RESUMO
Hyperactivation of the amygdala following chronic stress is believed to be one of the primary mechanisms underlying the increased propensity for anxiety-like behaviors and pathological states; however, the mechanisms by which chronic stress modulates amygdalar function are not well characterized. The aim of the current study was to determine the extent to which the endocannabinoid (eCB) system, which is known to regulate emotional behavior and neuroplasticity, contributes to changes in amygdalar structure and function following chronic stress. To examine the hypothesis, we have exposed C57/Bl6 mice to chronic restraint stress, which results in an increase in fatty acid amide hydrolase (FAAH) activity and a reduction in the concentration of the eCB N-arachidonylethanolamine (AEA) within the amygdala. Chronic restraint stress also increased dendritic arborization, complexity and spine density of pyramidal neurons in the basolateral nucleus of the amygdala (BLA) and increased anxiety-like behavior in wild-type mice. All of the stress-induced changes in amygdalar structure and function were absent in mice deficient in FAAH. Further, the anti-anxiety effect of FAAH deletion was recapitulated in rats treated orally with a novel pharmacological inhibitor of FAAH, JNJ5003 (50 mg per kg per day), during exposure to chronic stress. These studies suggest that FAAH is required for chronic stress to induce hyperactivity and structural remodeling of the amygdala. Collectively, these studies indicate that FAAH-mediated decreases in AEA occur following chronic stress and that this loss of AEA signaling is functionally relevant to the effects of chronic stress. These data support the hypothesis that inhibition of FAAH has therapeutic potential in the treatment of anxiety disorders, possibly by maintaining normal amygdalar function in the face of chronic stress.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/fisiologia , Tonsila do Cerebelo/patologia , Ansiedade/prevenção & controle , Estresse Psicológico/enzimologia , Amidoidrolases/deficiência , Amidoidrolases/genética , Tonsila do Cerebelo/metabolismo , Animais , Ansiedade/enzimologia , Ansiedade/etiologia , Ácidos Araquidônicos , Doença Crônica , Cicloexanóis/farmacologia , Dendritos/ultraestrutura , Avaliação Pré-Clínica de Medicamentos , Endocanabinoides/deficiência , Endocanabinoides/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alcamidas Poli-Insaturadas , Células Piramidais/patologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/fisiologia , Restrição Física/efeitos adversos , Estresse Psicológico/complicações , Estresse Psicológico/patologia , Estresse Psicológico/fisiopatologiaRESUMO
Recent data suggest that the endocannabinoid system (ECS) may be involved in the glial response in different types of brain injury. Both acute and chronic insults seem to trigger a shift in the pattern of expression of some elements of this system from neuronal to glial. Specifically, data obtained in human brain tissue sections from Alzheimer's disease patients showed that the expression of cannabinoid receptors of the CB(2) type is induced in activated microglial cells while fatty acid amide hydrolase (FAAH) expression is increased in reactive astrocytes. The present study was designed to determine the time-course of the shift from neuronal to glial induction in the expression of these proteins in Down's syndrome, sometimes referred to as a human model of Alzheimer-like beta-amyloid (Abeta) deposition. Here we present immunohistochemical evidence that both CB(2) receptors and FAAH enzyme are induced in Abeta plaque-associated microglia and astroglia, respectively, in Down's syndrome. These results suggest that the induction of these elements of the ECS contributes to, or is a result of, amyloid deposition and subsequent plaque formation. In addition, they confirm a striking differential pattern of distribution of FAAH and CB(2) receptors.
Assuntos
Amidoidrolases/metabolismo , Peptídeos beta-Amiloides/fisiologia , Síndrome de Down/metabolismo , Neuroglia/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Adulto , Encéfalo/patologia , Química Encefálica/fisiologia , Criança , Síndrome de Down/patologia , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Placa Amiloide/patologiaRESUMO
BACKGROUND AND PURPOSE: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction. EXPERIMENTAL APPROACH: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy. KEY RESULTS: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA. CONCLUSIONS AND IMPLICATIONS: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Ácidos Araquidônicos/metabolismo , Glicerídeos/metabolismo , Artéria Cerebral Média/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Amidoidrolases/antagonistas & inibidores , Compostos de Anilina/farmacologia , Animais , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Ácidos Araquidônicos/farmacologia , Benzamidas/farmacologia , Benzoxazinas/farmacologia , Carbamatos/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Artéria Cerebral Média/metabolismo , Artéria Cerebral Média/fisiologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Nimodipina/farmacologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto , Tromboxanos/farmacologia , Vasoconstritores/farmacologiaRESUMO
BACKGROUND: The endocannabinoid system and the hypothalamic-pituitary-adrenal axis are important neuromodulators of nausea and vomiting. This led us to hypothesize that patients with cyclic vomiting syndrome (CVS) have lower serum endocannabinoids (eCBs) and higher salivary cortisol and alpha amylase. METHODS: Serum eCBs and related lipids, N-oleoylethanolamine (OEA) and N-palmitoylethanolamide (PEA), and salivary cortisol, and alpha amylase (index of sympathetic nervous system activity) were measured in 22 CVS patients (age 40 ± 11, female = 17) in the well and sick phases and 12 matched controls (age 37 ± 12, female = 10). KEY RESULTS: Contrary to our hypothesis, serum concentrations of the eCBs were not different among the study groups. However, serum concentrations of OEA and PEA were significantly higher during the sick than well phase in CVS patients (p = 0.001 and p = 0.04). There were positive correlations between serum PEA and nausea scores in the sick phase (Pearson's rho = 0.48, p = 0.036) and between serum OEA and poor sleep quality in patients (Pearson's rho = 0.7, p = 0.0005). Salivary cortisol and alpha amylase were not different between patients and controls, but subgroup analysis revealed that both were significantly higher in marijuana users compared to non-users during the sick phase (p = 0.04 and 0.03, respectively). CONCLUSIONS & INFERENCES: These data demonstrate that eCB-related lipids, OEA and PEA, are mobilized in the sick phase of CVS and are positively correlated with several of the symptoms of a CVS episode. These data also suggest the hypothesis that chronic marijuana use results in enhanced stress responses during CVS.
Assuntos
Endocanabinoides/sangue , Etanolaminas/sangue , Hidrocortisona/análise , Ácidos Oleicos/sangue , Ácidos Palmíticos/sangue , alfa-Amilases Salivares/análise , Vômito/metabolismo , Adulto , Amidas , Feminino , Humanos , Sistema Hipotálamo-Hipofisário/fisiopatologia , Masculino , Pessoa de Meia-Idade , Sistema Hipófise-Suprarrenal/fisiopatologia , Saliva/química , Índice de Gravidade de Doença , Vômito/sangue , Vômito/diagnóstico , Vômito/fisiopatologiaRESUMO
The cannabinoids have been shown to affect both membrane lipid ordering and the activities of several membrane-associated proteins. We have investigated the effects of the cannabinoids on protein kinase C, a lipid-dependent enzyme that functions as an important regulator of signal-transduction processes in the brain. The naturally occurring cannabinoid delta 9-tetrahydrocannabinol (delta 9-THC) increased the activity of protein kinase C isolated from rat forebrain at concentrations of 10 microM and above. 11-OH-delta 9-THC, cannabinol and cannabidiol also increased protein kinase C activity in the same concentration range. delta 9-THC (10 microM) decreased the Kact of protein kinase C for calcium from 28 microM to 18 microM and had no effect on the phosphatidylserine concentration-stimulation relationship. At a concentration of 30 microM, delta 9-THC increased the binding of [3H]phorbol-12,13-dibutyrate ([3H]PDBu) to protein kinase C and decreased the Kd for [3H]PDBu from 8.2 nM to 5.4 nM. delta 9-THC also had effects on lipid ordering of PS micelles, producing a significant increase in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at a concentration of 10 microM. These data suggest that delta 9-THC activates protein kinase C via a novel mechanism, possibly as a result of effects on vesicle lipid physical characteristics.
Assuntos
Encéfalo/enzimologia , Canabinoides/farmacologia , Proteína Quinase C/metabolismo , Animais , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Dronabinol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Polarização de Fluorescência , Masculino , Fosfatidilserinas/farmacologia , Proteína Quinase C/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Arachidonoylethanolamide or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to activate cannabinoid receptors in the brain. Its metabolic inactivation by brain tissue has been investigated. Anandamide is hydrolyzed by the membrane fraction of rat brain homogenate to arachidonic acid and ethanolamine. The hydrolysis is temperature and pH- dependent (pH maximum at 8.5) and abolished by boiling. Anandamide hydrolysis is protein dependent in the range of 25-100 micrograms protein/ml; does not require calcium and is inhibited by phenylmethylsulfonylfluoride, diisopropylfluorophosphate, thimerosal and arachidonic acid. Hydrolysis of 10 microM anandamide by brain membranes follows first order kinetics; at 30 degrees C, the rate constant for anandamide catabolism is 0.34 min-1 mg protein-1. The Km for anandamide hydrolysis is 3.4 microM, and the Vmax is 2.2 nmol/min per mg protein. Hydrolysis occurs in all subcellular fractions except cytosol with the highest specific activity in myelin and microsomes. The distribution of anandamide hydrolytic activity correlates with the distribution of cannabinoid receptor-binding sites; the hippocampus, cerebellum and cerebral cortex exhibit the highest metabolic activity, while activity is lowest in the striatum, brain stem and white matter.
Assuntos
Amidoidrolases/metabolismo , Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Canabinoides/metabolismo , Amidoidrolases/antagonistas & inibidores , Animais , Ácido Araquidônico/metabolismo , Encéfalo/enzimologia , Citosol/metabolismo , Endocanabinoides , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Masculino , Microssomos/metabolismo , Alcamidas Poli-Insaturadas , Ratos , Ratos Sprague-Dawley , Receptores de Canabinoides , Receptores de Droga/metabolismo , Frações Subcelulares/metabolismoRESUMO
We have demonstrated previously that spontaneously diabetic BB-Wistar rats exhibit decreased adrenal medullary catecholamine secretion in response to splanchnic nerve terminal stimulation. We hypothesized that this abnormality is caused by changes in the sensitivity of the adrenomedullary chromaffin cells to acetylcholine (ACh). To study this hypothesis, we isolated adrenal glands from control and spontaneously diabetic BB-Wistar rats, perfused them with ACh, and measured catecholamine secretion. Adrenal catecholamine release in response to ACh was significantly decreased at 2, 8, and 16 weeks after the onset of diabetes compared with age-matched, nondiabetic control rats. Catecholamine release in response to perfusion with 20 mM K+ was the same in adrenals from diabetic and control rats. The decreased responsiveness of diabetic rat adrenals to perfusion with ACh was significantly correlated with a decrease in the release of catecholamines in response to splanchnic nerve stimulation. A similar defect in catecholamine secretion was also seen in adrenals harvested from nondiabetic BB-Wistar rats following a 3-h period of acute hypoglycemia; however, the adrenal response to potassium was also decreased as was the catecholamine content of the adrenal. Conversely, nondiabetic BB-Wistar rats made diabetic with streptozocin (STZ) and maintained in a hyperglycemic state did not exhibit catecholamine hyposecretion 2 weeks after STZ administration. Collectively, the data describe decreased adrenomedullary response to cholinergic stimulation in spontaneously diabetic rats as early as 2 weeks after the onset of diabetes and that a similar, although more severe, hyposecretion occurs after acute, severe hypoglycemia.
Assuntos
Medula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Acetilcolina/farmacologia , Medula Suprarrenal/efeitos dos fármacos , Medula Suprarrenal/fisiopatologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Estimulação Elétrica , Técnicas In Vitro , Insulina/farmacologia , Cinética , Potenciais da Membrana , Perfusão , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos BB , Valores de ReferênciaRESUMO
Many humans with IDDM eventually lose the capacity to secrete epinephrine from their adrenal medullae. The mechanism for this pathological change is unknown. We hypothesized that this abnormality is attributable to neuropathic changes in the greater splanchnic nerves or in the chromaffin cells that they innervate. To study this hypothesis, we isolated rat adrenal glands, perfused them ex vivo, and measured the epinephrine content of the perfusate under various conditions of stimulation. We used transmural electrical stimulation (20-80 V, at 10 Hz) to induce epinephrine secretion indirectly by selectively activating residual splanchnic nerve terminals within the isolated glands. Under these conditions, epinephrine secretion was severely attenuated in glands from female BB-Wistar rats with diabetes of 4 mo duration compared with their age-matched, nondiabetic controls. These perfused diabetic adrenal medullae also demonstrated decreased catecholamine release in response to direct chromaffin cell depolarization with 20 mM K+, evidence that a functional alteration exists within the chromaffin cells themselves. Nonetheless, total catecholamine content of adrenal medullae from these diabetic rats was not significantly different from controls, indicating that the secretory defect was not simply attributable to a difference in the amount of catecholamines stored and available for release. Herein, we also provide histological evidence of degenerative changes within the cholinergic nerve terminals that innervate these glands.
Assuntos
Medula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Medula Suprarrenal/inervação , Análise de Variância , Animais , Doença Crônica , Neuropatias Diabéticas/metabolismo , Feminino , Técnicas In Vitro , Ratos , Ratos Endogâmicos BB , Nervos Esplâncnicos/metabolismoRESUMO
Two topics are presented in this review. In the first section, we review data regarding the effects of the endocannabinoids (eCBs) and cannabinoid receptors on neuroimmune function. The function of eCBs in the interaction between the immune system and the central nervous system (CNS) is of particular interest, since the CNS itself is a rich source of eCBs while being exquisitely sensitive to inflammation. There are several sites at which cannabinoids can influence neuroinflammation. Microglial cells express both CB receptors and make eCBs. Activation of CB receptors on these cells seems to promote migration and proliferation but to reduce activation to macrophages. In several neurodegenerative diseases, up-regulation of microglial CB2 receptors have been observed. It is our hypothesis that microglial CB receptor activity is anti-inflammatory and could be exploited to manipulate neuroinflammatory processes with a minimum of unwanted effects. The second topic discussed suggests that the eCB/CB1 receptor pair is involved in the responses of animals to acute, repeated and variable stress. The roles of this pair are complex and dependent upon previous stress, among other things. Dysfunctional responding to stress is a component of several human neuropsychiatric disorders, including anxiety and panic disorders, post-traumatic stress disorders, premenstrual dysphoria and quite possibly, drug abuse. While it is too early to say with certainty, it is very possible that either inhibition or potentiation of endocannabinoid signaling will be an efficacious novel therapeutic approach to more than one human psychiatric disease.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Neuroimunomodulação/fisiologia , Estresse Fisiológico/metabolismo , Animais , Moduladores de Receptores de Canabinoides/metabolismo , HumanosRESUMO
Tissue concentrations of the endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are regulated by both synthesis and inactivation. The purpose of this review is to compile available data regarding three inactivation processes: fatty acid amide hydrolase, monoacylglycerol lipase, and cellular membrane transport. In particular, we have focused on mechanisms by which these processes are modulated. We describe the in vitro and in vivo effects of inhibitors of these processes as well as available evidence regarding their modulation by other factors.
Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Membrana Celular/metabolismo , Endocanabinoides , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/fisiologia , Animais , Ácidos Araquidônicos/metabolismo , Transporte Biológico , Humanos , Hidrólise , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/fisiologia , Alcamidas Poli-Insaturadas , Especificidade por SubstratoRESUMO
Endocannabinoids modulate a diverse array of functions including progenitor cell proliferation in the central nervous system, and odorant detection and food intake in the mammalian central olfactory system and larval Xenopus laevis peripheral olfactory system. However, the presence and role of endocannabinoids in the peripheral olfactory epithelium have not been examined in mammals. We found the presence of cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptor protein and mRNA in the olfactory epithelium. Using either immunohistochemistry or calcium imaging we localized CB1 receptors on neurons, glia-like sustentacular cells, microvillous cells and progenitor-like basal cells. To examine the role of endocannabinoids, CB1- and CB2- receptor-deficient (CB1(-/-)/CB2(-/-)) mice were used. The endocannabinoid 2-arachidonylglycerol (2-AG) was present at high levels in both C57BL/6 wildtype and CB1(-/-)/CB2(-/-) mice. 2-AG synthetic and degradative enzymes are expressed in wildtype mice. A small but significant decrease in basal cell and olfactory sensory neuron numbers was observed in CB1(-/-)/CB2(-/-) mice compared to wildtype mice. The decrease in olfactory sensory neurons did not translate to impairment in olfactory-mediated behaviors assessed by the buried food test and habituation/dishabituation test. Collectively, these data indicate the presence of an endocannabinoid system in the mouse olfactory epithelium. However, unlike in tadpoles, endocannabinoids do not modulate olfaction. Further investigation on the role of endocannabinoids in progenitor cell function in the olfactory epithelium is warranted.
Assuntos
Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Mucosa Olfatória/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Olfato/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Olfatória/citologia , Mucosa Olfatória/efeitos dos fármacos , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Neurônios Receptores Olfatórios/metabolismo , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genética , Técnicas de Cultura de TecidosRESUMO
CONTEXT: The endocannabinoid (eCB) system is involved in the regulation of food intake and of peripheral metabolism. Although the cross talk between energy metabolism and the circadian system is well documented, little is known about a potential circadian modulation of human eCB activity. OBJECTIVE: The objective of the study was to define the 24-hour profile of circulating levels of the most abundant endogenous ligand of the CB1 receptor, 2-arachidonoylglycerol (2-AG), in healthy young nonobese adults studied under controlled bedtime, dietary, and activity conditions. METHODS: Fourteen subjects participated in this 4-day laboratory study with fixed light-dark cycles, standardized meals, and bedtimes. Sleep was recorded each night. On the third day, blood sampling at 15- to 30-minute intervals began at 9:30 pm and continued for 24 hours. Cortisol, leptin, and ghrelin were assayed on all samples, whereas the levels of 2-AG and its structural analog, 2-oleoylglycerol (2-OG), were measured at 60-minute intervals. RESULTS: All participants exhibited a large circadian variation of 2-AG serum concentrations with a nadir around midsleep, coincident with the middle of the overnight fast. Levels of 2-AG increased continually across the morning, peaking in the early to midafternoon. Peak values represented, on average, a nearly 3-fold increase above nocturnal nadir levels. Concentrations of 2-OG followed a similar pattern, although with a shorter morning increase and lower amplitude. CONCLUSIONS: The findings demonstrate that activity of the eCB system is profoundly modulated by circadian rhythmicity and suggest that its impact on the regulation of food intake is suppressed during sleep and is maximal during early to midafternoon.
Assuntos
Ácidos Araquidônicos/sangue , Ritmo Circadiano/fisiologia , Ingestão de Alimentos/fisiologia , Endocanabinoides/sangue , Glicerídeos/sangue , Sono/fisiologia , Adolescente , Adulto , Feminino , Grelina/sangue , Humanos , Hidrocortisona/sangue , Leptina/sangue , Masculino , Adulto JovemRESUMO
Cerebellar granule cells (CGCs) express the CB(1) subtype of cannabinoid receptor. CB(1) receptor agonists Win 55212-2, CP55940 and HU210 inhibit KCl-induced activation of nitric oxide synthase (NOS) in CGCs. Win 55212-2 has no effect on either basal NOS activity or on activation by N-methyl-D-aspartate and its effect is abolished by pre-treatment of the cells with pertussis toxin. The CB(1) receptor antagonist/inverse agonist SR141716A both reverses the effects of Win 55212-2 and produces an increase in NOS activity that is additive with KCl. These results support the hypothesis that activation of the CB(1) receptor in CGCs results in a decreased influx of calcium in response to membrane depolarization, resulting in a decreased activation of neuronal NOS.
Assuntos
Cerebelo/enzimologia , Óxido Nítrico Sintase/metabolismo , Receptores de Droga/metabolismo , Animais , Benzoxazinas , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Interações Medicamentosas , Feminino , Masculino , Morfolinas/farmacologia , Naftalenos/farmacologia , Óxido Nítrico Sintase Tipo I , Toxina Pertussis , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Receptores de Canabinoides , Receptores de Droga/agonistas , Receptores de Droga/antagonistas & inibidores , Rimonabanto , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Earlier studies from this laboratory demonstrated that activation of nicotinic cholinergic receptors of cerebral cortical synaptosomes of the rat produced a decrease in the accumulation of [3H]tetraphenylphosphonium ([3H]TPP+) as a result of a decreased synaptosomal membrane potential. In the present study, the role of sodium in the effect of nicotine on the accumulation of [3H]TPP+ and the estimated potential difference was explored. Replacement of buffer sodium with either sucrose or N-methyl-D-glucamine (NMDG), attenuated the depolarization produced by the sodium channel activator, veratridine and had no effect on potassium-induced depolarization. The effect of nicotine on accumulation of [3H]TPP+ into cerebral cortical synaptosomes was abolished in sucrose buffer and attenuated in NMDG buffer. 1,1-Dimethyl-4-phenylpiperazinium iodide (DMP; 30 microM) produced a small increase in the influx of 22Na+ into cerebral cortical synaptosomes. The effect of DMPP on the influx of 22 Na+ was not blocked by tetrodotoxin. These results support the hypothesis that the nicotinic cholinergic receptor in the brain, functions as a sodium ionophore and further demonstrate that accumulation of synaptosomal [3H]TPP+ provides a simple tool with which to assess the effect of nicotine on sodium permeability through open nicotinic cholinergic receptor ionophores.
Assuntos
Córtex Cerebral/metabolismo , Fármacos Neuromusculares Despolarizantes/farmacologia , Nicotina/farmacologia , Sódio/fisiologia , Sinaptossomos/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Iodeto de Dimetilfenilpiperazina/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Colinérgicos/efeitos dos fármacos , Radioisótopos de Sódio , Veratridina/farmacologiaRESUMO
The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 120 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 120 min, was also increased in the contralateral hemisphere. 2-Arachidonoylglycerol content was unaffected by MCAo. In a second set of studies, injury was assessed 24 h after 2 h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY320135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55212-2 (0.1-1 mg/kg) did not affect either infarct volume or neurological score.
Assuntos
Ácidos Araquidônicos/metabolismo , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/prevenção & controle , Piperidinas/farmacologia , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Benzofuranos/administração & dosagem , Benzoxazinas , Pressão Sanguínea/efeitos dos fármacos , Química Encefálica/fisiologia , Infarto Encefálico/patologia , Cromatografia Líquida/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocanabinoides , Hemodinâmica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Ataque Isquêmico Transitório/etiologia , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Espectrometria de Massas/métodos , Morfolinas/administração & dosagem , Naftalenos/administração & dosagem , Exame Neurológico , Piperidinas/administração & dosagem , Alcamidas Poli-Insaturadas , Pirazóis/administração & dosagem , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/fisiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Rimonabanto , Sais de Tetrazólio , Fatores de TempoRESUMO
Delta-Tetrahydrocannabinol (delta 9-THC), the principal psychoactive constituent of Cannabis sativa, was found to increase glucagon activation of liver plasma membrane adenylate cyclase. In the presence of 30 microM delta 9-THC, the EC50 for glucagon was decreased by 60% from 7.6nM to 3.1 nM. 11-OH-delta 9-THC, a psychoactive metabolite of delta 9-THC, also increased glucagon activation of adenylate cyclase while two cannabinoids without marihuana-like psychoactive potency, cannabinol and cannabidiol, did not. At 30 microM, delta 9-THC either slightly decreased or had no effect on the activation of adenylate cyclase by GTP, Gpp(NH)p, fluoride ion, forskolin or ATP alone. Delta 9-THC had no effect on the binding of [125I] glucagon to liver plasma membranes. Arrhenius plots demonstrated that delta 9-THC and 11-OH-delta 9-THC, but not CBD, decreased the activation energy above the break temperature. Therefore, delta 9-THC increased the coupling of the glucagon receptor to adenylate cyclase apparently by removing a constraint on receptor-Ns coupling.
Assuntos
Adenilil Ciclases/metabolismo , Dronabinol/farmacologia , Fígado/enzimologia , Receptores de Superfície Celular/metabolismo , Animais , Canabinoides/farmacologia , Membrana Celular/enzimologia , Colforsina/farmacologia , Glucagon/farmacologia , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Cinética , Fígado/citologia , Masculino , Ratos , Ratos Endogâmicos , Receptores de Glucagon , Fluoreto de Sódio/farmacologiaRESUMO
Recent discoveries have opened new fields for research on the biochemistry and pharmacology of cannabinoids. Among them, and most importantly, are the characterization and molecular cloning of central and peripheral cannabinoid receptors as well as the isolation of the first putative endogenous ligands that bind to them, anandamide and 2-arachidonylglycerol. The enzyme that degrades these so-called "endocannabinoids" is an integral membrane protein, fatty acid amide hydrolase. Its distribution and biochemistry in rat brain suggest that it plays a critical role in the regulation of the endocannabinoid system. However, few data exist regarding its distribution and mechanism of action in human tissues. To that end, we have studied its cellular distribution in the human central nervous system by immunohistochemistry. Using an affinity-purified antibody, we report that fatty acid amide hydrolase is localized to specific and well-delimited cell populations, including cortical pyramidal neurons, subcortical white matter astrocytes, striatal and striatoefferent projecting neurons, hypothalamic and midbrain nuclei, granular and molecular layers of the cerebellum, Purkinje neurons, dentate cerebellar nucleus, inferior olivary nuclei and others. This distribution resembles that of the central cannabinoid receptors as well as that of the enzyme distribution in the rat brain. In summary, the cellular localization of the degradative enzyme of the endogenous cannabinoid ligands in human central nervous system reveals its presence on both neuronal and glial elements and shows a significant overlapping with that of central cannabinoid receptors, mainly in areas related with motor control, confirming the notion that the endocannabinoid system plays a critical role in the control of movement.
Assuntos
Amidoidrolases/metabolismo , Ácidos Araquidônicos/metabolismo , Astrócitos/metabolismo , Canabinoides/metabolismo , Sistema Nervoso Central/metabolismo , Glicerídeos/metabolismo , Neurônios/metabolismo , Receptores de Droga/metabolismo , Adulto , Astrócitos/citologia , Encéfalo/citologia , Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides , Sistema Nervoso Central/citologia , Endocanabinoides , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neurônios/citologia , Alcamidas Poli-Insaturadas , Receptores de Canabinoides , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [3H]PN-200. AEA competed for [3H]PN-200 binding with a K(I) of 40 +/- 4 microM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K(I) of AEA (48 +/- 6 microM). Arachidonic acid had a slightly higher K(I) (120 +/- 11 microM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-gamma-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [3H]PN-200 binding at concentrations as high as 10 microM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.
Assuntos
Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Di-Hidropiridinas/metabolismo , Amidas/metabolismo , Amidoidrolases/efeitos dos fármacos , Amidoidrolases/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Endocanabinoides , Etanolaminas/metabolismo , Masculino , Alcamidas Poli-Insaturadas , Ratos , Ratos Sprague-Dawley , Compostos de Tosil/farmacologiaRESUMO
Arachidonylethanolamide (N-2-hydroxyethyl-arachidonamide) or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to bind and activate cannabinoid receptors in the brain. Since other potent ligands for the cannabinoid receptor have an aromatic hydroxyl group, we investigated the hypothesis that replacement of the ethanolamine hydroxyl with an aromatic hydroxyl will increase the binding affinity for the cannabinoid receptor. Two novel congeners of anandamide containing aromatic hydroxyl groups were synthesized: N-2-(4-hydroxyphenyl)ethyl arachidonamide (HEA) and N-2-hydroxyphenyl arachidonamide (HPA). The affinity of these congeners for the brain cannabinoid receptor was determined by competition with [3H]CP55940. HEA competed for [3H]CP55940 binding with a Ki of 600 nM; HPA had a Ki of 2200 nM. These results indicate that increased size in the amide portion of anandamide decreases affinity for the receptor. Phenylmethylsulfonyl fluoride (PMSF), an inhibitor of anandamide catabolism by brain membranes, had no effect on the binding of either HEA or HPA. We conclude that these congeners are not substrates for the amidase that catabolizes anandamide.
Assuntos
Ácidos Araquidônicos/metabolismo , Receptores de Droga/metabolismo , Animais , Ácidos Araquidônicos/química , Ligação Competitiva , Membrana Celular/metabolismo , Endocanabinoides , Técnicas In Vitro , Masculino , Fluoreto de Fenilmetilsulfonil/farmacologia , Alcamidas Poli-Insaturadas , Ratos , Ratos Sprague-Dawley , Receptores de CanabinoidesRESUMO
The effects of 3 cannabinoids, delta 9-tetrahydrocannabinol (delta 9-THC), 11-OH-delta 9-tetrahydrocannabinol (11-OH-delta 9-THC) and cannabidiol (CBD) on the binding of [3H]dihydroalprenolol [( 3H]DHA) to mouse brain beta-adrenergic receptors were determined. In vitro, delta 9-THC and 11-OH-delta 9-THC increased the specific binding of [3H]DHA. The increased specific binding of [3H]DHA was due to an increase in receptor affinity as indicated by a decrease in the dissociation constant (Kd). CBD had no effect on binding. Chronic administration of delta 9-THC in vivo caused a decrease in the number of [3H]DHA binding sites with no change in Kd.