RESUMEN
In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citosina/análogos & derivados , Encefalomielitis Autoinmune Experimental/prevención & control , Animales , Citosina/farmacocinética , Citosina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Hidroxiquinolinas/farmacología , Inmunohistoquímica , Interferones/sangre , Ratones , Esclerosis Múltiple/tratamiento farmacológicoAsunto(s)
Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/métodos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/fisiología , Compartimento Celular , Células Cultivadas , Estudios de Evaluación como Asunto , Indicadores y Reactivos , Cinética , Análisis de los Mínimos Cuadrados , Modelos QuímicosRESUMEN
We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.
Asunto(s)
Citosina/análogos & derivados , Citosina/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inductores de Interferón/uso terapéutico , Animales , Citosina/farmacocinética , Relación Dosis-Respuesta a Droga , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inductores de Interferón/farmacocinética , Interferón gamma/sangre , Leucocitos/patología , Ratones , Médula Espinal/efectos de los fármacos , Médula Espinal/patologíaRESUMEN
Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2',7'-dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for the these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U-87,663, and the bulk of the ROS prevented detection of any protection of U-87,663 may offer.
Asunto(s)
Antioxidantes/farmacología , Derivados del Benceno/farmacología , Microscopía Confocal/métodos , Análisis Numérico Asistido por Computador , Oxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Fluoresceínas/química , Colorantes Fluorescentes/química , Radicales Libres , Ratones , Ratas , Células Tumorales CultivadasRESUMEN
Few inhibitors of the RNase H function associated with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U-34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles, and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules form stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted, the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.
Asunto(s)
Inhibidores Enzimáticos/farmacología , VIH-1/enzimología , Novobiocina/análogos & derivados , Ribonucleasa H/antagonistas & inhibidores , Fenómenos Químicos , Química Física , Emulsiones , Inhibidores Enzimáticos/química , Concentración de Iones de Hidrógeno , Conformación Molecular , Estructura Molecular , Novobiocina/química , Novobiocina/farmacología , Solubilidad , Relación Estructura-ActividadRESUMEN
We examined the efficacy of a group of drugs that stabilize the cell membrane and can potentially prevent cytotoxicity in cultured fetal chick cardiac myocytes exposed to hydrogen peroxide (H2O2). The effects of various membrane-protective agents were determined by analysis of the kinetics of lactic dehydrogenase (LDH) release. The kinetic parameters calculated from the data include a rate constant for release of LDH (kb) and the fraction of total LDH that is released from the cells (CIIMax). The CIIMaxs derived from a range of H2O2 concentrations reveal that the mean toxic concentration of H2O2 is 1.1 mM and that the pattern of toxicity is consistent with the damage being directly proportional to the concentration of the free radicals generated from the H2O2. Maximum nontoxic concentrations of three amphiphilic membrane protective agents had no effect upon cytotoxicity from H2O2. The slightly polar lipophilic agent, Trolox C, a vitamin E derivative, was also without protective effect at a maximum nontoxic concentration. The highly lipophilic agent, probucol, had a small protective effect at 50 microM, the maximum concentration we succeeded in solubilizing in the culture medium. However, the lipophilic 21-aminosteroid U74500, delivered to the cells in an emulsion, markedly reduced cytotoxicity from H2O2. The CII Max was significantly reduced and the protection was concentration dependent over a range of concentrations from 50-400 nmol/ml. Furthermore, the inhibition by U74500 was fully consistent with a mechanism of scavenging of free radicals formed during lipid peroxidation. In support of this hypothesis, a dose of 400 nmoles/ml completely prevented an increase in lipid peroxides due to H2O2 exposure, whereas there was a sixfold increase during exposure to H2O2 in untreated myocytes. Thus, a lipid soluble 21-aminosteroid prevented lipid peroxidation and reduced cardiac myocyte injury during exposure to H2O2, probably by scavenging of free radicals formed during lipid peroxidation in the cell membrane, whereas amphiphilic agents, which probably altered the physicochemical structure of the cell membrane but did not scavenge free radicals, were not protective.
Asunto(s)
Antioxidantes/farmacología , Membrana Celular/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Lípidos , Pregnatrienos/farmacología , Solubilidad , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Cromanos/farmacología , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres , Cinética , L-Lactato Deshidrogenasa/metabolismo , Lidocaína/farmacología , Peroxidación de Lípido/efectos de los fármacos , Probucol/farmacología , Propranolol/farmacologíaRESUMEN
N18-RE-105 neuronal hybridoma cells were used in a cell culture system to evaluate the protective effects of a novel 6-chromanol-containing antioxidant, U78517F. First, the incorporation of the compound into the cells was evaluated, using a serum albumin carrier. Then the cells were exposed to peroxide-generating compounds, and the cell injury was estimated from the loss of alpha-aminoisobutyric acid (AIB) transport. We found that U78517F only protected the cells significantly when the degree of oxidative insult was below a certain limit; the measurable protection of cells by U78517F against either cumene hydroperoxide or H2O2 was limited to a narrow range of concentrations of the reactive oxygen species generator. Additionally, the protection provided by U78517F was largely localized to the cell membrane and did not extend to protection of mitochondrial function. The action of U78517 was fully consistent with a direct radical scavenging in the cells. The results indicate that the following factors must be taken into account for evaluation of antioxidants in cell culture: (a) the delivery of a compound to cells, especially when the compound is lipophilic; (b) the nature and extent of the oxidative insult used to evaluate protection; and (c) the location of the protective agent in the cells.
Asunto(s)
Antioxidantes/farmacología , Cromanos/farmacología , Depuradores de Radicales Libres/farmacología , Estrés Oxidativo/efectos de los fármacos , Piperazinas/farmacología , Sistemas de Transporte de Aminoácidos , Ácidos Aminoisobutíricos/metabolismo , Animales , Derivados del Benceno/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cromanos/química , Hibridomas , Peróxido de Hidrógeno/farmacología , Ratones , Piperazinas/química , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.
Asunto(s)
Antioxidantes/metabolismo , Sistemas de Liberación de Medicamentos , Pregnatrienos/metabolismo , Animales , Antioxidantes/farmacología , Emulsiones , Cinética , Ratones , Neuroblastoma/metabolismo , Neuronas/metabolismo , Fosfatidilcolinas/metabolismo , Ratas , Termodinámica , Trioleína/metabolismo , Células Tumorales CultivadasRESUMEN
N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single-hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process.
Asunto(s)
Membrana Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/farmacología , Ácidos Aminoisobutíricos/metabolismo , Animales , Derivados del Benceno/farmacología , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Glucosa/metabolismo , Hibridomas , Peróxido de Hidrógeno/farmacología , Ratones , Mitocondrias/metabolismo , Oxidopamina/farmacología , Ratas , Coloración y Etiquetado , Sales de Tetrazolio , TiazolesRESUMEN
Primary in vivo challenge with the superantigen staphylococcal enterotoxin B (SEB) induces polyclonal proliferation of an unusually large proportion of circulating T-cells that bear the V beta 8-T-cell receptor (TcR) domain. Early and vigorous proliferation of V beta 8+ T-cells precedes their selective deletion, leaving the host unresponsive upon rechallenge with the native immunogen SEB. Nonetheless, this induction of anergy is incompletely understood. Recently we demonstrated that more cells than just V beta 8+ T-cells undergo clonal proliferation after challenge with SEB (Cell. Immunol. 154, 440, 1994). These findings suggested that non-V beta 8+ T-cells may have a role in the induction of superantigen-induced anergy. To further investigate this, we enumerated CD4+ and CD8+ T-cells in lymph nodes and spleens from Balb/c mice at various times after primary and secondary challenge with either a high or a low dose of SEB. Using these kinetic data we investigated whether challenge with SEB would modulate antigen-specific V beta 8-associated T-memory responses. To this end, the V beta 8+ T-cell-associated responses induced by SEB were compared with the V beta 8+ TcR-associated memory responses induced by the nominal antigen sperm whale myoglobin (SWM). Results indicated that challenge of SWM-primed mice with SEB abrogated the V beta 8-associated SWM-specific T-cell memory for an extended but transient period of time. Moreover, prechallenge with SEB blocked the establishment of de novo V beta + T-cell-mediated immunity. These findings suggest that administration of low and controlled doses of microbial superantigen could provide long-term suppression of antigen-specific cell-mediated immunity.
Asunto(s)
Toxinas Bacterianas , Linfocitos T CD8-positivos/inmunología , Anergia Clonal/inmunología , Memoria Inmunológica/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Superantígenos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Enterotoxinas/inmunología , Femenino , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Mioglobina/inmunologíaRESUMEN
The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.
Asunto(s)
Antioxidantes , Conservación de la Sangre/métodos , Fragilidad Osmótica/efectos de los fármacos , Pregnatrienos/farmacología , Dinoprost/metabolismo , Membrana Eritrocítica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Factores de Tiempo , Vitamina E/sangreRESUMEN
The in vivo challenge with exogenous superantigen, staphylococcal enterotoxin B (SEB), selectively induces vigorous polyclonal proliferation of T cells bearing the V beta 8+ TcR domain, whereafter the responsive cells become anergic. We used kinetic analyses to compare the effects of primary (1 degree) and secondary (2 degrees) challenge with a high and a low dose of SEB and the conventional antigen, sperm whale myoglobin, to determine the differential effects of in vivo challenge with a superantigen compared with a conventional antigen. We demonstrate that SEB induces very early activation-associated intralymphatic proliferation and trafficking of more T cells than can be accounted for by V beta 8+ T cells alone. Overall, this study indicates that challenge with SEB causes an apparent loss of CD4+ T-helper cell function and provides an essential foundation for the understanding of the mechanisms of peripheral tolerance induction and T cell "memory."
Asunto(s)
Enterotoxinas/inmunología , Activación de Linfocitos , Autotolerancia , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Femenino , Interleucina-2/farmacología , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Receptores de Antígenos de Linfocitos T alfa-beta/análisisRESUMEN
Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse beta-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies--N60, M15, and 27/21--showed any reactivity toward denatured NGF. Three experimental methods--radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots--detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodies to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor.
Asunto(s)
Epítopos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Unión Competitiva , Reacciones Cruzadas , Glicoproteínas de Membrana/metabolismo , Ratones , Modelos Moleculares , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/inmunología , Células PC12 , Fosforilación , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Receptor de Factor de Crecimiento Nervioso , Receptor trkARESUMEN
We examined the effects of 6-hydroxydopamine (6-OHDA) treatment on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the rat pheochromocytoma cell line, PC12. Structural and metabolic integrity was tested by measuring the ability of cells to transport the non-metabolizable amino acid analogue [3H]-alpha-aminoisobutyric acid (AIB). We determined that treatment with 6-OHDA at concentrations of 49 microM and 62 microM inhibited 50% of the AIB uptake in SY5Y and PC12 cells, respectively. Inhibition of AIB uptake was prevented by the addition of catalase, but was not influenced by the addition of 1 mM dopamine. This indicated that cell damage resulted from the generation of H2O2 and was independent of the catecholamine uptake system. Effects directly on the catecholamine uptake system were observed by measuring the uptake of 3H-dopamine. In contrast to the effects on amino acid uptake, dopamine uptake was significantly inhibited by 6-OHDA treatment, and this inhibition was not prevented by the addition of catalase. The results indicate a Ki of 430 microM for inhibition of dopamine uptake by 6-OHDA treatment of PC12 cells. The results are consistent with a competitive irreversible inhibition of the dopamine uptake sites by 6-OHDA or one of its metabolites. Thus, the lack of a catecholamine uptake-dependent cellular toxicity appears to result from the direct inactivation of catecholamine uptake sites. Similarly, the inhibition of dopamine uptake in vivo by 6-OHDA may be explained, at least in part, by direct inactivation of dopamine uptake sites rather than exclusively by intracellular transport and action of 6-OHDA.
Asunto(s)
Antagonistas de Dopamina , Dopamina/metabolismo , Oxidopamina/farmacología , Neoplasias de las Glándulas Suprarrenales/metabolismo , Ácidos Aminoisobutíricos/antagonistas & inhibidores , Ácidos Aminoisobutíricos/metabolismo , Animales , Unión Competitiva , Transporte Biológico , Catalasa/farmacología , Humanos , Neuroblastoma/metabolismo , Feocromocitoma/metabolismo , Ratas , Células Tumorales CultivadasAsunto(s)
Receptores de Factor de Crecimiento Nervioso , Secuencia de Aminoácidos , Animales , Expresión Génica , Humanos , Datos de Secuencia Molecular , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/fisiología , Distribución TisularRESUMEN
We generated a family of ultra-high affinity monoclonal antibodies (MAb) which inhibit competitively the binding of nerve growth factor (NGF) to its receptor. Preliminary experiments indicated that the dissociation constants (Kd) of some of the MAb:NGF complexes were substantially less than 0.1 nM. Conventional methods, such as ELISA and radioimmunoassays (RIA), were not sufficiently sensitive to measure the Kds of these MAb. Therefore, experimental conditions were developed to determine binding constants for these very high affinity MAb. The experiments establish that the Kds for our anti-NGF MAb range from 2.6 nM to 39 fM. Additionally, the inhibition of NGF binding to NGF-receptor by MAb is fully consistent with a purely competitive model but is not consistent with a model allowing the formation of a ternary complex of NGF, MAb, and NGF-receptor. One MAb, M4, immunoprecipitates NGF indicating interaction between each protomer of the NGF dimer and individual MAb molecules. We also evaluated the effects of mild denaturing conditions on the binding and biological activity of NGF and on recognition by the MAbs. Guanidine HCl or heat treatment of NGF resulted in only small, but significant, changes in binding or biological activity, in parallel with changes in recognition by the MAbs. However, binding, biological activity, and recognition by six of seven MAbs were completely eliminated by beta-mercaptoethanol reduction. Thus, our results are consistent with the MAbs interacting with the receptor recognition site on the surface of the NGF molecule. The high affinity MAbs will serve as sensitive probes of structural elements of NGF responsible for binding and biological activity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Factores de Crecimiento Nervioso/inmunología , Receptores de Superficie Celular/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática , Guanidina , Guanidinas , Calor/efectos adversos , Inmunodifusión , Inmunoglobulina G/análisis , Técnicas In Vitro , Análisis de los Mínimos Cuadrados , Mercaptoetanol , Ratones , Radioinmunoensayo , Receptores de Factor de Crecimiento NerviosoRESUMEN
We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than the Kd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd = 10(-9) M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF:NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.
Asunto(s)
Factores de Crecimiento Nervioso/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Unión Competitiva , Humanos , Cinética , Ratones , Microsomas/metabolismo , Factores de Crecimiento Nervioso/inmunología , Factores de Crecimiento Nervioso/metabolismo , Células PC12 , Receptores de Superficie Celular/metabolismo , Receptores de Factor de Crecimiento Nervioso , Relación Estructura-ActividadRESUMEN
In an effort to determine if PC12 cells have functional dopamine autoreceptors we found that carbachol-stimulated release of dopamine from undifferentiated PC12 cells was inhibited by the dopamine autoreceptor agonists apomorphine and U-68553B. Studies were conducted to determine the mechanism of this effect. The inhibition of dopamine release by apomorphine or U-68553B did not appear to result from effects on dopamine metabolism. When cells were exposed to the dopamine agonists for 2 minutes no changes in dopa, DOPAC or dopamine were observed. Over this same time interval, apomorphine and U68553B at 10 microM inhibited carbachol-stimulated dopamine release by 45.6% and 57.4% respectively. However, these drugs failed to inhibit the potassium dependent release of dopamine from cells suggesting no direct involvement with ion fluxes. Furthermore, haloperidol did not block the inhibitory effects of U-68553B on PC12 cells. This would appear to preclude activation of a dopamine autoreceptor as a possible mechanism. Kinetic analysis revealed that U-68553B is most likely a non-competitive inhibitor (Ki = 2 microM) of the nicotinic acetylcholine receptor. These data do not provide evidence for functional dopamine autoreceptors on undifferentiated PC12 cells but rather indicate that dopamine agonists may alter dopamine release by alternative mechanisms.
Asunto(s)
Dopaminérgicos/farmacología , Dopamina/metabolismo , Fenalenos , Animales , Apomorfina/farmacología , Carbacol/farmacología , Haloperidol/farmacología , Oxotremorina/farmacología , Compuestos Policíclicos/farmacología , Potasio/farmacología , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.
Asunto(s)
Mariposas Nocturnas/metabolismo , Factores de Crecimiento Nervioso/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Baculoviridae/genética , Southern Blotting , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/inmunología , Factores de Crecimiento Nervioso/fisiologíaRESUMEN
A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.