RESUMEN
Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.
Asunto(s)
Acetamidas/farmacología , Analgésicos Opioides/farmacología , Pirrolidinas/farmacología , Receptores Opioides kappa/agonistas , Acetamidas/química , Analgésicos Opioides/química , Animales , Estructura Molecular , Pirrolidinas/química , RatasRESUMEN
A proline scan at positions 2 and 3 of the opioid peptide dynorphin A(1-11)-NH(2) led to the discovery of the analogue [Pro(3)]Dyn A(1-11)-NH(2). This analogue possesses high affinity and selectivity for the kappa opioid receptor (K(i)(kappa) = 2.7 nM, K(i) ratio kappa/micro/delta = 1/2110/3260). The gain in selectivity is achieved through an overall reduction of opioid receptor affinity which is most pronounced at micro and delta receptors. The Pro(3) analogue exhibits antagonist properties. Despite its high kappa affinity, [Pro(3)]Dyn A(1-11)-NH(2) is a relatively weak antagonist in both the [(35)S]GTPgammaS assay (IC(50) = 380 nM) and the guinea pig ileum assay (K(e) = 244 nM). Discrepancies between GPI and binding assay have often been ascribed to differential kappa receptor subtypes prevailing in central vs peripheral neurons. Since the [(35)S]GTPgammaS assay uses the same membrane preparations as the binding assay, differential kappa subtypes can be ruled out as an explanation in this case, and the observed behavior rather seems to reflect an intrinsic property of the ligand.
Asunto(s)
Dinorfinas/síntesis química , Fragmentos de Péptidos/síntesis química , Receptores Opioides kappa/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Dinorfinas/química , Dinorfinas/metabolismo , Dinorfinas/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Humanos , Íleon/efectos de los fármacos , Íleon/fisiología , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
The antihyperalgesic properties of the opiate antidiarrheal agent loperamide (ADL 2-1294) were investigated in a variety of inflammatory pain models in rodents. Loperamide exhibited potent affinity and selectivity for the cloned micro (Ki = 3 nM) compared with the delta (Ki = 48 nM) and kappa (Ki = 1156 nM) human opioid receptors. Loperamide potently stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding (EC50 = 56 nM), and inhibited forskolin-stimulated cAMP accumulation (IC50 = 25 nM) in Chinese hamster ovary cells transfected with the human mu opioid receptor. The injection of 0.3 mg of loperamide into the intra-articular space of the inflamed rat knee joint resulted in potent antinociception to knee compression that was antagonized by naloxone, whereas injection into the contralateral knee joint or via the i.m. route failed to inhibit compression-induced changes in blood pressure. Loperamide potently inhibited late-phase formalin-induced flinching after intrapaw injection (A50 = 6 microgram) but was ineffective against early-phase flinching or after injection into the paw contralateral to the formalin-treated paw. Local injection of loperamide also produced antinociception against Freund's adjuvant- (ED50 = 21 microgram) or tape stripping- (ED50 = 71 microgram) induced hyperalgesia as demonstrated by increased paw pressure thresholds in the inflamed paw. In all animal models examined, the potency of loperamide after local administration was comparable to or better than that of morphine. Loperamide has potential therapeutic use as a peripherally selective opiate antihyperalgesic agent that lacks many of the side effects generally associated with administration of centrally acting opiates.
Asunto(s)
Analgésicos Opioides/farmacología , Antidiarreicos/farmacología , Hiperalgesia/tratamiento farmacológico , Loperamida/farmacología , Sistema Nervioso Periférico/efectos de los fármacos , Analgésicos Opioides/metabolismo , Animales , Antidiarreicos/metabolismo , Clonación Molecular , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Hiperalgesia/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Loperamida/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/metabolismoRESUMEN
Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.
Asunto(s)
Analgésicos Opioides/farmacología , Oligopéptidos/farmacología , Receptores Opioides mu/metabolismo , Animales , Células CHO , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Oocitos/metabolismo , Canales de Potasio/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , XenopusRESUMEN
Dynorphin A is an endogenous opioid peptide that activates the kappa opioid receptor (KOR) with high potency. Some studies also showed that the distribution and functional activity of dynorphin A are not completely correlated with those of KOR, suggesting that dynorphin A may interact with other receptors. To investigate the possibility that dynorphin A may serve as an agonist for other opioid receptors, we took the advantage of the cloning of the three major types of opioid receptors, mu (MOR), delta (DOR) and KOR, and examined their affinity for and their activation by dynorphin A. We used mammalian cells transfected with each of the cDNA clones for the human receptors hMOR, hDOR, hKOR and showed that dynorphin A displaced [3H]-diprenorphine binding with Ki values in the nanomolar range at all three receptors. We also showed that, when hMOR, hDOR or hKOR was coexpressed with a G protein-activated potassium channel in Xenopus oocytes, dynorphin A induced a potassium current with EC50 values in the nanomolar range for all three receptors. Furthermore, we showed that the human hORLI, an opioid receptor-like receptor that has been identified as a novel member of the opioid receptor gene family, displayed dynorphin A binding and functional activation. These results indicate that dynorphin A is capable of binding to and functional activation of all members of the opioid receptor family, suggesting that, as a potential endogenous agonist, its activity in humans may involve interaction with other members of the opioid receptor family in addition to kappa receptors.
Asunto(s)
Dinorfinas/metabolismo , Receptores Opioides/metabolismo , Animales , Dinorfinas/farmacología , Femenino , Humanos , Receptores Opioides/efectos de los fármacos , Receptores Opioides/genética , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Proteínas Recombinantes/metabolismo , XenopusRESUMEN
A series of compounds that inhibit the coupling of the alpha 2-adrenergic receptor and the beta 2-adrenergic receptor to the guanine nucleotide-binding proteins (G proteins) Gi and Gs, respectively, have been identified. This inhibition of G protein coupling was detected by the ability of the compounds to reduce the affinity of these receptors for agonists without affecting antagonist affinity. Analysis of the structure-activity relationships of these compounds revealed a requirement for regularly spaced anionic substituents on amphipathic structures for this inhibition to occur. The compounds do not interact at the ligand binding site of the receptor or at the GTP binding site of the G protein. The identification of compounds that can uncouple receptors from G proteins demonstrates the potential for the discovery of small molecule inhibitors of receptor-G protein interactions that act as allosteric antagonists at this site.
Asunto(s)
Proteínas de Unión al GTP/efectos de los fármacos , Receptores Adrenérgicos alfa/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Naftalenosulfonatos/farmacología , Unión Proteica/efectos de los fármacos , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Relación Estructura-Actividad , Simpaticolíticos/metabolismo , Simpatomiméticos/metabolismoAsunto(s)
Cetoácidos , Fenilbutiratos/farmacología , Receptores de Prostaglandina/efectos de los fármacos , Sulfonas , Aerosoles , Animales , Antígenos/inmunología , Bronquios/efectos de los fármacos , Cobayas , Humanos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Fenilbutiratos/administración & dosificación , Fenilbutiratos/metabolismo , Receptores de Leucotrienos , Receptores de Prostaglandina/metabolismo , Saimiri , Tráquea/efectos de los fármacos , Tráquea/inmunologíaAsunto(s)
Fenilbutiratos/farmacología , Receptores de Prostaglandina/efectos de los fármacos , Animales , Bronquios/efectos de los fármacos , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Contracción Muscular/efectos de los fármacos , Receptores de Leucotrienos , Receptores de Prostaglandina/metabolismo , SRS-A/metabolismoRESUMEN
Studies of the binding of tritiated sulfidopeptide leukotrienes (LTs) to a membrane preparation from rat lung tissue revealed a site specific for LTC4 with a dissociation constant of 4.1 X 10(-8)M. Similar experiments with a guinea pig lung preparation demonstrated binding specific for LTD4 with a dissociation constant of 2.1 X 10(-10)M. The divalent cations Ca++, Mg++, and Mn++ significantly enhanced the affinity of binding of the respective LTs to both sites. The binding of LTC4 to guinea pig lung and rat lung exhibited similar characteristics, but the former was observed only in the presence of the gamma-glutamyl transpeptidase inhibitor, serineborate complex. The binding affinities of various isomers of both sulfidopeptide LTs paralleled the potency of their pharmacologic effects, which supports the contention that the sites are receptors specific for LTC4 and LTD4. The slow-reacting substance of anaphylaxis antagonist, FPL 55712, had a higher affinity for the LTD4 receptor, which is consistent with its more effective antagonism of the LTD4-induced contractile response of guinea pig lung parenchymal strips. The ability of Na+ and guanosine triphosphate to inhibit the binding of LTD4 suggests that the action of LTD4 is associated with a depression of intracellular concentrations of cyclic adenosine monophosphate.
Asunto(s)
Pulmón/inmunología , Receptores de Superficie Celular/metabolismo , SRS-A/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Cationes Monovalentes , Membrana Celular/metabolismo , Cobayas , Humanos , Cinética , Leucotrieno E4 , Ratas , Receptores de Leucotrienos , SRS-A/análogos & derivados , Especificidad de la EspecieRESUMEN
[3H]Leukotriene D4 was found to bind, in a saturable manner and with exceedingly high affinity, to a membrane preparation from guinea pig lung. Measurement of saturation at equilibrium yielded Kd values of 5.46 +/- 0.31 X 10(-11) M at 20 degrees C and 2.12 +/- 0.37 X 10(-10) M at 0 degree C while the numbers of binding sites (Bmax) were 384 +/- 34 and 302 +/- 44 fmol/mg of protein at 20 and at 0 degree C, respectively. The time courses of both association and dissociation were slow but the rate of dissociation was accelerated by either NaCl or GTP. Binding was enhanced by Ca2+, Mg2+, and Mn2+ and inhibited by Na+ but not by Li+ or K+, suggesting that the binding of leukotriene D4 may be regulated by ions. Leukotriene E4, but not leukotriene C4, had a high affinity for the putative receptor, consistent with the pharmacological evidence that the actions of leukotrienes D4 and E4 are mediated by a receptor distinct from that for leukotriene C4. Affinities of stereoisomers and related compounds for the leukotriene D4 binding sites closely paralleled their contractile activities in guinea pig lung parenchymal strips. In addition, the antagonist of slow-reacting substance of anaphylaxis, FPL 55712, inhibited the binding of [3H]leukotriene D4 with a Ki value of 1 X 10(-7) M, which is in agreement with reported Kb values obtained in pharmacological studies.
Asunto(s)
Pulmón/metabolismo , Receptores de Superficie Celular/metabolismo , SRS-A/metabolismo , Animales , Cationes , Cobayas , Cinética , Masculino , Receptores de Leucotrienos , Temperatura , TritioRESUMEN
A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.
Asunto(s)
Pulmón/metabolismo , SRS-A/metabolismo , Animales , Unión Competitiva , Cationes Bivalentes/farmacología , Cromatografía Líquida de Alta Presión , Calor , Pulmón/efectos de los fármacos , Masculino , Membranas/metabolismo , Ratas , Ratas EndogámicasAsunto(s)
Acetilcolina/metabolismo , Colina/metabolismo , Hipocampo/metabolismo , Sinaptosomas/metabolismo , Animales , Bario/farmacología , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Magnesio/farmacología , Masculino , Fisostigmina/farmacología , Potasio/farmacología , Ratas , Sodio/farmacología , Estroncio/farmacología , Sinaptosomas/efectos de los fármacos , Tetrodotoxina/farmacología , Veratridina/farmacología , Vinblastina/farmacologíaRESUMEN
Placement of high frequency lesions in the medial habenular area results in a large depletion of acetylcholine (ACh) levels, choline acetyltransferase (ChAc) activity, and high affinity choline uptake in the interpeduncular nucleus area (IPN) at 1, 3 and 7 days post-lesion. Areas adjacent to the IPN did not have a reduction in ChAc activity. The reduction in high affinity choline uptake was selective in that there was no change in the uptake of L-[3H]tyrosine or L-[3H]glutamic acid. Unlike the cholinergic septal-hippocampal neurons, there was no rise in ACh levels in the IPN 1 h after placement of lesion, or 30 min after administration of pentobarbital. While the IPN probably has a much denser cholinergic innervation than the hippocampus (as evidenced by much higher ACh levels, ChAc activity and choline uptake levels), it has only one-fourth as many [3H]QNB binding sites (a measure of cholinergic muscarinic receptors). This, some of the habenulo-interpeduncular neurons are probably cholinergic, and they may have pharmacologic and functional differences compared to the septal-hippocampal neurons.