RESUMO
4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund's Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h - 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.
RESUMO
The tobacco cembranoid known as (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) has been shown to offer neuroprotection against conditions such as brain ischemia, systemic inflammation, Parkinson's disease, and organophosphate toxicity in rodents. Previous safety studies conducted on male and female Sprague Dawley rats revealed no significant side effects following a single injection of 4R at varying concentrations (6, 24, or 98 mg/kg of body weight). This study aimed to assess the potential of 4R for clinical trials in neurotherapy in male nonhuman primates. Ten macaques (Macacca mulatta) were randomly separated into two groups of 5 and then intravenously injected with 4R or vehicle for 11 consecutive days at a dose of 1.4 mg/kg. Throughout the study, we monitored brain activity by electroencephalogram, somatosensory evoked potentials, and transcranial motor evoked potentials on days 0, 4, 8, and 12 and found no significant changes. The spontaneous behavior of the primates remained unaffected by the treatment. Minor hematological and blood composition variations were also detected in the experimental animals but lacked clinical significance. In conclusion, our results reinforce the notion that 4R is non-toxic in nonhuman primates under the conditions of this study.
Assuntos
Isquemia Encefálica , Diterpenos , Ratos , Masculino , Feminino , Animais , Ratos Sprague-Dawley , NicotianaRESUMO
Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 µM, but the NP declined at higher concentrations (>3 µM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(-)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4ß2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.
Assuntos
Memantina , Fármacos Neuroprotetores , Hipocampo/metabolismo , Memantina/metabolismo , Memantina/farmacologia , N-Metilaspartato/toxicidade , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Nicotina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) interacts with nicotinic acetylcholine receptors, which results in neuroprotection against organophosphate toxicity, brain ischemia, and Parkinson's disease. The present study is a continuation of our previous research in which we applied a single dose of 4R 1 h before or 24 h after exposure to diisopropylfluorophosphate (DFP) (analog of the nerve agent sarin). The 4R dose robustly decreased neuroinflammation and neuronal death at both timepoints. Here, we investigated the toxicity of a single dose of 4R in male and female Sprague Dawley (SD) rats after a subcutaneous (s.c.) injection of 6, 24, or 98 mg/kg. Body weight was not affected by 4R during the 7-day observation period. No histopathologic changes in the organs were attributed to 4R. Minor hematological and blood composition variations were detected on Day 3 in the mid- and the high-dose males, but these were resolved by Day 8. At the area of the s.c. injection site, alopecia and dry skin were detected in both the 4R-treated males and females and in the female controls.
RESUMO
(1S, 2E, 4R, 6R,-7E, 11E)-2, 7, 11-cembratriene-4, 6-diol (4R) is one of the cembranoids found in tobacco leaves. Previous studies have found that 4R protected acute rat hippocampal slices against neurotoxicity induced by N-methyl-D-aspartate (NMDA) and against the toxic organophosphorus compounds paraoxon and diisopropylfluorophosphate (DFP). Furthermore, in vivo, 4R reduced the infarct size in a rodent ischemic stroke model and neurodegeneration caused by DFP. The present study expanded our previous study by focusing on the effect of 4R in Parkinson's disease (PD) and elucidating its underlying mechanisms using 6-hydroxydopamine (6-OHDA)-induced injury models. We found that 4R exhibited significant neuroprotective activity in the rat unilateral 6-OHDA-induced PD model in vivo. The therapeutic effect was evident both at morphological and behavioral levels. 4R (6 and 12 mg/kg) treatments significantly improved outcomes of 6-OHDA-induced PD in vivo as indicated by reducing forelimb asymmetry scores and corner test scores 4 weeks after injection of 6-OHDA (p < 0.05). The therapeutic effect of 4R was also reflected by decreased depletion of tyrosine hydroxylase (TH) in the striatum and substantia nigra (SN) on the side injected with 6-OHDA. TH expression was 70.3 and 62.8% of the contralateral side in striatum and SN, respectively, after 6 mg/kg 4R treatment; furthermore, it was 80.1 and 79.3% after treatment with 12 mg/kg of 4R. In the control group, it was 51.9 and 23.6% of the contralateral striatum and SN (p < 0.05). Moreover, 4R also protected differentiated neuro-2a cells from 6-OHDA-induced cytotoxicity in vitro. The activation of p-AKT and HAX-1, and inhibition of caspase-3 and endothelial inflammation, were involved in 4R-mediated protection against 6-OHDA-induced injury. In conclusion, the present study indicates that 4R shows a therapeutic effect in the rat 6-OHDA-induced PD model in vivo and in 6-OHDA-challenged neuro-2a cells in vitro.
RESUMO
Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of acetylcholine esterase and a surrogate of the organophosphorus (OP) nerve agent sarin. The neurotoxicity of DFP was assessed as a reduction of population spike (PS) area elicited by synaptic stimulation in acute hippocampal slices. Two classical antidotes, atropine, and pralidoxime, and two novel antidotes, 4R-cembranotriene-diol (4R) and a caspase nine inhibitor, were tested. Atropine, pralidoxime, and 4R significantly protected when applied 30 min after DFP. The caspase inhibitor was neuroprotective when applied 5-10 min before or after DFP, suggesting that early synaptic apoptosis is responsible for the loss of PSs. It is likely that apoptosis starts at the synapses and, if antidotes are not applied, descends to the cell bodies, causing death. The acute slice is a reliable tool for mechanistic studies, and the assessment of neurotoxicity and neuroprotection with PS areas is, in general, pharmacologically congruent with in vivo results and predicts the effect of drugs in vivo. 4R was first found to be neuroprotective in slices and later we demonstrated that 4R is neuroprotective in vivo. The mechanism of neurotoxicity of OPs is not well understood, and there is a need for novel antidotes that could be discovered using acute slices.
Assuntos
Inibidores da Colinesterase/farmacologia , Hipocampo/efeitos dos fármacos , Isoflurofato/farmacologia , Fármacos Neuroprotetores/farmacologia , Sinapses/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Atropina/farmacologia , Hipocampo/metabolismo , Masculino , Neuroproteção/fisiologia , Compostos de Pralidoxima/farmacologia , Ratos Sprague-Dawley , Sinapses/metabolismoRESUMO
Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyridostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 µM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 µM pyridostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.
Assuntos
Acetilcolinesterase/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Brometo de Piridostigmina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Células Cultivadas , Inibidores da Colinesterase/farmacologia , Células-Tronco Neurais/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/metabolismo , ParaoxonRESUMO
4R-cembranoid (4R) is a natural cyclic diterpenoid found in tobacco leaves that displays neuroprotective activity. 4R protects against NMDA, paraoxon (POX), and diisopropylfluorophosphate (DFP) damage in rat hippocampal slices and against DFP in rats in vivo. The purpose of this study was to examine the metabolism and pharmacokinetics of 4R as part of its preclinical development as a neuroprotective drug. 10 µM 4R was found to be very stable in plasma for up to 1 hr incubation. 4R metabolism in human microsomes was faster than in the rat. Ten metabolites of 4R were detected in the microsomal samples; 6 dihydroxy and 4 monohydroxy forms of 4R. Male rats received a single dose of 4R at 6 mg/kg i.v., i.m., or s.c. The i.v. group had the highest plasma concentration of 1017 ng/mL. The t1/2 was 36 min and reached the brain within 10 min. The brain peak concentration was 6516 ng/g. The peak plasma concentration in the i.m. group was 163 ng/mL compared to 138 ng/mL in the s.c. group. The t1/2 of 4R after i.m. and s.c. administration was approximately 1.5 hr. The brain peak concentration was 329 ng/g in the i.m. group and 323 ng/g for the s.c. group. The brain to plasma ratio in the i.v. group was 6.4, reached 10 min after dose, whereas in the i.m. and s.c. groups was 2.49 and 2.48, respectively, at 90 min after dose. Our data show that 4R crosses the BBB and concentrates in the brain where it exerts its neuroprotective effect.
Assuntos
Diterpenos/metabolismo , Diterpenos/farmacocinética , Animais , Diterpenos/sangue , Diterpenos/química , Feminino , Humanos , Masculino , Metaboloma , Microssomos Hepáticos/metabolismo , Ratos Sprague-DawleyRESUMO
(1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (4R) is a precursor to key flavor ingredients in leaves of Nicotiana species. The present study shows 4R decreased brain damage in rodent ischemic stroke models. The 4R-pretreated mice had lower infarct volumes (26.2±9.7 mm3) than those in control groups (untreated: 63.4±4.2 mm3, DMSO: 60.2±14.2 mm3). The 4R-posttreated rats also had less infarct volumes (120±65 mm3) than those in the rats of the DMSO group (291±95 mm3). The results from in vitro experiments indicate that 4R decreased neuro2a cell (neuroblastoma cells) apoptosis induced by oxygen-glucose deprivation (OGD), and improved the population spikes' (PSs) recovery in rat acute hippocampal slices under OGD; a phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, abolished the effect of 4R on PSs recovery. Furthermore, 4R also inhibited monocyte adhesion to murine brain-derived endothelial (bEND5) cells and upregulation of intercellular adhesion molecule-1(ICAM-1) induced by OGD/reoxygenation (OGD/R), and restored the p-Akt level to pre-OGD/R values in bEND5 cells. In conclusion, the present study indicates that 4R has a protective effect in rodent ischemic stroke models. Inhibition of ICAM-1 expression and restoration of Akt phosphorylation are the possible mechanisms involved in cellular protection by 4R.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Diterpenos/farmacologia , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glucose/deficiência , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Many organophosphorous esters synthesized for applications in industry, agriculture, or warfare irreversibly inhibit acetylcholinesterase, and acute poisoning with these compounds causes life-threatening cholinergic overstimulation. Following classical emergency treatment with atropine, an oxime, and a benzodiazepine, surviving victims often suffer brain neurodegeneration. Currently, there is no pharmacological treatment to prevent this brain injury. Here we show that a cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) ameliorates the damage caused by diisopropylfluorophosphate (DFP) in the hippocampal area CA1. DFP has been frequently used as a surrogate for the warfare nerve agent sarin. In rats, DFP is lethal at the dose used to cause brain damage. Therefore, to observe brain damage in survivors, the death rate was reduced by pre-administration of the peripherally acting antidotes pyridostigmine and methyl atropine or its analog ipratropium. Pyridostigmine bromide, methyl atropine nitrate, and ipratropium bromide were dissolved in saline and injected intramuscularly at 0.1mg/kg, 20mg/kg, and 23mg/kg, respectively. DFP (9mg/kg) dissolved in cold water was injected intraperitoneally. 4R (6mg/kg) dissolved in DMSO was injected subcutaneously, either 1h before or 5 or 24h after DFP. Neurodegeneration was assessed with Fluoro-Jade B and amino cupric silver staining; neuroinflammation was measured by the expression of nestin, a marker of activated astrocytes. Forty-eight hours after DFP administration, 4R decreased the number of dead neurons by half when injected before or after DFP. 4R also significantly decreased the number of activated astrocytes. These data suggest that 4R is a promising new drug that could change the therapeutic paradigm for acute poisoning with organophosphorous compounds by the implementation of a second-stage intervention after the classical countermeasure treatment.
Assuntos
Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/prevenção & controle , Inibidores da Colinesterase/intoxicação , Diterpenos/uso terapêutico , Isoflurofato/intoxicação , Fármacos Neuroprotetores/uso terapêutico , Acetilcolinesterase/metabolismo , Animais , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Morte Celular , Diterpenos/farmacologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamenteRESUMO
Diisopropylfluorophosphate (DFP) is an organophosphorous insecticide used as a surrogate for the more toxic chemical warfare nerve agent sarin. DFP produces neurotoxicity in vivo and irreversibly decreases the area of population spikes recorded from the CA1 region of acute hippocampal slices. (1S,2E,4R,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol (1) is a neuroprotective natural cembranoid that reverses DFP-induced damage both in vivo and in the hippocampal slice. Cembranoid 1 acts by noncompetitive inhibition of the α7 nicotinic acetylcholine receptor. This study aims at establishing a preliminary structure-activity relationship to define the neuroprotective cembranoid pharmacophores using the hippocampal slice assay and pharmacophore modeling. Fourteen natural, semisynthetic, or biocatalytic cembranoid analogues 2-15 related to 1 were tested for their capacity to protect the population spikes from DFP-induced damage and intrinsic toxicity. Twelve cembranoids caused significant reversal of DFP toxicity; only 3 active analogues displayed minor intrinsic toxicity at 10 µM. The C-4 epimer of 1 (2) and the 4-O-methyl ether analogue of 1 (3), were totally devoid of neuroprotective activity. The results suggested a model for cembranoid binding where the hydrophobic ring surface binds to a hydrophobic (Hbic) patch on the receptor molecule and an electronegative atom (oxygen or sulfur) in proper spatial relationship to the ring surface interacts with an electropositive group in the receptor binding site. A pharmacophore model consisting of 1 hydrogen bond acceptor (HBA), 2 Hbic, and 10 exclusion spheres was established using HipHop-REFINE and supported the above mentioned pharmacophoric hypothesis.
Assuntos
Diterpenos/química , Diterpenos/farmacologia , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/prevenção & controle , Animais , Diterpenos/síntese química , Feminino , Hipocampo/efeitos dos fármacos , Modelos Moleculares , Fármacos Neuroprotetores/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection has been implicated in the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases and hypoxic ischemic events as well as other diseases hallmarked by excitotoxic and apoptotic neuronal death. Several modalities of nicotinic neuroprotection have been reported. However, although this process generally involves α4ß2 and α7 subtypes, the underlying mechanisms are largely unknown. Interestingly, both activation and inhibition of α7 nAChRs have been reported to be neuroprotective. We have shown that inhibition of α7 nAChRs protects the function of acute hippocampal slices against excitotoxicity in an α4ß2-dependent manner. Neuroprotection was assessed as the prevention of the N-methyl-D-aspartate-dependent loss of the area of population spikes (PSs) in the CA1 area of acute hippocampal slices. Our results support a model in which α7 AChRs control the release of γ-aminobutyric acid (GABA). Blocking either α7 or GABA(A) receptors reduces the inhibitory tone on cholinergic terminals, thereby promoting α4ß2 activation, which in turn mediates neuroprotection. These results shed light on how α7 nAChR inhibition can be neuroprotective through a mechanism mediated by activation of α4ß2 nAChRs.
Assuntos
Antagonistas GABAérgicos/farmacologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Humanos , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/fisiologia , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The goal of this study was to characterize acute neuronal injury in a novel nonhuman primate (NHP) ischemic stroke model by using multiple outcome measures. Silk sutures were inserted into the M1 segment of the middle cerebral artery of rhesus macaques to achieve permanent occlusion of the vessel. The sutures were introduced via the femoral artery by using endovascular microcatheterization techniques. Within hours after middle cerebral artery occlusion (MCAO), infarction was detectable by using diffusion-weighted MRI imaging. The infarcts expanded by 24 h after MCAO and then were detectable on T2-weighted images. The infarcts seen by MRI were consistent with neuronal injury demonstrated histologically. Neurobehavioral function after MCAO was determined by using 2 neurologic testing scales. Neurologic assessments indicated that impairment after ischemia was limited to motor function in the contralateral arm; other neurologic and behavioral parameters were largely unaffected. We also used microarrays to examine gene expression profiles in peripheral blood mononuclear cells after MCAO-induced ischemia. Several genes were altered in a time-dependent manner after MCAO, suggesting that this ischemia model may be suitable for identifying blood biomarkers associated with the presence and severity of ischemia. This NHP stroke model likely will facilitate the elucidation of mechanisms associated with acute neuronal injury after ischemia. In addition, the ability to identify candidate blood biomarkers in NHP after ischemia may prompt the development of new strategies for the diagnosis and treatment of ischemic stroke in humans.
Assuntos
Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Transtornos Psicomotores/patologia , Acidente Vascular Cerebral/patologia , Animais , Western Blotting , Cateterismo , Citocinas/metabolismo , Técnicas de Diagnóstico Neurológico , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Técnicas Histológicas , Leucócitos Mononucleares/metabolismo , Macaca mulatta , Imageamento por Ressonância Magnética , Análise em Microsséries , Neurônios/patologia , Transtornos Psicomotores/etiologia , Acidente Vascular Cerebral/sangueRESUMO
BACKGROUND: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. PRINCIPAL FINDINGS: Bradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. CONCLUSIONS: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.
Assuntos
Bradicinina/toxicidade , N-Metilaspartato , Receptor B1 da Bradicinina/agonistas , Receptor B2 da Bradicinina/fisiologia , Animais , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Química Encefálica , Região CA1 Hipocampal , Carboxipeptidases/metabolismo , Fármacos Neuroprotetores , Ratos , Receptor B1 da Bradicinina/fisiologiaRESUMO
Many neurotoxic organophosphates (OPs) inhibit acetylcholinesterase (AChE) and as a result can cause a life threatening cholinergic crisis. Current medical countermeasures, which typically include atropine and oximes target the cholinergic crisis and are effective in decreasing mortality but do not sufficiently protect against delayed neurological deficits. There is, therefore, a need to develop neuroprotective drugs to prevent long-term neurological deficits. We used acute hippocampal slices to test the hypothesis that 4R,6R-cembratrienediol (4R) protects against functional damage caused by the OP paraoxon (POX). To assess hippocampal function, we measured synaptically evoked population spikes (PSs). Application of 4R reversed POX inhibition of PSs and the EC(50) of this effect was 0.8 µM. Atropine alone did not protect against POX neurotoxicity, but it did enhance protection by 4R. Pralidoxime partially regenerated AChE activity and protected against POX inhibition of PSs. 4R did not regenerate AChE suggesting that under our experimental conditions, the deleterious effect of POX on hippocampal function is not directly related to AChE inhibition. In conclusion, 4R is a promising neuroprotective compound against OP neurotoxins.
Assuntos
Inibidores da Colinesterase/toxicidade , Diterpenos/farmacologia , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Paraoxon/toxicidade , Acetilcolinesterase/metabolismo , Animais , Atropina/farmacologia , Reativadores da Colinesterase/farmacologia , Diterpenos/química , Relação Dose-Resposta a Droga , Hipocampo/citologia , Masculino , Fármacos Neuroprotetores/química , Parassimpatolíticos/farmacologia , Compostos de Pralidoxima/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Feocromocitoma/metabolismo , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células PC12 , Feocromocitoma/patologia , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 microM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 microM nicotine for 24 h in the presence of 1 microM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence.
Assuntos
Comportamento Animal/efeitos dos fármacos , Diterpenos/farmacologia , Nicotiana/química , Nicotina/efeitos adversos , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Síndrome de Abstinência a Substâncias/psicologia , Animais , Carbacol/efeitos adversos , Atividade Motora/efeitos dos fármacos , PlanáriasRESUMO
Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies.
Assuntos
Antozoários/química , Diterpenos/farmacologia , Nicotiana/química , Receptores Nicotínicos/efeitos dos fármacos , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Diterpenos/metabolismo , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores Nicotínicos/metabolismoRESUMO
In tobacco, there are two types of compounds that interact with neuronal nicotinic acetylcholine receptors (nnAChRs) in the brain. The first is the addictive component of tobacco and an agonist of these receptors, nicotine. The second are cyclic diterpenoids called cembranoids that non-competitively inhibit many types of nnAChRs. Nictotinic receptors composed of alpha4beta2 subunits are the predominant type of nicotinic receptors in the brain. These alpha4beta2 receptors are up-regulated upon chronic exposure to nicotine and have been implicated in nicotine addiction. The present study was designed to determine whether the inhibitory effects of two cembranoids from tobacco [(1S, 2E, 4R, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4R) and its diastereoisomer (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4S)] were comparable on acetylcholine (ACh) and nicotine-evoked currents through alpha4beta2 nnAChRs. alpha4beta2 nnAChRs from rat brain were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The dose-response curves for acetylcholine and nicotine were hyperbolic and bell-shaped, respectively. Although there was no difference in the potency between cembranoids 4R and 4S, both of these cembranoids more potently inhibited nicotine-induced currents than acetylcholine-induced currents. Furthermore, both cembranoids were more potent inhibitors of this receptor when they were preincubated for 1 min prior to application of agonist. The finding that cembranoids preferentially inhibit nicotine-induced currents over those elicited by the natural neurotransmitter acetylcholine may have important implications when developing strategies to prevent nicotine addiction and tobacco use.