RESUMEN
Human adipose-derived stem cells (hASCs) are a feasible source of stem cells for use in clinical applications. hASCs are typically cultured in medium containing fetal bovine serum (FBS); however, use of FBS is not recommended due to issues of clinical safety with regard to infections or immune response. Replacement of FBS with autologous human serum (autoHS) can eliminate these problems; however, their maintainability as potent ASCs in autoHS needs to be confirmed. Thus, we conducted an investigation of characterizations and functions of hASCs grown in medium containing autoHS compared to FBS. Cell counting and the WST-8 assay were used in assessment of the proliferation rate. In hASC cultured with culture medium plus autoHS or FBS, cell phenotypes were characterized by flow cytometry (CD13, CD29, CD31, CD34, and CD44) and expression of BDNF, HGF, IGF, LIF, NGF, and VEGF was determined by RTPCR. Adipogenic differentiation was confirmed by oil red O stain. hASC showed greater expansion in AutoHS than in FBS. Cell surface markers of hASCs grown in autoHS (autoHS-hASCs) were similar to markers of those grown in FBS (FBS-hASCs). AutoHS-hASCs also expressed multiple growth factors as well as FBS-hASCs. In addition, autoHS was effective in growth of hASCs as well as FBS and autoHS-hASCs retained their ability for adipogenic differentiation. In summary, autoHS-hASCs have multiple growth factor expressions with the same cell surface markers as FBShASCs in vitro. Our results suggest that autoHS can provide sufficient ex vivo expansion of hASCs.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Diferenciación Celular/fisiología , Medios de Cultivo , Técnicas Citológicas/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adipogénesis/fisiología , Animales , Compuestos Azo/química , Biomarcadores/metabolismo , Bovinos , Recuento de Células/métodos , Procesos de Crecimiento Celular/fisiología , Citometría de Flujo/métodos , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Coloración y Etiquetado/métodosRESUMEN
The huntingtin (htt) mutation causes a polyglutamine expansion in the N-terminal region of protein. Mutant N-htt proteolytic fragments aggregate and cause cell death in Huntington's disease (HD). The normal huntingtin also can be cleaved by calpain and produce N-terminal htt fragments following ischemic injury, but the fate of cleaved fragment in dead neurons in the brain are unclear. To determine the localization of huntingtin following proteolysis, we examined htt expression after transient ischemic injury. Huntingtin immunoreactivity in mixed cultures of neuronal and astrocytes-derived clonal cells showed alteration of immunoreactivity from neurons into astrocytes. In the brain, both focal and global ischemia induced reactive astrocytes that were co-immunoreactive for huntingtin with elevated GFAP expression. The immunoreactive huntingtin was 55kDa calpain-cleaved N-terminal fragment, which appeared initially in the process, and extended into the cytoplasm of astrocytes. The results showed, after ischemic injury, huntingtin accumulated in astrocytes indicating that astrocytes may play a role in uptake of cleaved N-htt fragments.
Asunto(s)
Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Calpaína/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Astrocitos/citología , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Huntingtina , Masculino , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.
Asunto(s)
Simulación por Computador , Modelos Químicos , Modelos Moleculares , Teoría Cuántica , Programas Informáticos , Carbohidratos/química , Biología Computacional , Lípidos/química , Ácidos Nucleicos/química , Péptidos/química , Proteínas/químicaRESUMEN
We have reexamined the heterogeneity shown by histidine in its uptake by the Ehrlich ascites tumor cell, in the face of a contradiction of our earlier interpretation. We again find the fraction of histidine uptake at neutral pH inhibitable by the model substrate for System A, 2-(methylamino)-isobutyric acid, to be fully dependent on the presence of Na+ or Li+. The small Na+ -independent component not attributable to System L can be identified with System Ly+ through its inhibitability by homoarginine. This component increases as the pH is lowered with an apparent pK' a of 6.1. The simultaneous decrease in the uptake by the neutral systems could be identified, for System L, with the same titration of histidine to its cationic form, but for System A the sharp decrease is identified with the protonation of a structure on the membrane rather than one on the substrate. The action of H+ in the latter case proved approximately non-competitive with Na+ when tested with ordinary substrates.
Asunto(s)
Carcinoma de Ehrlich/metabolismo , Histidina/metabolismo , Ácidos Aminoisobutíricos/análogos & derivados , Ácidos Aminoisobutíricos/farmacología , Animales , Transporte Biológico , Transporte Biológico Activo , Ácidos Carboxílicos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Litio/farmacología , Sodio/farmacologíaRESUMEN
Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Ácidos Grasos Insaturados/farmacología , Microsomas/enzimología , Estómago/enzimología , Animales , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Perros , ATPasa Intercambiadora de Hidrógeno-Potásio , Membranas Intracelulares/enzimología , Médula Renal/enzimología , Masculino , Ratas , Ratas Endogámicas , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
We have examined the interaction of omeprazole, a gastric antisecretory agent, with endogenous or exogenous sulfhydryl compounds in isolated rabbit gastric glands. The glands exposed to omeprazole (2 microM for 50 min) could recover acid secretory response to dibutyryl-cAMP upon addition of dithiothreitol, cysteine or glutathione. Washing the omeprazole-exposed glands free of the extracellular drug also led to a similar recovery of the acid secretory response. Depletion of cellular glutathione with 2-cyclohexen-1-one had no considerable effect on the secretory response of the glands to dibutyryl-cAMP, but prevented the reversal of the antisecretory effect of omeprazole upon washing or adding exogenous cysteine. Also, the antisecretory potency of omeprazole increased several fold in the glutathione-depleted glands. These observations indicate that cellular glutathione is essential to reactivate the omeprazole-modified enzyme(s), possibly (H+ + K+)-ATPase, in acid secretory process and led us to propose that omeprazole is an agent reacting with sulfhydryl groups.
Asunto(s)
Bencimidazoles/farmacología , Mucosa Gástrica/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , Animales , Ciclohexanonas/farmacología , Cisteína/farmacología , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Glutatión/metabolismo , Glutatión/farmacología , Técnicas In Vitro , Masculino , Omeprazol , ConejosRESUMEN
Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg+2 -dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na+ or K+. The first of these, previously described, shows little (Na+ + K+)-ATPase activity, and is characteristicallly stimulated by the presence of certain diamino acids with low pK2, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the (Na+ +K+)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the L than for the D isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of L-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg+ -dependent hydrolytic activity.
Asunto(s)
Adenosina Trifosfatasas/farmacología , Aminoácidos/farmacología , Carcinoma de Ehrlich/enzimología , Membrana Celular/enzimología , Ouabaína/farmacología , Animales , Fraccionamiento Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Ácido Desoxicólico/farmacología , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , Magnesio/farmacología , Ratones , Oligomicinas/farmacología , Potasio/farmacología , Sodio/farmacologíaRESUMEN
We have examined histamine- or carbachol-induced changes in rat gastric membranes enriched in K+-stimulated ATPase. Stimulation of secretion by both secretagogues in vivo produced a class of microsomal membranes which exhibited valinomycin-independent, KCl-dependent H+ transport. In contrast, membrane vesicles isolated from cimetidine inhibited resting mucosa exhibited largely the ionophore-dependent H+ transport. In addition, only in the carbachol-stimulated membranes a portion of the ionophore-independent H+ transport was refractory to cimetidine pretreatment. The gastric microsomal membranes were resolved into light and heavy fractions by centrifugation over isotonic 2H2O media. The ionophore-independent H+ transport was almost exclusively associated with the heavy microsomal fraction while the ionophore-dependent H+ transport was detected in the light fraction. Also, these fractions were considerably different from each other in their appearance in electron micrographs and SDS gel electrophoresis patterns. Secretagogue stimulation increased the population of the heavy microsomal membrane vesicles exhibiting the valinomycin-independent, K+-dependent H+ transport and their overall content of K+-stimulated ATPase. Cimetidine treatment, on the other hand, increased the ATPase activity associated with the light microsomes, and produced the heavy microsomal membranes showing only a marginal degree of the ionophore independent H+ accumulation, even though they were very similar to the carbachol-stimulated heavy membranes in the specific activity of K+-stimulated ATPase. SDS gel patterns and appearance in electron micrograph. These observations suggest that activation of secretion involves at least two distinctive events; transformation of the light to the heavy gastric membranes containing a K+-dependent H+ pump and an increased KCl permeability in the latter.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Carbacol/farmacología , Mucosa Gástrica/enzimología , Histamina/farmacología , Membranas Intracelulares/enzimología , Animales , Cimetidina/farmacología , Mucosa Gástrica/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio , Concentración de Iones de Hidrógeno , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/ultraestructura , Cinética , Masculino , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Microsomas/ultraestructura , Potasio/farmacología , Ratas , Ratas Endogámicas , Valinomicina/farmacologíaRESUMEN
The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with (H+ + K+)-ATPase. The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20 mM) was 50 microM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifluoperazine at 4 microM, verapamil and TMB-8 at 15 microM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutyryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on (H+ + K+)-ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited (H+ + K+)-ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Ácido Gálico/análogos & derivados , Mucosa Gástrica/enzimología , Hidrógeno/metabolismo , Trifluoperazina/farmacología , Verapamilo/farmacología , Animales , Ácido Gálico/farmacología , Mucosa Gástrica/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio , Cinética , Conejos , Porcinos , Factores de TiempoRESUMEN
Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.
Asunto(s)
Carbacol/farmacología , Cicloheximida/farmacología , Ácido Gástrico/metabolismo , Jugo Gástrico/efectos de los fármacos , Biosíntesis de Péptidos , Adenosina Trifosfatasas/biosíntesis , Animales , ATPasa Intercambiadora de Hidrógeno-Potásio , Cinética , Masculino , Microsomas/enzimología , Peso Molecular , Péptidos/aislamiento & purificación , Biosíntesis de Proteínas/efectos de los fármacos , Puromicina/farmacología , Ratas , Ratas EndogámicasRESUMEN
Previous reports suggested that Na,K-ATPase activity and Na(+)-pump capacity decrease with senescence in left atrial myocardium of F344 rats. Current experiments were designed to determine if this reduction in the Na(+)-pump affects free intracellular Na+ levels. Mean intracellular Na+ ion activity (aiNa) was measured with Na-selective microelectrodes in left atrial muscle isolated from hearts of 4-, 14- and 25-month-old F344 rats. Preparations were stimulated randomly at frequencies between 0 and 12 h. There were no age-associated differences in aiNa measured at any frequency or in the decay of Na+ activity following discontinuation of electrical stimulation. These data indicate that the aging-related decline in Na,K-ATPase does not result in elevated aiNa even at extremely high stimulation frequencies, thus suggesting that other routes of Na+ influx and efflux are also altered in atrial muscle.
Asunto(s)
Envejecimiento/fisiología , Miocardio/metabolismo , Sodio/metabolismo , Animales , Estimulación Eléctrica , Técnicas In Vitro , Líquido Intracelular/metabolismo , Masculino , Contracción Miocárdica , Ratas , Ratas Endogámicas F344 , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
The synthesis of N-substituted benzimidazole (H(+)-K+)-ATPase or proton-pump inhibitors is described. These compounds were prepared to function as prodrugs of the parent N-H compound and evaluated for their ability to inhibit gastric (H(+)-K+)-ATPase and gastric acid secretion. The prodrugs reported rely on either in vivo esterase hydrolysis for liberation of the parent compound (type I and type II) or require an acid environment for release of the active drug (type III and type IV). The N-(acyloxy)alkyl-substituted benzimidazoles 9, 11, and 24 showed improved chemical stability in the solid state and in aqueous solutions when compared to their parent N-H compounds. When given orally, 24 was found to be twice as potent as omeprazole in both the Shay rat and inactivation of gastric (H(+)-K+)-ATPase in the rat. The N-ethoxy-1-ethyl-substituted benzimidazoles 48-50 were found equally as effective as the N-H compound for inhibition of rat (H(+)-K+)-ATPase activity. In the Shay rat 48 at 10 mg/kg was approximately twice as active as parent timoprazole.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Bencimidazoles/síntesis química , Ácido Gástrico/metabolismo , Profármacos/síntesis química , 2-Piridinilmetilsulfinilbencimidazoles , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Fenómenos Químicos , Química , Estabilidad de Medicamentos , Esterasas/metabolismo , Femenino , Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio , Concentración de Iones de Hidrógeno , Masculino , Estructura Molecular , Omeprazol/farmacología , Conejos , Ratas , Ratas Endogámicas , PorcinosRESUMEN
(4RS)-1-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihyd roimidazo[1,5- a]pyrrolo[2,1-c]quinoxalin-10(11H)-one (1a), 5-benzoyl-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5- dihydroimidazo[1,5-a]quinoxaline (13b), and tert-butyl (4S)-12,12a-dihydroimidazo[1,5-a]pyrrolo[2,1- c]quinoxaline-1-carboxylate (1e), as well as other imidazo[1,5-a]quinoxaline amides and carbamates, represent a new series of compounds which bind with high affinity to the GABAA/benzodiazepine receptor. These compounds exhibit a wide range of intrinsic efficacies as measured by [35S]TBPS binding ratios. The synthesis of 1a begins with the addition of DL-glutamic acid to 1-fluoro-2-nitrobenzene, followed by reduction of the nitro group and subsequent ring closure to form 3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinoxalin-2-one, followed by a second ring closure to afford (4RS)-1,5-dioxo-1,2,3,4,5,6-hexahydropyrrolo[1,2-a]quinoxali ne as the key intermediate. Appendage of a substituted imidazo ring via the anion of 5-cyclopropyl-1,2,4-oxadiazol-3-yl gives 1a. The (-)- and (+)-isomers of 1a were prepared from 1-fluoro-2-nitrobenzene and L- and D-glutamic acid, respectively. 1a and its enantiomers demonstrated affinity for the [3H]flunitrazepam binding site with Ki's of 0.87, 0.62, and 0.65 nM, respectively.
Asunto(s)
Carbamatos/síntesis química , Imidazoles/síntesis química , Quinoxalinas/síntesis química , Receptores de GABA/metabolismo , Animales , Carbamatos/química , Carbamatos/metabolismo , Células Cultivadas , Antagonistas del GABA , Imidazoles/química , Imidazoles/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of tetracyclic imidazoquinoxaline analogs was developed which constrain the carbonyl group of the partial agonist 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(dimethylamino)carbonyl] - 4,5-dihydroimidazo[1,5-alpha]quinoxaline (2, U-91571) away from the benzene ring. These analogs orient the carbonyl group in the opposite direction of the previously reported full agonist 1-(5- cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihydroimidazo[1,5- alpha]pyrrolo [2,1-c]quinoxalin-10(11H)-one (3, U-89267). A number of approaches were utilized to form the "bottom" ring of this tetracyclic ring system including intramolecular cyclizations promoted by Lewis acids or base, as well as metal-carbenoid conditions. The size and substitution pattern of the additional ring was widely varied. Analogs within this series had high affinity for the benzodiazepine receptor on the alpha-aminobutyric acid A chloride ion channel complex. From TBPS shift and Cl- current assays, the in vitro efficacy of compounds within this class ranged from antagonists to partial agonists with only 18a identified as a full agonist. Additionally, several analogs were quite potent at antagonizing metrazole-induced seizures indicating possible anticonvulsant or anxiolytic activity. Unlike 3, analogs in this series did not have high affinity for the diazepam insensitive alpha 6 beta 2 delta 2 subtype. These results suggest that either constraining the carbonyl group away from the benzene ring or the greater planarity that results from the additional cyclic structure provides analogs with partial agonist properties and prevents effective interaction with the alpha 6 beta 2 delta 2 subtype.
Asunto(s)
Quinoxalinas/síntesis química , Receptores de GABA-A/metabolismo , Animales , Línea Celular , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Potenciales de la Membrana/efectos de los fármacos , Ratones , Conformación Molecular , Nucleopoliedrovirus/genética , Quinoxalinas/metabolismo , Quinoxalinas/uso terapéutico , Ratas , Receptores de GABA-A/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Convulsiones/tratamiento farmacológico , Espectrofotometría Infrarroja , Spodoptera , Relación Estructura-ActividadRESUMEN
A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.
Asunto(s)
Agonistas del GABA/síntesis química , Agonistas del GABA/farmacología , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , Receptores de GABA-A/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Benzodiazepinas/farmacología , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cloruros/metabolismo , Flunitrazepam/antagonistas & inhibidores , Flunitrazepam/metabolismo , Agonistas del GABA/química , Antagonistas del GABA/farmacología , Conformación Molecular , Estructura Molecular , Oxadiazoles/química , Pentilenotetrazol/farmacología , Quinoxalinas/química , RatasRESUMEN
A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.
Asunto(s)
Imidazoles/síntesis química , Quinoxalinas/síntesis química , Receptores de GABA/metabolismo , Animales , Anticonvulsivantes , Benzodiazepinas/antagonistas & inhibidores , Membrana Celular/metabolismo , Corteza Cerebral/metabolismo , Sinergismo Farmacológico , Etanol/farmacología , Imidazoles/metabolismo , Imidazoles/uso terapéutico , Ligandos , Masculino , Ratones , Conformación Molecular , Estructura Molecular , Pentilenotetrazol/antagonistas & inhibidores , Quinoxalinas/metabolismo , Quinoxalinas/uso terapéutico , Ratas , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Relación Estructura-Actividad , Trastornos Relacionados con SustanciasRESUMEN
A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.
Asunto(s)
Agonistas del GABA/síntesis química , Imidazoles/síntesis química , Piperazinas/síntesis química , Quinoxalinas/síntesis química , Receptores de GABA-A/metabolismo , Urea/análogos & derivados , Urea/síntesis química , Animales , Ansiolíticos/síntesis química , Ansiolíticos/química , Ansiolíticos/farmacología , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Ansiedad/metabolismo , Ansiedad/fisiopatología , Disponibilidad Biológica , Línea Celular , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Convulsivantes/toxicidad , GMP Cíclico/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Agonistas del GABA/química , Agonistas del GABA/farmacología , Imidazoles/química , Imidazoles/farmacología , Técnicas In Vitro , Ligandos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Pentilenotetrazol/toxicidad , Piperazinas/química , Piperazinas/farmacología , Quinoxalinas/química , Quinoxalinas/farmacología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/fisiopatología , Relación Estructura-Actividad , Urea/química , Urea/farmacologíaRESUMEN
1. A pair of benzamide analogues containing a pyrrolidinyl or piperidinyl group was examined for their anticonvulsant activity against the electroshock-induced seizures in mice and the ability to block the voltage-gated Na channel in N1E-115 cells, in comparison with the prototype compound, U-54494A, (+/-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-ben zam ide , a potent anticonvulsant and a Na channel blocker. 2. The pyrrolidinyl benzamide (U-49524E) was found to be effective against the electroshock-induced seizures (ED50 = 35 mg kg-1, i.p.) whereas the benzamide with a piperidinyl moiety (U-49132E) was inactive (ED50 greater than 100 mg kg-1). 3. Using whole-cell patch clamp techniques we found that U-49132E was several times less potent, with an IC50 of 396 microM as compared to 118 microM for U-49524E at the holding potential of -80 mV, and was much slower in blocking Na channels with a half-time of 10.7 +/- 1.1 min vs. 2.2 +/- 0.4 min for its counterpart. 4. Qualitatively, their general modes of interaction with Na channels were similar to each other and to that of U-54494A in that they interacted with the resting and slowly-inactivated states of the channels and exhibited a use-dependent inhibition because of a slow recovery from the inactivated state in the presence of the drugs. 5. Comparison of their physicochemical properties, shows the less potent and slowly acting U-49132E is more hydrophobic and bulkier than U-49524E, but has the same pKa. This suggests that the drugs approach the Na channel through a narrow and hydrophilic pathway.6. Overall, this study underscores the importance of inhibiting the Na channel to the anticonvulsant activity of the benzamide compounds.
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Anticonvulsivantes/farmacología , Ciclohexanos/farmacología , Piperidinas/farmacología , Pirrolidinas/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Inyecciones Intraperitoneales , Masculino , Ratones , Neuroblastoma , Convulsiones/tratamiento farmacológico , Sodio/metabolismo , Canales de Sodio/metabolismo , Células Tumorales CultivadasRESUMEN
The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH-SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)-induced GTPgamma(35)S binding and high affinity sites were 3 - 4 fold greater in SH-SY5Y than in HEK293 cells. N-type Ca(2+) channel currents present in SH-SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half-maximal inhibitory concentration of 0.15+/-0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPgamma(35)S-Bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galpha(i) and Galpha(o) subunits were highly labelled with GTPgamma(35)S in SH-SY5Y cells, but only Galpha(i) subunits were labelled in HEK293 cells. The additional G(o) coupling in SH-SY5Y cells could arise, at least in part, from the presence of G(o) coupled-effectors, such as the N-type Ca(2+) channel, and may contribute to robust agonist-induced GTPgamma(35)S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein-coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non-neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron-specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein-coupled receptors of various origins.
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Clonación Molecular/métodos , Riñón/metabolismo , Neuronas/metabolismo , Receptores de Dopamina D2/biosíntesis , Canales de Calcio/metabolismo , Células Cultivadas , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Riñón/citología , Neuroblastoma , Racloprida/farmacología , Proteínas Recombinantes/metabolismo , Espiperona/farmacología , Células Tumorales CultivadasRESUMEN
1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenesis of C114 to serine. 2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((-)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters 'a propyl cleft' on the receptor. The mutation hardly affected quinpirole-dependent [35S]-GTPgammaS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4(2) receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4(2) receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4(2) receptor, is highly susceptible to external oxidizing agents.