RESUMEN
Chimeric mice with humanized liver (PXB mice) have been generated by transplantation of urokinase-type plasminogen activator/severe combined immunodeficiency mice with human hepatocytes. The purpose of the present study was to clarify the protein expression levels of metabolizing enzymes and transporters in humanized liver of PXB mice transplanted with hepatocytes from three different donors, and to compare their protein expressions with those of human livers to validate this human liver model. The protein expression levels of metabolizing enzymes and transporters were quantified in microsomal fraction and plasma membrane fraction, respectively, by means of liquid chromatography-tandem mass spectrometry. Protein expression levels of 12 human P450 enzymes, two human UDP-glucuronosyltransferases, eight human ATP binding cassette (ABC) transporters, and eight human solute carrier transporters were determined. The variances of protein expression levels among samples from mice humanized with hepatocytes from all donors were significantly greater than those from samples obtained from mice derived from each individual donor. Compared with the protein expression levels in human livers, all of the quantified metabolizing enzymes and transporters were within a range of 4-fold difference, except for CYP2A6, CYP4A11, bile salt export pump (BSEP), and multidrug resistance protein 3 (MDR3), which showed 4- to 5-fold differences between PXB mouse and human livers. The present study indicates that humanized liver of PXB mice is a useful model of human liver from the viewpoint of protein expression of metabolizing enzymes and transporters, but the results are influenced by the characteristics of the human hepatocyte donor.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Glucuronosiltransferasa/biosíntesis , Hígado/metabolismo , Espectrometría de Masas en Tándem , Transportadoras de Casetes de Unión a ATP/análisis , Animales , Niño , Preescolar , Quimera , Cromatografía Liquida/métodos , Sistema Enzimático del Citocromo P-450/análisis , Femenino , Glucuronosiltransferasa/análisis , Hepatocitos/química , Hepatocitos/metabolismo , Humanos , Hígado/química , Masculino , Ratones , Ratones SCID , Espectrometría de Masas en Tándem/métodosRESUMEN
We constructed brain-derived neurotrophic factor (BDNF) expressing rat retinal pigment epithelial (RPE) cells by stable transfection of BDNF cDNA, and the RPE cells were cultured on a cross-linked collagen sheet (Coll-RPE-BDNF). BDNF expression of the Coll-RPE-BDNF was confirmed by western blot, and the Coll-RPE-BDNF was transplanted into the rabbit sclera. In vivo BDNF expression was confirmed by His expression that was linked to the expressing BDNF. The effect of the released BDNF was examined in a rabbit acute high intraocular pressure system by electroretinogram and histological examination. Statistically significant preservation of ERG b wave amplitude was observed in the rabbits treated by Coll-RPE-BDNF when compared to that of no treatment. Statistically significant preservation of the thickness of the inner nuclear layer at the transplanted area was observed in the rabbits treated by Coll-RPE-BDNF compared to that of no treatment. Intra-scleral Coll-RPE-BDNF transplantation may partially rescue retinal cells from acute high intraocular pressure.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trasplante de Células/métodos , Presión Intraocular/fisiología , Retina/fisiología , Enfermedades de la Retina/cirugía , Epitelio Pigmentado de la Retina/trasplante , Animales , Colágeno/farmacología , Reactivos de Enlaces Cruzados/farmacología , Electrorretinografía , Supervivencia de Injerto , Masculino , Conejos , Ratas , Retina/citología , Enfermedades de la Retina/etiología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Esclerótica/cirugíaRESUMEN
We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/µg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/µg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/µg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteómica/métodos , Receptores de Superficie Celular/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Animales , Transporte Biológico/fisiología , Encéfalo/citología , Cromatografía Liquida/métodos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
Tight junctions (TJs) are an important component of the blood-brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of brain capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 x 10(6) cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the brain capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-brain barrier in cooperation with claudin-5.
Asunto(s)
Encéfalo/citología , Separación Celular/métodos , Células Endoteliales/metabolismo , Expresión Génica , Magnetismo , Proteínas de la Membrana/genética , ARN Mensajero/metabolismo , Animales , Capilares/citología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 µg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1730-1737, 2016.
Asunto(s)
Dinoprost/análogos & derivados , Mutación , Retina/metabolismo , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/terapia , Rodopsinas Sensoriales/genética , Animales , Dinoprost/farmacología , Regulación de la Expresión Génica , Ratas , Ratas Mutantes , Retinitis Pigmentosa/genética , Rodopsinas Sensoriales/metabolismoRESUMEN
Cerebral clearance of amyloid-ß peptide (Aß), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aß1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aß1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAß1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAß1-40, suggesting that hAß1-40 and hANP share a common elimination process. Internalization of [125I]hAß1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAß1-40, suggesting that there is no direct interaction between Npr-C and hAß1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAß1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAß1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Encéfalo/citología , Células Endoteliales/efectos de los fármacos , Insulina/metabolismo , Insulisina/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Factor Natriurético Atrial/genética , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Insulisina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , ARN Interferente Pequeño/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Factores de TiempoRESUMEN
Controlled transscleral co-delivery of two drugs, edaravone (EDV) and unoprostone (UNO), using a platform that comprises a microfabricated reservoir, controlled-release cover, and drug formulations, which are made of photopolymerized poly(ethyleneglycol) dimethacrylates, shows synergistic retinal neuroprotection against light injury in rats when compared with single-drug-loaded devices. The device would offer a safer therapeutic method than intravitreal injections for retinal disease treatments.
Asunto(s)
Sistemas de Liberación de Medicamentos/instrumentación , Sistemas de Liberación de Medicamentos/métodos , Retina/metabolismo , Enfermedades de la Retina/tratamiento farmacológico , Administración Oftálmica , Animales , Antipirina/administración & dosificación , Antipirina/análogos & derivados , Antipirina/farmacocinética , Dinoprost/administración & dosificación , Dinoprost/análogos & derivados , Dinoprost/farmacocinética , Combinación de Medicamentos , Edaravona , Diseño de Equipo , Metacrilatos/química , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Prótesis e Implantes , Ratas , Retina/efectos de la radiación , Enfermedades de la Retina/etiología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/prevención & control , Esclerótica/cirugíaRESUMEN
Cynomolgus monkey has been used as a model for the prediction of drug disposition in human brain. The purpose of this study was to clarify protein expression levels of membrane proteins affecting drug distribution to brain, such as transporters, receptors, and junctional proteins, in cynomolgus monkey brain microvessels by using liquid chromatography tandem mass spectrometry. In adult monkeys, three ATP-binding cassette transporters (multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4)), six solute carrier transporters (glucose transporter 1 (GLUT1), GLUT3/14, monocarboxylate transporter 1 (MCT1), MCT8, organic anion transporting polypeptide 1A2, and equilibrative nucleoside transporter 1), two junctional proteins (claudin-5 and vascular endothelial cadherin), and two receptors (insulin receptor and low-density lipoprotein receptor-related protein 1) were detected. Comparison of the expression levels with those in mouse, which we reported previously, revealed a pronounced species difference. BCRP expression in monkey was greater by 3.52-fold than that in mouse, whereas MDR1 and MRP4 expression levels in monkey were lower by 0.304- and 0.180-fold, respectively, than that in mouse. This study also investigated the developmental changes in expression of membrane proteins in neonate and child monkeys. Expression of MDR1 was similar in neonate and adult monkeys, whereas in rat, P-glycoprotein expression was reported to be significantly lower in brain microvessels of neonate as compared with adult rat. These results will be helpful to understand and predict brain concentrations of drugs in different species and at different ages of primates.
Asunto(s)
Factores de Edad , Barrera Hematoencefálica , Proteínas de la Membrana/metabolismo , Animales , Cromatografía Liquida , Macaca fascicularis , Espectrometría de Masas en TándemRESUMEN
Cerebral atrial natriuretic peptide (ANP), which is generated in the brain, has functions in the regulation of brain water and electrolyte balance, blood pressure and local cerebral blood flow, as well as in neuroendocrine functions. However, cerebral ANP clearance is still poorly understood. The purpose of this study was to clarify the mechanism of blood-brain barrier (BBB) efflux transport of ANP in mouse. Western blot analysis showed expression of natriuretic peptide receptor (Npr)-A and Npr-C in mouse brain capillaries. The brain efflux index (BEI) method confirmed elimination of [(125)I]human ANP (hANP) from mouse brain across the BBB. Inhibition studies suggested the involvement of Npr-C in vivo. Furthermore, rapid internalization of [(125)I]hANP by TM-BBB4 cells (an in vitro BBB model) was significantly inhibited by Npr-C inhibitors and by two different Npr-C-targeted short interfering RNAs (siRNAs). Finally, treatment with 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) significantly increased Npr-C expression in TM-BBB4 cells, as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted absolute proteomics. Our results indicate that Npr-C mediates brain-to-blood efflux transport of ANP at the mouse BBB as a pathway of cerebral ANP clearance. It seems likely that levels of natriuretic peptides in the brain are modulated by 1,25(OH)(2)D(3) through upregulation of Npr-C expression at the BBB.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Barrera Hematoencefálica/fisiología , Química Encefálica/efectos de los fármacos , Péptido Natriurético Tipo-C/biosíntesis , Receptores del Factor Natriurético Atrial/fisiología , Animales , Western Blotting , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Capilares/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Células Endoteliales/metabolismo , Humanos , Radioisótopos de Yodo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño/genética , Receptores del Factor Natriurético Atrial/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
PURPOSE: To develop an absolute quantification method for membrane proteins, and to construct a quantitative atlas of membrane transporter proteins in the blood-brain barrier, liver and kidney of mouse. METHODS: Mouse tissues were digested with trypsin, and mixed with stable isotope labeled-peptide as a quantitative standard. The amounts of transporter proteins were simultaneously determined by liquid chromatography-tandem mass spectrometer (LC/MS/MS). RESULTS: The target proteins were digested in-silico, and target peptides for analysis were chosen on the basis of the selection criteria. All of the peptides selected exhibited a detection limit of 10 fmol and linearity over at least two orders of magnitude in the calibration curve for LC/MS/MS analysis. The method was applied to obtain the expression levels of 34 transporters in liver, kidney and blood-brain barrier of mouse. The quantitative values of transporter proteins showed an excellent correlation with the values obtained with existing methods using antibodies or binding molecules. CONCLUSION: A sensitive and simultaneous quantification method was developed for membrane proteins. By using this method, we constructed a quantitative atlas of membrane transporter proteins at the blood-brain barrier, liver and kidney in mouse. This technology is expected to have major implications for various fields of biomedical science.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteínas de Transporte de Membrana/análisis , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Barrera Hematoencefálica , Riñón/química , Hígado/química , Masculino , Ratones , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
The primitive nervous system in planula larvae of Hydractinia echinata (Cnidaria) has sensory neurons containing LWamide or RFamide neuropeptides. LWamides have been shown to induce metamorphosis of planula larvae into adult polyps. We report here that RFamides act antagonistically to LWamides. RFamides inhibit metamorphosis when applied to planula larvae during metamorphosis induction by treatment with LWamides (or other inducing agents such as CsCl ions, diacylglycerol and bacterial inducers). Our results show further that RFamides act downstream of LWamide release, presumably directly on target cells mediating metamorphosis. These observations support a model in which metamorphosis in H. echinata is regulated by sensory neurons secreting LWamides and RFamides in response to environmental cues.
Asunto(s)
Hidrozoos/crecimiento & desarrollo , Hormonas de Invertebrados/farmacología , Neuropéptidos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Hidrozoos/anatomía & histología , Hidrozoos/efectos de los fármacos , Hormonas de Invertebrados/fisiología , Cinética , Larva/anatomía & histología , Larva/efectos de los fármacos , Metamorfosis Biológica/efectos de los fármacos , Modelos Biológicos , Neuropéptidos/fisiologíaRESUMEN
Planula larvae of Hydractinia echinata (Cnidaria) settled on a substratum migrate toward light. We observed that planula migration is not a continuous process. Instead, it consists of repeating cycles of active migration (about 8 min on average) and inactive resting periods (about 26 min on average). This pattern of periodic migration is regulated by LWamide and RFamide neuropeptides. LWamide (10(-8) mol l(-1)) stimulates migration primarily by making the active periods longer, whereas RFamide (10(-7) mol l(-1)) inhibits migration by blocking the initiation and also shortening the length of the active periods. Since sensory neurons containing LWamides and RFamides are present in planula larvae, it appears likely that planula migration is regulated by the release of endogenous neuropeptides in response to environmental cues.