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Patients who initially survive the rupture and repair of a brain aneurysm often take a devastating turn for the worse some days later and die or suffer permanent neurologic deficits. This catastrophic sequela is attributed to a delayed phase of global cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage (aSAH), but we lack effective treatment. Here we present our view, based on 20 years of research, that the initial drop in blood flow at the time of rupture triggers genomic responses throughout the brain vasculature that manifest days later as increased vasoconstriction and decreased cerebral blood flow. We propose a novel treatment strategy to prevent DCI by early inhibition of the vascular mitogen-activated protein kinase (MAPK) pathway that triggers expression of vasoconstrictor and inflammatory mediators. We summarize evidence from experimental SAH models showing early treatment with MAPK inhibitors "switches off" these detrimental responses, maintains flow, and improves neurological outcome. This promising therapy is currently being evaluated in clinical trials.
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ABSTRACT: Effects of sex hormones on stroke outcome are not fully understood. A deleterious consequence of cerebral ischemia is upregulation of vasoconstrictor receptors in cerebral arteries that exacerbate stroke injury. Here, we tested the hypothesis that female sex hormones alter vasocontractile responses after experimental stroke in vivo or after organ culture in vitro, a model of vasocontractile receptor upregulation. Female rats with intact ovaries and ovariectomized (OVX) females treated with 17ß-estradiol, progesterone, or placebo were subjected to transient, unilateral middle cerebral artery occlusion followed by reperfusion (I/R). The maximum contractile response, measured my wire myography, in response to the endothelin B receptor agonist sarafotoxin 6c was increased in female arteries after I/R, but the maximum response was significantly lower in arteries from OVX females. Maximum contraction mediated by the serotonin agonist 5-carboxamidotryptamine was diminished after I/R, with arteries from OVX females showing a greater decrease in maximum contractile response. Contraction elicited by angiotensin II was similar in all arteries. Neither estrogen nor progesterone treatment of OVX females affected I/R-induced changes in endothelin B- and 5-carboxamidotryptamine-induced vasocontraction. These findings suggest that sex hormones do not directly influence vasocontractile alterations that occur after ischemic stroke; however, loss of ovarian function does impact this process.
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Infarto da Artéria Cerebral Média/fisiopatologia , Artéria Cerebral Média/fisiopatologia , Ovariectomia , Ovário/fisiopatologia , Vasoconstrição , Animais , Modelos Animais de Doenças , Estradiol/farmacologia , Terapia de Reposição de Estrogênios , Feminino , Infarto da Artéria Cerebral Média/metabolismo , Artéria Cerebral Média/efeitos dos fármacos , Artéria Cerebral Média/metabolismo , Técnicas de Cultura de Órgãos , Ovário/metabolismo , Progesterona/farmacologia , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Migraine is ranked as the second highest cause of disability worldwide and the first among women aged 15-49 years. Overall, the incidence of migraine is threefold higher among women than men, though the frequency and severity of attacks varies during puberty, the menstrual cycle, pregnancy, the postpartum period and menopause. Reproductive hormones are clearly a key influence in the susceptibility of women to migraine. A fall in plasma oestrogen levels can trigger attacks of migraine without aura, whereas higher oestrogen levels seem to be protective. The basis of these effects is unknown. In this Review, we discuss what is known about sex hormones and their receptors in migraine-related areas in the CNS and the peripheral trigeminovascular pathway. We consider the actions of oestrogen via its multiple receptor subtypes and the involvement of oxytocin, which has been shown to prevent migraine attacks. We also discuss possible interactions of these hormones with the calcitonin gene-related peptide (CGRP) system in light of the success of anti-CGRP treatments. We propose a simple model to explain the hormone withdrawal trigger in menstrual migraine, which could provide a foundation for improved management and therapy for hormone-related migraine in women.
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Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Estrogênios/metabolismo , Hormônios/metabolismo , Transtornos de Enxaqueca/metabolismo , Ocitocina/metabolismo , Adolescente , Adulto , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Migraine occurs 2-3 times more often in females than in males and is in many females associated with the onset of menstruation. The steroid hormone, 17ß-estradiol (estrogen, E2), exerts its effects by binding and activating several estrogen receptors (ERs). Calcitonin gene-related peptide (CGRP) has a strong position in migraine pathophysiology, and interaction with CGRP has resulted in several successful drugs for acute and prophylactic treatment of migraine, effective in all age groups and in both sexes. METHODS: Immunohistochemistry was used for detection and localization of proteins, release of CGRP and PACAP investigated by ELISA and myography/perfusion arteriography was performed on rat and human arterial segments. RESULTS: ERα was found throughout the whole brain, and in several migraine related structures. ERß was mainly found in the hippocampus and the cerebellum. In trigeminal ganglion (TG), ERα was found in the nuclei of neurons; these neurons expressed CGRP or the CGRP receptor in the cytoplasm. G-protein ER (GPER) was observed in the cell membrane and cytoplasm in most TG neurons. We compared TG from males and females, and females expressed more ER receptors. For neuropeptide release, the only observable difference was a baseline CGRP release being higher in the pro-estrous state as compared to estrous state. In the middle cerebral artery (MCA), we observed similar dilatory ER-responses between males and females, except for vasodilatory ERß which we observed only in female arteries. CONCLUSION: These data reveal significant differences in ER receptor expression between male and female rats. This contrasts to CGRP and PACAP release where we did not observe discernable difference between the sexes. Together, this points to a hypothesis where estrogen could have a modulatory role on the trigeminal neuron function in general rather than on the acute CGRP release mechanisms and vasomotor responses.
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Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Sistema Nervoso Central/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Feminino , Humanos , Masculino , Transtornos de Enxaqueca/fisiopatologia , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Recent clinical findings suggest that oxytocin could be a novel treatment for migraine. However, little is known about the role of this neuropeptide/hormone and its receptor in the trigeminovascular pathway. Here we determine expression, localization, and function of oxytocin and oxytocin receptors in rat trigeminal ganglia and targets of peripheral (dura mater and cranial arteries) and central (trigeminal nucleus caudalis) afferents. METHODS: The methods include immunohistochemistry, messenger RNA measurements, quantitative PCR, release of calcitonin gene-related peptide and myography of arterial segments. RESULTS: Oxytocin receptor mRNA was expressed in rat trigeminal ganglia and the receptor protein was localized in numerous small to medium-sized neurons and thick axons characteristic of A∂ sensory fibers. Double immunohistochemistry revealed only a small number of neurons expressing both oxytocin receptors and calcitonin gene-related peptide. In contrast, double immunostaining showed expression of the calcitonin gene-related peptide receptor component receptor activity-modifying protein 1 and oxytocin receptors in 23% of the small cells and in 47% of the medium-sized cells. Oxytocin immunofluorescence was observed only in trigeminal ganglia satellite glial cells. Oxytocin mRNA was below detection limit in the trigeminal ganglia. The trigeminal nucleus caudalis expressed mRNA for both oxytocin and its receptor. K+-evoked calcitonin gene-related peptide release from either isolated trigeminal ganglia or dura mater and it was not significantly affected by oxytocin (10 µM). Oxytocin directly constricted cranial arteries ex vivo (pEC50 â¼ 7); however, these effects were inhibited by the vasopressin V1A antagonist SR49059. CONCLUSION: Oxytocin receptors are extensively expressed throughout the rat trigeminovascular system and in particular in trigeminal ganglia A∂ neurons and fibers, but no functional oxytocin receptors were demonstrated in the dura and cranial arteries. Thus, circulating oxytocin may act on oxytocin receptors in the trigeminal ganglia to affect nociception transmission. These effects may help explain hormonal influences in migraine and offer a novel way for treatment.
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Neurônios/metabolismo , Ocitocina/metabolismo , Receptores de Ocitocina/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Artéria Basilar/metabolismo , Artérias Cerebrais/metabolismo , Dura-Máter/metabolismo , Masculino , Artérias Meníngeas/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the blood-brain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). METHODS: With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. RESULTS: Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C- and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. CONCLUSION: We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.
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Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Nós Neurofibrosos/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Axônios , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/metabolismo , Dura-Máter/metabolismo , Imuno-Histoquímica , Masculino , Transtornos de Enxaqueca/fisiopatologia , Fibras Nervosas/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Treatment of migraine is on the cusp of a new era with the development of drugs that target the trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) or its receptor. Several of these drugs are expected to receive approval for use in migraine headache in 2018 and 2019.â CGRP-related therapies offer considerable improvements over existing drugs as they are the first to be designed specifically to act on the trigeminal pain system, they are more specific and they seem to have few or no adverse effects. CGRP receptor antagonists such as ubrogepant are effective for acute relief of migraine headache, whereas monoclonal antibodies against CGRP (eptinezumab, fremanezumab and galcanezumab) or the CGRP receptor (erenumab) effectively prevent migraine attacks. As these drugs come into clinical use, we provide an overview of knowledge that has led to successful development of these drugs. We describe the biology of CGRP signalling, summarize key clinical evidence for the role of CGRP in migraine headache, including the efficacy of CGRP-targeted treatment, and synthesize what is known about the role of CGRP in the trigeminovascular system. Finally, we consider how the latest findings provide new insight into the central role of the trigeminal ganglion in the pathophysiology of migraine.
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Anticorpos Monoclonais/uso terapêutico , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Humanos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/imunologiaRESUMO
Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.
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Butadienos/farmacologia , Circulação Cerebrovascular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nitrilas/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Ratos , Ratos Wistar , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17ß-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1ß and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1ß and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1ß, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1ß and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection.
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Encéfalo/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Mitocôndrias/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/irrigação sanguínea , Feminino , Fatores de Transcrição Forkhead/metabolismo , Genômica , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Mitocôndrias/metabolismo , Fator 1 Relacionado a NF-E2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ovariectomia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND AND PURPOSE: Male-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation. EXPERIMENTAL APPROACH: Freshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1) and endothelin-1 type A and B (ETA and ETB) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression. KEY RESULTS: Vascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ETB receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT1B, AT1, and ETB receptors and in local expression of Ang II after organ culture. CONCLUSIONS AND IMPLICATIONS: In spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.
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Angiotensina II/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Endotelina-1/farmacologia , Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Vasoconstrição/efeitos dos fármacos , Artérias Cerebrais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miografia , Técnicas de Cultura de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor 5-HT1B de Serotonina/genética , Receptor 5-HT1B de Serotonina/metabolismo , Fatores Sexuais , Transdução de SinaisRESUMO
Mitochondria support the energy-intensive functions of brain endothelium but also produce damaging-free radicals that lead to disease. Previously, we found that estrogen treatment protects cerebrovascular mitochondria, increasing capacity for ATP production while decreasing reactive oxygen species (ROS). To determine whether these effects occur specifically in endothelium in vivo and also explore underlying transcriptional mechanisms, we studied freshly isolated brain endothelial preparations from intact and ovariectomized female mice. This preparation reflects physiologic influences of circulating hormones, hemodynamic forces, and cell-cell interactions of the neurovascular unit. Loss of ovarian hormones affected endothelial expression of the key mitochondrial regulator family, peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), but in a unique way. Ovariectomy increased endothelial PGC-1α mRNA but decreased PGC-1ß mRNA. The change in PGC-1ß correlated with decreased mRNA for crucial downstream mitochondrial regulators, nuclear respiratory factor 1 and mitochondrial transcription factor A, as well as for ATP synthase and ROS protection enzymes, glutamate-cysteine ligase and manganese superoxide dismutase. Ovariectomy also decreased mitochondrial biogenesis (mitochondrial/nuclear DNA ratio). These results indicate ovarian hormones normally act through a distinctive regulatory pathway involving PGC-1ß to support cerebral endothelial mitochondrial content and guide mitochondrial function to favor ATP coupling and ROS protection.
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Córtex Cerebral/irrigação sanguínea , Endotélio Vascular/metabolismo , Estrogênios/fisiologia , Mitocôndrias Musculares/metabolismo , Ovário/fisiologia , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Córtex Cerebral/metabolismo , DNA Mitocondrial/metabolismo , Regulação para Baixo , Endotélio Vascular/enzimologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ovariectomia , Ovário/cirurgia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
17ß-Estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized as playing central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effects of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemia-like condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis, and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI-182,780) and were mimicked by an ER agonist selective for ERα (PPT), but not by an ER agonist selective for ERß (DPN). OGD/reperfusion provoked mitochondrial dysfunction as manifested by an increase in cellular reactive oxygen species production, loss of mitochondrial membrane potential, and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI-182,780. Therefore, we conclude that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondrial dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.
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Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Mitocôndrias/fisiologia , Doenças Mitocondriais/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Citoproteção , Estradiol/análogos & derivados , Estradiol/farmacologia , Fulvestranto , Glucose/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenóis , Cultura Primária de Células , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The hormone melatonin (5-methoxy-N-acetyltryptamine) is synthesized primarily in the pineal gland and retina, and in several peripheral tissues and organs. In the circulation, the concentration of melatonin follows a circadian rhythm, with high levels at night providing timing cues to target tissues endowed with melatonin receptors. Melatonin receptors receive and translate melatonin's message to influence daily and seasonal rhythms of physiology and behavior. The melatonin message is translated through activation of two G protein-coupled receptors, MT(1) and MT(2), that are potential therapeutic targets in disorders ranging from insomnia and circadian sleep disorders to depression, cardiovascular diseases, and cancer. This review summarizes the steps taken since melatonin's discovery by Aaron Lerner in 1958 to functionally characterize, clone, and localize receptors in mammalian tissues. The pharmacological and molecular properties of the receptors are described as well as current efforts to discover and develop ligands for treatment of a number of illnesses, including sleep disorders, depression, and cancer.
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Receptores de Melatonina/classificação , Animais , Humanos , Receptores de Melatonina/química , Receptores de Melatonina/metabolismo , Terminologia como AssuntoRESUMO
Numerous studies show the neuroprotective effects of estrogen, but the underlying mechanism still remains unclear. Recent studies indicate that mitochondria are critically involved in estrogen-mediated neuroprotection. Mitochondria are the main sources of cellular energy and reactive oxygen species (ROS), they play an important role in signaling transduction and cellular life-death decisions. Estrogen exerts multiple effects on mitochondria under physiological and/or pathological conditions, these effects may include modulating ATP and ROS production, preserving mitochondria membrane potential, maintaining calcium homeostasis, and regulating mitochondrial gene and protein expression, etc. In this paper, we discussed the neuroprotective effects of estrogen, particularly focused on the underlying mechanisms related to mitochondria.
Assuntos
Estrogênios/fisiologia , Mitocôndrias/fisiologia , Fármacos Neuroprotetores , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Humanos , Potencial da Membrana Mitocondrial/fisiologiaRESUMO
Protective effects of estrogen against experimental stroke and neuronal ischemic insult are well-documented, but it is not known whether estrogen prevents ischemic injury to brain endothelium, a key component of the neurovascular unit. Increasing evidence indicates that estrogen exerts protective effects through mitochondrial mechanisms. We previously found 17beta-estradiol (E2) to improve mitochondrial efficiency and reduce mitochondrial superoxide in brain blood vessels and endothelial cells. Thus we hypothesized E2 will preserve mitochondrial function and protect brain endothelial cells against ischemic damage. To test this, an in vitro ischemic model, oxygen-glucose deprivation (OGD)/reperfusion, was applied to immortalized mouse brain endothelial cells (bEnd.3). OGD/reperfusion-induced cell death was prevented by long-term (24, 48 h), but not short-term (0.5, 12 h), pretreatment with 10 nmol/L E2. Protective effects of E2 on endothelial cell viability were mimicked by an estrogen-receptor (ER) agonist selective for ERalpha (PPT), but not by one selective for ERbeta (DPN). In addition, E2 significantly decreased mitochondrial superoxide and preserved mitochondrial membrane potential and ATP levels in early stages of OGD/reperfusion. All of the E2 effects were blocked by the ER antagonist, ICI-182,780. These findings indicate that E2 can preserve endothelial mitochondrial function and provide protection against ischemic injury through ER-mediated mechanisms.
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Isquemia Encefálica/patologia , Células Endoteliais/fisiologia , Mitocôndrias/fisiologia , Receptores de Estrogênio/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/fisiologia , Estrogênios/farmacologia , Glucose/deficiência , Hipóxia Encefálica/patologia , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Traumatismo por Reperfusão/patologia , Superóxidos/metabolismoRESUMO
Our previous studies show that long-term testosterone treatment augments vascular tone under physiological conditions and exacerbates endotoxin-induced inflammation in the cerebral circulation. However, testosterone can be metabolized by aromatase to estrogen, evoking a balance between androgenic and estrogenic effects. Therefore, we investigated the effect of the nonaromatizable androgen receptor agonist, dihydrotestosterone (DHT), on the inflammatory nuclear factor-kappaB (NFkappaB) pathway in cerebral blood vessels. Cerebral arteries were isolated from orchiectomized male rats treated chronically with DHT in vivo. Alternatively, pial arteries were isolated from orchiectomized males and were exposed ex vivo to DHT or vehicle in culture medium. DHT treatment, in vivo or ex vivo, increased nuclear NFkappaB activation in cerebral arteries and increased levels of the proinflammatory products of NFkappaB activation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Effects of DHT on COX-2 and iNOS were attenuated by flutamide. In isolated pressurized middle cerebral arteries from DHT-treated rats, constrictions to the selective COX-2 inhibitor NS398 or the selective iNOS inhibitor L-nil, [L-N6-(Iminoethyl)lysine], were increased, confirming a functional consequence of DHT exposure. In conclusion, activation of the NFkappaB-mediated COX-2/iNOS pathway by the selective androgen receptor agonist, DHT, results in a state of vascular inflammation. This effect may contribute to sex-related differences in cerebrovascular pathophysiology.
Assuntos
Doenças Arteriais Cerebrais/induzido quimicamente , Circulação Cerebrovascular/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Inflamação/induzido quimicamente , NF-kappa B/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Doenças Arteriais Cerebrais/metabolismo , Doenças Arteriais Cerebrais/patologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Di-Hidrotestosterona/sangue , Inflamação/metabolismo , Inflamação/patologia , Masculino , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Tamanho do Órgão , Próstata/efeitos dos fármacos , Ratos , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17beta-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17beta-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ERalpha, but not ERbeta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17beta-estradiol, but not 17alpha-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ERalpha, not ERbeta. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERalpha-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.
Assuntos
Células Endoteliais/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Mitocôndrias/efeitos dos fármacos , Aconitato Hidratase/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Citocromos c/genética , Células Endoteliais/metabolismo , Receptor alfa de Estrogênio/genética , Fumarato Hidratase/metabolismo , Humanos , Mitocôndrias/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Mitochondria are a major source of reactive oxygen species (ROS) and oxidative stress, key contributors to aging and neurodegenerative disorders. We report that gonadal hormones influence brain mitochondrial ROS production in both females and males. Initial experiments showed that estrogen decreases mitochondrial superoxide production in a receptor-mediated manner, as measured by MitoSOX fluorescence in differentiated PC-12 cells. We then assessed in vivo effects of gonadal hormones on brain mitochondrial oxidative stress in female and male rats. Brain mitochondria were isolated to measure a functional indicator of ROS, i.e., activity of the ROS-sensitive mitochondrial enzyme, aconitase. Gonadectomy of both males and females caused a decrease in aconitase activity, suggesting that endogenous gonadal hormones influence mitochondrial ROS production in the brain. In vivo treatment of gonadectomized animals with testosterone or dihydrotestosterone (DHT) had no effect, but estrogen replacement significantly increased aconitase activity in brain mitochondria from both female and male rats. This indicates that estrogen decreases brain mitochondrial ROS production in vivo. Sex hormone treatments did not affect protein levels of brain mitochondrial uncoupling proteins (UCP-2, 4, and 5). However, estrogen did increase the activity, but not the levels, of manganese superoxide dismutase (MnSOD), the mitochondrial enzyme that catalyzes superoxide radical breakdown, in brain mitochondria from both female and male rats. Thus, in contrast to the lack of effect of androgens on mitochondrial ROS, estrogen suppression of mitochondrial oxidative stress may influence neurological disease incidence and progression in both females and males.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético/fisiologia , Estrogênios/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Aconitato Hidratase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Metabolismo Energético/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Gônadas/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Caracteres Sexuais , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
1. Cerebral vessels express oestrogen receptors (ER) in both the smooth muscle and endothelial cell layers of cerebral blood vessels. Levels of ERalpha are higher in female rats chronically exposed to oestrogen, either endogenous or exogenous. 2. Chronic exposure to oestrogen, either endogenous (normally cycling females) or exogenous (ovariectomized with oestrogen replacement), results in cerebral arteries that are more dilated than arteries from ovariectomized counterparts when studied in vitro. This effect is primarily mediated by an increase in the production of vasodilator factors, including nitric oxide (NO) and prostacylin. In contrast, oestrogen appears to suppress the production of endothelial-derived hyperpolarizing factor. Oestrogen treatment increases cerebrovascular levels of endothelial nitric oxide synthase (eNOS), cyclo-oxygenase (COX)-1 and prostacyclin synthase. In addition, via activation of the phosphatidylinositol 3-kinase/Akt pathway, both acute and chronic oestrogen exposure increases eNOS phosphorylation, increasing NO production. 3. Oestrogen receptors have also been localized to cerebrovascular mitochondria and exposure to oestrogen increases the efficiency of energy production while simultaneously reducing mitochondrial production of reactive oxygen species. Oestrogen increases the production of mitochondrial proteins encoded by both mitochondrial and nuclear DNA, including cytochrome c, subunits I and IV of complex IV and Mn-superoxide dismutase. Oestrogen treatment increases the activity of citrate synthase and complex IV and decreases mitochondrial production of H(2)O(2). 4. Oestrogen also has potent anti-inflammatory effects in the cerebral circulation that may have important implications for the incidence and severity of cerebrovascular disease. Administration of lipopolysaccharide or interleukin-1beta to ovariectomized female rats induces cerebrovascular COX-2 and inducible nitric oxide synthase (iNOS) protein expression and increases prostaglandin E(2) expression. Levels of COX-2 and iNOS expression vary with the stage of the oestrous cycle, and the cerebrovascular inflammatory response is suppressed in ovariectomized animals treated with oestrogen. Interleukin-1beta induction of COX-2 protein is prevented by treatment with a nuclear factor (NF)-kappaB inhibitor, and oestrogen treatment reduces cerebrovascular NF-kappaB activity. 5. Cerebrovascular dysfunction and pathology contribute to the pathogenesis of stroke, brain trauma, oedema and dementias, such as Alzheimer's disease. A better understanding of the action of oestrogen on cerebrovascular function holds promise for the development of new therapeutic entities that could be useful in preventing or treating a wide variety of cerebrovascular diseases.
Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Vasodilatação , Vasodilatadores/metabolismo , Envelhecimento/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/fisiopatologia , Metabolismo Energético , Epoprostenol/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Transdução de SinaisRESUMO
Tissues from males can be regulated by a balance of androgenic and estrogenic effects because of local metabolism of testosterone and expression of relevant steroid hormone receptors. As a critical first step to understanding sex hormone influences in the cerebral circulation of males, we investigated the presence of enzymes that metabolize testosterone to active products and their respective receptors. We found that cerebral blood vessels from male rats express 5alpha-reductase type 2 and aromatase, enzymes responsible for conversion of testosterone into dihydrotestosterone (DHT) and 17beta-estradiol, respectively. Protein levels of these enzymes, however, were not modulated by long-term in vivo hormone treatment. We also showed the presence of receptors for both androgens (AR) and estrogens (ER) from male cerebral vessels. Western blot analysis showed bands corresponding to the full-length AR (110 kDa) and ERalpha (66 kDa). Long-term in vivo treatment of orchiectomized rats with testosterone or DHT, but not estrogen, increased AR levels in cerebral vessels. In contrast, ERalpha protein levels were increased after in vivo treatment with estrogen but not testosterone. Fluorescent immunostaining revealed ERalpha, AR, and 5alpha-reductase type 2 in both the endothelial and smooth muscle layers of cerebral arteries, whereas aromatase staining was solely localized to the endothelium. Thus, cerebral vessels from males are target tissues for both androgens and estrogen. Furthermore, local metabolism of testosterone might balance opposing androgenic and estrogenic influences on cerebrovascular as well as brain function in males.