RESUMO
Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.
Assuntos
Ansiolíticos/farmacologia , Anticonvulsivantes/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Erythrina/química , Ácido Glutâmico/metabolismo , Antagonistas Nicotínicos/farmacologia , Oócitos , Técnicas de Patch-Clamp , Potássio/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Canais de Sódio/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Xenopus laevis , Ácido gama-Aminobutírico/metabolismoRESUMO
A pheochromocytoma of the rat adrenal medulla derived (a.k.a. PC12) cell-based assay for dopamine measurement by luminescence detection was customized for the qualitative evaluation of agonists and antagonists of nicotinic acetylcholine receptors (nAChRs). The assay mechanism begins with ligand binding to transmembrane nAChRs, altering ion flow into the cell and inducing dopamine release from the cell. Following release, dopamine is oxidized by monoamine oxidase generating hydrogen peroxide that catalyzes a chemiluminescence reaction involving luminol and horseradish peroxidase, thus producing a detectable response. Results are presented for the action of nAChR agonists (acetylcholine, nicotine, and cytisine), and antagonists (α-conotoxins (α-CTxs) MII, ImI, LvIA, and PeIA) that demonstrate a luminescence response correlating to the increase or decrease of dopamine release. A survey of cell growth and treatment conditions, including nerve growth factor, nicotine, ethanol, and temperature, led to optimal assay requirements to achieve maximal signal intensity and consistent response to ligand treatment. It was determined that PC12 cells treated with a combination of nerve growth factor and nicotine, and incubated at 37 °C, provided favorable results for a reduction in luminescence signal upon treatment of cells with α-CTxs. The PC12 assay is intended for use as a fast, efficient, and economic qualitative method to assess the bioactivity of molecules that act on nAChRs, in which testing of ligand-nAChR binding hypotheses and computational predictions can be validated. As a screening method for nAChR bioactivity, lead compounds can be assessed for their likelihood of exhibiting desired bioactivity prior to being subjected to more complex quantitative methods, such as electrophysiology or live animal studies.
Assuntos
Dopamina/metabolismo , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Conotoxinas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ligantes , Fator de Crescimento Neural/farmacologia , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
RATIONALE: Cognitive decline and dementia are major concerns in today's aging society. As limited treatments are available, measures to prevent cognitive decline and dementia are needed. We previously demonstrated that matured hop bitter acids (MHBA), bitter components of beer, increase norepinephrine in the hippocampus and improve memory in amnesia model mice induced by scopolamine (SCP), an antagonist of muscarinic receptor. However, other neurotransmitters involved in the effects of MHBA on memory improvement remain unknown. OBJECTIVES: This study aimed to assess the role of acetylcholine receptors (AChR) in the effects of MHBA on memory. METHOD: The involvement of AChR on the effects of MHBA (10 mg/kg) on cognitive function was evaluated using AChR antagonists, SCP, mecamylamine hydrochloride (MEC), a non-competitive antagonist of nicotinic-AChR (nAChR), and methyllycaconitine citrate (MLA), an α7nAChR antagonist, for the Y-maze test and the novel object recognition test (NORT). A separate population of mice, which underwent vagotomy or sham operation, was subjected to NORT to elucidate further mechanism. In addition, the effect of MHBA on acetylcholinesterase (AChE) activity was measured in vitro. RESULTS: In accordance with previous reports, MHBA improved spontaneous alternations of the Y-maze test in SCP-induced amnesia mice and increased discrimination index evaluated by the NORT in normal mice. On the other hand, treatment with MEC or MLA attenuated the effects of MHBA on memory improvement in the Y-maze test and the NORT. Vagotomized mice also showed attenuated memory enhancement by MHBA in the NORT. In addition, MHBA did not alter AChE activity in vitro. CONCLUSIONS: The results support the involvement of nAChRs in memory improvement in mice by MHBA. MHBA is thus thought to activate the vagal nerve and enhance hippocampus-dependent memory via nAChRs.
Assuntos
Humulus , Memória/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores Nicotínicos/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Comportamento Espacial/efeitos dos fármacos , Animais , Cognição/efeitos dos fármacos , Cognição/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Antagonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Extratos Vegetais/isolamento & purificação , Reconhecimento Psicológico/fisiologia , Comportamento Espacial/fisiologiaRESUMO
CONTEXT: Nicotine, a bioactive component of tobacco, is highly addictive. Numerous therapies have been developed for smoking cessation, and all have met with limited success. Our laboratory has previously shown that an extract of Passiflora incarnata Linn. (Passifloraceae) antagonized the expression of nicotine locomotor sensitization in rats. OBJECTIVE: This study examined the ability of vitexin, a flavonoid found in P. incarnata, to ameliorate the signs of nicotine sensitization in rats. MATERIALS AND METHODS: Male Wistar rats were administered 0.4 mg/kg nicotine or vehicle (n = 16-18 per group) once a day for four consecutive days. Nicotine administration produces sensitization of locomotor activity. On the fifth day, locomotor activity was monitored as rats from each treatment group were administered either 30 or 60 mg/kg vitexin or its vehicle (n = 4-6 per group) 30 min before a challenge dose of 0.4 mg/kg nicotine. RESULTS: The challenge dose of nicotine resulted in locomotor activity in rats sensitized to nicotine for 4 days that was approximately twice that measured in rats treated with vehicle during the sensitization phase. Rats sensitized to nicotine and then treated with 60 mg/kg vitexin prior to the nicotine challenge exhibited a level of locomotor activity equivalent to the vehicle-treated controls. DISCUSSION: Vitexin antagonized the expression of nicotine locomotor sensitization in rats as the whole extract did in the previous study. CONCLUSION: Vitexin should be examined in future studies to evaluate its potential for treating nicotine addiction in humans.
Assuntos
Apigenina/farmacologia , Nicotina/farmacologia , Abandono do Hábito de Fumar/métodos , Animais , Locomoção/efeitos dos fármacos , Masculino , Antagonistas Nicotínicos/farmacologia , Passiflora/química , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Tabagismo/tratamento farmacológicoRESUMO
Several previous studies have demonstrated that the activity of neurotransmitters acting on ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR) can be altered by compounds binding to allosteric modulatory sites. In the case of α7 nAChRs, both positive and negative allosteric modulators (PAMs and NAMs) have been identified and have attracted considerable interest. A recent study, employing revised structural models of the transmembrane domain of the α7 nAChR in closed and open conformations, has provided support for an inter-subunit transmembrane allosteric binding site (Newcombe et al 2017). In the present study, we have performed virtual screening of the DrugBank database using pharmacophore queries that were based on the predicted binding mode of PAMs to α7 nAChR structural models. A total of 81 compounds were identified in the DrugBank database, of which the 25 highest-ranked hits corresponded to one of four previously-identified therapeutic compound groups (carbonic anhydrase inhibitors, cyclin-dependent kinase inhibitors, diuretics targeting the Na+-K+-Cl- cotransporter, and fluoroquinolone antibiotics targeting DNA gyrase). The top-ranked compound from each of these four groups (DB04763, DB08122, furosemide and pefloxacin, respectively) was tested for its effects on human α7 nAChR expressed in Xenopus oocytes using two-electrode voltage-clamp electrophysiology. These studies, conducted with wild-type, mutant and chimeric receptors, resulted in all four compounds exerting allosteric modulatory effects. While DB04763, DB08122 and pefloxacin were antagonists, furosemide potentiated ACh responses. Our findings, supported by docking studies, are consistent with these compounds acting as PAMs and NAMs of the α7 nAChR via interaction with a transmembrane site.
Assuntos
Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Furosemida/química , Furosemida/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Agonistas Nicotínicos/química , Antagonistas Nicotínicos/química , Oócitos , Pefloxacina/química , Pefloxacina/farmacologia , Conformação Proteica , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
BACKGROUND: Previous studies have demonstrated that oral administration of curcumin exhibited an anti-arthritic effect despite its poor bioavailability. The present study aimed to explore whether the gut-brain axis is involved in the therapeutic effect of curcumin. METHODS: The collagen-induced arthritis (CIA) rat model was induced by immunization with an emulsion of collagen II and complete Freund's adjuvant. Sympathetic and parasympathetic tones were measured by electrocardiographic recordings. Unilateral cervical vagotomy (VGX) was performed before the induction of CIA. The ChAT, AChE activities, and serum cytokine levels were determined by ELISA. The expression of the high-affinity choline transporter 1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) were determined by real-time PCR and immunohistochemical staining. The neuronal excitability of the vagus nerve was determined by whole-cell patch clamp recording. RESULTS: Oral administration of curcumin restored the imbalance between the sympathetic and parasympathetic tones in CIA rats and increased ChAT activity and expression of ChAT and VAChT in the gut, brain, and synovium. Additionally, VGX eliminated the effects of curcumin on arthritis and ACh biosynthesis and transport. Electrophysiological data showed that curcumin markedly increased neuronal excitability of the vagus nerve. Furthermore, selective α7 nAChR antagonists abolished the effects of curcumin on CIA. CONCLUSIONS: Our results demonstrate that curcumin attenuates CIA through the "gut-brain axis" by modulating the function of the cholinergic system. These findings provide a novel approach for mechanistic studies of anti-arthritic compounds with low oral absorption and bioavailability.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Encéfalo/metabolismo , Curcumina/uso terapêutico , Trato Gastrointestinal/metabolismo , Acetilcolina/antagonistas & inibidores , Acetilcolina/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/patologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Colina O-Acetiltransferase , Curcumina/farmacologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Antagonistas Nicotínicos/farmacologia , Gânglio Nodoso/efeitos dos fármacos , Gânglio Nodoso/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Vagotomia/tendências , Nervo Vago/cirurgiaRESUMO
Alpha-7 nicotinic acetylcholine receptor (α7nAChR) was reported to be involved in the modulation of neuropathic pain. Electroacupuncture (EA) has therapeutic effects on neuropathic pain induced by nerve injury, but the underlying mechanisms remain unclear. The present study was designed to investigate whether α7nAChR participates in the relieving effects of 2â¯Hz EA on neuropathic pain. Paw withdrawal threshold (PWT) was measured to study the EA-mediated analgesic effect in a rat model of spared nerve injury (SNI). The spinal α7nAChR and IL-1ß expression levels were determined by RT-PCR, Western blot analysis, and immunofluorescence staining. Additionally, immunofluorescence targeting the expression of CD11b, which is a molecular indicator of microglial activation. The results showed that 2â¯Hz EA stimulation significantly improved the expression of α7nAChR and reduced the production of IL-1ß and CD11b in the spinal cord of rats with SNI-induced neuropathic pain, along with the relief of mechanical hypersensitivity after EA treatment. Moreover, intrathecal injection of alpha-bungarotoxin (α-Bgtx), a selective antagonist for α7nAChR, at the dosage of 1.0⯵g/kg, not only suppressed the analgesic effect of EA in SNI rats, but also inhibited the enhancement of α7nAChR expression and the reduction of IL-1ß expression induced by EA. In conclusion, our study indicated that 2â¯Hz EA reduces SNI-induced mechanical hypersensitivity via upregulating α7nAChR and downregulating IL-1ß and CD11b in the spinal cord of SNI rats, which might be one of the mechanisms underlying its effectiveness in the neuropathic pain.
Assuntos
Eletroacupuntura , Hiperalgesia/metabolismo , Hiperalgesia/terapia , Neuralgia/metabolismo , Neuralgia/terapia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Bungarotoxinas/farmacologia , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Método Duplo-Cego , Eletroacupuntura/métodos , Hiperalgesia/patologia , Interleucina-1beta/metabolismo , Masculino , Neuralgia/patologia , Antagonistas Nicotínicos/farmacologia , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/patologia , Tato , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.
Assuntos
Nicotina/administração & dosagem , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Comportamento de Procura de Droga/efeitos dos fármacos , Masculino , Antagonistas Nicotínicos/farmacocinética , Ratos Sprague-Dawley , Receptores Nicotínicos/metabolismo , Reforço Psicológico , Autoadministração , Relação Estrutura-AtividadeRESUMO
Learning to associate a stimulus with reinforcement causes plasticity in primary sensory cortex. Neural activity caused by the associated stimulus is paired with reinforcement, but population analyses have not found a selective increase in response to that stimulus. Responses to other stimuli increase as much as, or more than, responses to the associated stimulus. Here, we applied population analysis at a new time point and additionally evaluated whether cholinergic receptor blockers interacted with the plastic changes in cortex. Three days of tone identification behavior caused responsiveness to increase broadly across primary auditory cortex, and target responses strengthened less than overall responsiveness. In pharmacology studies, behaviorally impairing doses of selective acetylcholine receptor blockers were administered during behavior. Neural responses were evaluated on the following day, while the blockers were absent. The muscarinic group, blocked by scopolamine, showed lower responsiveness and an increased response to the tone identification target that exceeded both the 3-day control group and task-naïve controls. Also, a selective increase in the late phase of the response to the tone identification stimulus emerged. Nicotinic receptor antagonism, with mecamylamine, more modestly lowered responses the following day and lowered target responses more than overall responses. Control acute studies demonstrated the muscarinic block did not acutely alter response rates, but the nicotinic block did. These results lead to the hypothesis that the decrease in the proportion of the population spiking response that is selective for the target may be explained by the balance between effects modulated by muscarinic and nicotinic receptors.
Assuntos
Córtex Auditivo/metabolismo , Percepção Auditiva/fisiologia , Neurônios/metabolismo , Reconhecimento Fisiológico de Modelo/fisiologia , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Córtex Auditivo/efeitos dos fármacos , Percepção Auditiva/efeitos dos fármacos , Mapeamento Encefálico , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Masculino , Mecamilamina/farmacologia , Microeletrodos , Antagonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Reconhecimento Fisiológico de Modelo/efeitos dos fármacos , Ratos Sprague-Dawley , Escopolamina/farmacologiaRESUMO
Traditional uses and current results highlight the neuroprotective properties of Rosmarinus officinalis L. The compelling need for novel strategies able to relieve neuropathic pain encouraged us to analyze different rosemary leaf extracts in rats following chronic constriction injury (CCI) of sciatic nerve. Ethanol, acetone, and the innovative ultrasound-hexane extractive methods were used to obtain: EE, AE, and for hexane extracts UREprel and URE. Extracts were characterized in terms of typical constituents and repeatedly administered to CCI-rats (13-days treatment, from the day of surgery). URE showed the best efficacy and potency in reducing hypersensitivity to noxious- and non-noxious stimuli and spontaneous pain. URE contained the higher quantity of the terpenoid carnosic acid (CA) and its efficacy was compared to pure CA. Histological analysis of the sciatic nerve revealed that URE prevented axon and myelin derangement, edema and inflammatory infiltrate. In the dorsal horn of the spinal cord, URE did not reduce astrocyte activation. Both the pain reliever and the neuroconservative effects of URE were significantly prevented by the nicotinic receptor (nAChR) antagonist mecamylamine. In conclusion, the hexane-ultrasound rosemary extract is able to reduce neuropathic hypersensitivity and protect nervous tissues. Effectiveness is mainly related to the terpenoid fraction by mechanisms involving nAChRs.
Assuntos
Neuralgia/tratamento farmacológico , Extratos Vegetais/química , Receptores Nicotínicos/metabolismo , Rosmarinus/química , Terpenos/farmacologia , Abietanos/análise , Abietanos/farmacologia , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Animais , Fracionamento Químico/métodos , Modelos Animais de Doenças , Mecamilamina/farmacologia , Neuralgia/etiologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Antagonistas Nicotínicos/farmacologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Solventes/química , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.
Assuntos
Acetilcolina/metabolismo , Ritmo Circadiano/fisiologia , Citocinas/sangue , Inflamação/sangue , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Aconitina/análogos & derivados , Aconitina/farmacologia , Aconitina/uso terapêutico , Animais , Ritmo Circadiano/efeitos dos fármacos , Corticosterona/sangue , Modelos Animais de Doenças , Antagonistas de Hormônios/farmacologia , Antagonistas de Hormônios/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/mortalidade , Rim/efeitos dos fármacos , Rim/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mifepristona/farmacologia , Mifepristona/uso terapêutico , Antagonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/uso terapêuticoRESUMO
In screening a library of plant extracts from ~1000 species native to the Southeastern United States, Lobelia cardinalis was identified as containing nicotinic acetylcholine receptor (nicAchR) binding activity which was relatively non-selective for the α4ß2- and α7-nicAchR subtypes. This nicAchR binding profile is atypical for plant-derived nicAchR ligands, the majority of which are highly selective for α4ß2-nicAchRs. Its potential therapeutic relevance is noteworthy since agonism of α4ß2- and α7-nicAchRs is associated with anti-inflammatory and neuroprotective properties. Bioassay-guided fractionation of L. cardinalis extracts led to the identification of lobinaline, a complex binitrogenous alkaloid, as the main source of the unique nicAchR binding profile. Purified lobinaline was a potent free radical scavenger, displayed similar binding affinity at α4ß2- and α7-nicAchRs, exhibited agonist activity at nicAchRs in SH-SY5Y cells, and inhibited [(3)H]-dopamine (DA) uptake in rat striatal synaptosomes. Lobinaline significantly increased fractional [(3)H] release from superfused rat striatal slices preloaded with [(3)H]-DA, an effect that was inhibited by the non-selective nicAchR antagonist mecamylamine. In vivo electrochemical studies in urethane-anesthetized rats demonstrated that lobinaline locally applied in the striatum significantly prolonged clearance of exogenous DA by the dopamine transporter (DAT). In contrast, lobeline, the most thoroughly investigated Lobelia alkaloid, is an α4ß2-nicAchR antagonist, a poor free radical scavenger, and is a less potent DAT inhibitor. These previously unreported multifunctional effects of lobinaline make it of interest as a lead to develop therapeutics for neuropathological disorders that involve free radical generation, cholinergic, and dopaminergic neurotransmission. These include neurodegenerative conditions, such as Parkinson's disease, and drug abuse.
Assuntos
Alcaloides/farmacologia , Lobelia/química , Antagonistas Nicotínicos/farmacologia , Quinolinas/farmacologia , Animais , Linhagem Celular , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Ensaios de Triagem em Larga Escala , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4ß2 * and decreases α6ß2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.
Assuntos
Preferências Alimentares/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Sacarose/metabolismo , Alcaloides/farmacologia , Animais , Azocinas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Masculino , Mecamilamina/farmacologia , Núcleo Accumbens/metabolismo , Quinolizinas/farmacologia , Ratos Wistar , Receptores Nicotínicos/metabolismo , Vareniclina/farmacologiaRESUMO
Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.
Assuntos
Acetilcolina/metabolismo , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Respiração , Acetilcolina/farmacologia , Animais , Animais Recém-Nascidos , Atropina/farmacologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/metabolismo , Agonistas Colinérgicos/farmacologia , Curare/farmacologia , Modelos Animais de Doenças , Feminino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Antagonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/crescimento & desenvolvimento , Nervo Frênico/metabolismo , Gravidez , Ratos Sprague-Dawley , Respiração/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Técnicas de Cultura de TecidosRESUMO
RATIONALE: The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. OBJECTIVES: We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. METHODS: Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. RESULTS: Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. CONCLUSIONS: Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.
Assuntos
Inibidores da Colinesterase/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Reflexo de Sobressalto/efeitos dos fármacos , Rivastigmina/farmacologia , Isolamento Social/psicologia , Acetilcolina/metabolismo , Estimulação Acústica , Animais , Química Encefálica/efeitos dos fármacos , Dopamina/metabolismo , Masculino , Mecamilamina/farmacologia , Camundongos , Antagonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptor 5-HT1A de Serotonina/efeitos dos fármacosRESUMO
Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4ß2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHßE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHßE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHßE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4ß2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4ß2 nAChR-mediated transmission.
Assuntos
Ingestão de Alimentos/fisiologia , Hipotálamo/metabolismo , Atividade Motora/fisiologia , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Di-Hidro-beta-Eritroidina/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Neurônios/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Orexinas/metabolismo , Ocitocina/metabolismo , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100 µM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56 mM K(+). Ikarisoside A (1-100 µM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3ß4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100 µM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100 µM and 10-100 µM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.
Assuntos
Acetilcolina/antagonistas & inibidores , Medula Suprarrenal/efeitos dos fármacos , Catecolaminas/antagonistas & inibidores , Flavonoides/farmacologia , Glicosídeos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Nicotínicos , Acetilcolina/toxicidade , Medula Suprarrenal/metabolismo , Animais , Canais de Cálcio/metabolismo , Catecolaminas/biossíntese , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Flavonoides/isolamento & purificação , Glicosídeos/isolamento & purificação , Ativação do Canal Iônico/fisiologia , Antagonistas Nicotínicos/isolamento & purificação , Antagonistas Nicotínicos/farmacologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de Planta , Receptores Nicotínicos/metabolismo , Canais de Sódio/metabolismo , Xenopus laevisRESUMO
Acetylcholine-activating pentameric nicotinic receptors (nAChRs) are an essential mode of neurotransmission in the enteric nervous system (ENS). In this study, we examined the functional development of specific nAChR subtypes in myenteric neurons using Wnt1-Cre;R26R-GCaMP3 mice, where all enteric neurons and glia express the genetically encoded calcium indicator, GCaMP3. Transcripts encoding α3, α4, α7, ß2, and ß4 nAChR subunits were already expressed at low levels in the E11.5 gut and by E14.5 and, thereafter, α3 and ß4 transcripts were the most abundant. The effect of specific nAChR subtype antagonists on evoked calcium activity in enteric neurons was investigated at different ages. Blockade of the α3ß4 receptors reduced electrically and chemically evoked calcium responses at E12.5, E14.5, and P0. In addition to the α3ß4 antagonist, antagonists to α3ß2 and α4ß2 also significantly reduced responses by P10-11 and in adult preparations. Therefore, there is an increase in the diversity of functional nAChRs during postnatal development. However, an α7 nAChR antagonist had no effect at any age. Furthermore, at E12.5 we found evidence for unconventional receptors that were responsive to the nAChR agonists 1-dimethyl-4-phenylpiperazinium and nicotine, but were insensitive to the general nicotinic blocker, hexamethonium. Migration, differentiation, and neuritogenesis assays did not reveal a role for nAChRs in these processes during embryonic development. In conclusion, there are significant changes in the contribution of different nAChR subunits to synaptic transmission during ENS development, even after birth. This is the first study to investigate the development of cholinergic transmission in the ENS.
Assuntos
Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Receptores Nicotínicos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Sistema Nervoso Entérico/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antagonistas Nicotínicos/farmacologia , Gravidez , Transmissão Sináptica/efeitos dos fármacosRESUMO
Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4ß2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4ß2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059).
Assuntos
Agonistas Nicotínicos/farmacologia , Triazóis/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Modelos Químicos , Estrutura Molecular , Agonistas Nicotínicos/síntese química , Agonistas Nicotínicos/química , Antagonistas Nicotínicos/síntese química , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Ensaio Radioligante , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Transfecção , Triazóis/síntese química , Triazóis/química , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Oxaliplatin, a platinum-based chemotherapy drug, often induces acute neuropathic pain, especially cold allodynia, even after a single administration. Subcutaneous injection of diluted bee venom (BV) into acupoints has been used to treat various pain symptoms in traditional oriental medicine. Although we previously demonstrated the suppressive effect of BV injection on oxaliplatin-induced cold allodynia in rats, its neurochemical mechanism remained unclear. This study investigates whether and how the cholinergic system mediates the relieving effect of BV injection on cold allodynia in oxaliplatin-administered rats. The behavioral signs of cold allodynia induced by an oxaliplatin administration (6 mg/kg, intraperitoneally (i.p.)) were evaluated by a tail immersion test in cold water (4°C). BV (0.25 mg/kg, subcutaneously (s.c.)) injection into the Yaoyangguan acupoint, located between the spinous processes of the fourth and fifth lumbar vertebrae, significantly alleviated the cold allodynia. This relieving effect of BV injection on oxaliplatin-induced cold allodynia was blocked by a pretreatment with mecamylamine (a non-selective nicotinic receptor antagonist, 2 mg/kg, i.p.), but not by atropine (a non-selective muscarinic receptor antagonist, 1 mg/kg, i.p.). Further, dihydro-ß-erythroidinehydrobromide (DHßE, an α4ß2 nicotinic antagonist, 5 mg/kg, i.p.) prevented the anti-allodynic effect of BV, whereas methyllycaconitine (an α7 nicotinic antagonist, 6 mg/kg, i.p.) did not. Finally, intrathecal administration of DHßE (10 nM) blocked the BV-induced anti-allodynic effect. These results suggest that nicotinic acetylcholine receptors, especially spinal α4ß2 receptors, but not muscarinic receptors, mediate the suppressive effect of BV injection on oxaliplatin-induced acute cold allodynia in rats.