RESUMEN
The effect of lovastatin, an inhibitor of 3-hydroxymethyl-3-glutaryl coenzyme A (HMG CoA) reductase, was examined on human vascular smooth muscle cells (HVSMC). Untransformed HVSMC were obtained from saphenous vein and in addition an SV-40 transformed immortalized cell line (HVTs-SM1) derived from saphenous vein smooth muscle was also used. HVTs-SM1 cell proliferation and DNA synthesis were measured, and cell cycle analysis was performed by flow cytometry. Apoptosis in both cell types was assessed by a combination of flow cytometry, terminal deoxynucleotidyl transferase (TUNEL) reagent-based immunocytochemistry, DAPI staining, and DNA agarose gel electrophoresis. Lovastatin had no effect on apoptosis of HVSMC over 96 h in serum-free conditions or after stimulation with platelet-derived growth factor (PDGF-BB), although PDGF-BB increased apoptosis in HVSMC, and this was prevented by lovastatin. In HVTs-SM1 cells lovastatin inhibited cell proliferation and DNA synthesis and induced apoptosis in a time- and concentration-dependent manner. The effects of lovastatin on cell proliferation, DNA synthesis, and apoptosis were prevented by coincubation with mevalonate and geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate. Lovastatin does not induce apoptosis in saphenous vein HVSMC in culture and inhibits PDGF-BB-induced DNA synthesis and apoptosis. In contrast, in SV40 transformed immortalized HVTs-SM1 cells, lovastatin induces apoptosis and inhibits cell proliferation and DNA synthesis. The pro-apoptotic effects of lovastatin in SV40 transformed HVTs-SM1 cells may be related to the enhanced rate of proliferation or deregulation of the cell cycle in this cell line.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Vena Safena/citología , Virus 40 de los Simios/fisiología , División Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Relación Dosis-Respuesta a Droga , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimologíaRESUMEN
Using an in vitro model of a conditionally immortalized cell line, we have investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein-derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive-temperature conditions (36 degrees C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or UV irradiation. Downregulation of LT antigen expression at a nonpermissive temperature (39 degrees C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21(WAF1/CIP1), increased murine double minute-2 promoter activity, and differential expression of murine double minute-2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cycle control but not necessarily in apoptosis. The established SMC line HVTs-SM1 may be a useful model for study of the processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.
Asunto(s)
Apoptosis , División Celular , Ciclinas/fisiología , Músculo Liso Vascular/citología , Proteínas Nucleares , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Antígenos Transformadores de Poliomavirus/análisis , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Fragmentación del ADN , Expresión Génica , Humanos , Mutación , Proteínas Proto-Oncogénicas c-mdm2 , Vena Safena , Transfección , Rayos UltravioletaRESUMEN
Using an in vitro model of a conditionally immortalized cell line, we investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators, such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein-derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive (ts) mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive temperature conditions (36 degrees C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT-antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or by UV irradiation. Downregulation of LT-antigen expression at the nonpermissive temperature (39 degrees C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21(WAF1/CIP1), increased MDM2-promoter activity, and differential expression of MDM2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cell cycle control but not necessarily apoptosis. The established SMC line HVTs-SM1 may be a useful model for the study of processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.
Asunto(s)
Apoptosis/fisiología , Ciclinas/metabolismo , Músculo Liso Vascular/enzimología , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Actinas/análisis , Antígenos Transformadores de Poliomavirus/análisis , Antígenos Transformadores de Poliomavirus/genética , Western Blotting , División Celular/fisiología , Línea Celular Transformada/citología , Línea Celular Transformada/enzimología , Línea Celular Transformada/efectos de la radiación , Supervivencia Celular/fisiología , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/análisis , Ciclinas/genética , Citoesqueleto/química , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Humanos , Técnicas In Vitro , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Cadenas Pesadas de Miosina/análisis , Fenotipo , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Vena Safena/citología , Virus 40 de los Simios/genética , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta , Vimentina/análisisRESUMEN
Vasoactive peptides like thrombin, angiotensin II and endothelin induce vascular smooth muscle cell (VSMC) contraction via activation of G-protein-coupled receptors. Recent studies have shown that they also induce VSMC migration and proliferation, processes which are important in the remodelling of the vasculature during embryogenesis and in the response to vascular injury. G-protein-coupled receptor-mediated mitogenic signals appear to be transmitted via a number of intracellular mechanisms which include proto-oncogene gene expression, G-protein-mediated protein translocation and tyrosine phosphorylation of growth factor receptor proteins, and activation of autocrine growth factor pathways. The ability of vasoactive peptides to have an impact on signalling cascades mediated by growth factor tyrosine kinase receptors may be important in the pathogenesis of diseases in the vasculature.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , División Celular/fisiología , Proteínas de Unión al GTP/fisiología , Humanos , Músculo Liso Vascular/citología , Proto-Oncogenes Mas , Receptores de Neuropéptido/fisiología , Transducción de Señal/fisiologíaRESUMEN
1. PDGF is a highly hydrophilic cationic glycoprotein (M(r) 28-35kDa) produced by platelets, monocyte/macrophages, endothelial cells and vascular smooth muscle cells under some conditions. 2. Since its original description, PDGF has attracted much attention and it is currently believed to play a role in atherosclerosis and other vascular pathologies. 3. This review describes the vascular biology of PDGF. It particularly focuses on recent findings regarding the intracellular signals activated by PDGF in the context of vascular smooth muscle cell proliferation, migration and, contraction.
Asunto(s)
Vasos Sanguíneos/fisiología , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Animales , HumanosRESUMEN
Treatment of human vascular smooth muscle cells (SMC) with human alpha-thrombin greatly increased DNA synthesis and cell proliferation. Both the integrity of the catalytic site and that of the anion binding exosite were required for expression of this activity. Experiments employing Northerns indicated induction of c-fos expression as well as a time-dependent induction of platelet-derived growth factor-A (PDGF-A) gene by thrombin. The thrombin mitogenic activity was potentiated by PDGF-BB, insulin and the vasoconstrictor peptide endothelin-1 suggesting synergism by convergence of intracellular growth-promoting signals. SMC treatment with pertussis toxin and forskolin indicated that the mitogenic activity of thrombin may be induced via signal transduction mechanism(s) involving changes in cAMP levels and activation of a Gi-like protein. These results suggest that thrombin may play a functional role in the regulation of human vascular SMC proliferation.
Asunto(s)
Regulación de la Expresión Génica , Mitógenos/farmacología , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Trombina/farmacología , Aorta/citología , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/análisis , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Endotelinas/farmacología , Humanos , Insulina/farmacología , Desarrollo de Músculos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/crecimiento & desarrollo , Toxina del Pertussis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Opioid receptors are currently classified as mu, delta, and kappa types, but various subtypes have also been proposed. We have investigated whether subtypes exist by using [3H]bremazocine. [3H]Bremazocine binds to twice as many naloxone-sensitive sites as other nonselective opioid agonists, as shown in four membrane types that have very different ratios of mu, delta, and kappa receptor types. [3H]Bremazocine binding is completely inhibited by an excess (in unlabeled form) of other opioid ligands, with Hill coefficients of 0.8-0.95. These paradoxes can be explained if there are high- and low-affinity states of the mu, delta, and kappa receptors and bremazocine binds with similar affinities to both states. We propose that these states are the guanine nucleotide-binding protein (G-protein)-coupled form and the uncoupled form of each receptor. As evidence for this proposal, the [3H]bremazocine binding suffered little or no loss with G-protein-uncoupling treatments, whereas binding of other opioid agonists was fully sensitive. We conclude that [3H]bremazocine offers a tool for the measurement of the total pools of coupled and uncoupled opioid receptors and that much of the previous characterization of opioid receptor subtypes reflects, instead, a significant pool of G-protein-uncoupled opioid receptors.
Asunto(s)
Analgésicos/metabolismo , Bencenoacetamidas , Benzomorfanos/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Opioides/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Cerebelo/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Encefalinas/metabolismo , Etorfina/metabolismo , Femenino , Cobayas , Humanos , Cinética , Ligandos , Placenta/metabolismo , Embarazo , Prosencéfalo/metabolismo , Unión Proteica , Pirrolidinas/metabolismo , Ratas , Receptores Opioides/clasificaciónRESUMEN
BACKGROUND: Vascular smooth muscle cell (VSMC) growth is responsible for intimal hyperplasia, a major cause of failure after vascular surgery and angioplasty. Heparin is the first described inhibitor of VSMC growth, but has not proved effective in the prevention of human intimal hyperplasia. Heparin is a heterogeneous substance, which may contain a mixture of components which differ in antiproliferative activity. Isolation of an active component may favourably influence its therapeutic profile. METHODS AND RESULTS: Growth of human VSMC cultured from operative specimens, assessed by cell counting and labelled thymidine incorporation, was used as a model of VSMC proliferation in intimal hyperplasia. Unfractionated (UFH) and low molecular weight (LMWH) heparins inhibit cell growth and thymidine uptake by human VSMCs in response to 15% foetal calf serum. UFHs are more active than LMWHs and this difference increases with increasing heparin dose. To confirm this effect, size-based fractions of heparin were prepared by gel permeation chromatography, and characterised by high performance liquid chromatography. High molecular weight fractions (MW > 21000) have higher activity than fractions of medium (MW 12000-21000) or low molecular weight (MW < 12000). These differences become more pronounced at higher dose, and are statistically significant at 100 micrograms/ml (Mann-Whitney, p < 0.05). CONCLUSIONS: The antiproliferative activity of heparin appears to be maximal in its high molecular weight component.
Asunto(s)
Heparina/farmacología , Músculo Liso Vascular/efectos de los fármacos , División Celular , Células Cultivadas , Constricción Patológica/patología , Relación Dosis-Respuesta a Droga , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Hiperplasia , Técnicas In Vitro , Músculo Liso Vascular/patología , Recurrencia , Túnica Íntima/patologíaRESUMEN
The guanine nucleotide analogue, 5'-p-fluorosulphonylbenzoyl guanosine (FSBG), can react covalently with GTP-binding proteins (G proteins). In rat brain membranes, FSBG causes a time-dependent loss of beta,gamma-imido[8-3H]guanosine 5'-triphosphate binding sites. Using 1 mM FSBG, the guanyl nucleotide modulation of opioid agonist binding is abolished, whereas the guanyl nucleotide sensitivity of neurotensin binding is retained. The action of FSBG can be prevented by the presence of opioid agonists, but not the antagonist naloxone. Iodoacetamide treatment of membranes in the presence of agonist, but not antagonist, can attenuate the action of FSBG in blocking guanyl nucleotide modulation of opioid agonist binding. These results suggest that FSBG covalently modifies essential thiol groups, whose exposure to the reagent is modified by agonist occupancy of the receptor, on a species of G protein linked to opioid receptors, but not on a species of G protein linked to neurotensin receptors. Thus, FSBG may have selectivity for the forms of Gi or Go, proteins associated with opioid receptors.
Asunto(s)
Marcadores de Afinidad/farmacología , Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanosina/análogos & derivados , Receptores Opioides/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Ditiotreitol/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Encefalina Leucina/farmacología , Leucina Encefalina-2-Alanina , Encefalinas/farmacología , Guanosina/farmacología , Guanilil Imidodifosfato/metabolismo , Guanilil Imidodifosfato/farmacología , Yodoacetamida/farmacología , Cinética , Datos de Secuencia Molecular , Naloxona/farmacología , Ratas , Receptores Opioides/efectos de los fármacosRESUMEN
Cholecystokinin (CCK) binding sites were solubilized from pig cerebral cortical membranes with digitonin (2%, w/v) in the presence of Na+ (120 mM) and Mg2+ (5 mM). Scatchard plot transformation of equilibrium binding data obtained with 125I-CCK-8S gave an apparent dissociation constant (Kd) of 0.6 nM, comparable to that obtained in membranes in the presence of these cations. Hill coefficients close to unity suggested the presence of a single population of receptor sites. Competitive inhibition studies with pentagastrin, gastrin(1-17)S and the CCKA receptor antagonist L-364,718 indicated that the solubilized receptor sites were of the B-type (CCKB), with the same pharmacological profile as that observed in membranes. Optimal specific binding of 125I-CCK-8S to membrane-bound and solubilized receptors was obtained in the presence of divalent cations. Both the membrane-bound and the solubilized receptor activity were attenuated by guanylyl-imidodiphosphate (Gpp(NH)p) indicating that the brain CCKB receptors are coupled to G proteins.
Asunto(s)
Corteza Cerebral/metabolismo , Receptores de Colecistoquinina/aislamiento & purificación , Animales , Unión Competitiva/efectos de los fármacos , Cationes/farmacología , Corteza Cerebral/efectos de los fármacos , Colecistoquinina/metabolismo , Cromatografía Líquida de Alta Presión , Detergentes , Digitonina , Nucleótidos de Guanina/farmacología , Técnicas In Vitro , Radioisótopos de Yodo , Cinética , Membranas/efectos de los fármacos , Membranas/metabolismo , Receptores de Colecistoquinina/metabolismo , Albúmina Sérica Bovina/metabolismo , Porcinos , Termodinámica , Factores de TiempoRESUMEN
Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Toxina del Pertussis , Receptores Opioides/metabolismo , Factores de Virulencia de Bordetella/farmacología , Animales , Cromatografía/métodos , Cromatografía de Afinidad , Digitonina , Magnesio , Membranas/metabolismo , Ratas , Solubilidad , Aglutininas del Germen de TrigoRESUMEN
Pertussis toxin-catalyzed ADP-ribosylation of the guanine nucleotide-binding proteins Gi and Go is shown to proceed in Mg2+-digitonin extracts from rat brain; the Mr 41,000 and Mr 39,000 peptides are labelled there as in the membranes. The ADP-ribosylation in detergent solution retains the differential sensitivity to guanine nucleotide analogues. This reaction also removes the partial inhibition by the guanine nucleotides of the binding of opioid agonists, as does the same treatment in the membranes. The partial inhibition of agonist binding by Na+, however, is left unchanged. The binding of the antagonist naloxone is little affected by Na+ or by guanine nucleotides in the treated membranes, but the treated soluble receptors show an enhanced binding in high-Na+ medium, although still guanine nucleotide insensitive. The data suggest that the toxin reaction in the absence of guanine nucleotides and agonist stabilizes the opioid receptor in a receptor-G-protein coupled state which is no longer sensitive to guanine nucleotides but retains its sensitivity to the Na+ ions.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Encéfalo/metabolismo , Digitonina , Guanosina Trifosfato/farmacología , Toxina del Pertussis , Receptores Opioides/metabolismo , Factores de Virulencia de Bordetella/metabolismo , Animales , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Leucina Encefalina-2-Alanina , Etorfina/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanilil Imidodifosfato/farmacología , Cinética , Naloxona/metabolismo , Oligopéptidos/metabolismo , Ratas , Receptores Opioides/efectos de los fármacos , Sodio/farmacología , SolubilidadRESUMEN
Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.
Asunto(s)
Corteza Cerebral/metabolismo , Neurotensina/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Cationes/farmacología , Bovinos , Detergentes , Estabilidad de Medicamentos , Membranas/metabolismo , Neurotensina/análogos & derivados , Solubilidad , TritioRESUMEN
The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of [3,11-tyrosyl-3,5-3H]neurotensin with an apparent affinity (Kd = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of Mr 72,000. Under nonreducing conditions the apparent Mr, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogeneous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.
Asunto(s)
Encéfalo/metabolismo , Neurotensina/metabolismo , Receptores de Neurotransmisores/metabolismo , Animales , Bovinos , Cromatografía de Afinidad , Cinética , Peso Molecular , Receptores de Neurotensina , Receptores de Neurotransmisores/aislamiento & purificaciónRESUMEN
The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of delta, kappa, mu 1, and mu 2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5'-(beta, gamma-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of "half-of-the-sites" reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous kappa-delta or mu 1-mu 2 receptor complexes is required for stabilization of the high-affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association-dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl]-methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of -SH/-SS- bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.
Asunto(s)
Encéfalo/metabolismo , Receptores Opioides/metabolismo , Sitio Alostérico , Animales , Cationes , Membrana Celular/metabolismo , Dihidromorfina/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Encefalina Leucina/farmacología , Leucina Encefalina-2-Alanina , Encefalinas/farmacología , Etorfina/metabolismo , Guanilil Imidodifosfato/farmacología , Cinética , Magnesio/farmacología , Masculino , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacos , Sodio/farmacologíaRESUMEN
Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.
Asunto(s)
Encéfalo/metabolismo , Receptores Opioides/metabolismo , Animales , Cationes , Membrana Celular/metabolismo , Fenómenos Químicos , Química Física , Cromatografía en Gel , Dihidromorfina/metabolismo , Dihidromorfina/farmacología , Dinorfinas/metabolismo , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Leucina Encefalina-2-Alanina , Etorfina/metabolismo , Guanilil Imidodifosfato/farmacología , Yodoacetamida/farmacología , Cinética , Magnesio/farmacología , Naloxona/metabolismo , Ratas , Receptores Opioides/efectos de los fármacos , Sodio/farmacología , SolucionesRESUMEN
The binding sites for opiates (agonist and antagonist) and opioid peptides can be solubilized from rat brain membranes with digitonin in the presence of Mg2+ (10 mM). High affinity and high capacity binding to the soluble delta, mu, and kappa receptors is obtainable when the membranes are treated in Mg2+ (30 degrees C, 60 min) prior to solubilization. The yields of solubilized binding sites extracted with digitonin, 40-90%, are higher than those obtained from Mg2+-pretreated membranes with other detergents commonly used for receptor solubilization. The stability of the digitonin-soluble opioid receptor at room temperature makes it useful for purification and characterization.
Asunto(s)
Química Encefálica , Receptores Opioides/aislamiento & purificación , Animales , Sitios de Unión , Membrana Celular/análisis , Precipitación Química , Detergentes , Digitonina , Estabilidad de Medicamentos , Dinorfinas , Endorfinas/metabolismo , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Leucina Encefalina-2-Alanina , Etorfina/metabolismo , Magnesio , Fragmentos de Péptidos/metabolismo , Polietilenglicoles , Ratas , Receptores Opioides/metabolismo , SolubilidadAsunto(s)
Receptores Opioides , Marcadores de Afinidad/farmacología , Animales , Sitios de Unión , Cationes/farmacología , Disulfuros , Guanosina Trifosfato/farmacología , Cinética , Ligandos , Ratas , Receptores Opioides/aislamiento & purificación , Receptores Opioides/metabolismo , Solubilidad , Compuestos de SulfhidriloRESUMEN
The concentration-dependent stimulation of adenylate cyclase by the photoaffinity reagent 2-[(2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon (glucagon-NAPS) and also its binding characteristics were compared with those of the native hormone. The derivative was found to be slightly more potent in stimulating adenylate cyclase than glucagon, in the presence of guanosine 5'-triphosphate (GTP). 125I-Labeled glucagon-NAPS or 125I-labeled glucagon bound rapidly to receptors and was competitively displaced by unlabeled glucagon or glucagon-NAPS. Glucagon-NAPS displaced bound radiolabeled hormone at a lower concentration than did glucagon in the absence of GTP. Scatchard analysis of the binding data obtained from displacement of bound radiolabeled ligand with unlabeled peptide demonstrated a heterogeneous population of saturable glucagon binding sites. Glucagon-NAPS displayed a higher affinity (0.7 nM) for the high-capacity sites (80-90% of total binding sites) than glucagon (4.0 nM) in the absence of GTP. In the presence of the nucleotide, both ligands had approximately the same affinity (0.5-0.6 nM). Hill plot analysis of the binding data suggested noncooperative interactions. Photoaffinity labeling of plasma membranes with glucagon-NAPS resulted in an irreversible activation of adenylate cyclase with a reduced response to further stimulation by glucagon, glucagon-NAPS, and NaF.