RESUMEN
BACKGROUND AND PURPOSE: Chronic pain and hyperalgesia can be difficult to treat with classical opioids acting predominately at the µ-opioid receptor. Buprenorphine and its active metabolite are believed to act through µ-, κ- and δ-receptors and may therefore possess different analgesic and anti-hyperalgesic effects compared with pure µ-receptor agonists, for example, fentanyl. Here, we have compared the analgesic and anti-hyperalgesic effects of buprenorphine and fentanyl. EXPERIMENTAL APPROACH: Twenty-two healthy volunteers were randomized to treatment with transdermal buprenorphine (20 µg·h(-1), 144 h), fentanyl (25 µg·h(-1), 72 h) or placebo patches in a double-blind, cross-over experimental pain study. The experimental pain tests (phasic pain, sensitization) involved pressure at the tibial bone, cutaneous electrical and thermal stimulation, intramuscular nerve growth factor, UVB light burn injury model and intradermal capsaicin-induced hyperalgesia. Pain testing was carried out at baseline, 24, 48, 72 and 144 h after application of the drugs. KEY RESULTS: Compared with placebo, buprenorphine, but not fentanyl, significantly attenuated pressure at the tibial bone as well as pressure pain in the primary hyperalgesic area induced by UVB light The two drugs were equipotent and better than placebo against cutaneous thermal pain stimulation), but failed to show significant analgesic effect to cutaneous electrical stimulation, nerve growth factor-induced muscle soreness and to capsaicin-induced hyperalgesia. CONCLUSIONS AND IMPLICATIONS: Buprenorphine, but not fentanyl, showed analgesic effects against experimentally induced, bone-associated pain and primary hyperalgesia compared with placebo. These tissue- and modality-differentiated properties may reflect the variable effects of opioid drugs observed in individual patients.
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Analgésicos Opioides/administración & dosificación , Buprenorfina/administración & dosificación , Fentanilo/administración & dosificación , Hiperalgesia/tratamiento farmacológico , Dolor/tratamiento farmacológico , Administración Cutánea , Analgésicos/administración & dosificación , Analgésicos/efectos adversos , Analgésicos Opioides/efectos adversos , Buprenorfina/efectos adversos , Capsaicina/farmacología , Estudios Cruzados , Método Doble Ciego , Estimulación Eléctrica/métodos , Fentanilo/efectos adversos , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Masculino , Factor de Crecimiento Nervioso/metabolismo , Dolor/metabolismo , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Piel/efectos de los fármacos , Piel/metabolismo , Adulto JovenRESUMEN
The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.
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Oligodesoxirribonucleótidos Antisentido/metabolismo , Péptidos/metabolismo , Tionucleótidos/metabolismo , Secuencia de Aminoácidos , Animales , Aorta , Células CHO , Permeabilidad de la Membrana Celular , Núcleo Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Cistina/química , Citoplasma/metabolismo , Perros , Electroforesis Capilar , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Fluorometría , Proteínas de Microfilamentos/genética , Microscopía Confocal , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/químicaRESUMEN
The subcommissural organ (SCO) is an enigmatic secretory gland of the brain, which is believed to be derived from ependymal (glial) precursor cells. We here developed a dispersed cell culture system of the bovine SCO as an approach to functional analyses of this brain gland. Tissue of the bovine SCO obtained from the slaughterhouse was papain dissociated either directly after dissection or after preparation of SCO explants. The latter had been maintained for 4-6 weeks in organ culture. The dispersed cells were cultured for up to 14 days and continuously tested for their secretory state by immunostaining of their secretory product. With respect to the morphology of the SCO cells (shape, processes, nucleus), no difference was found between the culture of freshly dissociated SCOs and that of dissociated SCO explants. In all cases, the dissociation caused a dedifferentiation; typical elongated cells were formed increasingly after 1 day of culture. Thereafter, only the cellular size increased, whereas the shape and the viability of the cells remained unchanged. Proliferating SCO cells were never observed. The culture obtained from fresh SCO tissue contained more glia cells and fibrocytes than the culture prepared from SCO explants. The proliferation of glia cells and fibrocytes was suppressed by blocking the mitotic activity with cytosine-beta-D-arabino furanoside (CAF). The cytophysiological features of the cultured dispersed cells of both origins did not differ as demonstrated by classical histology, by immunocytochemistry for the secretory products of the SCO, by the characteristics of calcium influx into the cytoplasm ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) after stimulation with adenosine-5-triphosphate, substance P or serotonin, and by the activation of the transcription factor cAMP-responsive element-binding protein. Because of the maintenance of their viability, their capacity to release the secretory product into the culture medium, their receptive capacity, and their signal transduction pathways, we conclude that the dispersed cell culture system, especially that obtained from SCO explants, represents an appropriate and useful model for functional studies of the mammalian SCO.
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Secreciones Corporales/fisiología , Células Cultivadas/citología , Órgano Subcomisural/citología , Adenosina Trifosfato/farmacología , Animales , Bromodesoxiuridina/farmacocinética , Calcio/metabolismo , Bovinos , Moléculas de Adhesión Celular Neuronal/inmunología , Moléculas de Adhesión Celular Neuronal/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Tamaño de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colforsina/farmacología , AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inmunohistoquímica , Neuroglía/citología , Neuroglía/metabolismo , Fosforilación/efectos de los fármacos , Órgano Subcomisural/efectos de los fármacos , Órgano Subcomisural/metabolismoRESUMEN
The subcommissural organ (SCO) is a conserved brain gland present throughout the vertebrate phylum. During ontogeny, it is the first secretory structure of the brain to differentiate. In the human, the SCO can be morphologically distinguished in 7- to 8-week-old embryos. The SCO of 3- to 5-month-old fetuses is an active, secretory structure of the brain. However, already in 9-month-old fetuses, the regressive development of the SCO-parenchyma is evident. In 1-year-old infants, the height of the secretory ependymal cells is distinctly reduced and they are grouped in the form of islets that alternate with cuboid non-secretory ependyma. The regression of the SCO continues during childhood, so that at the ninth year of life the specific secretory parenchyma is confined to a few islets of secretory ependymal cells. The human fetal SCO shares the distinct ultrastructural features characterizing the SCO of all other species, namely, a well-developed rough endoplasmic reticulum, with many of its cisternae being dilated and filled with a filamentous material, several Golgi complexes, and secretory granules of variable size, shape, and electron density. The human fetal SCO does not immunoreact with any of the numerous polyclonal and monoclonal antibodies raised against RF-glycoproteins of animal origin. This and the absence of RF in the human led to the conclusion that the human SCO does not secrete RF-glycoproteins. Taking into account the ultrastructural, lectin-histochemical, and immunocytochemical findings, it can be concluded that the human SCO, and most likely the SCO of the anthropoid apes, secrete glyco- protein(s) with a protein backbone of unknown nature, and with a carbohydrate chain similar or identical to that of RF-glycoproteins secreted by the SCO of all other species. These, as yet unidentified, glycoprotein(s) do not aggregate but become soluble in the CSF. Evidence is presented that these CSF-soluble proteins secreted by the human SCO correspond to (1) a 45-kDa compound similar or identical to transthyretin and, (2) a protein of about 500 kDa.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Feto/química , Órgano Subcomisural/metabolismo , Órgano Subcomisural/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Órgano Subcomisural/embriología , Órgano Subcomisural/crecimiento & desarrolloRESUMEN
We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.
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Endotelina-1/metabolismo , Proteínas Luminiscentes/metabolismo , Receptores de Endotelina/metabolismo , Animales , Línea Celular , Perros , Regulación hacia Abajo , Proteínas Fluorescentes Verdes , Microscopía Fluorescente , Receptor de Endotelina B , Receptores de Endotelina/análisis , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Vasopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrand's disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.
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AMP Cíclico/fisiología , Desamino Arginina Vasopresina/farmacología , Endotelio Vascular/efectos de los fármacos , Receptores de Vasopresinas/fisiología , Factor de von Willebrand/metabolismo , Arginina Vasopresina/farmacología , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Pulmón/metabolismo , ARN Mensajero/análisis , Receptores de Vasopresinas/análisis , Receptores de Vasopresinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.
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Endosomas/fisiología , Endotelina-1/metabolismo , Lisosomas/fisiología , Receptores de Endotelina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Regulación hacia Abajo , Ligandos , Microscopía Fluorescente , Datos de Secuencia Molecular , Fagocitosis , Receptor de Endotelina BRESUMEN
We have previously shown a conserved glutamate/dileucine motif ((335)ELRSLL(340)) in the intracellular C terminus of the vasopressin V(2) receptor (V(2) receptor) to be essential for receptor transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The motif may represent a transport signal that is recognized by a component of ER to Golgi vesicles. Alternatively, it may be necessary for transport-competent receptor folding to pass the quality-control system of the ER. To assess these two possibilities, we constructed a receptor fragment that allows transport studies independent of full-length receptor folding. Transmembrane domains II-VII were deleted, thereby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introduced, and receptor fragments were localized in transiently transfected HEK 293 cells. All mutant receptor fragments were detectable at the plasma membrane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly suggest that this motif is required for transport-competent folding of the full-length receptor. To assess the underlying conformational features, a three-dimensional homology model of the V(2) receptor was computed. Our model predicts that the glutamate/dileucine motif contributes to a U-like loop within the intracellular C terminus. Residue Leu(339) may be required for folding back the intracellular C terminus to residue Leu(62) of the first cytoplasmic loop. We characterized the naturally occurring L62P and DeltaL62-R64 mutations in the first cytoplasmic loop and show that they lead to transport-defective full-length V(2) receptors that are retained in the ER, consistent with the structure model.
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Receptores de Vasopresinas/química , Receptores de Vasopresinas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Retículo Endoplásmico/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Aparato de Golgi/metabolismo , Leucina/genética , Leucina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Pliegue de Proteína , Receptores de Vasopresinas/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.
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Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Transporte Biológico Activo , Línea Celular , Secuencia Conservada , Cisteína/química , Cartilla de ADN/genética , Diabetes Insípida Nefrogénica/genética , Disulfuros/química , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Receptores de Vasopresinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.
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Túbulos Renales Distales/química , Túbulos Renales Distales/citología , Receptores de Vasopresinas/genética , Adenilil Ciclasas/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Sondas de ADN , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/fisiopatología , Perros , Expresión Génica/fisiología , Humanos , Túbulos Renales Distales/enzimología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/análisis , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Transfección , Tritio , Cromosoma XRESUMEN
Cultured renal epithelial cells rapidly downregulate expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2). Our aim was to define conditions that favor maintenance of AQP-2 expression in vitro without genetic manipulation. We show here that primary cultures of rat inner medullary collecting duct (IMCD) cells retain AQP-2 expression for at least 6 days when grown with dibutyryl cAMP (DBcAMP) supplementation. We also found that coating the culture dishes with type IV collagen, rather than rat-tail collagen, retards AQP-2 downregulation. Immunofluorescence and biochemical studies indicate a shuttling of AQP-2-bearing vesicles after stimulation with vasopressin or forskolin. Rab3 proteins, known to be involved in regulated exocytosis, were detected only in cells grown in the presence of DBcAMP. Using the adenylyl cyclase assay, we confirmed the functional integrity of the vasopressin V2 receptor in a broken cell preparation. Our data show that cAMP supplementation is sufficient for the maintenance of AQP-2 expression in primary cultured cells. The model system established here allows the study of the regulation of genes encoding the antidiuretic machinery at the cellular level.
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Acuaporinas/biosíntesis , Túbulos Renales Colectores/metabolismo , Animales , Acuaporina 2 , Acuaporina 6 , Bucladesina/metabolismo , Células Cultivadas , Colágeno/metabolismo , RatasRESUMEN
Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.
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Ácido Glutámico/metabolismo , Leucina/metabolismo , Receptores de Vasopresinas/metabolismo , Secuencia de Aminoácidos , Animales , Arginina Vasopresina/metabolismo , Sitios de Unión , Transporte Biológico , Células COS/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Pliegue de Proteína , Receptores de Vasopresinas/genética , Homología de Secuencia de Aminoácido , Transfección , TritioRESUMEN
Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined.
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Acuaporinas , Diabetes Insípida Nefrogénica/etiología , Secuencia de Aminoácidos , Acuaporina 2 , Acuaporina 6 , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/terapia , Genes Recesivos , Ligamiento Genético , Humanos , Canales Iónicos/genética , Canales Iónicos/fisiología , Datos de Secuencia Molecular , Mutación/genética , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/fisiología , Vasopresinas/fisiología , Cromosoma XRESUMEN
We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C-terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild-type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin-sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c-myc epitope- or green fluorescent protein-tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C-terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length-dependent reduction of cell surface expression. Replacement of positively charged residues within the C-terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C-terminus replaced by that of the beta2-adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N-terminal part of the C-terminus are necessary for correct folding and that C-terminal residues are important for efficient cell surface expression.
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Pliegue de Proteína , Receptores de Vasopresinas/química , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Arginina Vasopresina/farmacología , Células CHO , Células COS , Técnicas de Cultivo de Célula , Cricetinae , Perros , Relación Dosis-Respuesta a Droga , Humanos , Mutación , Conformación Proteica , Receptores de Vasopresinas/efectos de los fármacos , Receptores de Vasopresinas/genética , Proteínas Recombinantes de FusiónRESUMEN
We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.
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Receptores de Vasopresinas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , ADN Complementario , Perros , Proteínas Fluorescentes Verdes , Riñón/metabolismo , Proteínas Luminiscentes/metabolismo , Microinyecciones , Plásmidos , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónAsunto(s)
Acuaporinas , Diabetes Insípida Nefrogénica , Secuencia de Aminoácidos , Acuaporina 2 , Acuaporina 6 , Arginina Vasopresina/fisiología , Desamino Arginina Vasopresina/farmacología , Deshidratación/etiología , Diabetes Insípida Nefrogénica/complicaciones , Diabetes Insípida Nefrogénica/diagnóstico , Diabetes Insípida Nefrogénica/epidemiología , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/historia , Diabetes Insípida Nefrogénica/fisiopatología , Resistencia a Medicamentos , Femenino , Efecto Fundador , Tamización de Portadores Genéticos , Heterogeneidad Genética , Historia del Siglo XVII , Historia del Siglo XVIII , Humanos , Incidencia , Lactante , Recién Nacido , Discapacidad Intelectual/etiología , Canales Iónicos/deficiencia , Canales Iónicos/genética , Canales Iónicos/fisiología , Irlanda/etnología , Fallo Renal Crónico/etiología , Masculino , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Nefronas/fisiopatología , Nueva Escocia/epidemiología , Diagnóstico Prenatal , Prevalencia , Conformación Proteica , Receptores de Vasopresinas/química , Receptores de Vasopresinas/deficiencia , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/fisiología , Cromosoma X/genéticaRESUMEN
The vasopressin V2 receptor (V2R) and the aquaporin-2 genes of two unrelated male patients with congenital nephrogenic diabetes insipidus were analyzed. The V2R gene of the patient of family 1 had the wild-type sequence. Consequently, the coding region of the aquaporin-2 gene including the exon-intron junctions was sequenced. A novel G to T transversion at codon 202, predictive of an exchange of tryptophan 202 by cysteine, was identified. As the mutation occurs at G-1 of the 5' splice donor site of intron 3, aberrant splicing is also likely. The mutation involves one of the supposed water pore-forming loops. Therefore, both aberrant splicing and amino acid substitution are likely to result in a functionally defective protein. Sequencing of the complete V2R gene of the male patient of family 2 revealed a novel single-base deletion at codon 310 (delta C1001), shifting the reading frame to give an altered amino acid sequence beginning at codon 311. The mutation is unique in predicting a C-terminally extended protein (termination after codon 434 in the mutant receptor instead of codon 371 in the wild-type). The deduced mutant protein is likely to be nonfunctional since the amino acid sequence of the seventh transmembrane domain and the C-terminus is altered.
Asunto(s)
Acuaporinas , Diabetes Insípida Nefrogénica/genética , Canales Iónicos/genética , Mutación , Receptores de Vasopresinas/genética , Secuencia de Aminoácidos , Acuaporina 2 , Acuaporina 6 , Análisis Mutacional de ADN , Femenino , Eliminación de Gen , Humanos , Lactante , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active. Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing. The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.