RESUMEN
Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na+ + K+)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.
Asunto(s)
Membrana Celular/ultraestructura , Corteza Renal/ultraestructura , Animales , Fraccionamiento Celular/métodos , Perros , Corteza Renal/citología , Microscopía Electrónica , Microvellosidades/ultraestructura , ATPasa Intercambiadora de Sodio-Potasio/análisisRESUMEN
Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.
Asunto(s)
Membrana Basal/metabolismo , Bicarbonatos/metabolismo , Proteínas Portadoras/metabolismo , Cloruros/metabolismo , Túbulos Renales Proximales/metabolismo , Animales , Proteínas de Transporte de Anión , Transporte Biológico , RatasRESUMEN
The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Renal/metabolismo , Microvellosidades/metabolismo , Reactivos de Sulfhidrilo/farmacología , 4-Cloromercuribencenosulfonato/farmacología , Animales , Proteínas de Transporte de Anión , Perros , Etilmaleimida/farmacología , Cinética , Cloruro de Mercurio/farmacología , Microvellosidades/efectos de los fármacos , Ácido p-Aminohipúrico/metabolismoRESUMEN
We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.
Asunto(s)
Riñón/metabolismo , Microvellosidades/metabolismo , Niacinamida/análogos & derivados , Protones , Naranja de Acridina , Animales , Transporte Biológico/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas Portadoras/metabolismo , Cationes , Perros , Electroquímica , Gramicidina/farmacología , Cinética , Niacinamida/metabolismo , Nigericina/farmacología , Potasio/farmacología , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno , Espectrofotometría , Valinomicina/farmacologíaRESUMEN
Mutations in the Neuroserpin gene have been reported to cause familial presenile dementia. We describe a new family in which the S52R Neuroserpin mutation is associated with progressive myoclonus epilepsy in 2 siblings. The proband presented myoclonus and epilepsy at age 24, his brother and mother presented a similar disorder when they were 25. A clinical diagnosis of progressive myoclonus epilepsy was made on the proband and his brother. Skin and liver biopsies did not reveal the presence of cytological alterations in the proband. His neurological status worsened over the subsequent 19 yr during which he became demented and had uncontrollable seizures. He died at 43 yr of age from aspiration pneumonia. Neuropathologically, eosinophilic bodies, which were positive for periodic acid-Schiff and immunoreactive with antibodies against human neuroserpin, were present in the perikarya and cell processes of the neurons. They were found in large numbers in the cerebral cortex and substantia nigra and to a lesser extent, in most subcortical gray areas, spinal cord, and dorsal root ganglia. By electron microscopy, the intracytoplasmic bodies were contained within the membranes of the rough endoplasmic reticulum. Occasionally neuroserpin immunopositivity was seen throughout the cytoplasm, even without the presence of well-defined bodies. Our study characterizes for the first time the neuropathologic phenotype associated with hereditary progressive myoclonus epilepsy caused by the S52R Neuroserpin mutation.
Asunto(s)
Sistema Nervioso Central/metabolismo , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Mutación/fisiología , Neuronas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Serpinas/genética , Serpinas/metabolismo , Adulto , Sistema Nervioso Central/patología , Epilepsias Mioclónicas/patología , Femenino , Humanos , Masculino , Microscopía Electrónica , Linaje , NeuroserpinaRESUMEN
BACKGROUND: We recently discovered an autosomal dominant disease causing a progressive dementia. The disease is caused by a point mutation in the gene coding for the serine protease inhibitor (ie, serpin) neuroserpin. The mutation results in an unstable neuroserpin protein that readily aggregates into intraneuronal inclusions that we identify as Collins bodies. The bodies are distributed throughout the cerebral hemispheres but are significantly more numerous in the cortex and the substantia nigra. We have named the disease familial encephalopathy with neuroserpin inclusion bodies (FENIB). OBJECTIVES: To describe the cognitive and neurophysiological changes exhibited by individuals with FENIB and to correlate the phenotypic expression of the disease with the neuropathological findings. DESIGN: Multiple case studies using neuropsychological assessment, electroencephalography (EEG), magnetic resonance imaging (MRI), and single-photon emission computed tomographic (SPECT) studies of family members were performed. Using these measures, we also compared family members in whom the mutation is present with family members in whom the mutation was absent to control for nonspecific familial factors. SUBJECTS: Nine individuals (5 women, aged 31-64 years; 4 men, aged 43-67 years) from 2 generations of family members related to the first reliably identified individual with symptoms of this disease. Symptoms, by self-report and reports of other family members, ranged from asymptomatic to severe dementia. Six of the 9 individuals carried the disease mutation. RESULTS: All subjects with the mutation demonstrated some cognitive changes, with the greatest demonstrated by subjects older than 40 years. The changes included restricted attention, concentration, and response regulation functions, reduced controlled oral fluency (word-list generation), and restricted visuospatial organization. In general, recall memory was not as affected as other cognitive domains. The most severely affected subject demonstrated global dementia with prominent frontal lobe features. Findings on SPECT showed anomalies limited to frontal areas in the less affected subjects and more global, patchy areas of hypoperfusion in the more severely affected subjects. The 3 oldest and most affected subjects demonstrated slowing on EEG findings. The MRI findings were noncontributory except in the 2 most severe cases, which showed global cortical atrophy. CONCLUSIONS: Cognitive changes in mildly to moderately affected subjects were characterized by deficits in frontal and frontal-subcortical area-dependent processes. Continued progressive deterioration of cerebral functions with relative sparing of recall memory suggests a unique dementia associated with this disease.
Asunto(s)
Trastornos del Conocimiento/genética , Trastornos del Conocimiento/patología , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Neuropéptidos/genética , Serpinas/genética , Adulto , Trastornos del Conocimiento/diagnóstico por imagen , Electroencefalografía , Salud de la Familia , Femenino , Trastornos Heredodegenerativos del Sistema Nervioso/diagnóstico por imagen , Humanos , Cuerpos de Inclusión/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Mutación Puntual , Tomografía Computarizada de Emisión de Fotón Único , NeuroserpinaRESUMEN
Hypersensitivity reactions to trimethoprim/sulfamethoxazole occur with a high frequency in human immunodeficiency virus (HIV)-infected patients. This study tested whether differences in oxidative metabolism and plasma reductive capacity correlate with sulfonamide intolerance in patients with HIV. Eighteen stable outpatients with HIV were prospectively studied. Nine patients had documented histories of hypersensitivity reactions to trimethoprim/sulfamethoxazole and nine did not. Urinary caffeine metabolite ratios assessed the activity of two oxidative enzymatic pathways: cytochrome P-450 1A2 (demethylation) and 8-hydroxylation. Plasma cyst(e)ine was used as a measure of reductive capacity. The trimethoprim/sulfamethoxazole-intolerant group showed greater rates of 8-hydroxylation, lower rates of demethylation, and lower cyst(e)ine levels. The results of this pilot study extend previous observations of differences in oxidative metabolism and reductive capacity that exist within the population of HIV-infected individuals. In addition, these findings lay the groundwork for future interventional studies that could use agents to inhibit sulfonamide oxidation and increase reductive capacity in sulfonamide-intolerant patients with HIV when rechallenged with trimethoprim/sulfamethoxazole.
Asunto(s)
Antiinfecciosos/efectos adversos , Hipersensibilidad a las Drogas/metabolismo , Infecciones por VIH/metabolismo , Sulfonamidas/efectos adversos , Adulto , Cafeína/farmacocinética , Cafeína/orina , Cisteína/sangre , Femenino , Infecciones por VIH/fisiopatología , Humanos , Masculino , Oxidación-Reducción , Inhibidores de Fosfodiesterasa/farmacocinética , Inhibidores de Fosfodiesterasa/orina , Proyectos Piloto , Estudios Prospectivos , Combinación Trimetoprim y Sulfametoxazol/efectos adversosRESUMEN
Transport for organic cations has been described in both luminal and antiluminal membranes. The two transport systems difer from one another on the basis of kinetic parameters and countertransport. By using N1-[3H]methylnicotinamide as the indication cation, a series of organic cations (which were known to be secreted) were tested for their capacity to stimulate countertransport. The phenomenon of countertransport was symmetrical in that it was observed for both influx and efflux in both membranes. However, under appropriate conditions, certain organic cations were effective in producing "uphill" transport of N1-[3H]methylnicotinamide in the luminal membrane and the effect was limited to that membrane exclusively. Detailed analysis of countertransport in the luminal membrane showed quantitative differences in the maximal stimulation produced by the various cations: giving rise to the relationship that the concentration of organic cation which gave one-half maximal N1-[3H]methylnicotinamide countertransport approximated its affinity constant. The data were interpreted to suggest that all the members of the organic cation series tested are translocated across the luminal membrane by the same carrier but that they are moved at different rates.
Asunto(s)
Cationes/metabolismo , Colina/metabolismo , Corteza Renal/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Animales , Transporte Biológico , Transporte Biológico Activo , Membrana Celular/metabolismo , Perros , Corteza Renal/ultraestructuraRESUMEN
In the winter flounder, Pseudopleuronectes americanus, renal clearance experiments showed that the model organic cations, tetraethylammonium (TEA) and N'-methylnicotinamide (NMN), were strongly secreted; organic cation-to-polyethylene glycol (glomerular filtration rate marker) clearance ratios averaged 130 and 30, respectively. TEA uptake by isolated renal tubular masses was concentrative and saturable. Transport was inhibited by competitor organic cations and reduced by exposure to NaCN,2,4-dinitrophenol, ouabain, and HgCl2. Organic anions did not reduce TEA uptake. NMN was the poorest inhibitor of TEA uptake of all the organic cations tested. In addition, the rate of NMN uptake was slower than that of TEA, and the steady-state tissue-to-medium ratio was lower (5 for NMN vs. 10 for TEA; both at 25 microM). The data show the presence of an organic cation secretory system in flounder tissue that resembles the mammalian systems in several respects.
Asunto(s)
Cationes/metabolismo , Peces Planos/fisiología , Lenguado/fisiología , Riñón/metabolismo , 2,4-Dinitrofenol , Animales , Transporte Biológico Activo , Dinitrofenoles/farmacología , Tasa de Filtración Glomerular , Cloruro de Mercurio/farmacología , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Ouabaína/farmacología , Valores de Referencia , Cianuro de Sodio/farmacología , Compuestos de Tetraetilamonio/metabolismoRESUMEN
The active step in the renal secretion of the organic cation N1-methylnicotinamide has been proposed to be the translocation across the luminal membrane. The presence of a H+:N1-methylnicotinamide antiport system has been found in luminal membrane vesicles isolated from dog kidney cortex but not in the antiluminal membrane. The conclusion reached is that N1-methylnicotinamide secretion is "secondarily active" and is driven by a proton gradient (urine to cell). The ph gradient in turn is created by the Na+:H+ antiport which is present in the luminal membrane. Therefore, two functionally linked antiport systems supply the energy for the active secretion of cationic drugs.
Asunto(s)
Concentración de Iones de Hidrógeno , Corteza Renal/metabolismo , Niacinamida/análogos & derivados , Animales , Transporte Biológico , Cationes , Membrana Celular/metabolismo , Perros , Litio/metabolismo , Niacinamida/metabolismo , Sodio/metabolismoRESUMEN
The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.
Asunto(s)
Aldehídos/farmacología , Arginina , Butanonas/farmacología , Diacetil/farmacología , Dietil Pirocarbonato/farmacología , Formiatos/farmacología , Histidina , Corteza Renal/metabolismo , Microvellosidades/metabolismo , Fenilglioxal/farmacología , Animales , Ditiotreitol/farmacología , Perros , Hidroxilamina , Hidroxilaminas/farmacología , Cinética , Ácido p-Aminohipúrico/metabolismoRESUMEN
This study delineates the various operational modes catalyzed by the organic anion exchanger present in the canine renal brush-border membrane. The experiments examined the carrier-mediated effects of various organic and inorganic anions on the transport of either p-[3H]aminohippuric acid ([3H]PAH) or 36Cl-. [3H]PAH countertransport was significantly stimulated by PAH, urate, Cl-, Br-, HCO3-, and by a pH gradient. This pH stimulation remained in the absence of HCO3- (i.e., under N2), implying PAH-OH- exchange. Furosemide, bumetanide, penicillin, and probenecid inhibited countertransport of [3H]PAH. Likewise, the above anions produced cis inhibition of [3H]PAH transport. The cis and trans effects of SO4(-2) and formate were minimal. 36Cl- countertransport was stimulated by PAH, Cl-, Br-, HCO3-, formate, and by a pH gradient that was effective even in the absence of HCO3- (i.e., under N2), implying Cl- -OH- exchange. Cl- -OH- and Cl- -Cl- exchange was inhibited by PAH. In each instance, the trans-stimulation of 36Cl- efflux was insensitive to maneuvers that created an inside-positive membrane potential, demonstrating electroneutral mediated exchange. We conclude that the organic anion transporter can operate in three distinct exchange modes: organic-organic, organic-inorganic, and inorganic-inorganic.
Asunto(s)
Proteínas Portadoras/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Transporte Biológico Activo , Antiportadores de Cloruro-Bicarbonato , Perros , Canales Iónicos/metabolismo , Intercambio Iónico , Microvellosidades/metabolismo , Ácido p-Aminohipúrico/metabolismoRESUMEN
The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.
Asunto(s)
Cationes/metabolismo , Disulfuros/análisis , Corteza Renal/ultraestructura , Compuestos de Sulfhidrilo/análisis , Animales , Transporte Biológico Activo/efectos de los fármacos , Ditiotreitol/farmacología , Perros , Etilmaleimida/farmacología , Glucosa/metabolismo , Corteza Renal/metabolismo , Microvellosidades/análisis , Microvellosidades/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Concentración Osmolar , Fenciclidina/análogos & derivados , Fenciclidina/farmacología , Factores de TiempoRESUMEN
The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.
Asunto(s)
Bicarbonatos/metabolismo , Corteza Renal/ultraestructura , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Membrana Basal/metabolismo , Cloruros/farmacología , Furosemida/farmacología , Concentración de Iones de Hidrógeno , Hidróxidos , Radical Hidroxilo , Masculino , Ratas , Ratas Endogámicas , Sodio/metabolismoRESUMEN
The molecular mechanism of the electroneutral organic cation/H+ antiporter in renal brush border membrane vesicles was studied utilizing the prototypic organic cation N1-methylnicotinamide. The hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD), inactivated organic cation transport irreversibly with an IC50 of 2.6 microM at pH 7.5 and 40 nM at pH 6.0. On the other hand, the hydrophilic reagents, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, did not affect organic cation transport. Substrate did not affect the rate of the DCCD inactivation which followed pseudo-first-order-kinetics. A double logarithmic plot of the apparent rate constants vs. the DCCD concentration gave a straight line with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of DCCD inactivates one carboxylate group per active transport unit and that the carboxylate group is critical for transport.
Asunto(s)
Carbodiimidas/farmacología , Diciclohexilcarbodiimida/farmacología , Riñón/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Etildimetilaminopropil Carbodiimida/farmacología , Técnicas In Vitro , Riñón/metabolismo , Microvellosidades/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacología , Protones , Quinolinas/farmacología , Sodio/metabolismoRESUMEN
We examined the effects of the neurotoxins 1-methyl-4-phenyl-pyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the transport of the prototypic organic cation, N1-[3H]methylnicotinamide ([3H]NMN), in canine renal brush border membrane vesicles. A dose-response curve for MPTP and its oxidized metabolite, MPP+, revealed IC50 values of 160 and 16 microM, respectively. MPTP (5 mM) and MPP+ (5 mM), maximally inhibited H+-driven NMN (50 microM) uptake by 86.0 and 86.6%, respectively. Additionally, serotonin (0.5 mM) and harmaline (0.5 mM) inhibited NMN transport by 65.8 and 87.1%, respectively. Mepiperphenidol (Darstine) (0.5 mM), a classical organic cation competitor, inhibited NMN transport by 80.6%. However, dopamine (0.5 mM) was not as effective and resulted in only 24.9% inhibition. The cationic specificity was demonstrated by showing that MPP+ and MPTP had no effect on the transport of the organic anion, p-aminohippurate. Additionally, the effect of MPP+ on NMN was not due to either a voltage effect or vesicle disruption. In countertransport studies, MPTP acted as an elicitor of NMN counterflow and produced trans stimulation whereas MPP+ did not and resulted in trans inhibition. Inasmuch as transport involves both binding and translocation we speculate that MPTP binds and is translocated; in contrast MPP+ binds and is not translocated. These reagents may serve as a basis for elucidating the structure-activity requirements of the organic cation/H+ antiporter.
Asunto(s)
Cationes/metabolismo , Corteza Renal/metabolismo , Niacinamida/análogos & derivados , Piridinas/farmacología , Compuestos de Piridinio/farmacología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , 1-Metil-4-fenilpiridinio , Animales , Transporte Biológico/efectos de los fármacos , Perros , Microvellosidades/metabolismo , Neurotoxinas , Niacinamida/metabolismo , Piridinas/metabolismo , Compuestos de Piridinio/metabolismo , Relación Estructura-Actividad , Valinomicina/farmacología , Ácido p-Aminohipúrico/metabolismoRESUMEN
Organic cation (OC+)/H+ exchangers are found in several mammalian tissues and in numerous organisms. In the kidney OC+/H+ exchange activity is localized to the brush border membrane of the proximal tubule cells of the nephron and is believed to be responsible for the efflux of numerous xenobiotics from the tubule cell into the tubular fluid. The objective of the present study was to identify the OC+/H+ exchanger in brush border membrane vesicles isolated from dog kidney by photoaffinity labeling. The results show that [3H]azidopine is an ideal photoaffinity labeling reagent; in the dark it binds reversibly, but irreversibly after photoactivation. The photoaffinity labeling reaction is efficient, specific and sensitive. Our findings are consistent with the conclusions that a 41-kDa protein is the exchanger and that it is present at a concentration of 780 +/- 140 fmol/mg membrane protein (n = 4). A 49-kDa protein is labeled to some extent as well. The relationship between the 41- and 49-kDa proteins has not been resolved.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Renal/metabolismo , Microvellosidades/metabolismo , Naranja de Acridina/farmacología , Marcadores de Afinidad , Animales , Azidas/metabolismo , Unión Competitiva , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Dihidropiridinas/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Cinética , Sustancias Macromoleculares , Peso Molecular , Especificidad por SustratoRESUMEN
The active secretion of organic ions by the kidney may be described by the following models: 1)binding to a carrier protein and 2) a translocation process across the membrane. The feasibility of such a model was tested by measuring binding of either an organic cation, N1-methylnicotinamide (NMN) or an organic anion p-aminohippuric acid (PAH) to particulate material obtained from dog renal cortex tissue. The method employed was one in which the bound and free forms of the ligand were separated by centrifugation through a gel matrix. Binding of NMN and PAH was found to be tissue specific. In addition, binding was pH, time, temperature, protein-concentration and ligand-concentration dependent. Saturation of binding for either ligand was observed at concentrations greater than 50 mM, suggesting low affinity. Interestingly, a positive cooperative effect was observed for binding of either NMN or PAH to the particulate material. Although binding was associated only with particulate material, the binding proteins were released from the membrane system(s) by treatment with the nonionic detergent Lubrol WX. These studies show that NMN and PAH binding share many features in common but that the two processes are independent of each other. The results are consistent with, but do not prove, the model.
Asunto(s)
Ácidos Aminohipúricos/metabolismo , Riñón/metabolismo , Niacinamida/análogos & derivados , Animales , Unión Competitiva , Perros , Estabilidad de Medicamentos , Femenino , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Riñón/ultraestructura , Ligandos , Masculino , Niacinamida/metabolismo , Especificidad de Órganos , Concentración Osmolar , Presión Osmótica , Proteínas/metabolismo , Fracciones Subcelulares , Temperatura , Factores de TiempoRESUMEN
The effect of potential difference on the organic cation/H+ antiport system located in brush border membrane vesicles was examined. Potential difference was generated using K+ gradients and a K+-specific ionophore, valinomycin. Transport of a prototypic organic cation, 1N-[3H]methylnicotinamide (NMN), was assessed under conditions where K+ diffusion potentials generated transient intravesicular negative and positive states. The results demonstrate that NMN transport is independent of potential difference. The organic cation/H+ coupling ratio was studied by imposing transmembrane H+ and NMN gradients of varying magnitudes and measuring net flux of NMN for a 5-sec period. Consistent with an equilibrium thermodynamics model, no net NMN flux is observed when the NMN gradient from out to in is equal to the H+ gradient from out to in ([NMN]o/[NMN]i equals [H+]o/[H+]i). This suggests that the carrier is at equilibrium and that the stoichiometry for the organic cation/H+ antiport is 1:1.