RESUMO
CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2beta ligands in the regulation of CRF R2beta expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2beta mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2beta mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2beta mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2beta expression in the aorta-derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2beta mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2beta mRNA expression. The multifactorial regulation of CRF R2beta mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.
Assuntos
Sistema Cardiovascular/metabolismo , Hormônio Liberador da Corticotropina/fisiologia , Citocinas/fisiologia , Glucocorticoides/fisiologia , RNA Mensageiro/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Hormônio Adrenocorticotrópico/farmacologia , Animais , Aorta/metabolismo , Corticosterona/farmacologia , Corticosterona/fisiologia , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/farmacologia , Citocinas/farmacologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Miocárdio/metabolismo , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Restrição Física , UrocortinasRESUMO
The synthesis, purification, chemical characterization, and binding properties of two 125I-labeled analogues of dermorphin and deltorphin-I are described. Native deltorphin-I and [Lys7] dermorphin sequences were elongated by an aminopentyl chain on their C-terminal amide function and alkylated with the 125I-labeled monoiodinated derivative of Bolton-Hunter reagent (BH*). The resulting radiolabeled peptides, epsilon-BH* [Lys7] dermorphin 5-aminopentylamide and omega-BH* deltorphin-I 5-aminopentylamide, have kept most of the original properties of the parent peptides. They bind with high selectivity and specificity to the mu- (dermorphin analogue) or delta- (deltorphin-I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the mu and delta receptors. The autoradiographic distribution of specific binding sites for the 125I-labeled dermorphin and deltorphin-I analogues in rat brain is in complete agreement with previously reported localizations of mu- and delta-opioid receptors. The two radiolabeled peptides are the best ligands of mu- and delta-opioid receptors currently available in terms of sensitivity, specificity, and selectivity.
Assuntos
Analgésicos Opioides/farmacologia , Oligopeptídeos/farmacologia , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Sequência de Aminoácidos , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Animais , Autorradiografia , Ligação Competitiva/fisiologia , Química Encefálica/fisiologia , Radioisótopos do Iodo , Ligantes , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos Opioides , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificação , Precursores de Proteínas/síntese química , Ratos , Sensibilidade e EspecificidadeRESUMO
The binding and internalization of radioiodinated and fluorescent mu and delta opioid peptides in mammalian cells were quantitatively studied by biochemical techniques and directly visualized by confocal microscopy. The labeled peptides were prepared by inserting either a 125I-Bolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and [Lys7]dermorphin. The purified derivatives kept most of their specificity and selectivity toward delta and mu opioid receptors, respectively. Biochemical and confocal microscopy data showed that both mu and delta opioid peptides were internalized in mammalian cells transfected with the corresponding opioid receptor according to a receptor-mediated mechanism. The internalization process was time- and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both delta and mu ligands occurred from a single large cap at one pole of the cell, indicating that polymerization of ligand-receptor complexes preceeded internalization. Finally, green and red fluorescent analogues of deltorphin-I and [Lys7]dermorphin, respectively, were found to internalize through partly distinct endocytic pathways in cells co-transfected with mu and delta receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.
Assuntos
Analgésicos Opioides/metabolismo , Oligopeptídeos/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Animais , Compostos de Boro , Encéfalo/metabolismo , Células COS , Linhagem Celular , Corantes Fluorescentes , Radioisótopos do Iodo , Cinética , Mamíferos , Ensaio Radioligante , Ratos , Receptores Opioides delta/biossíntese , Receptores Opioides mu/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Succinimidas , TransfecçãoRESUMO
A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position alpha (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.
Assuntos
Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Animais , Células COS , Células Cultivadas , Fluorescência , Microscopia Confocal , Receptores de Somatostatina/classificação , Receptores de Somatostatina/genética , TransfecçãoRESUMO
Radiolabeled analogues of neuromedin N have been prepared by acylation of the alpha, epsilon 1, and epsilon 2 amino groups of [Lys2]neuromedin N (Lys-Lys-Pro-Tyr-Ile-Leu) either with the 125l-labeled Bolton-Hunter reagent or with N-succinimidyl[2,3-3H]propionate. The binding properties of the purified analogues toward newborn mouse brain homogenate or toward membranes of cells transitorily (COS) or permanently (AA1) transfected with the cloned rat brain neurotensin receptor cDNA were evaluated and compared with those of radiolabeled neurotensin. The alpha-modified analogue of [Lys2]neuromedin N behaves exactly like neurotensin in these binding experiments, whereas the epsilon 1- and epsilon 2-modified analogues selectively recognize the fraction of neurotensin binding sites that is sensitive to GTP gamma S. The proportion of neurotensin receptors coupled to GTP binding proteins is approximately 50% in membranes of newborn mouse brain or of AA1 cells that respond to neurotensin by an increase of the intracellular inositol trisphosphate concentration. By contrast, membranes of transitorily transfected COS cells that do not respond to neurotensin exhibit very low levels of GTP-sensitive receptors labeled with the epsilon 1- or epsilon 2-modified analogues. These radiolabeled peptides offer new tools to selectively detect active neurotensin receptors.
Assuntos
Química Encefálica/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Receptores de Neurotensina/metabolismo , Animais , Animais Recém-Nascidos , Autorradiografia , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/metabolismo , Indicadores e Reagentes , Radioisótopos do Iodo , Ligantes , Proteínas de Membrana/metabolismo , Camundongos , Neurotensina/análogos & derivados , Neurotensina/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores de Neurotensina/química , SuccinimidasRESUMO
Polypeptide hormones and growth factors have long been known to internalize into peripheral target cells as a result of their interaction with cell surface receptors. Studies in culture have suggested that certain neuropeptides might undergo a similar type of translocation in neurons. To investigate this possibility in adult mammalian brain, we have examined by confocal laser microscopy the events that follow the binding of fluorescein-tagged derivatives of the tridecapeptide neurotensin to basal forebrain cholinergic cells. Our results demonstrate a selective time- and temperature-dependent internalization of fluo-neurotensin in these cells. This internalization is receptor mediated, proceeds from the entire somatodendritic membrane of the cells, and utilizes endosome-like organelles which are mobilized from dendrites to perikarya and from the periphery of the cell to its perinuclear region. Parallel studies carried out on Sf9 insect cells expressing the rat neurotensin receptor from a recombinant baculovirus indicated that the internalization process involves receptor-ligand complexes and not merely the fluorescent peptide itself. These data suggest that receptor internalization plays a role in neuropeptide signaling in the brain and that it can be harnessed for selective identification of neuropeptide target cells.
Assuntos
Fluoresceínas/metabolismo , Neurotensina/análogos & derivados , Neurotensina/metabolismo , Prosencéfalo/metabolismo , Receptores de Neurotensina/metabolismo , Animais , Baculoviridae , DNA Complementar , Corantes Fluorescentes , Técnicas In Vitro , Cinética , Masculino , Microscopia Confocal , Prosencéfalo/citologia , Ratos , Ratos Sprague-Dawley , Receptores de Neurotensina/análise , Receptores de Neurotensina/biossíntese , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the 125I-labeled Bolton-Hunter reagent. This 125I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (KD = 0.5 nM). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with 125I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.
Assuntos
Encéfalo/metabolismo , Neurotensina/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Autorradiografia/métodos , Sítios de Ligação , Ligação Competitiva , Radioisótopos do Iodo , Cinética , Camundongos , Neurotensina/análogos & derivados , Especificidade de Órgãos , Ratos , Especificidade da Espécie , SuccinimidasRESUMO
This report describes the kinetics and molecular aspects of neurotensin internalization in neurons. Incubation of alpha-125I-Bolton-Hunter neurotensin-(2-13) with cortical neurons at 37 degrees C was followed by a rapid internalization of the peptide bound to its receptors. This process was completed within 20 min and was inhibited either irreversibly by the general endocytosis inhibitor phenylarsine oxide or reversibly by incubation at low temperature (0-4 degrees C). The discrepancy of maximal binding capacities observed in the presence (150 fmol/mg of protein) or in the absence (250 fmol/mg of protein) of internalization inhibitors could be attributed to the appearance of a new pool of neurotensin binding sites on the cell surface rather than a recycling of internalized receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in denaturing conditions revealed that three different protein subunits of 50, 60, and 100 kDa were covalently labeled at 37 degrees C with a radioactive photoreactive analogue of neurotensin. The 50- and 60-kDa subunits remained labeled when internalization was blocked, whereas the specific labeling of the 100-kDa protein was abolished. These results suggest that neurotensin-induced internalization of the 50- and 60-kDa subunits initially present on the cell surface triggers insertion of the 100-kDa subunit into the membrane from an intracellular compartment. Subcellular fractionation experiments have shown that, in the absence of neurotensin, the 100-kDa protein is located in an intracellular vesicular fraction of neurons.
Assuntos
Córtex Cerebral/metabolismo , Neurônios/metabolismo , Neurotensina/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Ligação Competitiva , Diferenciação Celular , Cinética , Camundongos , Receptores de NeurotensinaRESUMO
Incorporation of N-succinimidyl-3(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) or its 125I-labeled derivative into peptides can be selectively directed towards either alpha- or epsilon-amine functions by modifying the pH of the reaction. Acylation of alpha-amino groups is favored at pH 6.5 whereas epsilon-amino groups react more readily at pH 8.5. We have taken advantage of this result to prepare two new 125I-labeled analogues of substance P and neurotensin that bind selectively and reversibly to their respective receptors. The method described here is of general interest and can be used to incorporate various reporter groups into peptide structures.