RESUMEN

We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mm) yields several 2'-OH acylations per RNA strand in as little as 10 min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.

Asunto(s)

Azidas/farmacología , ARN/efectos de los fármacos , Acilación/efectos de los fármacos , Azidas/antagonistas & inhibidores , Azidas/química , Estructura Molecular , Fosfinas/química , Fosfinas/farmacología , Pliegue del ARN/efectos de los fármacos

RESUMEN

The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H(2)O(2)) absorbance decrease at 240 nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H(2)O(2)-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5 nM for heCAT, 5mM for H(2)O(2), and 10mM for GSH, and the incubation time was 10 min. Nitrite (NO(2)(-)) was found to be an uncompetitive inhibitor of heCAT activity (IC(50)=9 µM) and of CAT activity in hemolysate (IC(50)∼750 µM). Nitrate (NO(3)(-)) at concentrations up to 100 µM did not inhibit heCAT activity. Azide (N(3)(-)) was found to be a very strong inhibitor of the heCAT (IC(50)=0.2 nM) but a relatively weak CAT inhibitor (IC(50)∼10 µM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.

Asunto(s)

Catalasa/análisis , Cromatografía Líquida de Alta Presión/métodos , Eritrocitos/enzimología , Glutatión/análisis , Peróxido de Hidrógeno/análisis , Espectrofotometría Ultravioleta/métodos , Azidas/antagonistas & inhibidores , Pruebas de Enzimas/métodos , Eritrocitos/química , Glutatión/sangre , Humanos , Concentración de Iones de Hidrógeno , Nitritos/antagonistas & inhibidores , Oxidación-Reducción , o-Ftalaldehído/química

RESUMEN

Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K(d) < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant delta subunit-containing receptors, binding of the alcohol antagonist [(3)H]Ro15-4513 is inhibited by low concentrations of EtOH (K(i) approximately 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors.

Asunto(s)

Azidas/antagonistas & inhibidores , Azidas/metabolismo , Benzodiazepinas/antagonistas & inhibidores , Benzodiazepinas/metabolismo , Etanol/farmacología , Antagonistas de Receptores de GABA-A , Receptores de GABA-A/metabolismo , Animales , Azidas/química , Azidas/farmacología , Benzodiazepinas/química , Benzodiazepinas/farmacología , Unión Competitiva/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Humanos , Ligandos , Estructura Molecular , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Receptores de GABA-A/genética

RESUMEN

It has been hypothesized that mitochondrial respiratory chain dysfunction leads to a pyrimidine deficiency since the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase is coupled to the electron transport chain. The uridine prodrug triacetyluridine (PN401) is neuroprotective in several models of neurodegenerative disease involving respiratory chain toxins. Therefore, the therapeutic effects of PN401 might involve the correction of a pyrimidine deficiency secondary to respiratory chain impairment. We infused mice with the cytochrome c oxidase inhibitor azide, which inhibited brain complex IV activity. Chronic infusion of azide for 2 or 14 days induced significant toxicity and mortality but did not cause a pyrimidine deficit in the brain. In contrast, the pyrimidine synthesis inhibitor N-phosphonoacetyl-l-aspartate (PALA) produced a pyrimidine deficit with minimal mortality. Treatment with 6% PN401 decreased mortality and cerebrocortical apoptosis caused by azide. Previously, we found that optimal neuroprotection against mitochondrial complex II inhibition required 4-6% PN401. PN401 at 1, 3, 6 and 10% in chow induced nonlinear increases in plasma uridine with 6% PN401 elevating plasma uridine up to 80 muM, and these higher micromolar uridine levels were also required for neuroprotection in chemical hypoxia models in vitro. Our results indicate that severe complex IV inhibition in vivo does not lead to a pyrimidine deficiency, and therefore the protective effect of PN401 in the azide toxin model is not mediated through the correction of a pyrimidine deficiency. Furthermore, supraphysiological levels of uridine are required to produce optimal protective effects in disorders involving impairment of mitochondrial respiratory complex II or IV.

Asunto(s)

Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fármacos Neuroprotectores , Profármacos/farmacología , Pirimidinas/metabolismo , Uridina/análogos & derivados , Uridina/farmacología , Acetatos , Animales , Apoptosis/efectos de los fármacos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Azidas/antagonistas & inhibidores , Azidas/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Transporte de Electrón/efectos de los fármacos , Fibroblastos/metabolismo , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ácido Fosfonoacético/análogos & derivados , Ácido Fosfonoacético/farmacología , Profármacos/administración & dosificación , Uridina/administración & dosificación , Uridina/metabolismo

RESUMEN

Stopped-flow FTIR spectroscopy was used to monitor continuously the pre-steady- and steady-state phases of azide reduction by nitrogenase and the accompanying hydrolysis of ATP. This was characterized by a ca. 1.3 s lag phase that is explained by the number of Fe protein cycles required to effect the reductions of azide to N(2) + NH(3), N(2)H(4) + NH(3), or 3NH(3). Extrapolation of the steady-state time course for azide reduction to zero time showed that one azide binds within 200 ms to each FeMo cofactor. Inhibition of azide reduction by CO was established at times <400 ms, which was faster than the appearance of the first observable IR band assigned to CO (1904 cm(-)(1) detectable at ca. 1 s with maximum amplitude at ca. 7 s). IR bands associated with the rapidly formed (<400 ms) CO species that inhibits azide reduction were not observed over the range 1700-2100 cm(-)(1). This suggests either that the CO is initially bridging two or more Fe atoms or that a rapid reduction of CO to a formyl state occurs by insertion into a metal-hydride bond. The frequencies and time courses for the appearance and loss of the CO bands under hi- and lo-CO conditions were essentially unaffected by the presence of 20 mM azide, consistent with CO being a noncompetitive inhibitor of azide reduction and with azide and CO binding to different sites on the FeMo cofactor.

Asunto(s)

Adenosina Trifosfato/química , Azidas/antagonistas & inhibidores , Azidas/química , Monóxido de Carbono/química , Nitrogenasa/química , Adenosina Difosfato/química , Adenosina Monofosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Monóxido de Carbono/metabolismo , Hidrólisis , Cinética , Klebsiella pneumoniae/enzimología , Magnesio/química , Modelos Químicos , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Oxidación-Reducción , Fosfatos/química , Unión Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Especificidad por Sustrato , Factores de Tiempo

RESUMEN

Human P-glycoprotein (Pgp) is as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. The aim of this study is to evaluate whether Pgp modulators can be engineered to exhibit high-affinity binding using polyvalency. Five bivalent homodimeric polyenes based on stipiamide linked with polyethylene glycol ethers in the range of 3-50 A were synthesized and quantitatively characterized for their effect on Pgp function. The stipiamide homodimers displaced [(125)I]iodoarylazidoprazoin (IAAP), an analogue of the Pgp substrate prazosin. A minimal spacer of 11 A is necessary for inhibition of IAAP labeling, beyond which there is an inverse correlation between the length of the spacer and the IC(50) for the displacement of IAAP. ATP hydrolysis by Pgp on the other hand is stimulated by the dimers with spacers of up to 22 A, whereas dimers with longer spacers inhibit ATP hydrolysis. Finally, the homodimers reverse Pgp-mediated drug efflux in intact cells overexpressing Pgp, and 11 A is a threshold beyond which the effectiveness of the homodimers increases exponentially and levels off at 33 A. We demonstrate that dimerization and identification of an optimal spacer length increase by 11-fold the affinity of stipiamide, and this is reflected in the efficacy with which Pgp-mediated drug efflux is reversed. These results suggest that polyvalency could be a useful strategy for the development of more potent Pgp modulators.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Polienos/química , Polienos/metabolismo , Prazosina/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Sitios de Unión , Unión Competitiva , Transporte Biológico , Compuestos de Boro/metabolismo , Dimerización , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Fluoresceínas/metabolismo , Humanos , Hidrólisis , Radioisótopos de Yodo/metabolismo , Ratones , Células 3T3 NIH , Etiquetas de Fotoafinidad/metabolismo , Polienos/síntesis química , Prazosina/antagonistas & inhibidores , Prazosina/metabolismo

RESUMEN

ATP-sensitive potassium (K(ATP)) channels are under complex regulation by intracellular ATP and ADP. The potentiatory effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We have previously reported that Kir6.2 can be directly labeled by 8-azido-[gamma-(32)P]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 was low probably due to modification at 8' position of adenine. Here we demonstrate that Kir6.2 can be directly photoaffinity labeled with higher affinity by [gamma-(32)P]ATP-[gamma]4-azidoanilide ([gamma-(32)P]ATP-AA), containing an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-AA is not affected by the presence of Mg(2+), consistent with Mg(2+)-independent ATP inhibition of K(ATP) channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/análogos & derivados , Azidas/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Azidas/antagonistas & inhibidores , Azidas/farmacología , Unión Competitiva , Células COS , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Magnesio/farmacología , Oocitos , Etiquetas de Fotoafinidad/farmacología , Potasio/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Ratas , Receptores de Droga/genética , Receptores de Droga/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Especificidad por Sustrato , Receptores de Sulfonilureas , Transfección , Xenopus laevis

RESUMEN

This study demonstrates that astemizole, a non-sedating anti-histaminergic drug with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR). Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 microM in a dose-dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytofluorographic efflux experiments with doxorubicin. Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. Moreover, it also inhibits the photolabeling of P-glycoprotein by [3H]azidopine in a dose-dependent manner. These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.

Asunto(s)

Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Astemizol/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Células K562/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Astemizol/administración & dosificación , Astemizol/metabolismo , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Colorimetría , Dihidropiridinas/antagonistas & inhibidores , Dihidropiridinas/metabolismo , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Células K562/metabolismo , Etiquetas de Fotoafinidad/metabolismo

RESUMEN

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Secuencias de Aminoácidos/efectos de los fármacos , Secuencias de Aminoácidos/genética , Sustitución de Aminoácidos/genética , Anticuerpos Monoclonales/metabolismo , Asparagina/genética , Ácido Aspártico/genética , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Sitios de Unión/genética , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Temperatura Corporal , Membrana Celular/enzimología , Membrana Celular/metabolismo , Secuencia de Consenso/efectos de los fármacos , Secuencia de Consenso/genética , Activación Enzimática/efectos de los fármacos , Congelación , Células HeLa , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Radioisótopos de Fósforo/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Mutación Puntual , Prazosina/análogos & derivados , Prazosina/antagonistas & inhibidores , Prazosina/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vanadatos/farmacología , Verapamilo/farmacología

RESUMEN

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.

Asunto(s)

Adenosina Trifosfato/metabolismo , Creatina Quinasa/metabolismo , Nucleotidiltransferasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Animales , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Química Encefálica , Cromatografía Líquida de Alta Presión , Creatina/química , Creatina/metabolismo , Creatina Quinasa/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glicina/análogos & derivados , Humanos , Yodoacetatos/química , Isoenzimas , Músculo Esquelético/enzimología , Nucleotidiltransferasas/antagonistas & inhibidores , Oxidación-Reducción , Péptidos/metabolismo , Radioisótopos de Fósforo , Etiquetas de Fotoafinidad/metabolismo , Conejos , Especificidad por Sustrato , Temperatura , Factores de Tiempo , Trometamina , Tripsina/metabolismo

RESUMEN

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antagonistas de Dopamina/farmacología , Flupentixol/farmacología , Fenilalanina/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Alanina/genética , Sustitución de Aminoácidos/genética , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Antagonistas de Dopamina/química , Flupentixol/química , Células HeLa , Humanos , Hidrólisis/efectos de los fármacos , Radioisótopos de Yodo/metabolismo , Mutagénesis Sitio-Dirigida , Fenilalanina/genética , Etiquetas de Fotoafinidad/metabolismo , Prazosina/análogos & derivados , Prazosina/antagonistas & inhibidores , Prazosina/metabolismo , Receptores Dopaminérgicos/metabolismo , Estereoisomerismo , Especificidad por Sustrato/efectos de los fármacos , Células Tumorales Cultivadas , Verapamilo/farmacología

RESUMEN

P-glycoprotein (Mdr1p) is an ATP-dependent drug efflux pump that is overexpressed in multidrug-resistant cells and some cancers. Mdr1p is also expressed in normal tissues like the kidney, where it can mediate transepithelial drug transport. A human urinary compound that reverses multidrug resistance and blocks [3H]azidopine photolabeling of P-glycoprotein was purified to homogeneity and identified by 1H-NMR and mass spectrometry as the synthetic surfactant nonylphenol ethoxylate (NPE). Multidrug-resistant Chinese hamster ovary (CHO) C5 cells accumulated less [3H]NPE than parental drug-sensitive Aux-B1 cells, and Mdr1p substrates, verapamil and cyclosporin A, increased this surfactant's accumulation in C5 cells. NPE blocked the net transepithelial transport (basolateral to apical) of [3H]cyclosporin A in epithelia formed by Madin-Darby canine kidney (MDCK) cells. Net transepithelial transport (basal to apical) of [3H]NPE was demonstrated in MDCK cells and was inhibited by cyclosporin A. These findings show NPE is a Mdr1p substrate excreted into urine by kidney P-glycoprotein. NPE is a widely used surfactant and a known hormone disrupter that is readily absorbed orally or topically. The current findings indicate the function of kidney Mdr1p may be to eliminate exogenous compounds from the body.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Glicoles de Etileno/orina , Marcadores de Afinidad , Animales , Azidas/antagonistas & inhibidores , Células CHO/metabolismo , Permeabilidad de la Membrana Celular , Cromatografía Líquida de Alta Presión , Cricetinae , Ciclosporina/metabolismo , Dihidropiridinas/antagonistas & inhibidores , Perros , Resistencia a Múltiples Medicamentos , Glicoles de Etileno/química , Glicoles de Etileno/metabolismo , Glicoles de Etileno/farmacología , Humanos , Espectroscopía de Resonancia Magnética

RESUMEN

We have found that azide-resistant mutants of Salmonella typhimurium and of other bacteria studied produce a substance which inactivates the azide. The production of this substance was proved by the demonstration of a satellite growth of azide-sensitive cells around colonies of azide-resistant mutants and by testing azide inactivating properties of culture filtrates of the azide-resistant strains. The same substance was found to be present in lower concentrations in culture filtrates of wild-type sensitive strains. In both cultures of sensitive strains, it was apparently produced by the resistant mutants and not by the sensitive cells. The substance does not pass across a dialysis membrane and is heat stable. It has a high molecular weight but is not a protein.

Asunto(s)

Azidas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Salmonella typhimurium/metabolismo , Azidas/farmacología , Resistencia a Medicamentos , Salmonella typhimurium/efectos de los fármacos , Azida Sódica

RESUMEN

The potency of 30 benzodiazepine binding site ligands from 14 different structural classes for inhibition of [3H]Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate) binding to human embryonic kidney (HEK) 293 cells transiently transfected with alpha 4 beta 3 gamma 2S or alpha 1 beta 3 gamma 2S subunits of GABAA receptors was investigated. Most of these compounds were unable to significantly inhibit [3H]Ro 15-4513 binding to alpha 4 beta 3 gamma 2S receptors under conditions where they potently inhibited binding to alpha 1 beta 3 gamma 2S receptors. Nevertheless, compounds from four different structural classes were identified which exhibited a high affinity for alpha 4 beta 3 gamma 2S receptors. Variation of the structure of these compounds could lead to new ligands selectively interacting with alpha 4 beta 3 gamma 2S receptors. Compounds interacting with alpha 4 beta 3 gamma 2S receptor were also able to inhibit [3H]Ro 15-4513 binding to receptors consisting of alpha 4 gamma 2S subunits with comparable potency. These results support the conclusion that the alpha subunit is a major determinant of the benzodiazepine binding site properties of GABAA receptors containing alpha and gamma subunits.

Asunto(s)

Receptores de GABA-A/metabolismo , Animales , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Benzodiazepinas/antagonistas & inhibidores , Benzodiazepinas/metabolismo , Sitios de Unión , Células Cultivadas , Clonación Molecular , Embrión de Mamíferos , Humanos , Técnicas In Vitro , Riñón/citología , Riñón/metabolismo , Ligandos , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad

RESUMEN

1. In this study we investigated the role of catalase in relaxation induced by hydroxylamine, sodium azide, glyceryl trinitrate and hydrogen peroxide in isolated rings of rat aorta. 2. Hydrogen peroxide (1 microM-1 mM)-induced concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings. In endothelium-denuded rings, however, higher concentrations (30 microM-1 mM) of hydrogen peroxide were required to produce relaxation. The endothelium-dependent component of hydrogen peroxide-induced relaxation was abolished following pretreatment with N(O)-nitro-L-arginine methyl ester (L-NAME, 30 microM). L-NAME (30 microM) had no effect, however, on hydrogen peroxide-induced relaxation in endothelium-denuded rings. 3. Pretreatment of endothelium-denuded rings with catalase (1000 u ml-1) blocked relaxation induced by hydrogen peroxide (10 microM-1 mM). The ability of catalase to inhibit hydrogen peroxide-induced relaxation was partially blocked following incubation with 3-amino-1,2, 4-triazole (AT, 50 mM) for 30 min and completely blocked at 90 min. 4. Pretreatment of endothelium-denuded rings with methylene blue (MeB, 30 microM) inhibited relaxation induced by hydrogen peroxide (10 microM-1 mM), sodium azide (1-300 nM), hydroxylamine (1-300 nM) and glyceryl trinitrate (1-100 nM) suggesting that each acted by stimulation of soluble guanylate cyclase. 5. Pretreatment of endothelium-denuded rings with AT (1-50 mM, 90 min) to inhibit endogenous catalase blocked relaxation induced by sodium azide (1-300 nM) and hydroxylamine (1-300 nM) but had no effect on relaxation induced by hydrogen peroxide (10 microM-1 mM) or glyceryl trinitrate (1-100 nM). 6. In a cell-free system, incubation of sodium azide (10 microM-3 mM) and hydroxylamine (10 microM-30 mM) but not glyceryl trinitrate (10 microM-1 mM) with catalase (1000 u ml-1) in the presence of hydrogen peroxide (1 mM) led to production of nitrite, a major breakdown product of nitric oxide. AT (1-100 mM) inhibited, in a concentration-dependent manner, the formation of nitrite from azide in the presence of hydrogen peroxide. 7. These data suggest that metabolism by catalase plays an important role in the relaxation induced by hydroxylamine and sodium azide in isolated rings of rat aorta. Relaxation appears to be due to formation of nitric oxide and activation of soluble guanylate cyclase. In contrast, metabolism by catalase does not appear to be involved in the relaxant actions of hydrogen peroxide or glyceryl trinitrate.

Asunto(s)

Amitrol (Herbicida)/farmacología , Aorta/efectos de los fármacos , Azidas/antagonistas & inhibidores , Catalasa/antagonistas & inhibidores , Hidroxilaminas/antagonistas & inhibidores , Vasodilatadores/farmacología , Animales , Femenino , Peróxido de Hidrógeno/farmacología , Hidroxilamina , Técnicas In Vitro , Azul de Metileno/farmacología , Nitritos/química , Nitroglicerina/farmacología , Ratas , Ratas Wistar , Azida Sódica

RESUMEN

P-glycoprotein (P-gp) is expressed at high levels in a variety of non-cancerous tissues such as the endothelial cells of the blood-brain barrier (BBB) capillaries. These thin capillaries tightly regulate the movement of substrates from the circulating blood into the brain. P-gp may be involved in the exclusion of various drugs from the capillary endothelial cells, blocking their entry into the brain. However, interactions of drugs with P-gp expressed in brain capillaries remain to be characterized. We have performed photoaffinity labeling studies using [125I]arylazidoprazosin (IAAP) to evaluate the inhibitory efficiency of various compounds. Cyclosporin A (CsA) and its derivative PSC 833 (PSC) were the most effective inhibitors of IAAP binding among the drugs tested. The magnitude of inhibition was: PSC > CsA > quinidine > vinblastine > verapamil < actinomycin D > colchicine > reserpine > bilirubin > doxorubicin > progesterone. Cremophor El, the vehicle used to administer CsA and PSC intravenously, was also able to inhibit IAAP photolabeling of P-gp. Labeling experiments were also performed using a photoactivatable [3H]CsA derivative. Photolabeling of P-gp with this compound was abolished almost completely by CsA and PSC. In vivo studies were also performed by treating rats with CsA [10 mg/(kg.day) for 10 days]. Following this treatment, no alteration in the level of P-gp expression in brain capillaries was observed. These results suggest that, at the proper dosage, administration of CsA to cancer patients could help to enhance the response of brain tumors to chemotherapeutic agents without modifying the intrinsic level of P-gp expression in this tissue.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Ciclosporina/farmacología , Ciclosporinas/farmacología , Marcadores de Afinidad/metabolismo , Animales , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Capilares/efectos de los fármacos , Capilares/metabolismo , Interacciones Farmacológicas , Glicerol/análogos & derivados , Glicerol/farmacología , Técnicas In Vitro , Radioisótopos de Yodo , Masculino , Prazosina/análogos & derivados , Prazosina/antagonistas & inhibidores , Prazosina/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Tritio

RESUMEN

Benzodiazepines modulate gamma-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABAA receptor-chloride channel complex. The heterogeneity of diazepam-sensitive benzodiazepine binding sites (type I and type II) has been identified by pharmacological approaches both with native receptors and recombinant receptors coexpressing alpha, beta and gamma subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites are found, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of alpha 6 with beta 2 and gamma 2L subunits creates a pharmacologically similar benzodiazepine receptor to the diazepam-insensitive site observed in cerebellum, however, there is no evidence regarding the possible subunit combination forming the DI site in cerebral tissues. Here we report the cloning of the human alpha 4 cDNA and its pharmacology by coexpression of this alpha 4 subunit with beta 2 and gamma 2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenill, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-beta-carboline-3-carboxylate) and Ro15-4513 as determined by [3H]Ro15-4513 binding. However, it is insensitive to the benzodiazepine type I selective compounds CL218.872 (3-methyl-6-[3-(trifluoromethyl)[phenyl]-1,2,4-triazolo[4.3-b]pyridazine ) and zolpidem as well as the benzodiazepine full agonists diazepam, halazolam and midazolam. In addition, the benzodiazepine receptor ligands DMCM, beta-CCE (beta-carboline-3-carboxylate ethyl ester), Beta-CCM (beta-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for alpha 4 beta 2 gamma 2L. The pharmacology of the alpha 4 beta 2 gamma 2L receptor complex appears to resemble those of the diazepam-insensitive site found in the cerebral cortex. Our study thus suggests that this subpopulation of diazepam-insensitive GABAA receptors may be composed of alpha 4 beta 2 gamma 2L subunits.

Asunto(s)

Receptores de GABA-A/biosíntesis , Receptores de GABA-A/química , Secuencia de Aminoácidos , Azidas/antagonistas & inhibidores , Azidas/química , Benzodiazepinas/antagonistas & inhibidores , Benzodiazepinas/química , Sitios de Unión , Células Cultivadas , Clonación Molecular , ADN Complementario/análisis , Humanos , Datos de Secuencia Molecular , Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido

RESUMEN

Using Salmonella typhimurium strains TA 100 and TA 1535, the mutagenicity and anti-mutagenicity of extracts of several spices were checked. Spices like pepper, pippali, ginger and mustard increased the number of revertants indicating their mutagenic potential. Garlic extract on the other hand was found to inhibit the mutagenicity produced by direct acting mutagens such as N-methyl N'-nitro-N-nitrosoguanidine and sodium azide. Asafoetida and turmetic extract were found to inhibit microsomal activation dependent mutagenicity of 2-acetamidofluorene. Similar results were also obtained using curcumin and eugenol which are phenolics present in turmeric and clove respectively. These results indicated that some of the spices may ameliorate the effect of environmental mutagens especially present in the food.

Asunto(s)

Antimutagênicos/farmacología , Mutágenos/farmacología , Mutágenos/toxicidad , Especias/toxicidad , 2-Acetilaminofluoreno/antagonistas & inhibidores , 2-Acetilaminofluoreno/toxicidad , Animales , Azidas/antagonistas & inhibidores , Azidas/toxicidad , Masculino , Metilnitronitrosoguanidina/toxicidad , Pruebas de Mutagenicidad , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Azida Sódica

RESUMEN

We studied the restoration of doxorubicin accumulation and sensitivity by verapamil and quinine in a variant of the human erythroleukemia cell line K562 selected for resistance to doxorubicin and presenting a multidrug-resistance (MDR) phenotype. Verapamil was able to completely restore doxorubicin accumulation in the resistant cells to the level obtained in sensitive cells, but only partially reversed doxorubicin resistance. Quinine, in contrast, had a relatively weak effect on doxorubicin accumulation but was able to completely restore doxorubicin sensitivity in the resistant cells. In addition, verapamil was able to decrease azidopine binding to P-glycoprotein, whereas quinine was not. Quinine also modified the intracellular tolerance to doxorubicin, which suggests that it is able to modify drug distribution within the cells. Confocal microscopy revealed that verapamil and quinine were able to restore nuclear fluorescence staining of doxorubicin in resistant cells; since this was obtained for quinine without significant increase of doxorubicin accumulation, this observation confirms that quinine acts principally on doxorubicin redistribution within the cells, allowing the drug to reach its nuclear targets. When used in association, verapamil and quinine reversed doxorubicin resistance in a synergistic fashion. We conclude that verapamil and quinine do not share the same targets for reversal of MDR in this cell line; whereas verapamil directly interferes with P-glycoprotein and mainly governs drug accumulation, quinine has essentially intracellular targets involved in drug redistribution from sequestration compartments.

Asunto(s)

Doxorrubicina/farmacología , Leucemia Eritroblástica Aguda/tratamiento farmacológico , Leucemia Eritroblástica Aguda/metabolismo , Quinina/farmacología , Verapamilo/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Marcadores de Afinidad/metabolismo , Azidas/antagonistas & inhibidores , Azidas/metabolismo , Dihidropiridinas/antagonistas & inhibidores , Dihidropiridinas/metabolismo , Doxorrubicina/farmacocinética , Resistencia a Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Cinética , Microscopía Confocal , Microscopía Fluorescente , Fenotipo , Fracciones Subcelulares/metabolismo , Células Tumorales Cultivadas

RESUMEN

The effects of two new phthalazinone derivatives, azelastine (AZ) and flezelastine (FZ), on the reversal of resistance to doxorubicin (dox) were studied using two variants of the rat C6 glioblastoma cell line, selected with dox (C6 0.5) or with vincristine (C6 1V). Both lines presented a multidrug-resistant phenotype which was, in the case of C6 0.5 cells, likely to be accompanied by an additional mechanism leading to intracellular tolerance of the drug. Both AZ and FZ reversed dox resistance in a concentration-dependent manner, and FZ was shown to be at least three times more potent than AZ. FZ was able, at a relatively high concentration (30 microM), to completely restore dox sensitivity in both cell lines. Both drugs were able to virtually restore dox accumulation to the level reached in sensitive cells, and, interestingly, this complete restoration occurred at lower concentrations of modulator than required for complete reversal of resistance. FZ was able to reverse dox intracellular tolerance of C6 0.5 cells and to restore dox accumulation at the IC50 to the level observed in sensitive cells. AZ and FZ both inhibited azidopine binding to membrane preparations of C6 0.5 and C6 1V cells, although FZ was more potent. Both drugs more successfully inhibited azidopine binding to membranes prepared from C6 1V cells (which express the mdr1b gene product) than to membranes from C6 0.5 cells (which express the mdr1a gene product). In view of its potent activity on MDR, further preclinical evaluation of FZ is warranted.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Azepinas/farmacología , Resistencia a Múltiples Medicamentos , Ftalazinas/farmacología , Animales , Azidas/antagonistas & inhibidores , Dihidropiridinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Ratas , Células Tumorales Cultivadas/efectos de los fármacos , Vincristina/farmacología