RESUMEN
A multigenerational family complex with an admixture of essential tremor (ET) and PD is presented. Medical information obtained either by historic documentation and/or examination was available for five generations and included 36 members. Of these, 11 family members had tremor of the limbs and/or head. In all these instances ET made its first appearance at an early age, usually prior to the second decade of life. In one case focal dystonia of the hand, a possible prelude to PD occurred, while in three brothers of the third generation, two of them identical twins, classical Parkinson's disease (PD) developed. They had ET develop at an early age, which persisted and in their 50s began showing evidence of PD. Two decades later the twin brothers succumbed to cancer of the colon and at autopsy typical findings of PD with cell loss in the substantia nigra and Lewy-body formation positive for alpha-synuclein by immunohistochemistry was found. Additionally, more than the usual number of senile plaques and neurofibrillatory tangles were present without clinical evidence of dementia or significant decline in cognitive function. This unusual set of clinical and pathological circumstances can hardly be attributed to chance occurrence and raise the question of a specific genetic mutation and/or clustering, which may link ET with PD.
Asunto(s)
Temblor Esencial/complicaciones , Enfermedad de Parkinson/complicaciones , Adulto , Anciano , Autopsia , Ganglios Basales/patología , Encéfalo/patología , Temblor Esencial/patología , Familia , Femenino , Humanos , Hipertensión/complicaciones , Hipertensión/fisiopatología , Cuerpos de Lewy/patología , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/patología , Tamaño de los Órganos , Enfermedad de Parkinson/patología , Linaje , Placa Amiloide/patología , Sustancia Negra/patologíaRESUMEN
Intestinal epithelial cells derive from stem cells at the base of the crypt and migrate along the crypt-lumen axis. Their life is terminated as they reach the luminal surface where they detach and are shed. Intestinal epithelial cells show evidence of apoptosis in the region of shedding, and cell death is thought to resemble a form of apoptosis called detachment-induced cell death, or anoikis. Human intestinal epithelial cells die rapidly in vitro due to loss of anchorage during isolation, making primary culture of these cells a goal that has not yet been reached. However, the molecular mechanisms underlying this process of anoikis are largely unknown. In this study, a novel protocol for the rapid, temperature-controlled isolation of highly purified human colonic epithelial cells from surgical specimens is described. Using this method, early molecular events of anoikis in nontransformed epithelial cells were studied. Intestinal epithelial cells were isolated at the beginning of the apoptotic cascade, before the activation of caspase 3 family members and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Elucidating the molecular mechanisms of detachment-induced cell death may facilitate the establishment of long-term primary cultures of human intestinal epithelial cells and enhance our understanding of homeostasis in the intestinal epithelium.
Asunto(s)
Apoptosis , Caspasas , Separación Celular/métodos , Neoplasias del Colon/patología , Mucosa Intestinal/patología , Western Blotting , Caspasa 3 , Adhesión Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/ultraestructura , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Inmunohistoquímica , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/ultraestructura , Microscopía Electrónica , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/metabolismo , Coloración y EtiquetadoRESUMEN
Epithelial cell lines from the proximal tubule of SHR and WKY rats were generated by microdissection, cell growth on 3T3 cell feeder layers, and transduction of the SV40 large T-antigen gene. The cell lines that formed confluent, electrically-resistive monolayers (basal conductance 1 to 20 mS/cm2) were selected for further study. Of these, cell lines generated from one rat did not show evidence of T-antigen expression or integration, and apparently immortalized spontaneously. Cell lines from three other rats expressed high levels of T-antigen, and showed evidence of integration of one or more copies of T-antigen. All cell lines formed polarized monolayers with apical microvilli, tight junctional complexes, and convolutions of the basolateral plasma membrane. Most cell lines grew in the absence of extracellular glucose indicating a capacity for gluconeogenesis. Sodium succinate cotransport and P2-purinergic receptor mediated signaling were demonstrated in all lines tested. The cell lines also showed that Na/H exchanger activity is regulated by angiotensin II. The results indicate that these cell lines express a proximal tubular phenotype, and are morphologically and functionally similar to primary cultures. These rat cell lines represent a new, potentially useful cell model for elucidating the cellular and molecular mechanisms of genetic differences in proximal tubule Na+ reabsorption.
Asunto(s)
Hipertensión/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Células 3T3 , Angiotensina II/farmacología , Animales , Línea Celular , Transporte Iónico , Masculino , Ratones , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Transducción de Señal , Sodio/metabolismoRESUMEN
Recent reports indicate that alpha 1-Na,K-ATPase from Dahl salt-sensitive (DS) rats contains a glutamine for leucine substitution associated with increased Na-K coupling at unchanged maximal velocity. Genetic analyses suggest that alpha 1-Na,K-ATPase is a potential hypertension gene. Therefore, we investigated whether renal Na+ metabolism could constitute a pathophysiological link between the molecular/functional change in Na,K-ATPase and hypertension. We simulated the consequences of increased Na-K coupling on overall Na-bicarbonate reabsorption in a proximal tubular transport model that incorporates apical Na-H exchanger and basolateral Na-bicarbonate cotransporter, K+ channel, and Na,K-ATPase. As expected, increases in the levels of the former three transport pathways yielded higher Na+ reabsorption. In contrast, increases in the maximal velocity of the Na,K-ATPase with a normal 3:2 (Na-K) coupling ratio did not increase Na+ reabsorption when apical Na-H exchange activity was limiting overall absorption. However, an increase in the Na-K coupling from 3:2 to 3:1, reported for the mutant alpha 1-Na,K-ATPase in DS rats, was associated with greater Na+ reabsorption. This increase is a consequence of lower cytosolic pH and secondary stimulation of the Na-H exchanger at its allosteric H+ site. Decreased pH results from activation of Na-bicarbonate cotransport by Na,K-ATPase-dependent membrane hyperpolarization due to greater charge movement in 3:1 Na-K coupling. Thus, an increase in the Na-K coupling ratio results in an altered set point for cellular Na+ metabolism, with higher sodium reabsorption at unchanged Na,K-ATPase levels. The simulations thereby lend support for a unifying explanation for the salt sensitivity of DS rats, which has been proposed to stem from a mutation in the alpha 1-Na,K-ATPase.
Asunto(s)
Hipertensión/fisiopatología , Túbulos Renales Proximales/fisiopatología , Riñón/fisiopatología , Modelos Cardiovasculares , Sodio/metabolismo , Animales , Bicarbonatos/metabolismo , Proteínas Portadoras/metabolismo , Humanos , Hipertensión/enzimología , Hipertensión/genética , Riñón/metabolismo , Riñón/fisiología , Túbulos Renales Proximales/fisiología , Cinética , Matemática , Ratas , Ratas Mutantes , Simportadores de Sodio-Bicarbonato , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to measure H+ and anion transport in vesicles reconstituted with purified uncoupling protein and carried out a comparative study of the effects of laurate and its close analogue, undecanesulfonate. Undecanesulfonate was transported by uncoupling protein with a Km value similar to that observed for laurate as it activated H+ transport. Both laurate and undecanesulfonate inhibited Cl- with competitive kinetics. Undecanesulfonate inhibited laurate-induced H+ transport with competitive kinetics. Undecanesulfonate and laurate differed in two important respects. (i) Laurate caused uncoupling protein-mediated H+ transport, whereas undecanesulfonate did not. (ii) Lauric acid was rapidly transported across the bilayer by nonionic diffusion, whereas undecanesulfonic was not. We infer that the role of uncoupling protein in H+ transport is to transport fatty acid anions and that fatty acids induce H+ transport because they can diffuse electroneutrally across the membrane. According to this hypothesis, uncoupling protein is a pure anion porter and does not transport protons; rather it is designed to enable fatty acids to behave as cycling protonophores.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Protones , Desacopladores/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Cloruros/metabolismo , Cricetinae , Colorantes Fluorescentes , Canales Iónicos , Transporte Iónico , Mesocricetus , Proteínas Mitocondriales , Proteolípidos , Proteína Desacopladora 1RESUMEN
The uncoupling protein generates heat by catalyzing electrophoretic proton transport across the inner membrane of brown adipose tissue mitochondria. It also transports Cl- and other monovalent anions, and both proton and anion transport are inhibited by purine nucleotides. Several long-standing hypotheses bear on specific aspects of Cl- transport, H+ transport, and nucleotide gating mechanisms in uncoupling protein. We reevaluated these hypotheses in mitochondria and liposomes reconstituted with purified uncoupling protein; GDP inhibition is strictly noncompetitive with Cl- and unaffected by either transmembrane electrical potential or fatty acids. The Km and Vmax values for Cl- are independent of pH, arguing against a common binding site for Cl- and OH- ions. Cl- transport was inhibited by fatty acids and stimulated by fatty acid removal, refuting the consensus hypothesis that there is no interaction between fatty acids and anion transport through uncoupling protein. These results support a mechanism in which the transport pathway for anions is identical with the fatty acid binding site and distinct from the nucleotide binding site.
Asunto(s)
Proteínas Portadoras/farmacología , Cloruros/metabolismo , Ácidos Grasos/farmacología , Guanosina Difosfato/farmacología , Proteínas de la Membrana/farmacología , Mitocondrias/metabolismo , Desacopladores/farmacología , Tejido Adiposo Pardo/metabolismo , Animales , Transporte Biológico , Cricetinae , Concentración de Iones de Hidrógeno , Canales Iónicos , Mesocricetus , Proteínas Mitocondriales , Albúmina Sérica Bovina/farmacología , Proteína Desacopladora 1RESUMEN
6-Methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) is a fluorophore that is collisionally quenched by halide anions and is widely used to measure chloride ion transport across cellular and liposomal membranes. We report a new finding that SPQ fluorescence is also quenched by the zwitterionic hydrogen ion buffers introduced by Good et al. [(1966) Biochemistry 5, 467-477]. Although buffer quenching interferes with chloride ion measurements using SPQ, it can be turned to good advantage for measurements of proton flux. The basis for this application is that, for most buffers, the anion quenches and the zwitterion does not. Accordingly, buffer quenching of SPQ can be used to assay proton transport across liposomal membranes. We describe application of the technique to liposomes in which proton transport was mediated by ionophores and by the purified, reconstituted uncoupling protein of brown adipose tissue mitochondria. Because SPQ detects changes in buffer anion concentration, it can be used to measure changes in total acidity, which is the parameter desired when measuring net proton transport. Furthermore, this technique can be used to measure proton transport under conditions in which pH changes are minimized with buffers, and, consequently, effects of pH on proton transport can be dissociated from the transport itself.
Asunto(s)
Liposomas , Protones , Espectrometría de Fluorescencia/métodos , Aniones , Tampones (Química) , Proteínas Portadoras/metabolismo , Cationes , Estudios de Evaluación como Asunto , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Canales Iónicos , Transporte Iónico , Ionóforos , Cinética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Concentración Osmolar , Compuestos de Quinolinio , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/estadística & datos numéricos , Temperatura , Proteína Desacopladora 1RESUMEN
L-DOPA is frequently used to relieve symptoms of Parkinson's disease (PD), but its use in patients with more advanced PD is complicated by on-off phenomena. We used simultaneous microdialysis of striatum and ipsilateral substantia nigra to characterize changes in extracellular fluid (ecf) levels of dopamine (DA) following systemic treatment with L-DOPA (25 mg/kg as methylester) in awake, normal rats and those with partial (less than 99%) or complete (greater than 99%) DA depleting unilateral lesions of the nigrostriatal pathway (nsp). In normal rats, nigral ecf DA rose 17-fold above baseline after L-DOPA, compared to a 2.6-fold increase in normal striata. Striatal ecf DA rose equally after L-DOPA in all three groups, whereas peak nigral ecf DA in completely lesioned rats was three times that in normal or partially lesioned animals. Peak nigral ecf DA in completely lesioned rats exceeded striatal ecf DA in all groups by almost 2-fold. Activity after L-DOPA was biphasic ("hyperkinetic/bradykinetic") in completely lesioned but not in normal or partially lesioned animals, and the reduced activity occurred 2.5-4 h after L-DOPA at a time when both nigral and striatal ecf DA levels were still elevated. L-DOPA-induced increases in activity were predictable by greater elevations in nigral compared to striatal ecf DA in animals with complete lesions of the nigrostriatal pathway. Post-DOPA reduced activity might result from desensitization of synaptic events mediated by DA receptors; this may underlie DOPA-related on-off phenomena in patients with advanced PD.
Asunto(s)
Cuerpo Estriado/metabolismo , Levodopa/metabolismo , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Animales , Diálisis , Dopamina/metabolismo , Espacio Extracelular/metabolismo , Masculino , Ratas , Ratas EndogámicasRESUMEN
Small mammals, including human infants, rely on nonshivering thermogenesis for a substantial portion of their body heat during exposure to cold. This thermogenesis is mediated in large part by the uncoupling protein, which is found exclusively within the inner membrane of brown adipose tissue mitochondria. The sole function of uncoupling protein is to provide a regulated transport pathway for electrophoretic back-flux of H+ ions into the mitochondrial matrix, thereby dissipating the protonmotive force and producing heat. Thus, uncoupling protein is unique with respect to both its physiological role and its tissue expression. We have now achieved high level expression of rat uncoupling protein in yeast, with import into yeast mitochondria at levels, 70-100 micrograms/mg of mitochondrial protein, similar to those observed in brown adipose tissue mitochondria from cold-adapted rats. When the expressed protein was purified and reconstituted into liposomes, the proteoliposomes exhibited GDP-sensitive proton and chloride uniports that were inhibited by GDP with Ki values similar to those obtained with native protein. Moreover, the molecular activities of the expressed protein with respect to Cl- and H+ transport were indistinguishable from those of native protein. The availability of unlimited amounts of functional, expressed uncoupling protein will now permit application of site-directed mutagenesis to the many intriguing aspects of uncoupling protein structure and function.
Asunto(s)
Proteínas Portadoras , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/genética , Saccharomyces cerevisiae/genética , Tejido Adiposo Pardo/metabolismo , Animales , Secuencia de Bases , Transporte Biológico , Western Blotting , Cloruros/metabolismo , Electroforesis en Gel de Poliacrilamida , Genes Fúngicos , Guanosina Difosfato/farmacología , Hidrógeno/metabolismo , Canales Iónicos , Proteínas Mitocondriales , Datos de Secuencia Molecular , Ácido Oléico , Ácidos Oléicos/farmacología , Fosforilación Oxidativa , Oxígeno/metabolismo , Ratas , Saccharomyces cerevisiae/metabolismo , Proteína Desacopladora 1RESUMEN
The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Bromuros/metabolismo , Proteínas Portadoras , Cloruros/metabolismo , Guanosina Difosfato/farmacología , Yoduros/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Aniones , Cricetinae , Colorantes Fluorescentes , Canales Iónicos , Cinética , Liposomas/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Mesocricetus , Proteínas Mitocondriales , Peso Molecular , Proteolípidos/metabolismo , Compuestos de Quinolinio , Proteína Desacopladora 1RESUMEN
Parkinson's disease arises from extensive premature loss of dopamine (DA)-utilizing midbrain neurons whose axons form the nigrostriatal pathway and densely innervate the striatum (caudate-putamen). The clinical symptoms of Parkinson's disease are improved by treatment with DA receptor agonists, including apomorphine. DA agonists modulate basal ganglia outflow at multiple sites and may affect synaptic activity of multiple basal ganglia neurotransmitters, including substance-P peptide (SP). With microdialysis in the substantia nigra reticulata (s.n.r.), a major striatal projection area, we monitored the extracellular level of SP-like immunoreactivity (SP-LI) in awake rats with neurotoxin-induced destruction of nigral DA neurons, before and after treatment with apomorphine (1 mg/kg i.p.). Loss of DA neurons produced a 60% increase in baseline extracellular SP-LI levels. Apomorphine treatment increased baseline extracellular SP-LI levels up to 60% in control rats and 100% in rats with loss of nigral DA neurons. Striatonigral SP endings increase SP release in response to loss of nigral target DA neurons, and DA agonist acting on supersensitive DA receptors, perhaps on SP terminals in the nigra, further increases synaptic SP concentrations.
Asunto(s)
Apomorfina/farmacología , Enfermedad de Parkinson Secundaria/metabolismo , Sustancia P/metabolismo , Sustancia Negra/metabolismo , Animales , Diálisis , Modelos Animales de Enfermedad , Dopamina/metabolismo , Ganglios Simpáticos/efectos de los fármacos , Hidroxidopaminas , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidopamina , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Endogámicas , Sustancia Negra/efectos de los fármacosRESUMEN
Decarboxylation of an unnatural analogue of DOPA by brain aromatic amino acid decarboxylase was measured by HPLC with electrochemical detection. Incubation of striatal slices with DL-2,4-dihydroxyphenylalanine (2,4-DOPA) resulted in biosynthesis and accumulation of 2,4-dihydroxyphenylethylamine, which was completely blocked by carbidopa. 2,4-DOPA induced a significant decrease in endogenous dopamine, but the loss was not prevented by carbidopa. Since 2,4-DOPA is a potential pro-drug for melanoma chemotherapy, further evaluation of its neurobiologic properties is needed.