RESUMEN
The influence of cytosol acidification on the uptake of two-chain tetanus toxin (TeTX)1 by neurohybridoma cells NG 108-15 and NBr-10A was investigated with two established techniques, the NH4Cl pulse method and the pH-clamp method. With the former, the extracellular pH is maintained at its physiological value, but is set to different values with the latter. Acidification of the cytoplasm with an NH4Cl pulse retarded the uptake of TeTX by both NG 108-15 and NBr-10A cells. This result provides further evidence for a vesicular endocytotic uptake of TeTX. In contrast, acidification of both the external medium and the cytoplasm (pH-clamp method) resulted in a net increase of toxin uptake. This result is explained as follows: Acidification of the extracellular environment has been shown to facilitate the uptake of tetanus toxin, and under pH clamp conditions, this effect is stronger than the simultaneous retardation of the toxin uptake by acidification also of the cytosol.
Asunto(s)
Citosol/metabolismo , Hibridomas/efectos de los fármacos , Toxina Tetánica/metabolismo , Transferrina/metabolismo , Cloruro de Amonio/farmacología , Animales , Bovinos , Caballos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ionóforos/metabolismo , Nigericina/metabolismoRESUMEN
A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocratic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean +/- S.D.): 3.148 +/- 0.259 ng (8.22%), 12.594 +/- 0.571 ng (4.53%), and 25.016 +/- 0.658 ng (2.63%) with C.V. values in parentheses for n = 5. The accuracy of the assay ranged from -7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5 +/- 0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.
Asunto(s)
Azatioprina/sangre , Cromatografía Líquida de Alta Presión/métodos , Adulto , Azatioprina/farmacocinética , Humanos , Masculino , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of beta-adrenoceptors with "dipping" autoradiography and a modified trichrome stain. Incubation of 16 microns cryosections of rat lung tissue with the iodinated, photoaffinity labeling, non-selective, beta-adrenergic agonist [125I]-cyanopindololazide II ([125I]-CYPA II) (100 pmol/l) in the absence or presence of 1 mumol (+/-)-propranolol revealed strong, specific beta-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photoemulsion by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of beta-adrenoceptors with [125I]-CYPA II allows the use of "dipping" autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiographic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.
Asunto(s)
Autorradiografía/métodos , Compuestos Azo , Eosina Amarillenta-(YS) , Pulmón/metabolismo , Verde de Metilo , Receptores Adrenérgicos beta/metabolismo , Marcadores de Afinidad , Animales , Azidas , Colorantes , Crioultramicrotomía , Epitelio/metabolismo , Masculino , Músculo Liso/metabolismo , Fotograbar , Pindolol/análogos & derivados , Ratas , Ratas Sprague-DawleyRESUMEN
The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.
Asunto(s)
Proteínas/administración & dosificación , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Compartimento Celular , Reactivos de Enlaces Cruzados , Endocitosis , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Fragmentos Fab de Inmunoglobulinas/metabolismo , Leucemia Eritroblástica Aguda , Maleimidas/química , Toxoide Tetánico/metabolismo , Células Tumorales CultivadasRESUMEN
1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.
Asunto(s)
Células Eucariotas/inmunología , Fragmentos Fab de Inmunoglobulinas , Antitoxina Tetánica/inmunología , Aciclovir/inmunología , Peroxidasa de Rábano Silvestre/inmunología , Inmunotoxinas/inmunología , Transferrina/inmunología , Aglutininas del Germen de Trigo/inmunologíaRESUMEN
Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.
Asunto(s)
Calcio/fisiología , Gránulos Cromafines/metabolismo , Exocitosis/efectos de los fármacos , Antitoxina Tetánica/metabolismo , Toxina Tetánica/farmacología , Animales , Bovinos , Gránulos Cromafines/efectos de los fármacos , Gangliósidos/fisiología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Norepinefrina/metabolismo , Toxina Tetánica/metabolismoRESUMEN
Herpesvirus type 1 could be propagated most efficiently in cultured fetal neurons, to a lesser extent in NG108-15 neurohybridoma cells, and with the lowest titer in glial cells. Herpesvirus type 2 could not be cultured in neurohybridoma cells, and in fetal neurons a titer 100-fold lower than for herpesvirus type 1 was obtained. Cells infected with herpesvirus type 1 were used in an infectivity assay for acyclovir dose-response studies. The ED50 values were 7.4 nmol/l for fetal neurons, 180 for neurohybridoma cells, and 275 nmol/l for glial cells.
Asunto(s)
Aciclovir/farmacología , Neuroglía/microbiología , Neuronas/microbiología , Simplexvirus/efectos de los fármacos , Animales , Células Cultivadas , Efecto Citopatogénico Viral , Hibridomas , Ratones , Simplexvirus/crecimiento & desarrollo , Médula Espinal/citología , Médula Espinal/embriología , Cultivo de VirusRESUMEN
An HPLC method for the detection of demeton-S-methyl-sulfoxide (Metasystox R) in human plasma was developed. DSMSO was extracted by passing the acidified plasma sample through an Extrelut 3 column and eluted from the column with dichloromethane. An aliquot of the extract was injected onto the HPLC nitrile reversed-phase column. The mobile phase was a solution of 3% acetonitrile in 0.01M phosphoric acid. The eluent was monitored at 215 nm. With this method, plasma concentrations between 0.5 and 50 mg/L were determined. Within this range, the concentration-response relation was linear (r = 0.9991). The within-run and between-run coefficients of variation were concentration dependent, with maximal values less than 10%. The analysis took about 45 minutes.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Insecticidas/sangre , Compuestos Organotiofosforados/sangre , HumanosRESUMEN
Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.
Asunto(s)
Acetilcolina/metabolismo , Hibridomas/metabolismo , Toxina Tetánica/metabolismo , Células Tumorales Cultivadas/metabolismo , Animales , Bucladesina/farmacología , Relación Dosis-Respuesta a Droga , Glioma/metabolismo , Neuraminidasa/metabolismo , Neuroblastoma/metabolismo , Factores de TiempoRESUMEN
A new forequarters preparation, the in situ perfused cervical spinal cord of the adult rat, is described with respect to its suitability for pharmacological and toxicological investigations. The dose-dependent actions of strychnine, p-chloromercuriphenylsulfonate, ouabain, dinitrophenol, bicuculline, and (-)-nipecotic acid ethyl ester on mass reflex discharges were studied. The study was extended to sodium-lithium exchange and calcium reduction in the perfusion medium. With the intent to discriminate between primary central actions of substances and ion changes and their secondary metabolic effects, the flow rate, the sodium and potassium concentrations, the pH, the pO2, and the pCO2 of the perfusion medium were measured. The new perfused forequarters preparation, including the intact cervical spinal cord, has a number of advantages over an experimental design using an intact animal dependent on its physiological circulation. These advantages are: the composition of fluids supplying the preparation is under control; the oxygen supply to the cord is no longer dependent on the cardiovascular function, which may be impaired by the substance under study; a complete dose-response curve may be obtained from each animal; washout experiments may be performed; the action of substances can be studied in the presence of extreme concentrations of drugs, ions, etc.
Asunto(s)
Calcio/metabolismo , Litio/metabolismo , Sodio/metabolismo , Médula Espinal/metabolismo , 4-Cloromercuribencenosulfonato/farmacología , Animales , Bicuculina/farmacología , Dinitrofenoles/farmacología , Estimulación Eléctrica , Masculino , Ácidos Nipecóticos/farmacología , Ouabaína/farmacología , Perfusión , Ratas , Ratas Endogámicas , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiología , Estricnina/farmacologíaRESUMEN
Formula are derived for the calculation of the amounts of drug which, during a defined time interval, are eliminated from the body with and without the help of hemodialysis or hemoperfusion (HD/HP). A programmable pocket calculator like the TI-59 suffices to perform all calculations. The pharmacokinetic system constants of the drug, the effective plasma flow, and the HD/HP plasma extraction rate are entered into the program. Additional information can be obtained if the plasma concentration of the drug at the onset of HD/HP is known. Worked examples are presented.
Asunto(s)
Hemoperfusión , Modelos Biológicos , Preparaciones Farmacéuticas/sangre , Diálisis Renal , Intoxicación Alcohólica/terapia , Velocidad del Flujo Sanguíneo , Proteínas Sanguíneas/metabolismo , Humanos , Cinética , Matemática , Unión ProteicaRESUMEN
Radioactivities were measured in serum, urine, and bile of dogs at different times after intravenous injection of 14C-methyl-gamma-amanitin (14C-A) and 3H-O-methyl-dehydroxymethyl-alpha-amanitin (3H-A). For either substance, the relation between the specific plasma activity C and the time t could be best described with the function C = C1 X e- lambda 1 X t + C2 X e- lambda 2 X t. Therefore the linear open two-compartment system was selected as an adequate toxicokinetic model. Most important, the distribution volumes (in the steady state) were in the range of the extracellular space, and the total body clearances were in the range of the dog creatinine clearance. In accordance with former findings for 3H-A, 14C-A was not bound to plasma proteins. More than 80% of 14C-A was eliminated in the urine; less than 10% was found in the bile. From these data, two suggestions may be derived for the therapy of Amanita intoxication in man. First, detection in the urine of amatoxins 2 or 3 days after mushroom ingestion points to an ongoing amatoxin absorption or reabsorption from the intestine, and should lead to therapy with adsorbents and, in the absence of diarrhea, with laxatives. Second, hemoperfusion will remove significant amounts of amatoxins during the time of ongoing absorption or reabsorption and a few hours thereafter.
Asunto(s)
Amanitinas/sangre , Amanitinas/orina , Animales , Bilis/metabolismo , Sitios de Unión , Diálisis , Perros , Sangre Fetal/metabolismo , Semivida , Hemoperfusión , Cinética , Albúmina Sérica Bovina/metabolismoRESUMEN
Intrathecally administered free anti-tetanus immunoglobulin G (IgG) diffuses through the spinal cord but does not enter nerve cells. In order to facilitate entry into neurons, 125I-labeled anti-tetanus IgG was entrapped in liposomes. After injection into the cerebrospinal fluid of rats, however, only a very low specific radioactivity of the spinal cord could be calculated from gross counts and no neuronal labeling was seen in autoradiographs. Therefore, it was assumed that the liposomes were unable to cross the basement membrane of the spinal cord surface. To circumvent this barrier the liposome-entrapped [125I]IgGs were injected directly into the grey matter. Histoautoradiographs then showed marked accumulations of radioactivity in neurons. Direct intraspinal injection of free [125I]IgG, on the other hand, failed to produce heavy neuronal labeling.
Asunto(s)
Inmunoglobulina G/administración & dosificación , Médula Espinal/metabolismo , Antitoxina Tetánica/administración & dosificación , Animales , Autorradiografía , Inyecciones Espinales , Liposomas , Conejos/inmunología , Ratas , Ratas EndogámicasRESUMEN
A method was developed for the perfusion of the cervical spinal cord of adult rats through the supplying arteries. A medium containing perfluorotributylamine as an oxygen carrier was used. The electrophysiological responsiveness was continuously monitored; pO2, pCO2, pH, Na+ and K+ were intermittently measured in the medium. The perfused cord maintained its responsiveness for more than 5 h. No changes in its structure became apparent in electron micrographs.
Asunto(s)
Médula Espinal/fisiología , Animales , Sustitutos Sanguíneos , Emulsiones , Fluorocarburos , Concentración de Iones de Hidrógeno , Masculino , Microscopía Electrónica , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Oxígeno , Presión Parcial , Perfusión , Ratas , Ratas Endogámicas , Médula Espinal/ultraestructuraRESUMEN
The EMIT-dau for methadone and the EMIT-ST for opiates (both EMITs from Syva-Merck) gave false-positive results when applied to urine samples of a patient suffering from a moderate doxylamine monointoxication. The error was not detected in routine TLC. Gas chromatography and mass spectrometry unequivocally indicated the presence in urine and plasma of doxylamine and its metabolites and the definite absence of methadone. Opiates could not be detected with the Abuscreen RIA (Roche), which is a more sensitive method than EMIT-ST.
Asunto(s)
Doxilamina/envenenamiento , Metadona/sangre , Narcóticos/sangre , Piridinas/envenenamiento , Adulto , Cromatografía de Gases/métodos , Cromatografía en Capa Delgada/métodos , Reacciones Falso Positivas , Femenino , Humanos , Técnicas para InmunoenzimasRESUMEN
The pharmacokinetics of Tl+ were studied in 9 patients who underwent myocardial scintigraphy with 201Tl+. The time course of the 201Tl+ concentration fitted to an open two-compartment model. The following values were calculated: k12 = 0.164 +/- 0.025 min-1, k21 = 0.011 +/- 0.004 min-1, k10 = 0.005 +/- 0.004 min-1, t1/2(lambda 1) = 3.92 +/- 0.54 min, t 1/2(lambda 2) = 3108 +/- 896 min, CL = 0.08 +/- 0.03 l/min, V1 = 0.26 +/- 0.13 l/kg, Vz = 4.36 +/- 0.65 l/kg, Vss = 4.23 +/- 0.67 l/kg. Using previously published dialysance values the influence of hemodialysis on Tl+ elimination was calculated. Hemodialysis in Tl+ intoxications should be moderately effective.
Asunto(s)
Talio/metabolismo , Semivida , Humanos , Cinética , Diálisis RenalRESUMEN
A method is outlined to estimate from hemodialysis or hemoperfusion data, respectively, the rate constants, the distribution volume in the beta-phase, and other pharmacokinetic parameters of toxic substances. All calculations are based on the assumption that the usual linear open two-compartment system is also the appropriate model in a particular intoxication. No prior knowledge of the time of application or of the dose of the toxic substance is needed. The pharmacokinetic parameters may be helpful to estimate the benefit of hemodialysis or hemoperfusion, respectively, in future intoxications.
Asunto(s)
Hemoperfusión , Preparaciones Farmacéuticas/metabolismo , Diálisis Renal , Humanos , Cinética , MatemáticaRESUMEN
A fatal monointoxication with diphenhydramine in a male 28 year old is reported. The patient went into hyperpyrexia and tachycardia and died from sudden cardiac arrest. Hemorrhagic pulmonary edema and renal shock were the most prominent pathomorphological findings. At the time of death, the concentration of diphenhydramine was 5 mg/l plasma and was particularly high in the lungs (55 mg/kg) and kidneys (50 mg/kg).
Asunto(s)
Difenhidramina/envenenamiento , Adulto , Cromatografía de Gases , Difenhidramina/análisis , Estudios de Seguimiento , Cromatografía de Gases y Espectrometría de Masas , Humanos , MasculinoRESUMEN
The dose-dependency of Tl+ kinetics was studied in rabbits by intravenous injection first of a 201Tl+ tracer dose and 2 weeks later of a 5.5 mumol/kg dose. For either dose level, an open three compartment model was appropriate to describe the course of the plasma concentration curve. With the dosage increase a slight to moderate decrease in the distribution volumes (11.2 l/kg versus 9.7 l/kg) and in the plasma clearance (13 ml/min versus 9 ml/min) was found. Two days after injection of both the high and the low dose considerable amounts of Tl+ were found in the intestinal contents and only small amounts were found in the bile.
Asunto(s)
Talio/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Cinética , Tasa de Depuración Metabólica , Modelos Biológicos , Conejos , Distribución TisularRESUMEN
Tetanus toxin was tritiated with various amounts of N-succinimidyl-[2,3-3H]propionate. A tenfold molar excess of reagent over toxin was used for the introduction of about 5 nmol of [2,3-3H]propionate into 1 nmol of toxin, corresponding to a specific activity of about 48 MBq/mg protein. The labelling procedure did not change significantly the immunoreactivity of tetanus toxin, but reduced the toxicity in mice from 12 ng/kg to 16 ng/kg. Tetanus toxin-[2,3-3H]propionamid was carried through the peripheral nerves into the spinal cord. It could be used instead of 125I-labelled toxin for the detection of anti-tetanus toxin antibodies with a detection limit of 0.25 mU/ml of serum.