septide-(IC50's 0.2 +/- 0.06 nM and 70 +/- 18 nM) than SP-(IC50's 6.7 +/- 1.3 nM and 1.95 +/- 0.4 microM) induced IL-6 secretion. These data support the existence, also in human U373 MG cells, of a septide-sensitive NK1 receptor subtype(s) and/or epitope(s) blocked with high affinity by NK1 antagonist.
Asunto(s)
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Interleucina-6/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Fragmentos de Péptidos/antagonistas & inhibidores , Sustancia P/análogos & derivados , Unión Competitiva/efectos de los fármacos , Humanos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ácido Pirrolidona Carboxílico/análogos & derivados , Sustancia P/antagonistas & inhibidores , Sustancia P/metabolismo , Sustancia P/farmacología , Células Tumorales CultivadasRESUMEN
We have used one peptide (FK888) and two non-peptide ((+/-)-CP-96,345 and RP 67580) antagonists, along with the preferred endogenous agonist, substance P, to compare the pharmacological (binding) profile of NK1 receptors expressed by human B lymphoblastoma (IM9) and astrocytoma (U373 MG) cells. Of the ligands tested, substance P was the most potent in both cell lines: binding affinities were 0.1 nM for IM9 cells, and 0.3 nM for U373 MG cells, respectively. The high-affinity dipeptide antagonist, FK888, bound to NK1 receptors in both cell lines with similar potencies: Ki values were 1.2 nM and 3.6 nM for IM9 cells and U373 MG cells, respectively. Of the non-peptide antagonists, as expected, (+/-)-CP-96,345 displayed higher affinity (0.4 nM in IM9 cells, and 1.2 nM in U373 MG cells) than did RP 67580 (33 nM and 223 nM in IM9 cells and U373 MG cells, respectively) in both cell lines. We conclude that the pharmacological profile of NK1 receptors is similar in the human lymphoblastoma and astrocytoma cells, i.e. if NK1 receptor subtypes exist in humans, these cell lines are likely to express a similar subtype. Because IM9 cells grow faster and are easier to maintain, this cell line may be preferable to the astrocytoma cells as a primary screen to identify NK1 receptor antagonists.
Asunto(s)
Compuestos de Bifenilo/metabolismo , Dipéptidos/metabolismo , Indoles/metabolismo , Receptores de Neuroquinina-1/análisis , Sustancia P/antagonistas & inhibidores , Astrocitoma/metabolismo , Humanos , Isoindoles , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Tumorales CultivadasRESUMEN
Membranes obtained from post-mortem human spinal cord specimens bound [3H]resiniferatoxin (RTX) with an affinity of 11 nM in a non-cooperative fashion. This binding behaviour contrasted with the high affinity [3H]RTX binding (Kd = 24 pM) to rat spinal cord membranes which displayed apparent positive cooperativity (cooperativity index = 1.8) but was in accord with the low affinity (Kd = 5 nM) non-cooperative RTX binding to guinea pig spinal cord preparations. We conclude that the [3H]RTX binding assay utilizing post-mortem human spinal cord membranes affords a novel biochemical approach to explore structure-activity relations at human vanilloid receptors.
Asunto(s)
Diterpenos/metabolismo , Neurotoxinas/metabolismo , Receptores de Droga/metabolismo , Médula Espinal/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Capsaicina/análogos & derivados , Capsaicina/farmacocinética , Cobayas , Humanos , Técnicas In Vitro , Cinética , Membranas/metabolismo , Forbol 12,13-Dibutirato/farmacocinética , Proteína Quinasa C/metabolismo , RatasRESUMEN
1. The potential role of capsaicin-sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2. Capsaicin (1 microM) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mM). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin-pretreated rats. 3. Capsaicin-induced relaxation of NA-precontracted REUS was mimicked by calcitonin gene-related peptide (CGRP, 0.3-10 microM), but not by substance P (1 microM), vasoactive intestinal polypeptide (VIP, 1 microM), alpha-beta methylene ATP (10 microM), gamma-aminobutyric acid (GABA, 3 mM) or galanin (1 microM). A cross-tachyphylaxis between capsaicin (1 microM) and CGRP (1 microM) was observed. Both capsaicin and CGRP-induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8-37) (10 microM). 4. Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 microM) and CGRP (0.01-1 microM) produced an almost complete inhibition of EFS-induced tonic contraction. A cross-tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8-37) (10 microM). 5. [3H]-resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pM and a Bmax of 40 fmol mg-1 protein. This binding was inhibited by capsaicin with a Ki of 0.6 microM and it was reduced by approximately 80% in preparations taken from rats that had undergone surgical ablation of the major pelvic ganglion 4 days earlier.6. In conclusion we have demonstrated the existence of vanilloid receptors on capsaicin-sensitive nerves innervating the rat urethra mainly through the major pelvic ganglion. The activation of this set of nerves could lead to a local release of CGRP that in turn elicits a remarkable urethral relaxation. Such a mechanism could be of relevance in physiological conditions to facilitate urine expulsion during micturition and in pathological conditions to help removal of noxious stimuli following mechanical/chemical irritation of the lower urinary tract.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/farmacología , Capsaicina/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Receptores de Droga/fisiología , Uretra/efectos de los fármacos , Uretra/inervación , Animales , Péptido Relacionado con Gen de Calcitonina/fisiología , Diterpenos/metabolismo , Estimulación Eléctrica , Técnicas In Vitro , Cinética , Masculino , Músculo Liso/fisiología , Neurotransmisores/fisiología , Norepinefrina/antagonistas & inhibidores , Norepinefrina/farmacología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de Droga/efectos de los fármacos , Receptores de Droga/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Tritio , Uretra/fisiologíaRESUMEN
Resiniferatoxin (RTX) induced a dose-dependent loss of vanilloid receptors (specific [3H]RTX-binding sites) in tissues containing peripheral (urinary bladder) and central (spinal cord) endings of capsaicin-sensitive neurons. This receptor loss in the spinal cord was entirely due to a reduction in the Bmax. When examined 24 h after s.c. RTX treatment, receptor loss required somewhat less RTX in the urinary bladder (ED50 = 10 micrograms/kg) than in the spinal cord (ED50 = 50 micrograms/kg), whereas the loss of the xylene-induced neurogenic inflammatory response in the bladder displayed an approximate ED50 of 5 micrograms/kg. In the bladder of rats pretreated with 30 micrograms/kg RTX, both receptor binding and neurogenic inflammatory response recovered almost completely within 2 month after treatment. In the bladder of rats that received a 10-fold higher RTX dose, a 50% recovery of binding and a 70% recovery of the Evans' blue extravasation response were found. By contrast, no recovery of specific [3H]RTX binding to spinal cord membranes was observed at either dose. These findings suggest that vanilloid receptor loss after RTX treatment can be either reversible (desensitization) or irreversible (most likely reflecting neurotoxicity), and that peripheral and central terminals of capsaicin-sensitive neurons have a differential sensitivity to these long-term vanilloid actions.
Asunto(s)
Diterpenos/farmacología , Receptores de Droga/efectos de los fármacos , Médula Espinal/metabolismo , Vejiga Urinaria/metabolismo , Animales , Cistitis/inducido químicamente , Cistitis/metabolismo , Diterpenos/farmacocinética , Azul de Evans , Femenino , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , XilenosRESUMEN
Capsazepine was reported to block capsaicin- and resiniferatoxin (RTX)-induced responses both in vivo and in vitro with Schild plots suggesting a competitive mechanism of action. We have used the [3H]RTX binding assay, thought to represent the vanilloid (capsaicin) receptor, to explore the inhibitory mechanism of capsazepine at the receptor level in the rat. In competition assays, capsazepine inhibited [3H]RTX binding by spinal cord, dorsal root ganglion (DRG) and urinary bladder membranes with similar Ki values of 4.0 +/- 0.3, 3.5 +/- 0.5 and 5.0 +/- 1.0 microM (mean +/- S.E.M.; three determinations), respectively. By contrast, capsazepine was 35- to 50-fold more potent for inhibiting specific [3H]RTX binding in the airways (Ki = 0.12 +/- 0.02 microM; mean +/- S.E.M.; four determinations). In experiments in which the concentration of [3H]RTX was varied, 10 microM capsazepine reduced the affinity of the vanilloid receptor expressed by DRG and spinal cord membranes for [3H]RTX from 15 +/- 3 to 43 +/- 5 pM, and from 20 +/- 3 to 80 +/- 5 pM (mean +/- S.E.M.; three determinations), respectively, without a measurable change in Bmax or in cooperativity index; these shifts in affinity yield Ki values of 5.2 and 3.3 microM for DRG and spinal cord membranes, respectively. Capsaicin inhibited [3H]RTX binding by spinal cord, DRG and urinary bladder membranes with a 6- to 13-fold higher potency than did capsazepine; the Ki values were 0.3 +/- 0.1, 0.6 +/- 0.4 and 0.5 +/- 0.2 microM (mean +/- S.E.M.; three determinations), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)