RESUMEN
The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon, a mechanism critical to its role in maintaining overall K+ balance. Previous studies demonstrated that basolateral NKCC1 was up-regulated by aldosterone in the distal colon specifically to support K+ secretion-which is distinct from the more well-established role of NKCC1 in supporting luminal Cl- secretion. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by aldosterone. Furthermore, delineating such region-specific effects has important implications for the management of various gastrointestinal pathologies. Experiments were therefore designed to determine whether aldosterone similarly up-regulates NKCC1 in the proximal colon to support K+ secretion. Using dietary Na+ depletion as a model of secondary hyperaldosteronism in rats, we found that proximal colon NKCC1 expression was indeed enhanced in Na+-depleted (i.e., hyperaldosteronemic) rats. Surprisingly, electrogenic K+ secretion was not detectable by short-circuit current (ISC) measurements in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non-selective K+ channel blocker), despite enhanced K+ secretion in Na+-depleted rats, as measured by 86Rb+ fluxes. Expression of BK and IK channels was also found to be unaltered by dietary Na+ depletion. However, bumetanide-sensitive basal and agonist-stimulated Cl- secretion (ISC) were significantly enhanced by Na+ depletion, as was CFTR Cl- channel expression. These data suggest that NKCC1-dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon. Development of therapeutic strategies in treating pathologies related to aberrant colonic K+/Cl- transport-such as pseudo-obstruction or ulcerative colitis-may benefit from these findings.
Asunto(s)
Aldosterona , Bumetanida , Animales , Ratas , Aldosterona/farmacología , Aldosterona/metabolismo , Bumetanida/farmacología , Bumetanida/metabolismo , Cloruros/metabolismo , Colon , Potasio/metabolismo , Sodio/metabolismoRESUMEN
Previous studies have confirmed the relationship between chloride homeostasis and pain. However, the role of sodium potassium chloride co-transporter isoform 1 (NKCC1) in dorsal horn and dorsal root ganglion neurons (DRGs) in spinal cord injury (SCI)-induced neuropathic pain (NP) remains inconclusive. Therefore, we aimed to explore whether suppression of NKCC1 in the spinal cord and DRGs alleviate the NP of adult rats with thoracic spinal cord contusion. Thirty adult female Sprague-Dawley rats (8 week-old, weighing 250-280 g) were randomly divided into three groups with ten animals in each group (sham, SCI, and bumetanide groups). The paw withdrawal mechanical threshold and paw withdrawal thermal latency were recorded before injury (baseline) and on post-injury days 14, 21, 28, and 35. At the end of experiment, western blotting (WB) analysis, quantitative real-time Polymerase Chain Reaction (PCR) and immunofluorescence were performed to quantify NKCC1 expression. Our results revealed that NKCC1 protein expression in the spinal cord and DRGs was significantly up-regulated in rats with SCI. Intraperitoneal treatment of bumetanide (an NKCC1 inhibitor) reversed the expression of NKCC1 in the dorsal horn and DRGs and ameliorated mechanical ectopic pain and thermal hypersensitivities in the SCI rats. Our study demonstrated the occurrence of NKCC1 overexpression in the spinal cord and DRGs in a rodent model of NP and indicated that changes in the peripheral nervous system also play a major role in promoting pain sensitization after SCI.
Asunto(s)
Neuralgia , Traumatismos de la Médula Espinal , Ratas , Femenino , Animales , Ratas Sprague-Dawley , Bumetanida/metabolismo , Bumetanida/farmacología , Ganglios Espinales/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Neuralgia/metabolismo , Médula Espinal/metabolismo , Hiperalgesia/metabolismoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation-chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl-]i by accumulating and extruding Cl-, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aß1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aß1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aß1-42, but NKCC1 expression increased 30-days post-Aß1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aß1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aß1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aß1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aß1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.
Asunto(s)
Bumetanida , Hipocampo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Simportadores , Péptidos beta-Amiloides , Animales , Bumetanida/metabolismo , Bumetanida/farmacología , Cloruros/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/metabolismoRESUMEN
OBJECTIVES: The loop diuretic bumetanide has been proposed previously as an adjunct treatment for neonatal seizures because bumetanide is thought to potentiate the action of γ-aminobutyric acid (GABA)ergic drugs such as phenobarbital by preventing abnormal intracellular accumulation of chloride and the subsequent "GABA shift." However, a clinical trial in neonates failed to demonstrate such a synergistic effect of bumetanide, most likely because this drug only poorly penetrates into the brain. This prompted us to develop lipophilic prodrugs of bumetanide, such as the N,N-dimethylaminoethyl ester of bumetanide (DIMAEB), which rapidly enter the brain where they are hydrolyzed by esterases to the parent compound, as demonstrated previously by us in adult rodents. However, it is not known whether esterase activity in neonates is sufficient to hydrolyze ester prodrugs such as DIMAEB. METHODS: In the present study, we examined whether esterases in neonatal serum of healthy term infants are capable of hydrolyzing DIMAEB to bumetanide and whether this activity is different from the serum of adults. Furthermore, to extrapolate the findings to brain tissue, we performed experiments with brain tissue and serum of neonatal and adult rats. RESULTS: Serum from 1- to 2-day-old infants was capable of hydrolyzing DIMAEB to bumetanide at a rate similar to that of serum from adult individuals. Similarly, serum and brain tissue of neonatal rats rapidly hydrolyzed DIMAEB to bumetanide. SIGNIFICANCE: These data provide a prerequisite for further evaluating the potential of bumetanide prodrugs as add-on therapy to phenobarbital and other antiseizure drugs as a new strategy for improving pharmacotherapy of neonatal seizures.
Asunto(s)
Encéfalo/enzimología , Bumetanida/metabolismo , Esterasas , Ésteres/metabolismo , Profármacos/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Femenino , Humanos , Recién Nacido , Masculino , Ratas , Suero/enzimología , Suero/metabolismoRESUMEN
Monocarboxylate transporter 6 (MCT6; SLC16A5) has been recognized for its role as a xenobiotic transporter, with characterized substrates probenecid, bumetanide, and nateglinide. To date, the impact of commonly ingested dietary compounds on MCT6 function has not been investigated, and therefore, the objective of this study was to evaluate a variety of flavonoids for their potential MCT6-specific interactions. Flavonoids are a large group of polyphenolic phytochemicals found in commonly consumed plant-based products that have been recognized for their dietary health benefits. The uptake of bumetanide in human MCT6 gene-transfected Xenopus laevis oocytes was significantly decreased in the presence of a variety of flavonoids (e.g., quercetin, luteolin, phloretin, and morin), but was not significantly affected by flavonoid glycosides (e.g., naringin, rutin, phlorizin). The IC50 values of quercetin, phloretin, and morin were determined to be 25.3 ± 3.36, 17.3 ± 2.37, and 33.1 ± 3.29 µM, respectively. The mechanism of inhibition of phloretin was reversible and competitive, with a Ki value of 22.8 µM. Furthermore, typical MCT substrates were also investigated for their potential interactions with MCT6. Substrates of MCTs 1, 2, 4, 8, and 10 did not cause any significant decrease in MCT6-mediated bumetanide uptake, suggesting that MCT6 has distinct compound selectivity. In summary, these results suggest that dietary aglycon flavonoids may significantly alter the pharmacokinetics and pharmacodynamics of bumetanide and other MCT6-specific substrates, and may represent potential substrates for MCT6.
Asunto(s)
Flavonoides/metabolismo , Luteolina/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Floretina/metabolismo , Quercetina/metabolismo , Animales , Bumetanida/metabolismo , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oocitos/metabolismo , Xenopus laevisRESUMEN
Endolymph is the specialised extracellular fluid present inside the inner ear. In mammals, disruptions to endolymph homeostasis can result in either collapse or distension of the endolymphatic compartment in the cochlea, with concomitant hearing loss. The zebrafish little ears (lte) mutant shows a collapse of the otic vesicle in the larva, apparently owing to a loss of endolymphatic fluid in the ear, together with an over-inflation of the swim bladder. Mutant larvae display signs of abnormal vestibular function by circling and swimming upside down. The two available alleles of lte are homozygous lethal: mutant larvae fail to thrive beyond 6 days post-fertilisation. Patterning of the otic vesicle is apparently normal. However, the expression of several genes thought to play a role in endolymph production is downregulated, including the sodium-potassium-chloride cotransporter gene nkcc1 (slc12a2) and several Na(+)/K(+)-ATPase channel subunit genes. We show here that lte mutations correspond to lesions in nkcc1. Each allele has a point mutation that disrupts splicing, leading to frame shifts in the coding region that predict the generation of truncated products. Endolymph collapse in the lte/nkcc1 mutant shows distinct parallels to that seen in mouse Nkcc1 mutants, validating zebrafish as a model for the study of endolymph disorders. The collapse in ear volume can be ameliorated in the to27d allele of lte by injection of a morpholino that blocks splicing at an ectopic site introduced by the mutation. This exemplifies the use of morpholinos as potential therapeutic agents for genetic disease.
Asunto(s)
Sacos Aéreos/metabolismo , Oído Interno/metabolismo , Endolinfa/metabolismo , Isoformas de Proteínas/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra , Sacos Aéreos/anatomía & histología , Empalme Alternativo , Animales , Secuencia de Bases , Tipificación del Cuerpo/fisiología , Bumetanida/metabolismo , Oído Interno/anatomía & histología , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Furosemida/metabolismo , Regulación del Desarrollo de la Expresión Génica , Homeostasis , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Isoformas de Proteínas/genética , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Miembro 2 de la Familia de Transportadores de Soluto 12 , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The intracellular ion homeostasis in cockroach salivary acinar cells during salivation is not satisfactorily understood. This is mainly due to technical problems regarding strong tissue autofluorescence and ineffective ion concentration quantification. For minimizing these problems, we describe the successful application of two-photon (2P) microscopy partly in combination with fluorescence lifetime imaging microscopy (FLIM) to record intracellular Na(+) and Cl(-) concentrations ([Na(+)](i), [Cl(-)](i)) in cockroach salivary acinar cells. Quantitative 2P-FLIM Cl(-) measurements with the dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide indicate that the resting [Cl(-)](i) is 1.6 times above the Cl(-) electrochemical equilibrium but is not influenced by pharmacological inhibition of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and anion exchanger using bumetanide and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid disodium salt. In contrast, rapid Cl(-) reuptake after extracellular Cl(-) removal is almost totally NKCC mediated both in the absence and presence of dopamine. However, in physiological saline [Cl(-)](i) does not change during dopamine stimulation although dopamine stimulates fluid secretion in these glands. On the other hand, dopamine causes a decrease in the sodium-binding benzofuran isophthalate tetra-ammonium salt (SBFI) fluorescence and an increase in the Sodium Green fluorescence after 2P excitation. This opposite behavior of both dyes suggests a dopamine-induced [Na(+)](i) rise in the acinar cells, which is supported by the determined 2P-action cross sections of SBFI. The [Na(+)](i) rise is Cl(-) dependent and inhibited by bumetanide. The Ca(2+)-ionophore ionomycin also causes a bumetanide-sensitive [Na(+)](i) rise. We propose that a Ca(2+)-mediated NKCC activity in acinar peripheral cells attributable to dopamine stimulation serves for basolateral Na(+) uptake during saliva secretion and that the concomitantly transported Cl(-) is recycled back to the bath.
Asunto(s)
Cloruros/metabolismo , Cucarachas/citología , Cucarachas/metabolismo , Microscopía Fluorescente/métodos , Sodio/metabolismo , Animales , Bumetanida/metabolismo , Dopamina/metabolismo , Fluorescencia , Colorantes Fluorescentes/metabolismo , Compuestos de Quinolinio/metabolismo , Glándulas Salivales/citología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismoRESUMEN
The serous acini of airway submucosal glands are important for fluid secretion in the lung. Serous cells are also sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. However, the mechanisms of serous cell fluid secretion remain poorly defined. In this study, serous acinar cells were isolated from porcine bronchi and studied using optical techniques previously used to examine fluid secretion in rat parotid and murine nasal acinar cells. When stimulated with the cholinergic agonist carbachol, porcine serous cells shrank by approximately 20% (observed via DIC microscopy) after a profound elevation of intracellular [Ca(2+)] ([Ca(2+)](i); measured by simultaneous fura 2 fluorescence imaging). Upon removal of agonist and relaxation of [Ca(2+)](i) to resting levels, cells swelled back to resting volume. Similar results were observed during stimulation with histamine and ATP, and elevation of [Ca(2+)](i) was found to be necessary and sufficient to activate shrinkage. Cell volume changes were associated with changes in [Cl(-)](i) (measured using SPQ fluorescence), suggesting that shrinkage and swelling are caused by loss and gain of intracellular solute content, respectively, likely reflecting changes in the secretory state of the cells. Shrinkage was inhibited by niflumic acid but not by GlyH-101, suggesting Ca(2+)-activated secretion is mediated by alternative non-CFTR Cl(-) channels, possibly including Ano1 (TMEM16A), expressed on the apical membrane of porcine serous cells. Optimal cell swelling/solute uptake required activity of the Na(+)K(+)2Cl(-) cotransporter and Na(+)/H(+) exchanger, both of which are expressed on the basolateral membrane of serous acini and likely contribute to sustaining transepithelial secretion.
Asunto(s)
Líquidos Corporales/metabolismo , Bronquios/citología , Calcio/metabolismo , Glándulas Exocrinas/metabolismo , Animales , Anoctamina-1 , Bronquios/metabolismo , Bumetanida/metabolismo , Carbacol/metabolismo , Tamaño de la Célula , Células Cultivadas , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Agonistas Colinérgicos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Ácido Niflúmico/metabolismo , Ratas , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , PorcinosRESUMEN
Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl(-) through a bumetanide- and Na(+)-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl(-) during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl(-) concentration (i.e., [Cl(-)](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na(+) concentration. In contrast, the recovery on depletion (i.e., [Cl(-)](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na(+). The experimental observations could be reproduced by a mathematical model for intracellular Cl(-) kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl(-)-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl(-)](i), whereas lower NKCC1 activity in MNs allows large [Cl(-)](i) variations during neuronal activity.
Asunto(s)
Cloruros/metabolismo , Ganglios Espinales/metabolismo , Neuronas Motoras/metabolismo , Receptores de GABA-A/metabolismo , Simportadores de Cloruro de Sodio-Potasio/fisiología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bumetanida/metabolismo , Bumetanida/farmacología , Células Cultivadas , Electrofisiología , Embrión de Mamíferos , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/embriología , Gramicidina/metabolismo , Gramicidina/farmacología , Inmunohistoquímica , Cinética , Modelos Estadísticos , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas , Receptores de GABA/metabolismo , Receptores de GABA-A/fisiología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.
Asunto(s)
Aniones/metabolismo , Antiportadores/genética , Antiportadores/metabolismo , Encéfalo/metabolismo , Sinaptosomas/metabolismo , Secuencia de Aminoácidos , Animales , Arilsulfonatos/metabolismo , Bumetanida/metabolismo , Calcio/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/metabolismo , Colorantes Fluorescentes/metabolismo , Gluconatos/metabolismo , Concentración de Iones de Hidrógeno , Ionóforos/metabolismo , Ácido Niflúmico/metabolismo , Potasio/metabolismo , Ratas , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/metabolismo , Sulfatos/metabolismoRESUMEN
To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.
Asunto(s)
Proteínas Portadoras/metabolismo , Enfermedades Renales/metabolismo , Médula Renal/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Obstrucción Ureteral/metabolismo , Animales , Transporte Biológico Activo , Bumetanida/metabolismo , Membrana Celular/enzimología , Furosemida/farmacología , Enfermedades Renales/etiología , Cinética , NAD/metabolismo , Ouabaína/farmacología , Consumo de Oxígeno/efectos de los fármacos , Conejos , Valores de Referencia , Simportadores de Cloruro de Sodio-PotasioRESUMEN
Recently, it has been suggested that bumetanide, an inhibitor of the Na-K-2Cl co-transporter (NKCC1), may be useful in the treatment of central nervous system (CNS) disorders. However, from a physicochemical perspective, bumetanide may not cross the blood-brain barrier to the extent that is necessary for it to be an effective brain NKCC1 inhibitor in vivo. High plasma-protein binding, potentially high brain-tissue binding and putative efflux transporters including organic anion transporter 3 (OAT3) contribute to the poor pharmacokinetic profile of bumetanide. Bidirectional permeability assays are an in vitro method to determine the impact of plasma-protein/brain tissue binding, as well as efflux transport, on the permeability of a compound. We established and validated a cell line stably overexpressing human OAT3 using lentiviral cloning techniques for use in in vitro bidirectional permeability assays. Using efflux transport studies, we show that bumetanide is a transported substrate of human OAT3, exhibiting a transport ratio of ≥1.5, which is attenuated by OAT3 inhibitors. Bidirectional permeability assays were carried out in the presence and absence of either albumin or brain homogenate to elucidate the effect of plasma-protein/brain tissue binding. These tests confirmed the pharmacokinetic limitations for brain delivery of bumetanide. In this experiment, bumetanide is 53% bound to albumin, 77% bound to brain tissue and accumulates in brain cells. Moreover, we conclusively established that bumetanide is a transported substrate of OAT3. Taken together, these bidirectional permeability studies highlight the potential of efflux transporter inhibition as an augmentation strategy for enhanced delivery of bumetanide to the CNS.
Asunto(s)
Bumetanida/metabolismo , Bumetanida/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Barrera Hematoencefálica/metabolismo , Bumetanida/farmacocinética , Células HEK293 , Humanos , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , PermeabilidadRESUMEN
Population-based studies have demonstrated that children with a history of febrile seizure (FS) perform better than age-matched controls at hippocampus-dependent memory tasks. Here, we report that FSs induce two distinct structural reorganizations in the hippocampus and bidirectionally modify future learning abilities in an age-dependent manner. Compared with age-matched controls, adult mice that had experienced experimental FSs induced by hyperthermia (HT) on postnatal day 14 (P14-HT) performed better in a cognitive task that requires dentate granule cells (DGCs). The enhanced memory performance correlated with an FS-induced persistent increase in the density of large mossy fiber terminals (LMTs) of the DGCs. The memory enhancement was not observed in mice that had experienced HT-induced seizures at P11 which exhibited abnormally located DGCs in addition to the increased LMT density. The ectopic DGCs of the P11-HT mice were abolished by the diuretic bumetanide, and this pharmacological treatment unveiled the masked memory enhancement. Thus, this work provides a novel basis for age-dependent structural plasticity in which FSs influence future brain function.
Asunto(s)
Fiebre/complicaciones , Memoria/fisiología , Neurogénesis/fisiología , Plasticidad Neuronal/fisiología , Convulsiones/fisiopatología , Envejecimiento , Animales , Bumetanida/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/fisiopatología , Ratones , Convulsiones/etiología , Convulsiones/metabolismoRESUMEN
We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and [3H]BSTBA binds to intact duck red cells with a high affinity similar to that of [3H]bumetanide (K 1/2 congruent to 0.1 microM). We incubated duck red cells with [3H]BSTBA, then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0 degree C to prevent dissociation of the [3H]BSTBA. The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H2O2-digested gel slices revealed [3H]BSTBA to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by [3H]BSTBA in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1) Photolabelling of this protein by [3H]BSTBA is blocked when 10 microM unlabelled bumetanide is included in the initial incubation medium with [3H]BSTBA; (2) Photoincorporation of [3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (0.06 microM) similar to that for the reversible, saturable binding of [3H]BSTBA and [3H]bumetanide to duck red cells; and (4) [3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [3H]bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl- in the medium containing the radiolabelled diuretic.
Asunto(s)
Marcadores de Afinidad/análisis , Benzofenonas/análisis , Proteínas Portadoras/análisis , Membrana Eritrocítica/análisis , Animales , Benzofenonas/farmacología , Bumetanida/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Cloruros/metabolismo , Perros , Patos/sangre , Riñón/análisis , Potasio/metabolismo , Sodio/metabolismo , Simportadores de Cloruro de Sodio-Potasio , Sulfanilamidas/análisis , Sulfanilamidas/farmacologíaRESUMEN
Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 microM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic abd interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 microM, DTONB 141 microM) and for cholate uptake (DTSNB 250 microM, DTONB 115 microM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Bumetanida/metabolismo , Hígado/efectos de los fármacos , Reactivos de Sulfhidrilo/farmacología , 4-Cloromercuribencenosulfonato , Marcadores de Afinidad , Animales , Ácidos y Sales Biliares/antagonistas & inhibidores , Bumetanida/antagonistas & inhibidores , Células Cultivadas , Disulfuros , Ácido Ditionitrobenzoico/análogos & derivados , Ditiotreitol , Etilmaleimida , Hígado/metabolismo , Masculino , Nitrobenzoatos , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/metabolismo , TritioRESUMEN
We examined the properties of Na+/K+/2Cl- cotransport in cultured mouse mTAL cells with respect to its kinetics, the contribution of K/K exchange to K fluxes mediated by the cotransporter, and [3H]bumetanide binding and turnover numbers in media with varying osmolality. The addition of bumetanide, the replacement of external Na+ or the replacement of external Cl- resulted in an almost identical (approx. 50%) decrease in K+ influx, suggesting that Na(+)-dependent, Cl(-)-dependent, BS K+ influx was a measure of Na+/K+/2Cl- cotransport. The kinetics of the BS K+ influx revealed a high affinity for external Na+ (apparent Km 7 mM) and external K+ (apparent Km 1.3 mM), but a very low affinity for external Cl- (apparent Km 67 mM with a two-site model). Of interest was the finding that none of the K+ (86Rb+) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [3H]bumetanide binding was a saturable function of free bumetanide concentration with a Kd of 0.20 microM and maximum binding (Bmax) of 0.63 pmol/mg, or about 53,000 sites per cell. Simultaneous transport and bumetanide binding assays yielded a turnover number of 255 min-1. The omission of external Na+, K+ or Cl- reduced specific [3H]bumetanide binding to values indistinguishable from zero. Changing medium osmolarity resulted in a co-ordinate change in BS K+ influx and bumetanide binding, with a monotonic increase in both transport and bumetanide binding with increase in osmolality from 200 to 400 mosmol/kg. About 85% of the cotransporter sites were located on the apical side, as in the intact mTAL tubule. The simultaneous measurement of BS ion transport and [3H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na+/K+/2Cl- cotransport in this nephron segment.
Asunto(s)
Bumetanida/metabolismo , Cloruros/metabolismo , Electrólitos/metabolismo , Médula Renal/metabolismo , Túbulos Renales/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Animales , Transporte Biológico , Bumetanida/farmacología , Polaridad Celular , Células Cultivadas , Cloruros/farmacología , Cinética , Ratones , Potasio/farmacología , Radioisótopos de Potasio , Radioisótopos de Rubidio , Sodio/farmacología , TritioRESUMEN
The loop diuretic bumetanide which inhibits hepatic bile acid uptake competitively according to its transport kinetics has been proposed to serve as a substrate of a multispecific bile acid transport system in liver parenchymal cells. However, when the in vitro transcripts of two cloned hepatic bile acid uptake carriers, the Ntcp (Na+/taurocholate cotransporting polypeptide) and the oatp (organic anion transporting polypeptide), was expressed for three days in Xenopus laevis oocytes [3H]bumetanide uptake was not increased although bile acid uptake was stimulated. The data presented show that bumetanide is taken up by a third organic anion transport system which is different from the cloned bile acid transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Bumetanida/metabolismo , Proteínas Portadoras/metabolismo , Diuréticos/metabolismo , Hígado/metabolismo , Animales , Proteínas de Transporte de Anión , Transporte Biológico , Ácido Glicocólico/metabolismo , Transporte Iónico , Oocitos/metabolismo , ARN sin Sentido/metabolismo , ARN sin Sentido/farmacología , ARN Mensajero/metabolismo , Ratas , Sodio/farmacología , Xenopus laevisRESUMEN
In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aniones/metabolismo , Membrana Celular/metabolismo , Vertebrados/metabolismo , Animales , Axones/metabolismo , Transporte Biológico , Aves , Bumetanida/metabolismo , Calcio/farmacología , Calmodulina/fisiología , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patología , Proteínas Portadoras/sangre , Cationes , Línea Celular , Cloruros/metabolismo , Cloruros/farmacología , Eritrocitos/citología , Eritrocitos/metabolismo , Etilmaleimida/farmacología , Humanos , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Hígado/metabolismo , Linfocitos/metabolismo , Músculos/metabolismo , Neuroglía/metabolismo , Fosfatos/metabolismo , Potasio/sangre , Potasio/farmacología , Simportadores de Cloruro de Sodio-Potasio , Sulfatos/metabolismoRESUMEN
The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human-shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2-7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.
Asunto(s)
Bumetanida/farmacología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diuréticos/farmacología , Animales , Sitios de Unión/fisiología , Bumetanida/metabolismo , Proteínas Portadoras/metabolismo , Células Cultivadas , Cloruros/metabolismo , Diuréticos/metabolismo , Humanos , Riñón/citología , Cinética , Mutagénesis Sitio-Dirigida/fisiología , Sondas de Oligonucleótidos , Potasio/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Radioisótopos de Rubidio , Tiburones , Sodio/metabolismo , Simportadores de Cloruro de Sodio-Potasio , Especificidad de la EspecieRESUMEN
OBJECTIVES: Recent data highlight the potential of bumetanide as a treatment for neonatal seizures and autism, as it facilitates the excitatory to inhibitory switch in gamma-aminobutyric acid signalling. This study examines the extent of blood-brain barrier (BBB) permeation of bumetanide, a key determinant of the efficacy of centrally acting drugs. Furthermore, the impact of efflux transporter organic anion transporter 3 (oat3) inhibition on bumetanide pharmacokinetics was investigated. METHODS: Bumetanide levels in extracellular fluid (ECF) and plasma in the presence and absence of oat3 inhibitor probenecid were monitored using integrated microdialysis. KEY FINDINGS: Following a bumetanide bolus/continuous infusion of 10 mg/kg and 6 mg/kg/h, bumetanide was detected in hippocampal ECF at the estimated concentration of 131 ± 55 ng/ml. Plasma bumetanide levels were â¼20 mg/l at steady state. Coadministration of probenecid resulted in an increase in bumetanide levels in both ECF and plasma, indicating that oat3 inhibition influences the pharmacokinetics of bumetanide primarily in the periphery. CONCLUSION: Although bumetanide reached detectable levels in hippocampal ECF, bumetanide concentration in ECF was low relative to systemic concentration. Oat3 inhibition by probenecid resulted in increased bumetanide concentrations in brain and plasma. As an acute treatment in neonatal seizures, the bumetanide/probenecid combination may hold therapeutic potential.