RESUMO
Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
Assuntos
Proteínas Aviárias , Compartimento Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Transporte Biológico , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Moléculas de Adesão Celular/metabolismo , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrinas/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Trombina/farmacologia , Tirosina/metabolismoRESUMO
By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Células CHO , Membrana Celular/diagnóstico por imagem , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Interações Medicamentosas , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Humanos , Isótopos/farmacocinética , Ligação Proteica/efeitos dos fármacos , Cintilografia , Saponinas/farmacologiaRESUMO
The binding of aluminium ion to [Leu5]-enkephalin has been investigated by 1H, 13C and 27Al NMR spectroscopy in dimethyl sulphoxide solution at different peptide/metal ratio. Analysis of the spectra suggests that Al3+ binds at two metal-binding sites. The binding of Al3+ at the first site involves the Tyr1CO, and Leu5COO- groups to give a 2:1 species in a tetracoordinated structure; whereas the binding of Al3+ at the second site utilizes the NH2 terminal groups of the tyrosine moiety in a 2:2 species. 27Al chemical shift values strongly suggest that the second type of aluminium atom displays an octahedral environment. On this basis, we discuss our data in terms of the coordination of aluminium with [Leu5]-enkephalin.
Assuntos
Alumínio/metabolismo , Encefalina Leucina/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância Magnética , TemperaturaRESUMO
The H-2Db peptide sequence SMIENLEYM was manipulated (N- and C-terminus truncation and alanine substitution) to determine the role of structural elements (peptide ends and residue side chains) in binding to H-2Db. We found that good binding affinity could be obtained by compensating the minimal binding condition for one element by the optimal condition of the other element. In particular, we showed, that although the minimal binding sequence could be as short as a heptamer (deletion of positions 1 and 2), it needed the presence of optimal amino acids at other positions (IENLEYM). Conversely, the structurally minimal peptide would accept multiple alanine residues, but required the optimal nonameric length (AAAENAEAA). Positions 1, 2, 3, 4, 5, 7 and 9, but not 6 and 8, were involved in the H-2Db-peptide interaction. Most residues interacted directly with the MHC molecule via their main chain (amino and carboxyl) atoms (positions 1 and 2), their side chains (positions 3 and 5), or both (position 9). Positions 4 and 7 were found to play an indirect role, probably by influencing the secondary structure. At the C-terminus, the presence of a residue at position 9, but not the hydrophobic nature of its side chain, was mandatory for binding. At the N-terminus, the role of the residue at position 1 was of either minor or critical importance depending on the presence or not of a strong auxiliary anchor at position 3. The indirect contribution of residue side chains at positions 4 and 7 reflected the importance of dynamic components in the binding process. Based on these results, we designed a series of high-affinity, H-2Db selective peptides derived from the sequence X1 AIX4NAEAL, where X1 = Y or K and X4 = E or K. After radioiodination or fluorescent (FITC) labelling, these peptides bound strongly and specifically to the surface of viable H-2Db-expressing cells. Rationally designed synthetic peptides, either alone or in a stable complex with MHC, might be of value for controlling CTL activity.
Assuntos
Antígenos H-2/metabolismo , Oligopeptídeos/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Desenho de Fármacos , Antígeno de Histocompatibilidade H-2D , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Ligação Proteica , Engenharia de ProteínasRESUMO
A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.
Assuntos
Anticorpos Monoclonais , Glutamatos/metabolismo , Tubulina (Proteína)/análise , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Astrócitos/química , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Pulmão/química , Masculino , Camundongos , Dados de Sequência Molecular , Neurônios/química , Baço/química , Testículo/químicaRESUMO
The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Inibidores Enzimáticos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Técnicas Biossensoriais , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/química , Ciclinas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.
Assuntos
Dinorfinas/farmacologia , Peptídeos Opioides/farmacologia , Receptores Opioides kappa/fisiologia , Receptores Opioides/fisiologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Membrana Celular/metabolismo , Cricetinae , Diprenorfina/metabolismo , Dinorfinas/química , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos Opioides/química , Peptídeos/farmacologia , Receptores Opioides/biossíntese , Receptores Opioides kappa/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/biossíntese , Alinhamento de Sequência , Relação Estrutura-Atividade , Receptor de Nociceptina , NociceptinaRESUMO
Commercial aqueous 25% glutaraldehyde solutions contain no stable derivative of this aldehyde, but compounds of variable molecular weight which easily revert to glutaraldehyde. The effect of pH on the reaction of glutarldehyde with amino acids and on the stability of the products under acid conditions, shows the importance of the structure modification of the dialdehyde which occurs when pH increases, and even leads to precipitation in highly alkaline solutions. This precipitate results from aldol condensation of glutaraldehyde molecules. It contains aldehyd groups conjugated with ethylenic double bonds. Such a structure reacts with amino groups to give an imino bond, stabilized by resonance with the ethylenic bond, and does not undergo Michael-type addition reactions. Therefore, glutaraldehyde does not react with proteins under its free form, but as an unsaturated polymer, which gives imino bonds stabilized by conjugation.
Assuntos
Aldeídos , Glutaral , Lisina , Proteínas , Fenômenos Químicos , Química , Cromatografia em Gel , Cromatografia em Camada Fina , Crotonatos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Propilaminas , Espectrofotometria InfravermelhoRESUMO
Specific receptors for the octapeptide FLFQPQRFamide (NPFF), a mammalian FMRFamide-like neuropeptide with anti-opiate properties have been identified in rat central nervous system. However, exploration of the biological role of this peptide requires a peptidase-resistant agonist. In this study, the stability and binding characteristics of [125I]DYLMeFQPQRFamide, a radioiodinated analogue of NPFF, on rat spinal cord tissue were determined and compared with those of [125I]YLFQPQRFamide, the reference ligand which previously permitted to characterize NPFF binding sites. In a binding assay, [125I]DYLMeFQPQRFamide remained intact in the presence of membranes without peptidase inhibitors, whereas [125I]YLFQPQRFamide was completely hydrolysed. The specific binding was time-dependent, dose-dependent, saturable and reversible. [125I]DYLMeFQPQRFamide shared the same binding characteristics as [125I]YLFQPQRFamide (Kd = 0.07 nM; Bmax = 14.7 fmol/mg protein). Binding was not affected by various spinal cord opioids or peptides. Autoradiographic studies indicated that binding sites were mainly located in the most external layers of dorsal horn where high densities of NPFF binding sites have previously been described. [125I]YLFQPQRFamide and [125I]DYLMeFQPQRFamide binding sites were both GTP-regulated. These findings indicate that DYLMeFQPQRFamide should be of value in studies on NPFF-mediated actions in vivo.
Assuntos
Receptores de Neuropeptídeos/agonistas , Medula Espinal/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Cromatografia Líquida de Alta Pressão , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/fisiologia , Técnicas In Vitro , Radioisótopos do Iodo , Masculino , Membranas/metabolismo , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Neuropeptídeos/metabolismoRESUMO
Acid sensing ion channel 3 (ASIC3) is a cation channel gated by extracellular protons. It is highly expressed in sensory neurons, including small nociceptive neurons and has been proposed to participate in pain perception associated with tissue acidosis and in mechanoperception. Neuropeptide FF (NPFF) and FMRFamide have been shown to potentiate proton-gated currents from cultured sensory neurons and acid sensing ion channel (ASIC) cDNA transfected cells. In this study, we report that another mammalian peptide neuropeptide SF (NPSF), derived from the same precursor, also considerably increases the amplitude of the sustained current of heterologously expressed ASIC3 (12-fold vs. 19- and nine-fold for FMRFamide and NPFF, respectively) with an EC(50) of approximately 50 microM. Similar effects were also observed on endogenous ASIC3-like sustained current recorded from DRG neurons although of smaller amplitudes (two-, three- and seven-fold increase for NPSF, NPFF and FMRFamide, respectively), and essentially related to a slowing down of the inactivation rate. Importantly, this modulation induced changes in neuronal excitability in response to an electrical stimulus applied during extracellular acidification. ASIC3-mediated sustained depolarisation, and its regulation by neuropeptides, could thus be important in regulating polymodal neuron excitability particularly under inflammatory conditions where the expression levels of both NPFF precursor and ASIC3 are increased.
Assuntos
Proteínas de Membrana , Proteínas do Tecido Nervoso , Neurônios Aferentes/efeitos dos fármacos , Neuropeptídeos/farmacologia , Canais de Sódio/fisiologia , Canais Iônicos Sensíveis a Ácido , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Neurônios Aferentes/fisiologia , Ratos , Ratos WistarRESUMO
Twenty neuropeptide FF (NPFF) analogs having various lengths were synthesized by solid-phase peptide synthesis to gain more information on the role of N-terminal residues for the NPFF receptor affinity. The affinities were evaluated in the rat spinal cord membrane preparations, and the biological activities were measured on morphine analgesia in the mouse tail-flick test. Shortening of the NPFF sequence from the N-terminal produced only a moderate decrease in affinity until NPFF (4-8) was reached. In the same way, NPFF(3-8) significantly decreased morphine analgesia, while NPFF(4-8) had no significant effect at a dose of 22 nmol. The introduction in the N-terminal part of NPFF of a D-enantiomer at positions 2 and 1 or the presence of an N-methyl group on position 3 did not modify affinity and activity. Substitution of proline5 by the D-isomer decreased the affinity of NPFF analogs whatever their length, and [Tyr1,D-Pro5]NPFF(1-8) was 2.5-fold less potent than [Tyr1]NPFF(1-8) in reversing morphine-induced analgesia. In contrast, the presence of a glycine residue in position 5 did not influence the affinity toward NPFF receptors. Data provide evidence that the N-terminal segment of neuropeptide FF is responsible for high-affinity binding.
Assuntos
Oligopeptídeos/química , Sequência de Aminoácidos , Analgesia , Animais , Membrana Celular/metabolismo , Glutamina , Masculino , Camundongos , Dados de Sequência Molecular , Morfina , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Prolina , Ratos , Receptores de Neuropeptídeos/metabolismo , Medula Espinal/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Dynorphin A, which displays a wide variety of physiological effects, binds to opioid receptors preferentially at the kappa receptor type. kappa-selective antagonists would be very useful as pharmacological and biochemical probes to study and better understand the action of dynorphin A at its preferred receptor. However, the development of such molecules has been elusive, and very few are known at this time. Taking these features into account, we have synthesized by the solid-phase procedure several analogues of dynorphin A containing various D-amino acid substitutions. The binding properties of the peptides have been examined at three main opioid binding sites (mu, delta, and kappa) and their kappa selectivity determined. Their biological activities have been tested in three specific pharmacological assays for agonist and/or antagonist properties. Introduction of D-Trp substitution leads to analogues, in particular [D- Trp2,8,D-Pro10]-, [D-Trp5,8,D-Pro10]-, and [D-Trp2,4,8,D-Pro10]dynorphin(1-11), showing antagonist properties in the isolated rabbit vas deferens preparation, a kappa specific bioassay. The antagonism against dynorphin A is weak, as indicated by the observed Ke values (433, 199, and 293 nM, respectively), and not very selective (kappa vs. mu). Such peptide analogues derived from the endogenous ligand and endowed with antagonist properties are the first ones reported to date and could open a promising way in designing more potent and selective kappa opioid antagonists.
Assuntos
Dinorfinas/síntese química , Antagonistas de Entorpecentes/síntese química , Receptores Opioides/efeitos dos fármacos , Animais , Dinorfinas/metabolismo , Dinorfinas/farmacologia , Cobaias , Técnicas In Vitro , Antagonistas de Entorpecentes/metabolismo , Antagonistas de Entorpecentes/farmacologia , Ratos , Receptores Opioides kappa , Receptores Opioides mu , Relação Estrutura-AtividadeRESUMO
In a previous paper we demonstrated that the heptapeptide [Met]enkephalyl-Arg6-Phe7 was released from rat striatal slices by high K+ concentration and rapidly degraded by peptidases, even in the presence of the neutral endopeptidase 24.11 ("enkephalinase")-inhibitor, thiorphan (0.1 microM), the angiotensin-converting enzyme inhibitor, captopril (1 microM), and the aminopeptidase inhibitor, bestatin (20 microM). In this study the pattern of degradation of exogenous [3H]heptapeptide by rat striatal slices has been studied. The angiotensin-converting enzyme and aminopeptidase(s) were partly responsible for this degradation. In addition an enzymatic activity that cleaved the Phe4-Met5 bond was involved in the degradation of the heptapeptide by striatal slices. This activity was inhibited by the dipeptide Leu-Arg (1 mM) and the tripeptide Leu-Arg-Leu (1 mM). The simultaneous presence of thiorphan (0.1 microM), captopril (1 microM), bestatin (20 microM) and Leu-Arg (1 mM) almost completely inhibited the degradation of [3H]heptapeptide by striatal slices. In the presence of these peptidase inhibitors a concomitant release of [Met]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8 was evoked by KCl or veratridine. The K+-evoked release was by a Ca2+-dependent mechanism and the release evoked by veratridine was blocked by tetrodotoxin. In both cases the ratio of [Met]enkephalin to heptapeptide amounts released was close to that found in their common precursor, proenkephalin. Thus the enkephalinergic neuron appears to be capable of synthesizing, from a unique precursor, four different putative opioid neurotransmitters, namely [Met]enkephalin, [Leu]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8, to store these peptides and to release them upon depolarization.
Assuntos
Corpo Estriado/metabolismo , Encefalinas/metabolismo , Precursores de Proteínas/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Corpo Estriado/análise , Masculino , Inibidores de Proteases/farmacologia , Ratos , Ratos Endogâmicos , Veratridina/farmacologiaRESUMO
The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively. By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS. The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord. The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.
Assuntos
Química Encefálica , Receptores de Neuropeptídeos/análise , Medula Espinal/química , Animais , Autorradiografia , Células CHO , Cricetinae , Humanos , Radioisótopos do Iodo , Rim/citologia , Masculino , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Ligação Proteica , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeos/metabolismoRESUMO
A series of overlapping heptapeptides representing the entire amino acid sequences of the light subunits of the two Lathyrus ochrus isolectins were synthesized, coupled to carrier proteins, and the conjugates were reacted with polyclonal antibodies directed against the whole isolectins. Ten conjugates corresponding to two linear peptides localized respectively to the N- and C-terminal ends, reacted significantly with the polyclonal antibodies. The localization of two continuous epitopes at both ends of the chains is consistent with the regions of the chains predicted as putative antigenic determinants, according to their hydrophilicity, accessibility and flexibility.
Assuntos
Lectinas/imunologia , Oligopeptídeos/imunologia , Sequência de Aminoácidos , Epitopos/isolamento & purificação , Fabaceae/imunologia , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/isolamento & purificação , Lectinas de Plantas , Plantas MedicinaisRESUMO
A series of 66 overlapping heptapeptides covering the complete amino acid sequences of the heavy subunits (beta chains) of the two Lathyrus ochrus isolectins (LoLI and LoLII) were synthesised and coupled to bovine serum albumin used as a carrier protein, and the conjugates were allowed to react with polyclonal antibodies directed against the whole isolectins. A similar approach was previously carried out with a series of 21 peptides corresponding to the light subunits (alpha chains) of the two isolectins. Of this total of 87 conjugates, 54 reacted significantly with the polyclonal antibodies. They corresponded to 8 linear peptides laid out along the entire sequences of both subunits, most of them being localised along the heavy subunits. All these continuous/sequential epitopes fall into regions predicted to be putative determinants, according to their hydrophilicity, flexibility and solvent accessibility.
Assuntos
Epitopos , Lectinas/imunologia , Lectinas de Plantas , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Soros Imunes , Dados de Sequência Molecular , Peptídeos/síntese química , CoelhosRESUMO
1. In the search for kappa-opioid antagonists, we have designed two N,N-diallyl substituted analogues of the kappa-selective peptide [D-Pro10]dynorphin A (1-11)(DPDYN). In this study, we have examined (i) the binding properties of N,N-diallyl-DPDYN (analogue 1) and N,N-diallyl-[Aib2,3]DPDYN (analogue 2) at the three main types (mu, delta, kappa) of opioid binding sites, (ii) their binding sensitivity to Na+ ions (120 mM NaCl) and guanine nucleotide (50 microM Gpp(NH)p) at mu- and kappa-binding sites and (iii) their biological activity in two pharmacological bioassays specific for mu- and kappa-(guinea-pig ileum) and kappa-(rabbit vas deferens) opioid receptors. 2. Steric hindrance resulting from incorporation of two bulky allyl groups at the tyrosal nitrogen atom greatly altered the binding properties of DPDYN. A dramatic fall in apparent affinity for the three types (mu, delta, kappa) of site as well as selectivity for kappa-sites was observed for the two N,N-diallyl-substituted peptide analogues. 3. At kappa-sites of guinea-pig cerebellum and mu-sites of rabbit cerebellum, N,N-diallyl-substitution led to a complete loss of binding sensitivity to the inhibitory effect of 120 mM NaCl + 50 microM Gpp(NH)p compared to the high sensitivity of DPDYN. This may therefore suggest that the N,N-diallyl-DPDYN analogues are endowed with opioid antagonist properties. 4. No agonist activity of the analogues was observed in guinea-pig myenteric plexus and rabbit vas deferens organ preparations. In contrast, both of the diallyl-substituted peptides displayed similar antagonist properties against the kappa-agonist DPDYN in both preparations. In the guinea-pig ileum, the affinities of the antagonist peptides against the mu-agonist Tyr-D-Ala-Gly-MePhe- NH(CH2)20H(DAGOL) were approximately half that observed against DPDYN. 5. These results show that N,N-diallyl-tyrosyl substitution leads to analogues of DPDYN which act in vitro as pure opioid antagonists and exhibit a reasonable affinity at, but a weak selectivity for, the K-opioid receptors.
Assuntos
Dinorfinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores Opioides/efeitos dos fármacos , Tirosina/farmacologia , Compostos Alílicos/metabolismo , Compostos Alílicos/farmacologia , Animais , Dinorfinas/metabolismo , Guanilil Imidodifosfato/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Fragmentos de Peptídeos/metabolismo , Coelhos , Ratos , Receptores Opioides/metabolismo , Receptores Opioides delta , Receptores Opioides kappa , Receptores Opioides mu , Sódio/farmacologia , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.
Assuntos
Oligopeptídeos/metabolismo , Receptores de Neuropeptídeos/metabolismo , Equorina , Animais , Ligação Competitiva , Células CHO , Cálcio/metabolismo , Clonagem Molecular , Colforsina/antagonistas & inibidores , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Toxina Pertussis , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/genética , Especificidade por Substrato , Termodinâmica , Fatores de Virulência de Bordetella/farmacologiaRESUMO
[125I]17alpha-hydroxy-20alpha-yohimban-16beta-(N-4-p6 hydroxyphenethyl)carboxamide or [125I]rauwolscine-OHPC, a new radioiodinated probe derived from rauwolscine was synthesized and its binding characteristics investigated on sections of the mouse caudate putamen. [125I]rauwolscine-OHPC binding was saturable and revealed interaction with a single class of binding sites (KD= 0.171 nM, Bmax = 3082 pCi/mg of tissue). The kinetically derived affinity was in close agreement with the affinity evaluated by saturation experiments: k(-1)/k(+1)(0.0403 min(-1)/114 10(6) M(-1) min(-1))=0.35 nM. Competition studies revealed interaction with one single class of binding sites for each of the twelve compounds tested. The rank of potency suggested an interaction with alpha2 adrenoceptors (atipamezole > or = RX 821002 > yohimbine > (-)epinephrine). Moreover, the good affinity of [125I] rauwolscine-OHPC binding sites for spiroxatrine, yohimbine, WB 4101, the relatively good affinity for prazosin (Ki =37.4 nM) and the affinity ratio prazosin/oxymetazoline (37.4/43.4=0.86) were consistent with an alpha2C selective labelling of [125I]rauwolscine-OHPC. The distribution of [125I]rauwolscine-OHPC binding sites in mouse brain was characterized by autoradiography. The density of binding sites was high in the islands of Calleja, accumbens nucleus, caudate putamen and olfactory tubercles, moderate in the hippocampus, amygdala and anterodorsal nucleus of the thalamus. These findings demonstrated that [125I]rauwolscine-OHPC is a useful radioiodinated probe to label alpha2C adrenoceptors in mouse brain.
Assuntos
Encéfalo/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Ioimbina/análogos & derivados , Animais , Autorradiografia , Sítios de Ligação , Ligação Competitiva , Núcleo Caudado/metabolismo , Epinefrina/farmacologia , Radioisótopos do Iodo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Especificidade de Órgãos , Oximetazolina/farmacologia , Prazosina/farmacologia , Putamen/metabolismo , Receptores Adrenérgicos alfa 2/análise , Ioimbina/síntese química , Ioimbina/farmacocinética , Ioimbina/farmacologiaRESUMO
The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.