RESUMO
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.
Assuntos
Hipocampo/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Piperidinas/farmacologia , Quinolonas/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Potenciais de Ação , Animais , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Hipocampo/metabolismo , Humanos , Injeções Intraperitoneais , Injeções Subcutâneas , Fosfatos de Inositol/metabolismo , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/farmacocinética , Técnicas de Patch-Clamp , Permeabilidade , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Quinolonas/administração & dosagem , Quinolonas/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes/agonistas , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND AND PURPOSE: Central 5-HT-containing pathways are known to be important in cardiovascular regulation and a crucial area for this regulation is the nucleus tractus solitarius (NTS), which contains many of the known 5-HT receptor subtypes. In this study the role of 5-HT(1B) and 5-HT(1D) receptors, targets for the antimigraine drugs known collectively as triptans, was examined in the NTS. EXPERIMENT APPROACH: Extracellular recordings were made, in anaesthetized rats, from 109 NTS neurones that were excited by electrical stimulation of the vagus and drugs were applied ionophoretically to these neurones. KEY RESULTS: The 5-HT(1B/1D) receptor agonist sumatriptan applied to 64 neurones produced a 64% reduction in the firing rate of 54 of these neurones. Ketanserin, a 5-HT(1D/2A) receptor antagonist, alone had little effect, but co-applied with sumatriptan significantly attenuated this inhibition, whilst co-application of the 5-HT(1B) receptor antagonist GR55562 resulted in potentiation of this inhibition. Sumatriptan also caused a 25% reduction in vagal afferent evoked activity as well as that caused by stimulation of cardiopulmonary afferents. In another 41 neurones the 5-HT(1B) receptor agonist CP-93 129 produced a doubling of the background firing rate in 31 of these neurones and a significant increase in both vagal afferent evoked activity and that evoked by cardiopulmonary afferent activation. CONCLUSIONS AND IMPLICATIONS: Activation of 5-HT(1B) and 5-HT(1D) receptors have opposing actions on NTS neurones of excitation and inhibition, respectively. As both receptors are negatively coupled to adenylate cyclase this would indicate that they have different anatomical locations within NTS.
Assuntos
Neurônios/efeitos dos fármacos , Agonistas do Receptor 5-HT1 de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Núcleo Solitário/efeitos dos fármacos , Nervo Vago/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Sinergismo Farmacológico , Estimulação Elétrica , Iontoforese , Ketanserina/farmacologia , Masculino , Inibição Neural/efeitos dos fármacos , Neurônios/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Piridinas/farmacologia , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Núcleo Solitário/citologia , Núcleo Solitário/metabolismo , Sumatriptana/farmacologia , Fatores de Tempo , Nervo Vago/citologiaRESUMO
BACKGROUND: Outbred Sprague-Dawley (SD) rats are a commonly used strain in preclinical pain research. Here, we established empirically how SD rats obtained from different vendors might vary in sensitivity to injury and pharmacotherapy. METHODS: Chronic Constriction Injury (CCI) or complete Freund's adjuvant (CFA) hindpaw inflammation was induced in male SD rats sourced from three to four different vendors, respectively. Neuropathic hypersensitivity was evaluated over 58 days using von Frey filaments, pinprick stimulation and the hot plate test. Pharmacological sensitivity was evaluated by treatment with gabapentin (100 mg/kg, p.o.) or morphine (3 mg/kg, s.c.). CFA-induced hyperalgesia and sensitivity to morphine (0.3-6 mg/kg, s.c.) was measured using a digital Randall-Selitto device. In addition, paw weight gain was used as an index of peripheral oedema. RESULTS: Significant differences between the vendor-supplied SD rats in relation to onset, magnitude and resolution of hypersensitivity after CCI were observed. Although all sub-strains eventually developed a robust and reversible neuropathic hypersensitivity to mechanical stimulation, the thermal hypersensitivity varied. Whereas pharmacological response to gabapentin varied enormously, the response to morphine was both robust and much more consistent between sub-strains. Despite a similar degree of CFA-induced hypersensitivity, the paw oedema level differed between sub-strains. Here, morphine dose-dependently alleviated the CFA-induced hypersensitivity, with only a subtle difference in sensitivity between sub-strains observed. CONCLUSIONS: Our data reveal that the source of vendor used to obtain SD rats may be one key factor responsible for 'between laboratory variation' in reproducing sensitivity to some drugs targeting various pathophysiological mechanisms in specific animal pain models. SIGNIFICANCE: The choice of vendor used to source the same strain of rat for use in preclinical pain research can profoundly affect the level of nociceptive hypersensitivity and response to reference analgesics in neuropathic versus inflammatory models.
Assuntos
Aminas/uso terapêutico , Analgésicos/uso terapêutico , Ácidos Cicloexanocarboxílicos/uso terapêutico , Hiperalgesia/fisiopatologia , Morfina/uso terapêutico , Neuralgia/fisiopatologia , Ácido gama-Aminobutírico/uso terapêutico , Animais , Adjuvante de Freund , Gabapentina , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Inflamação/tratamento farmacológico , Masculino , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Medição da Dor , Fenótipo , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Resultado do TratamentoRESUMO
BACKGROUND AND PURPOSE: Amyloid-ß (Aß) aggregation into synaptotoxic, prefibrillar oligomers is a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The pharmacological and neuroprotective properties of a novel Aß aggregation inhibitor, SEN1269, were investigated on aggregation and cell viability and in test systems relevant to synaptic function and memory, using both synthetic Aß(1-42) and cell-derived Aß oligomers. EXPERIMENTAL APPROACH: Surface plasmon resonance studies measured binding of SEN1269 to Aß(1-42) . Thioflavin-T fluorescence and MTT assays were used to measure its ability to block Aß(1-42) -induced aggregation and reduction in cell viability. In vitro and in vivo long-term potentiation (LTP) experiments measured the effect of SEN1269 on deficits induced by synthetic Aß(1-42) and cell-derived Aß oligomers. Following i.c.v. administration of the latter, a complex (alternating-lever cyclic ratio) schedule of operant responding measured effects on memory in freely moving rats. KEY RESULTS: SEN1269 demonstrated direct binding to monomeric Aß(1-42) , produced a concentration-related blockade of Aß(1-42) aggregation and protected neuronal cell lines exposed to Aß(1-42) . In vitro, SEN1269 alleviated deficits in hippocampal LTP induced by Aß(1-42) and cell-derived Aß oligomers. In vivo, SEN1269 reduced the deficits in LTP and memory induced by i.c.v. administration of cell-derived Aß oligomers. CONCLUSIONS AND IMPLICATIONS: SEN1269 protected cells exposed to Aß(1-42) , displayed central activity with respect to reducing Aß-induced neurotoxicity and was neuroprotective in electrophysiological and behavioural models of memory relevant to Aß-induced neurodegeneration. It represents a promising lead for designing inhibitors of Aß-mediated synaptic toxicity as potential neuroprotective agents for treating AD.
Assuntos
Aminofenóis/farmacologia , Peptídeos beta-Amiloides/toxicidade , Diaminas/farmacologia , Piridazinas/farmacologia , Pirimidinas/farmacologia , Sinapses/efeitos dos fármacos , Animais , Linhagem Celular , Diaminas/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Masculino , Memória/efeitos dos fármacos , Estrutura Molecular , Ligação Proteica , Piridazinas/química , Ratos , Ratos Sprague-DawleyRESUMO
It has been demonstrated previously that the vagal bradycardia evoked by activation of pulmonary C-fibres is not respiratory modulated. Experiments were carried out in alpha-chloralose anaesthetized cats to determine if these cardiac vagal preganglionic neurones (CVPNs) in the nucleus ambiguus (NA), which have respiratory modulated activity, can be activated when pulmonary C-fibre afferents are stimulated by right atrial injections of phenylbiguanide (PBG). Eleven CVPNs with B-fibre axons in the right cardiac vagal branches were identified and found to be localized within or ventrolateral to the nucleus ambiguus. Ionophoretic application of a high current of dl-homocysteic acid (DLH) induced a vagally mediated bradycardia and hypotension in six of eight sites from which CVPNs were recorded. The activity of B-fibre CVPNs, whether spontaneous (n = 4) or induced by ionophoresis of DLH (n = 7) was respiratory modulated, firing perferentially during post-inspiration and stage 2 expiration. This activity also correlated with the rising phase of the arterial blood pressure wave consistent with these CVPNs receiving an arterial baroreceptor input. Right atrial injections of PBG excited nine of eleven CVPNs tested. In eight of these activated neurones the onset latency of the excitation was within the pulmonary circulation time, consistent with being activated only by pulmonary C-fibre afferents. In two neurones the PBG-evoked excitation still occurred when central inspiratory drive was inhibited, as indicated by the disappearance of phrenic nerve activity. In conclusion, B-fibre respiratory modulated CVPNs can be activated following stimulation of pulmonary C-fibre afferents.