RESUMEN
The effects of the non-peptide NK1 receptor antagonists, CP 96,345 and RP-67,580, were investigated in a model using anaesthetized pigs. Both the blood flow in the internal maxillary and the bronchial artery (ultrasonic flowmetry) and the superficial blood flow in nasal mucosa and the skin (laser-Doppler flowmetry) were monitored simultaneously. Vasodilation induced by substance P administered i.v. systemically was blocked by pretreatment with CP-96,345, 3 mg kg-1 but not by RP-67,580. CP-96,345 had no effects on the vasodilation induced by calcitonin gene-related peptide or vasoactive intestinal polypeptide. The capsaicin-induced vasodilation in the superficial blood flow of the nasal mucosa and the skin, was reduced after the CP-96,345 pretreatment. The vasodilation induced by capsaicin infusion in the internal maxillary or the bronchial artery was not affected by the CP-96,345 pretreatment. Electrical stimulation of the vagal nerve induced a vasodilation in the bronchial circulation which was not attenuated by pretreatment with CP-96,345. In the nasal mucosa and the skin NK1 receptors seem to be involved in the vasodilation in the superficial small vessels, due to chemical activation of sensory C-fibre afferents. Furthermore, CP-96,345 is a useful tool in the evaluation of NK1 receptor-mediated responses. RP-67,580 which has been shown to have NK1 antagonistic properties in the rat has no such effects in the domestic pig.
Asunto(s)
Arterias Bronquiales/fisiología , Mucosa Nasal/irrigación sanguínea , Fibras Nerviosas/fisiología , Antagonistas del Receptor de Neuroquinina-1 , Neuronas Aferentes/fisiología , Piel/irrigación sanguínea , Vasodilatación/efectos de los fármacos , Vías Aferentes/fisiología , Animales , Compuestos de Bifenilo/farmacología , Arterias Bronquiales/efectos de los fármacos , Estimulación Eléctrica , Indoles/farmacología , Isoindoles , Flujometría por Láser-Doppler , Especificidad de Órganos , Flujo Sanguíneo Regional/efectos de los fármacos , Sustancia P/antagonistas & inhibidores , Sustancia P/farmacología , Porcinos , Vasodilatación/fisiologíaAsunto(s)
Compuestos de Bifenilo/farmacología , Bronquios/fisiología , Indoles/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Neuronas Aferentes/fisiología , Nervios Periféricos/fisiología , Sustancia P/farmacología , Uréter/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Bronquios/efectos de los fármacos , Capsaicina/farmacología , Estimulación Eléctrica , Guanetidina/farmacología , Cobayas , Isoindoles , Nervios Periféricos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/fisiología , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Sustancia P/antagonistas & inhibidores , Uréter/efectos de los fármacosAsunto(s)
Presión Sanguínea/efectos de los fármacos , Broncoconstricción/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/farmacología , Capsaicina/análogos & derivados , Capsaicina/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Receptores de Péptido Relacionado con el Gen de Calcitonina/fisiología , Receptores de Taquicininas/fisiología , Animales , Compuestos de Bifenilo/farmacología , Proteínas Sanguíneas/metabolismo , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Femenino , Glicopéptidos/farmacología , Cobayas , Masculino , Metaloendopeptidasas/antagonistas & inhibidores , Receptores de Taquicininas/antagonistas & inhibidoresAsunto(s)
Péptido Relacionado con Gen de Calcitonina/fisiología , Piel/irrigación sanguínea , Sustancia P/fisiología , Vasodilatación/fisiología , Animales , Péptido Relacionado con Gen de Calcitonina/antagonistas & inhibidores , Péptido Relacionado con Gen de Calcitonina/farmacología , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Indoles/farmacología , Isoindoles , Masculino , Neuronas Aferentes/fisiología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Piel/inervación , Fenómenos Fisiológicos de la Piel , Sustancia P/antagonistas & inhibidores , Vasodilatación/efectos de los fármacosRESUMEN
The effects of the non-peptide NK1 receptor antagonist, CP-96,345, on cardiovascular homeostasis were investigated in conscious and anaesthetized rats in vivo and on heart function and muscle tonicity of vessels in vitro. CP-96,345 and its enantiomer, CP-96,344, which does not exhibit NK1 receptor-blocking activity when tested at a concentration of 1 microM, significantly decreased blood pressure in conscious rats at a dose of 0.32 mg/kg i.v. CP-96,345 and CP-96,344 additionally reduced heart rate at doses of 1 and 3.2 mg/kg, respectively. Studies in anaesthetized rats showed that ganglionic blockade did not modify the decreases in blood pressure and heart rate elicited by CP-96,345. In the isolated guinea-pig heart, CP-96,345 and CP-96,344 exerted negative chronotropic effects at 10(-7) M; negative inotropic effects were observed at 10(-6) M. At 10(-5) M, both CP-96,345 and CP-96,344 decreased the amplitude of contraction of the rat portal vein, whereas at 10(-4) M, both compounds increased the frequency of contraction of this vessel. CP-96,345, at 5 x 10(-8) M, caused relaxation of precontracted pig coronary arteries. Since both CP-96,345 and CP-96,344 produced similar changes in haemodynamics and in the contractility of vascular and cardiac tissue, the cardiovascular effects of CP-96,345 are probably not related to NK1 receptor antagonism. As only the enantiomer with NK1 antagonistic activity inhibited cigarette smoke-induced plasma protein extravasation in rat trachea, CP-96,345 remains a useful tool for elucidating NK1 receptor-mediated responses, provided CP-96,344 is included as control.
Asunto(s)
Compuestos de Bifenilo/farmacología , Músculo Liso Vascular/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Receptores de Neurotransmisores/efectos de los fármacos , Humo/efectos adversos , Sustancia P/antagonistas & inhibidores , Animales , Proteínas Sanguíneas/efectos de los fármacos , Extravasación de Materiales Terapéuticos y Diagnósticos/etiología , Cobayas , Hemodinámica/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Contracción Muscular/efectos de los fármacos , Plantas Tóxicas , Ratas , Ratas Sprague-Dawley , Receptores de Taquicininas , Porcinos , Nicotiana , Tráquea/efectos de los fármacosRESUMEN
CGRP is released from capsaicin-sensitive sensory neurons in a Ca(2+)-dependent manner in a variety of peripheral organs as well as from central terminals. The mechanisms for CGRP release by low concentrations of capsaicin, electrical antidromic nerve stimulation, and bradykinin have several similar characteristics regarding sensitivity to TTX, CTX, and alpha 2-adrenoceptor activation. High capsaicin concentration and nicotine evoke CGRP release via other mechanisms. The effects of capsaicin, resiniferatoxin, and SO2 are blocked by RR, which probably inhibits ion fluxes associated with capsaicin receptor activation. CGRP released upon irritation of peripheral branches of primary afferents may evoke a variety of cardiovascular actions and influence motility in the gastrointestinal and urogenital tracts.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Neuronas Aferentes/metabolismo , Animales , Capsaicina/farmacología , Neuronas Aferentes/efectos de los fármacos , Nicotina/farmacologíaRESUMEN
In the present study, dactinomycin (10(-5) M) inhibited the non-adrenergic, non-cholinergic bronchoconstriction upon antidromic vagal nerve stimulation (1 Hz for 1 min) in the isolated perfused guinea-pig lung by 84%. The release of calcitonin gene-related peptide was unchanged, however, suggesting a postjunctional action. Dactinomycin (10(-5), 5 x 10(-5) M) also reduced non-adrenergic non-cholinergic bronchial contractions (maximally by 75%) induced by electrical field stimulation or capsaicin, while the cholinergic component and non-adrenergic non-cholinergic relaxation remained intact. The neurokinin-2 receptor antagonist L-659,877 (10(-6) M) had a similar effect as dactinomycin, inhibiting the non-adrenergic non-cholinergic bronchial contractions by 69%, while the neurokinin-1 receptor antagonist CP-96,345 (10(-6) M) had no effect. The bronchoconstriction evoked by neurokinin A, the selective neurokinin-2 receptor agonist Nle10neurokinin A (4-10) and capsaicin was markedly inhibited by dactinomycin while the contraction induced by substance P (SP), the selective neurokinin-1 receptor agonist Sar9Met(O2)11SP, endothelin-1 and acetylcholine was not affected. In autoradiographic experiments on guinea-pig lung, [125I]neurokinin A-labelled sections showed dense binding in the bronchial smooth muscle layer. Dactinomycin inhibited the specific binding of [125I]neurokinin A in a concentration-dependent manner (IC50 = 6.3 x 10(-6) M) and 66% of [125I]neurokinin A total binding was inhibited by 10(-4) M dactinomycin. In the rat colon, [125I]neurokinin A binding to neurokinin-2 sites on circular smooth muscle was inhibited by dactinomycin with an IC50 value of 7.9 x 10(-6) M. Dactinomycin failed to reduce increased nerve-evoked contractions or those caused by Nle10neurokinin A (4-10) per se in the rat vas deferens, which are considered to be mediated by neurokinin-2 receptor activation. In the rat portal vein, dactinomycin did not influence the contractions caused by the neurokinin-3 selective agonist Pro7neurokinin B. In conclusion, dactinomycin selectively inhibited neurokinin-2 receptor activation in guinea-pig lung and rat colon, but not in rat vas deferens, which may depend on the existence of different neurokinin-2 receptor subtypes. Neurokinin A is most likely the main endogenous excitatory non-adrenergic non-cholinergic transmitter in guinea-pig bronchi.
Asunto(s)
Broncoconstricción/efectos de los fármacos , Dactinomicina/farmacología , Neuroquinina A/metabolismo , Acetilcolina/farmacología , Animales , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Broncoconstricción/fisiología , Estimulación Eléctrica , Endotelinas/farmacología , Femenino , Cobayas , Técnicas In Vitro , Masculino , Neuroquinina A/farmacología , Receptores de Neuroquinina-2 , Receptores de Neurotransmisores/antagonistas & inhibidores , Nervio Vago/fisiologíaRESUMEN
Dactinomycin, a peptide antibiotic from Streptomyces spp., is a classical agent which inhibits DNA replication. In the present study, dactinomycin inhibited specific [125I]NKA binding in rat colon membranes (KI = 1.05 x 10(-5) M) in a competitive manner. Furthermore, dactinomycin caused a parallel rightward shift of the concentration-response curve for the contractions in the rat colon induced by the NK-2 receptor agonist [Nle10]-NKA(4-10). A selective inhibition of NK-2 receptors by dactinomycin was supported by the absence of inhibition of NK-1 receptors activation in guinea pig vas deferens and of NK-3 receptors in rat portal vein. The structural similarity of the cyclic peptide moieties of dactinomycin to L-659,877, a known NK-2 receptor antagonist, can probably account for the present observations.
Asunto(s)
Colon/fisiología , Dactinomicina/farmacología , Músculo Liso Vascular/fisiología , Músculo Liso/fisiología , Neuroquinina A/metabolismo , Receptores de Neurotransmisores/fisiología , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Femenino , Cobayas , Técnicas In Vitro , Cinética , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Neuroquinina A/farmacología , Vena Porta/efectos de los fármacos , Vena Porta/fisiología , Ratas , Ratas Endogámicas , Receptores de Neuroquinina-2 , Receptores de Neurotransmisores/antagonistas & inhibidores , Receptores de Neurotransmisores/efectos de los fármacos , Taquicininas/farmacología , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiologíaAsunto(s)
Compuestos de Bifenilo/uso terapéutico , Edema Pulmonar/prevención & control , Receptores de Neurotransmisores/antagonistas & inhibidores , Fumar/efectos adversos , Animales , Proteínas Sanguíneas/metabolismo , Azul de Evans , Masculino , Edema Pulmonar/etiología , Ratas , Ratas Endogámicas , Receptores de Neuroquinina-2RESUMEN
The peptidase-resistance and bioavailability of BUBU [H-Tyr-D.Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)-OH], a highly selective and potent agonist of the delta opioid receptor, have been investigated in vitro and in vivo. In vitro at 37 degrees C, the peptide was fully resistant to degradation by rat serum and strongly resistant to degradation by rat brain membranes. In vivo 0.065% of the dose of [3H]BUBU injected intravenously to the mouse was present 15 min later in the brain. The percentage determined for [3H]DAGO [H-Tyr-D.Ala-Gly-(NMe)Phe-Gly-ol], a selective ligand for mu sites, was 0.038%. Specific binding to mouse brain membranes, determined after intracerebroventricular injection of [3H]BUBU, was saturable and a high affinity (KDapp = 25 pmol) was evaluated for the delta-agonist. Competition experiments showed that BUBU is a selective ligand for delta receptors in vivo. Comparison of the analgesic potency (hot plate test) of ICV or IV administered increasing doses of BUBU and DAGO with their in vivo binding properties supports the preferential involvement of mu receptors in supraspinal analgesia. BUBU also induced an increase in spontaneous locomotion after IV administration at a dose lower than that which produced analgesia. The quantitative results obtained in the present study demonstrate that BUBU and DAGO could be used to characterize the pharmacological responses induced by selective stimulation of delta and mu receptors after systemic administration.
Asunto(s)
Encéfalo/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides/fisiología , Analgésicos , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Metionina/metabolismo , Encefalinas/metabolismo , Inyecciones Intravenosas , Inyecciones Intraventriculares , Cinética , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Oligopéptidos/sangre , Oligopéptidos/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta , Receptores Opioides muRESUMEN
The binding of [3H]Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET), selective agonists for mu- and delta-opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris-HCl, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs-HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20-microns tissue slices were used, whatever the brain area studied. The dissociation rate of [3H]DTLET was clearly dependent on the tissue preparation used, because the koff value of this ligand in Krebs-HEPES was 2.5-fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs-HEPES was 6.5-fold greater than that measured in Tris-HCl. In intact cells, the koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs-HEPES and similar to that observed in membrane preparations in Tris-HCl supplemented with 30 mM NaCl. These data suggest (a) that the koff constant of [3H]DTLET was regulated by the ionic environment of the delta-opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.
Asunto(s)
Encéfalo/metabolismo , Receptores Opioides/metabolismo , Animales , Autorradiografía , Sitios de Unión , Tampones (Química) , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , HEPES , Soluciones Isotónicas , Cinética , Oligopéptidos/metabolismo , Ratas , Ratas Endogámicas , Receptores Opioides delta , Receptores Opioides mu , TrometaminaRESUMEN
The binding properties of [(3)H]DSTBULET, a new potent and specific ligand for ?-opioid binding sites, were characterized using slices from rat brain striatum. Specific [(3)H]DSTBULET binding reached steady-state within 60 min at 20 degrees C, was saturable and reversible and the ligand appeared to interact with a single class of binding sites, characterized as ? by competition experiments. Scatchard analysis of the saturation isotherm plots yielded a K(D) value of 5.73 nM. At this concentration the specific binding was 94% of the total binding. Unlabeled DSTBULET had a low affinity (K(1) = 422 nM) for ? binding sites labeled by [(3)H]DAGO and at 5 nM [(3)H]DSTBULET interacted with a maximum of 1.2% of total ? sites. [(3)H]DSTBULET was therefore used for a detailed quantitative radioautographic analysis of the distribution of rat brain ?-opioid binding sites in order to clarify some inconsistencies in the relative densities of these sites previously reported using either various non specific ligands or [(3)H]DPDPE, a cyclic enkephalin endowed with high ? selectivity but relatively low affinity. In addition to the claustrum, basolateral and posteromedial cortical amygdaloid nuclei, nucleus accumbens ahd caudate putamen, regions well-known to contain very high levels of ? sites, a [(3)H]DSTBULET labeling was also found in the intermediodorsal, rhomboid and mediodorsal (lat.) thalamic nuclei, cortex frontoparietal (layers II + III and V + VI), ventroposterior (parvocellular) and reuniens thalamic nuclei, and anterior olfactory nucleus (posterior part). In the thalamus, the mediodorsal, central medial and centrolateral nuclei and the posterior thalamic nucleus group were moderately labeled. The superior colliculus (superficial gray layer), the interpeduncular nucleus, the stria terminalis and the nucleus solitary tractus were moderately labeled. Lower labeling was found in the medial habenula, ventromedial, laterodorsal and ventroposterior (median) thalamic nuclei, dentate gyrus and the pyramidal cell layer of the hippocampus. In the globus pallidus, ventromedial hypothalamic nucleus, ventroposterior (lateralis) thalamic nucleus, substantia nigra, ventral tegmental area, periaqueductal gray matter and dorsal raphe the labeling was scarce but significant. At all levels in the spinal cord, a low labeling was found, restricted to the substantia gelatinosa. Interestingly ?-opioid receptors are highly concentrated in the more recently developed areas of the brain (cortex, neostriatum), where they could be involved in modulation of integrational processes mainly through regulation of aminergic (especially dopamine) pathways. Therefore, owing to its high affinity and good selectivity, [(3)H]DSTBULET seems to be the most appropriate radiolabeled probe currently available to investigate the ?-opioid receptors and to quantify these binding sites by radioautography with a high sensitivity and accuracy. Accordingly, labeling was found in several areas (ventral tegmental area, substantia nigra, nucleus tractus solitarius) where a mismatch between the site of action of ?-opioids and the presence of ? sites has been previously hypothesized.
RESUMEN
The possible changes in neutral endopeptidase EC 3.4.24.11 ("enkephalinase", NEP), mu and delta opioid binding sites, were investigated using in vitro quantitative radioautography in various regions of the central nervous system of the Freund's adjuvant-induced arthritic rat, a model of chronic pain. Enkephalinase was labeled by a specific tritiated inhibitor, [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly), while mu and delta opioid binding sites were selectively labelled with [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLFT), respectively. As compared to controls, no significant modifications were found in NEP, mu or delta binding sites at both supraspinal and spinal levels of arthritic rats. These results suggest that the enhanced efficiency of exogenous opioids or endogenous enkephalins, reported to occur in this model of chronic inflammatory pain, are not directly related to changes in mu and delta opioid binding sites or steady state levels of NEP.
Asunto(s)
Artritis Experimental/metabolismo , Artritis/metabolismo , Encéfalo/metabolismo , Neprilisina/metabolismo , Receptores Opioides/metabolismo , Médula Espinal/metabolismo , Animales , Artritis Experimental/enzimología , Autorradiografía , Densitometría , Masculino , Ratas , Ratas Endogámicas , Receptores Opioides delta , Receptores Opioides muRESUMEN
The selectivity and potency of two new enkephalin-derived delta-opioid receptor agonists, DSTBULET ([D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6) and BUBU ([D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6(O-t-butyl] were determined with functional tests in vitro of mu-, delta- and kappa-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 microM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD2 7.6-7.9), an effect exclusively mediated by delta-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible delta-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 microM, the peptides did not affect striatal [3H]dopamine (DA) release nor cortical [3H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating kappa and mu-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD2 8.2-8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by mu- and/or delta-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only mu-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional delta-opioid receptors in the brain.
Asunto(s)
Adenilil Ciclasas/metabolismo , Encéfalo/enzimología , Encefalinas/farmacología , Isotiocianatos , Neurotransmisores/metabolismo , Oligopéptidos/farmacología , Alquilantes/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Dopamina/metabolismo , Estimulación Eléctrica , Fentanilo/análogos & derivados , Fentanilo/farmacología , Masculino , Ratas , Ratas EndogámicasRESUMEN
The in vivo binding properties of cerebral mu and delta opioid receptors were investigated in mice after the intrastriatal injection of [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or [3H][D-Thr2,Leu5]enkephalyl-Thr (DTLET). Both peptides exhibited similar diffusion kinetics in the brain and 30-40% of [3H]DAGO or [3H]DTLET was shown to be present in the tissue 15 min after injection when maximal binding was observed. The specific binding of both agonists, defined as the fraction of the radioactivity bound to brain membranes which was displaced by 10 nmol of cold ligand, was reversible, saturable and displayed a pharmacological profile similar to that found in in vitro experiments. At doses producing a similar analgesic effect in the hot-plate test in mice, DTLET occupied 64% of delta sites and DAGO 15% of mu sites. However, because of the residual cross-reactivity of DTLET for mu sites, it appeared that both ligands occupied a similar number of mu receptors at their ED50 values, thus supporting a preferential involvement of mu opioid binding sites in the supraspinal pain control. [Met5]enkephalin inhibited the in vivo binding of both agonists only when the peptide was protected from degradation by the co-administration of a mixed inhibitor of enkephalin degrading enzymes RB38A (N[3(R)(hydroxyaminocarbonyl)-2-benzyl-1-oxopropyl]- L-phenylalanine). Unlike thiorphan, 5 nmol RB38A alone was able to inhibit [3H]DAGO binding by 60%. This result is the first direct demonstration of the existence of an in vivo tonic control of mu opioid receptor occupation by endogenous opioid peptides.
Asunto(s)
Encefalinas/metabolismo , Receptores Opioides/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cuerpo Estriado , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/efectos adversos , Encefalinas/farmacología , Ácidos Hidroxámicos/metabolismo , Técnicas In Vitro , Inyecciones , Cinética , Masculino , Ratones , Morfina/farmacología , Oligopéptidos/efectos adversos , Oligopéptidos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Receptores Opioides delta , Receptores Opioides mu , Tiorfan/farmacologíaRESUMEN
Neutral endopeptidase (NEP) and mu- and delta-opioid receptor densities were measured in sections of rat striatum by in vitro radioautography with the selective ligands: 3 nM [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([ 3H]HACBO-Gly), 3 nM [3H]Tyr-D.Ala-Gly-(NMe)Phe-Gly-ol ([ 3H]DAGO) and 3 nM [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([ 3H]DTLET), respectively. Haloperidol treatment (2 mg/kg/day, i.p., 3 weeks), which has been reported to increase enkephalin levels in the striatum, induced a 23% decrease in striatal (posterior level A= +8.4-8.6 mm) NEP labeling (but no change of mu- and delta-sites). In contrast, no change in NEP occurred after chronic morphine (40-160 mg/kg/day, s.c., 4 days) or kelatorphan (10 nmol/h, i.c.v., 7 days), a mixed inhibitor of enkephalin-degrading peptidases.
Asunto(s)
Cuerpo Estriado/enzimología , Haloperidol/farmacología , Neprilisina/metabolismo , Receptores Opioides/metabolismo , Animales , Autorradiografía , Cuerpo Estriado/efectos de los fármacos , Dipéptidos/farmacología , Masculino , Morfina/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacosRESUMEN
A series of linear conformationally constrained opioid peptides was designed in an attempt to develop highly selective and potent agonists for the delta opioid receptors. These enkephalin analogues corresponding to the general formula Tyr-D-X(OY)-Gly-Phe-Leu-Thr(OZ) were obtained by incorporating bulky residues (X = Ser or Thr; Y = tert-butyl or benzyl; Z = tert-butyl) into the sequence of the previously reported delta specific agonists DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr). In binding studies based on displacement of mu and delta opioid receptor selective radiolabeled ligands from rat brain membranes, the two constrained hexapeptides, Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr (1, DSTBULET) (KI(mu) = 374 nM, Kr(delta) = 6.14 nM, KI(delta)/KI(mu) = 0.016) and in particular Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr(O-t-Bu) (7, BUBU) (KI(mu) = 475 nM, KI(delta) = 4.68 nM, KI(delta)/KI(mu) = 0.010) were shown to be among the most potent and selective delta probes reported to date. A roughly similar pattern of selectivity was obtained with the guinea pig ileum and mouse vas deferens bioassays. In addition, the analgesic potency (hot-plate test) of these peptides intracerebroventricularly administered in mice was shown to be significantly related to their mu-receptor affinity.
Asunto(s)
Encefalinas/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Animales , Unión Competitiva , Encéfalo/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Ratones , Oligopéptidos/síntesis química , Conformación Proteica , Receptores Opioides deltaRESUMEN
Insertion of bulky tertiobutyl groups into the sequence of [D-Ser2,Leu5]enkephalyl-Thr6 leads to a conformationally induced large increase in selectivity toward rat brain delta-opioid binding sites, as shown by the ratio of apparent affinities for mu and delta receptors of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6,KI(mu)/KI(delta) = 130, and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (O-tert-butyl),KI(mu)/KI(delta) = 280. In addition to a selectivity similar to that of the cyclic compounds [D-Pen2, D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin, the affinity of [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 for the delta sites of rat brain membranes is significantly better (KD = 2.2 nM) than that of [3H][D-Pen2,D-Pen5]enkephalin (KD approximately 8.5 nM). Therefore, [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 seems to be the most appropriate delta-probe currently available for binding studies. Moreover, the lipophilic and protected peptide [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl) behaves as the most specific ligand for the delta-opioid binding sites and appears appropriate for in vivo investigations. The inactive analogue [D-Thr2(O-tert-butyl),Leu5]enkephalyl-Thr6 might serve as a negative control in biochemical or pharmacological studies.
Asunto(s)
Encefalinas/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Animales , Encéfalo/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalina Metionina/metabolismo , Cobayas , Cinética , Membranas/metabolismo , Ratas , Ratas Endogámicas , Receptores Opioides deltaRESUMEN
The previous rules proposed for selective recognition of mu and delta opioid receptors by modified enkephalins were investigated through an extensive structure-activity study. Thus, modifications of the sequence of TRIMU 4 (Tyr-D-Ala-Gly-NHCH(CH3)CH2CH(CH3)2, a peptide that exhibits mu selectivity close to that of DAGO (Try-D-Ala-Gly-N(Me)Phe-Gly.ol), were performed for two positions, 2 and 4, critical for mu recognition. The drastic loss of potency following introduction of L-Ala or Aib in position 2 emphasizes the importance of the stereochemistry and the steric size of the X2 amino acid for optimal mu binding. The enhancement of the intrinsic flexibility of the C-terminal alkyl chain of TRIMU 4 through removal of a methyl group leads to TRIMU 5 (Tyr-D-Ala-Gly-NHCH2CH2CH(CH3)2), a peptide with a mu selectivity similar to that of DAGO. In contrast, introduction of an O-tert-butyl Ser2 residue increases affinity for delta receptors. In the hexapeptide series derived from DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a D-Thr2 moiety was shown to be very efficient in improving delta recognition and delta selectivity appeared also to be modulated by the nature of the sixth residue. The potencies of the 24 peptides studied to inhibit the electrically evoked contractions of the GPI or MVD are relatively well correlated with their affinities for brain mu or delta receptors labeled with [3H]DAGO or [3H]DSLET, respectively. Moreover, the analgesic potency (hot plate test) of the peptides is related to their affinity for rat brain mu receptors. The wide range of receptor affinities exhibited by the compounds reported here could be useful to study the physiological role of mu and delta receptors.
Asunto(s)
Encefalina Leucina/análogos & derivados , Encefalinas/farmacología , Receptores Opioides/efectos de los fármacos , Analgésicos/farmacología , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Cobayas , Ratones , Contracción Muscular/efectos de los fármacos , Oligopéptidos/metabolismo , Receptores Opioides delta , Receptores Opioides mu , Relación Estructura-ActividadRESUMEN
The cellular localization of the rat brain neutral endopeptidase (NEP, EC 3.4.24.11) was investigated by quantitative autoradiography of the enzyme inhibitor [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly) after lesions of the striatum, nigrostriatal and corticostriatal pathways. The effect of these lesions on NEP levels was compared with that on delta and mu opioid receptors, selectively labeled with [3H]Tyr-D-Thr-Gly-Leu-Thr ([3H]DTLET) and [3H]Tyr-D-Ala-Gly-MePhe-Glycinol ([3H]DAGO), respectively. Twenty-one days after injection of kainate in the caudate putamen (CP), the NEP level was locally decreased (52%) but the time course of this decrease was different from that of mu and delta opioid receptors: [3H]DAGO binding was diminished by 40% from day 2 whereas that of [3H]DTLET was reduced by 51% from day 7. Kainic acid injection in the CP induced in the globus pallidus (GP) and substantia nigra (SN) a distant reduction of the 3 opioid markers. Likewise after injection of colchicine in the CP, [3H]HACBO-Gly binding was decreased in the GP (60%) and SN (58%), [3H]DTLET binding was reduced by 54 and 55% in the GP and SN, respectively and [3H]DAGO labeling was diminished by 49% in the GP, and 58% in the SN. Finally, lesion of the nigrostriatal dopamine pathway by 6-hydroxydopamine did not induce any change of NEP level in the CP and GP whereas delta and mu opioid receptor levels were diminished respectively by 25 and 29% in the CP, and 45 and 39% in the GP, a new finding of the present study. Taken together these data suggest that NEP is in part associated with striatal intrinsic neurons. In the GP and SN, a large part of NEP seems to be presynaptically associated with nerve terminals endowed with mu and delta opioid receptors, which originate from efferent striatal neurons. In contrast to opioid receptors in the CP, the NEP appears not to be associated with dopaminergic nerve terminals originating from the SN. Cortical ablation did not affect any of the opioid markers.