RESUMEN
BACKGROUND AND PURPOSE: M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtype-selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M(1) mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. EXPERIMENTAL APPROACH: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. KEY RESULTS: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. CONCLUSIONS AND IMPLICATIONS: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M1 mAChR.
Asunto(s)
Hipocampo/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Piperidinas/farmacología , Quinolonas/farmacología , Receptores Muscarínicos/efectos de los fármacos , Potenciales de Acción , Animales , Células CHO , Señalización del Calcio/efectos de los fármacos , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Hipocampo/metabolismo , Humanos , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Fosfatos de Inositol/metabolismo , Agonistas Muscarínicos/administración & dosificación , Agonistas Muscarínicos/farmacocinética , Técnicas de Placa-Clamp , Permeabilidad , Piperidinas/administración & dosificación , Piperidinas/farmacocinética , Quinolonas/administración & dosificación , Quinolonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes/agonistas , Factores de Tiempo , TransfecciónRESUMEN
Two distinct types of glycine transporter, GlyT-1 and GlyT-2, have been characterised. GlyT-1 and GlyT-2 are known to be differentially expressed amongst CNS areas, but direct functional evidence for their relative contributions to high-affinity glycine uptake by brain tissues is lacking. In the present study, we have used the selective GlyT-1 inhibitor N[3-(4"-fluorophenyl)-3-(4"-phenylphenoxy)propyl]sarcosine (NFPS) to investigate the role of GlyT-1 in mediating glycine uptake. HEK293 cells expressing human GlyT-1c or GlyT-2 showed high levels of Na(+)-dependent glycine uptake, with K(m) values of 117+/-13 and 200+/-22 microM, respectively. NFPS potently inhibited uptake in GlyT-1c cells (IC(50) value 0.22+/-0.03 microM), being around 500-fold more potent than glycine or sarcosine, but had no effect on uptake in GlyT-2 cells (IC(50) >10 microM). Efflux of pre-loaded [3H]-glycine from GlyT-1c cells was increased by glycine or sarcosine, whereas NFPS had no effect on its own but blocked the effects of glycine or sarcosine. These results confirm that NFPS is a potent, selective and non-transportable GlyT-1 inhibitor. Rat cortex and cerebellum synaptosomes also showed a high-affinity Na(+)-dependent component of glycine uptake, with affinities similar to those observed for uptake in GlyT-1c or GlyT-2 cells. In cortex synaptosomes, NFPS and sarcosine produced the same maximal inhibition of uptake as glycine itself. However, in cerebellum synaptosomes, the maximal inhibition produced by NFPS and sarcosine was only half that produced by glycine. In both tissues NFPS was around 1000-fold more potent than glycine or sarcosine. Overall, our findings indicate that high-affinity glycine uptake in cerebral cortex occurs predominantly via GlyT-1. However, in cerebellum, only a part of the high-affinity uptake is mediated by GlyT-1, with the remaining NFPS-insensitive component most likely mediated by GlyT-2.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Portadoras/fisiología , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Glicina/metabolismo , Receptores de Glicina/metabolismo , Sarcosina/farmacología , Sinaptosomas/metabolismo , Animales , Cerebelo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática , Humanos , Técnicas In Vitro , Ratas , Receptores de Glicina/efectos de los fármacos , Sarcosina/análogos & derivados , Sinaptosomas/efectos de los fármacosRESUMEN
GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.
Asunto(s)
Hormonas Hipotalámicas/metabolismo , Neuropéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Unión Competitiva , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/biosíntesis , Humanos , Hormonas Hipotalámicas/farmacología , Radioisótopos de Yodo , Cinética , Datos de Secuencia Molecular , Neuropéptidos/farmacología , Hormona Liberadora de Prolactina , RatasRESUMEN
SAR studies around a series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides, identified by high-throughput screening at the novel SB-204269 binding site, have provided compounds such as 13, 29-33 with high affinity and excellent anticonvulsant activity in animal models.
Asunto(s)
Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Benzamidas/química , Benzamidas/farmacología , Diseño de Fármacos , Isoquinolinas/química , Isoquinolinas/farmacología , Animales , Anticonvulsivantes/síntesis química , Anticonvulsivantes/metabolismo , Benzamidas/síntesis química , Benzamidas/metabolismo , Benzopiranos/metabolismo , Sitios de Unión , Química Encefálica , Evaluación Preclínica de Medicamentos , Isoquinolinas/síntesis química , Isoquinolinas/metabolismo , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Ensayo de Unión Radioligante , Ratas , Convulsiones/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
The present study describes the solubilisation of the novel anticonvulsant, SB-204269, binding site from pig cortical membranes. Throughout the study the binding of a close analogue of this compound, [125I]-SB-217644 (trans 6- Acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b ]pyran-3R-ol) was used to monitor the success of the solubilisation procedure. [125I]-SB-217644 was an ideal mechanistic tool for quantifying the binding to this novel anticonvulsant site, with a high specific activity and affinity (K(D) of 3 nmol/l). Optimum conditions for the solubilisation of this anticonvulsant binding site were investigated using a multifactorial experimental design to assess a large number of variables. Detergent type, detergent-protein ratio, absence of Mg2+ and temperature were deemed to be important factors. However, the increases observed in binding site specific activity were minimal compared with those achieved for yields. Maximum percentage yields of binding activity (25%) were achieved with a low concentration of the zwitterionic detergent, CHAPS, in the presence of a low protein concentration. This yield was further enhanced on combining mixtures of detergents. The highest recovery (37%) was achieved with a 50:50 (v:v; 1.5 x critical micelle concentration) mixture of the ionic detergent, sodium cholate, and the non-ionic detergent, MEGA-10. In summary, we report the successful solubilisation of a novel anticonvulsant binding site, identified by its selective affinity for SB-204269 and its analogues. The recovery of nearly 40% of the target binding sites from the starting material should provide a good starting point for the purification of this protein.
Asunto(s)
Anticonvulsivantes/metabolismo , Benzamidas/metabolismo , Benzopiranos/metabolismo , Corteza Cerebral/metabolismo , Animales , Sitios de Unión , Ácidos Cólicos/farmacología , Solubilidad , PorcinosRESUMEN
SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [(3)H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [(125)I]-labelled form of SB-217644 (trans-6-acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [(125)I]-SB-217644 shows a similar binding profile to that of [(3)H]-SB-204269, with a maximum specific binding capacity (B(max)) of 286+/-12 fmol/mg protein, but has twenty-fold higher affinity (K(d) value 1.7+/-0.1 nM). The high affinity and high specific activity of [(125)I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [(125)I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K(d) value of 2.7+/-0.3 nM and a B(max) value of 55+/-11 fmol/mg protein, with a 7.3+/-0.3% yield of solubilised binding sites. [(125)I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [(125)I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [(125)I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.
Asunto(s)
Benzamidas/metabolismo , Benzopiranos/metabolismo , Benzopiranos/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Línea Celular , Detergentes , Radioisótopos de Yodo , Cinética , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
Optimisation of novel cis- and trans-4-(substituted-amido)benzopyran-3-ol derivatives has led to the identification of SB-220453 20 with an in vivo pre-clinical CNS profile predictive of potential antimigraine activity.
Asunto(s)
Benzamidas/farmacología , Benzopiranos/farmacología , Trastornos Migrañosos/tratamiento farmacológico , Animales , Benzamidas/química , Relación Dosis-Respuesta a Droga , Metilcelulosa/farmacología , Ratones , Ratas , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Sumatriptán/farmacología , Temperatura , Factores de TiempoRESUMEN
A series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides was identified by high-throughput screening at the novel SB-204269 binding site. SAR studies have provided compounds 4 and 14 with high affinity and good anticonvulsant activity in animal models.
Asunto(s)
Anticonvulsivantes/química , Benzamidas/química , Quinolinas/química , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Benzamidas/farmacología , Benzamidas/uso terapéutico , Benzopiranos/farmacología , Sitios de Unión , Ratones , Modelos Moleculares , Quinolinas/farmacología , Quinolinas/uso terapéutico , Convulsiones/prevención & control , Relación Estructura-ActividadRESUMEN
1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1-30 mg Kg-1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MEST)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg-1, p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1-10 microM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg-1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB-204269 of > 31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (> or = 10 microM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 microM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.
Asunto(s)
Anticonvulsivantes/farmacología , Benzamidas/farmacología , Benzopiranos/farmacología , Epilepsias Parciales/tratamiento farmacológico , Epilepsia Generalizada/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
1. SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]-SB-204269 in rat forebrain membranes. 2. Specific [3H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (KD) of 32 +/- 1 nM and a maximum binding capacity (Bmax) of 253 +/- 18 fmol mg-1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. 3. Specific [3H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. 4. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. 5. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.
Asunto(s)
Anticonvulsivantes/metabolismo , Benzamidas/metabolismo , Benzopiranos/metabolismo , Encéfalo/metabolismo , Animales , Sitios de Unión , Cinética , Masculino , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , TritioRESUMEN
We have characterised the Ca2+ channel blocking properties of a new non-peptide Ca2+ channel antagonist, SB 201823-A, in cultures of rat sensory neurones. The IC50 for SB 201823-A against total Ca2+ current in sensory neurones was 4.9 microM. SB 201823-A showed little selectivity for sub-types of neuronal Ca2+ channel but was selective for Ca2+ channels over Na+ and K+ channels. Efficacy against other types of cation channel such as agonist gated channels was not assessed. SB 201823-A was neuroprotective in vivo when administered post-ischaemia in one focal and one global model of neuronal ischaemia. In the rat photothrombotic focal lesion model, SB 201823-A administered i.p. 10 min post-ischaemia resulted in a dramatic reduction in lesion volume. In the gerbil bilateral carotid artery occlusion global model, SB 201823-A dosed i.p. 30 min post-occlusion resulted in both histological and functional improvements when compared to vehicle treated animals. These data suggest that such novel neuronal Ca2+ channel antagonists may have potential in ameliorating both the pathological and functional consequences of stroke in man.
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Isquemia Encefálica/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/uso terapéutico , Neuronas Aferentes/metabolismo , Piperidinas/uso terapéutico , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Trombosis de las Arterias Carótidas/fisiopatología , Electrofisiología , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Gerbillinae , Neuronas Aferentes/efectos de los fármacos , Potasio/farmacología , Ratas , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
In the long-term castrated rat the negative feedback effect of testosterone is markedly reduced and the raised levels of plasma LH seen in the castrated animals are not suppressed by physiological concentrations of plasma testosterone. In this study we have measured pituitary gonadotrophin-releasing hormone (GnRH) receptor content as well as plasma and pituitary LH on days 1, 10 and 40 after castration and noted the effect of testosterone replacement on these parameters. We found that the negative feedback effect of physiological concentrations of testosterone on plasma and pituitary LH, pituitary GnRH receptor content and response to exogenous GnRH was attenuated 10 and 40 days after castration. It is suggested that the lack of effect of testosterone in the long-term castrated rat is due to its inability to reduce the pituitary GnRH receptor content. On increasing testosterone to supraphysiological levels, the negative feedback effect was reinstated. We also found that in rats 40 days after castration, physiological and subphysiological concentrations of testosterone significantly increased pituitary GnRH receptor content and this may explain the previous findings that low concentrations of testosterone can enhance the effect of GnRH and increase plasma LH levels.
Asunto(s)
Receptores de Superficie Celular/efectos de los fármacos , Testículo/fisiología , Testosterona/farmacología , Animales , Retroalimentación , Hormona Luteinizante/sangre , Hormona Luteinizante/metabolismo , Masculino , Orquiectomía , Tamaño de los Órganos , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Endogámicas , Receptores LHRH , Estimulación Química , Testosterona/sangre , Factores de TiempoRESUMEN
We have examined variations in hypothalamic dopamine D-2 receptor levels occurring during sexual maturation in male and female rats, using a [3H]domperidone radioligand binding assay. A major decrease in D-2 receptor levels was observed during sexual development, and was accompanied by the appearance of a sexual dimorphism in receptor levels, which appeared to be the result of neonatal sexual differentiation. These changes may be linked with the alterations in hormone levels which occur during sexual maturation.
Asunto(s)
Hipotálamo/análisis , Receptores Dopaminérgicos/análisis , Maduración Sexual , Animales , Animales Recién Nacidos/fisiología , Femenino , Hormonas Esteroides Gonadales/fisiología , Masculino , Ratas , Ratas Endogámicas , Receptores de Dopamina D2 , Caracteres Sexuales , Diferenciación Sexual , Testosterona/farmacologíaRESUMEN
The effects of castration with or without testosterone replacement in the adult male rat were studied to investigate possible hypothalamic mechanisms by which changes in gonadotrophin secretion occur at different times after castration, with particular reference to the continuing LH rise and its lack of suppression by testosterone in the long-term castrated rat. Castrated rats received either subcutaneous silicone elastomer implants containing testosterone or empty implants at the time of castration, and a sham-operated group served as controls. At 1, 10 and 40 days after castration, there were six-, 15- and 25-fold rises respectively in LH and 1.5-, two- and fivefold rises in FSH. However, there were no significant changes in hypothalamic noradrenaline concentration and turnover or in alpha-adrenoceptor density and affinity at any time after castration. Testosterone implants were effective in suppressing gonadotrophin release at 1 and 10 days, but not at 40 days after castration, and did not significantly affect hypothalamic noradrenaline turnover or alpha-adrenoceptors at any time. Neither acute inhibition of the noradrenergic system, using either the alpha-adrenoceptor blockers phenoxybenzamine and phentolamine or the synthesis inhibitor alpha-methyl-p-tyrosine, nor chronic depletion of hypothalamic noradrenaline by 6-hydroxydopamine had any significant effect on the normal rise in LH levels seen on days 10 and 40 after castration, and did not alter the ability of testosterone to suppress LH levels. This indicates that, in the long-term castrated rat, the noradrenergic system may not be involved in the control of gonadotrophin release. However, at 16 h after castration, alpha-adrenoceptor blockers and alpha-methyl-p-tyrosine did reduce LH levels, indicating that the noradrenergic system is likely to be involved in the short-term response to castration.
Asunto(s)
Castración , Hormona Folículo Estimulante/sangre , Hipotálamo/metabolismo , Hormona Luteinizante/sangre , Receptores Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Hormona Folículo Estimulante/metabolismo , Hidroxidopaminas/farmacología , Hormona Luteinizante/metabolismo , Masculino , Metiltirosinas/farmacología , Norepinefrina/metabolismo , Oxidopamina , Ratas , Testosterona/metabolismo , Testosterona/farmacología , Factores de Tiempo , alfa-MetiltirosinaRESUMEN
The radioligands [3H]-dihydroergocryptine and [3H]-dihydroalprenolol were used to characterise alpha- and beta-adrenergic binding sites, respectively, in membrane fractions of sheep cerebral cortex. In terms of affinity, density and specificity these sites possess properties similar to those previously characterised in rat brain. Further, in preliminary studies, these sites also appear to be responsive to treatment with estradiol/progesterone as well as to photoperiod. Thus, estrogen treatment can elevate both alpha- and beta-adrenergic binding sites in cortical tissue of sheep kept in natural light. In contrast, artificial light either has no effect or inhibits binding in response to estrogen.
Asunto(s)
Corteza Cerebral/análisis , Estradiol/farmacología , Luz , Progesterona/farmacología , Receptores Adrenérgicos alfa/análisis , Receptores Adrenérgicos beta/análisis , Receptores Adrenérgicos/análisis , Animales , Dihidroalprenolol/metabolismo , Dihidroergotoxina/metabolismo , Periodicidad , OvinosRESUMEN
Previous work has identified an effect of circulating estrogens on the number of central adrenergic binding sites. We have further characterized this effect by performing the experiments in vitro and have taken advantage of a well-described hypothalamic preparation in which diethylstilbestrol (DES), is known to elevate cAMP levels through a pathway which involves adrenoreceptors. We show that DES induces a reciprocal change in the numbers of alpha- and beta-adrenergic binding sites in incubated hypothalami obtained from intact immature female rats as well as from ovariectomized adult rats. The alpha-adrenergic binding sites are reduced by 25-30% whereas the beta-adrenergic sites are increased by 60-100%. The effect is maximal at 3 h in vitro (20 microM DES) and largely reversible following a 2 h wash in the absence of DES. Using the change in beta-adrenergic binding sites as a probe, we were further able to show that estradiol (100 microM) and 2-hydroxyestradiol (50 microM) had no effect. Further, the effect of DES was not blocked by the anti-estrogens clomiphene or tamoxifen. Since DES is able to elevate beta-adrenergic binding sites in hypothalamus and amygdala (brain areas known to contain high levels of estrogen receptors) but has no effect in cerebellum, we conclude that we have observed an effect of DES not shared by estradiol but which may be confined to estrogen target areas of the brain.