RESUMEN
Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) >> guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Receptores Adrenérgicos alfa 2/metabolismo , Adenilil Ciclasas/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Vectores Genéticos , Humanos , Ligandos , Ratones , Unión Proteica , Quinolizinas/metabolismo , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
RS 57639, by being a partial agonist in rat esophagus but a competitive antagonist in guinea-pig ileum, is one of several ligands which operationally discriminate among 5-HT4 receptors in different tissues. The discovery of splice variants of the 5-HT4 receptor, 5-HT4S and 5-HT4L, raises the possibility that this functional heterogeneity among 5-HT4 receptors may be due to differences in the interaction of ligands with different isoforms of the receptor. To test this idea, the functional and binding interactions of RS 57639 with rat 5-HT4S and 5-HT4L receptors were characterized. RS 57639 stimulated adenylate cyclase in cells expressing 5-HT4S or 5-HT4L receptors with similar potency (pEC50 = 7.9 +/- 0.1 and 7.6 +/- 0.1) and efficacy (71 +/- 3 and 59 +/- 4% of 5-HT). [3H]RS 57639 also bound to 5-HT4S and 5-HT4L receptors with similar affinity (Kd = 0.09 +/- 0.01 and 0.11 +/- 0.01 nM) and specificity (SB204070 > GR113808 > SDZ 205557 > cisapride > renzapride > alpha me-5-HT > 5-CT). Therefore, the operational differences among 5-HT4 receptors, detected with RS 57639, are not explained by differences in the interaction of the ligand with 5-HT4S and 5-HT4L receptors. [3H]RS 57639 binding to guinea-pig striatal membranes was also characterized. [3H]RS 57639 bound with high affinity (Kd = 0.25 +/- 0.07 nM) and a specificity similar to that of the 5-HT4 receptor antagonist, [3H]GR113808. Therefore, while the mechanism by which RS 57639 operationally distinguishes among 5-HT4 receptors was not determined, [3H]RS 57639 was shown to specifically label native and cloned 5-HT4 receptors. As the first selective agonist radioligand to be described for this receptor, [3H]RS 57639 may prove useful in further studies of receptor coupling and ligand interactions.
Asunto(s)
Aminobenzoatos/metabolismo , Neostriado/metabolismo , Piperidinas/metabolismo , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Animales , Clonación Molecular , AMP Cíclico/biosíntesis , Cobayas , Cinética , Membranas/efectos de los fármacos , Membranas/metabolismo , Neostriado/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/genética , Termodinámica , Células Tumorales CultivadasRESUMEN
5-HT4 receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC50) of 8.0 +/- 0.1 (n = 50) and 7.8 +/- 0.1 (n = 12), respectively. 5-HT4 receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT4 receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT4 receptors mediate relaxation. GR 113808 ¿[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl] methyl1-methyl-1H-indole-3-carboxylate¿, RS 39604 ¿1-[4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxy)phenyl]-3[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 ¿(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1, 4-benzodioxane-5-carboxylate¿ antagonized 5-HT responses with pA2 values of 9.1 +/- 0.1, 9.0 +/- 0.2 and 11.0 +/- 0.1, respectively. These affinity values were similar to those obtained at 5-HT4 receptors in isolated rat oesophagus (9.0+/- 0.4, 9.3 +/- 0.1 and 10.6 +/- 0.1 respectively). Despite these operational similarities between 5-HT4 receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT4 receptors in the two tissues. For example, the substituted benzoate, RS 23597 ¿3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 +/-0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA2 = 7.8 +/- 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 ¿(S)-6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl) 2,3-dihydro-1H-benz[de] isoquinoline-1,3-dione hydrochloride¿, was a potent (pEC50 = 7.9 +/- 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pKB) of 9.4 +/- 0.1. Furthermore, several novel, selective, 5-HT4 compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532. It is concluded that these differences are inconsistent with differences in 5-HT4 receptor reserves, and may suggest that 5-HT4 receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.
Asunto(s)
Colon/efectos de los fármacos , Esófago/efectos de los fármacos , Receptores de Serotonina/efectos de los fármacos , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Animales , Colon/fisiología , Esófago/fisiología , Cobayas , Técnicas In Vitro , Indoles/farmacología , Masculino , Ratas , Receptores de Serotonina/fisiología , Serotonina/farmacología , Sulfonamidas/farmacologíaRESUMEN
Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.
Asunto(s)
Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Regulación Alostérica , Animales , Línea Celular , Cricetinae , Interacciones Farmacológicas , Homeostasis , Humanos , Riñón/citología , Riñón/embriología , Cinética , Ratones , Ensayo de Unión RadioliganteRESUMEN
The role of secretory antibody in protection against respiratory syncytial virus (RSV) infection was examined by using monoclonal immunoglobulin A (IgA) antibody for intranasal passive immunization of mice. Eight anti-RSV IgA hybridomas were produced by fusing myeloma cells with lung lymphocytes from RSV-immunized mice. Five IgA antibodies recognized RSV strains of both the A and the B subgroups, and two of these neutralized virus in a plaque reduction assay. Monoclonal IgA antibody HNK20, which bound to F glycoprotein, was most effective, reducing plaques by 50% at a concentration of 0.1 microgram/ml for both subgroup A and subgroup B strains. HNK20 also neutralized all of eight clinical isolates of RSV tested. When delivered intranasally to mice 24 h prior to RSV challenge, HNK20 reduced virus titers in the lungs by nearly 100-fold. Maximal protection occurred at a dose of 0.5 mg/kg of body weight. Significant protection against lung infection was seen when the interval between antibody treatment and challenge was as long as 72 h. HNK20 also decreased virus titers in the nose approximately 10-fold when given 1 h, but not 24 h, before challenge. When mice were treated with HNK20 intranasally 3 days after challenge, viral titers were reduced in the lungs but not the nose. The results indicate that topical application of relatively small amounts of monoclonal IgA can protect against both upper and lower respiratory tract infections caused by RSV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Inmunoglobulina A Secretora/inmunología , Virus Sincitiales Respiratorios/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Femenino , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
Recent findings have suggested a relationship between 5-hydroxytryptamine (5-HT)4 receptors and sigma binding sites. To test this idea, the affinity of 5-HT4 receptor ligands for sigma binding sites was examined. In contrast to the 5-HT4 receptor ligands BIMU-1 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3- dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride] and BIMU-8 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3- yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-benzamidazole-1-carbox ami de hydrochloride], DAU 6215 ]N-(endo-8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-2,3-dihydro-2-oxo-1H- benzimidazole-1-carboxamide hydrochloride], 5-HT and 5-methoxytryptamine had low affinity for sigma binding sites (pKi < 6). Conversely, the sigma ligands haloperidol and pentazocine had low affinity for 5-HT4 receptors. Thus, no relationship was found between the affinity of ligands at 5-HT4 receptors and sigma binding sites. However, one potent 5-HT4 receptor antagonist, RS-23597-190 [3-(piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride], had high affinity for sigma-1 (pKi = 8.4) but not sigma-2 (pKi = 6.2) binding sites. [3H]RS-23597-190 bound to a saturable site with the pharmacology of a sigma-1 binding site: (pIC50) haloperidol (9.0) > (+)-pentazocine (8.8) > (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine (8.2) > 1,3-di-o-tolyl-guanidine (8.0) > (-)-pentazocine (7.8) = (+)-SKF 10,047 [N-allylnormetazocine] > (-)-SKF 10,047 (6.2) > BIMU-1 (5.3) > 5-HT and 5-methoxytryptamine. The distribution of [3H]RS-23597-190 binding sites was similar to that described for other sigma radioligands, with the greatest binding densities in cranial nerve nuclei, the tegmental nucleus and in the mamillary nucleus. In contrast to (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine, [3H]RS-23597-190 binding was not allosterically modulated by phenytoin. These studies do not support the notion of an obvious relationship between sigma and 5-HT4 receptors, but they provide additional insight into the structure/affinity relationship of ligands at specific sigma binding sites, and they uncover a novel sigma-1 receptor ligand whose binding is insensitive to the action of phenytoin.