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BACKGROUND: Multiple sclerosis (MS) is a degenerative disease of the central nervous system (CNS) that disrupts walking function and results in other debilitating symptoms. This study compares the effects of 'task-oriented exercise' against 'generalized resistance and aerobic exercise' and a 'stretching control' on walking and CNS function in people with MS (PwMS). We hypothesize that task-oriented exercise will enhance walking speed and related neural changes to a greater extent than other exercise approaches. METHODS: This study is a single-blinded, three-arm randomized controlled trial conducted in Saskatchewan, Canada. Eligible participants are those older than 18 years of age with a diagnosis of MS and an expanded Patient-Determined Disease Steps (PDDS) score between 3 ('gait disability') and 6 ('bilateral support'). Exercise interventions are delivered for 12 weeks (3 × 60-min per week) in-person under the supervision of a qualified exercise professional. Interventions differ in exercise approach, such that task-oriented exercise involves weight-bearing, walking-specific activities, while generalized resistance and aerobic exercise uses seated machine-based resistance training of major upper and lower body muscle groups and recumbent cycling, and the stretching control exercise involves seated flexibility and relaxation activities. Participants are allocated to interventions using blocked randomization that stratifies by PDDS (mild: 3-4; moderate: 5-6). Assessments are conducted at baseline, post-intervention, and at a six-week retention time point. The primary and secondary outcome measures are the Timed 25-Foot Walk Test and corticospinal excitability for the tibialis anterior muscles determined using transcranial magnetic stimulation (TMS), respectively. Tertiary outcomes include assessments of balance, additional TMS measures, blood biomarkers of neural health and inflammation, and measures of cardiorespiratory and musculoskeletal fitness. DISCUSSION: A paradigm shift in MS healthcare towards the use of "exercise as medicine" was recently proposed to improve outcomes and alleviate the economic burden of MS. Findings will support this shift by informing the development of specialized exercise programming that targets walking and changes in corticospinal excitability in PwMS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05496881, Registered August 11, 2022. https://classic. CLINICALTRIALS: gov/ct2/show/NCT05496881 . Protocol amendment number: 01; Issue date: August 1, 2023; Primary reason for amendment: Expand eligibility to include people with all forms of MS rather than progressive forms of MS only.
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Although acute COVID-19 is known to cause cardiac damage in some cases, there is still much to learn about the duration and relative permanence of the damage that may occur. Long COVID is a condition that can occur when COVID-19 symptoms remain in the postviral acute period. Varying accounts of long COVID have been described across the literature, however, cardiac impairments are sustained in many individuals and cardiovascular assessment is now considered to be an expected follow-up examination. The purpose of this review and proof of concept is to summarize the current research related to the assessment of cardiac function, including echocardiography and blood biomarker data, during the follow-up period in patients who recovered from COVID-19. Following a literature review, it was found that right ventricular dysfunction along with global longitudinal strain and diastolic dysfunction are common findings. Finally, more severe acute myocardial injury during the index hospitalization appears to exacerbate cardiac function. The available literature implies that cardiac function must be monitored in patients recovered from COVID-19 who remain symptomatic and that the impairments and severity vary from person-to-person. The proof-of-concept analysis of patients with cardiac disease and respiratory disease in comparison to those with sustained symptoms from COVID-19 suggests elevated systolic time interval in those with sustained symptoms from COVID-19, thus reducing heart performance indices. Future research must consider the details of cardiac complications during the acute infection period and relate this to the cardiac function in patients with long COVID during mid- and long-term follow-up.
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COVID-19 , Disfunção Ventricular Direita , COVID-19/complicações , Ecocardiografia , Humanos , Sístole , Síndrome de COVID-19 Pós-AgudaRESUMO
The pool of ß-Amyloid (Aß) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for Aß peptides. We examined how a naturally occurring variant, e.g. Aß(1-38), interacts with the AD-related variant, Aß(1-42), and the predominant physiological variant, Aß(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that Aß(1-38) interacts differently with Aß(1-40) and Aß(1-42) and, in general, Aß(1-38) interferes with the conversion of Aß(1-42) to a ß-sheet-rich aggregate. Functionally, Aß(1-38) reverses the negative impact of Aß(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an Aß(1-42) phenotype in Caenorhabditis elegans. Aß(1-38) also reverses any loss of MTT conversion induced by Aß(1-40) and Aß(1-42) in HT-22 hippocampal neurons and APOE ε4-positive human fibroblasts, although the combination of Aß(1-38) and Aß(1-42) inhibits MTT conversion in APOE ε4-negative fibroblasts. A greater ratio of soluble Aß(1-42)/Aß(1-38) [and Aß(1-42)/Aß(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that Aß(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant Aß(1-42).
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Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos adversos , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Caenorhabditis elegans , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismoRESUMO
OBJECTIVE: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P<0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe-/-S100a9-/- mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. CONCLUSIONS: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.
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Calgranulina B/fisiologia , Complicações do Diabetes/etiologia , Vesículas Extracelulares/fisiologia , Macrófagos/fisiologia , Receptor para Produtos Finais de Glicação Avançada/fisiologia , Calcificação Vascular/etiologia , Animais , Diabetes Mellitus Experimental/complicações , Humanos , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/etiologiaRESUMO
Preserved labile tissues (e.g., skin, muscle) in the fossil record of terrestrial vertebrates are increasingly becoming recognized as an important source of biological and taphonomic information. Here, we combine a variety of synchrotron radiation techniques with scanning electron and optical microscopy to elucidate the structure of 72 million-year-old squamous (scaly) skin from a hadrosaurid dinosaur from the Late Cretaceous of Alberta, Canada. Scanning electron and optical microscopy independently reveal that the three-dimensionally preserved scales are associated with a band of carbon-rich layers up to a total thickness of â¼75 microns, which is topographically and morphologically congruent with the stratum corneum in modern reptiles. Compositionally, this band deviates from that of the surrounding sedimentary matrix; Fourier-transform infrared spectroscopy and soft X-ray spectromicroscopy analyses indicate that carbon appears predominantly as carbonyl in the skin. The regions corresponding to the integumentary layers are distinctively enriched in iron compared to the sedimentary matrix and appear with kaolinite-rich laminae. These hosting carbonyl-rich layers are apparently composed of subcircular bodies resembling preserved cell structures. Each of these structures is encapsulated by calcite/vaterite, with iron predominantly concentrated at its center. The presence of iron, calcite/vaterite and kaolinite may, independently or collectively, have played important roles in the preservation of the layered structures.
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Context/Objectives: Spinal cord injury (SCI) results in significant neuronal and glial cell death resulting in impaired neurological and motor function. Uncontrolled Ca2+ entry results in excitotoxicity and cell death. In this study, we examine the use of a BK channel activator, Isopimaric acid (ISO), as a neuroprotective agent post-SCI as this channel is involved in regulating Ca2+ entry. Design:By using a 25-g clip compression at the T6 level, we generated a SCI event in wistar rats. At 1 h post-injury we administered ISO (BK channel activator), the BK channel inhibitor iberiotoxin (IbTx), or a vehicle control for 4 weeks via mini osmotic pump (pump capacity). For 8 weeks post-injury, gait analysis of motor function was performed. At the end of 8 weeks, the extent of myelination in the spinal cord was assessed in addition to the electrophysiological profile. Results:Our immunohistological data suggests that ISO treatment leads to an increase or preservation of myelinated axonal tracts. This was further supported by our electrophysiological studies which demonstrate higher compound action potential amplitudes and speed of transmission in ISO-treated animals compared to inj-non-treated. Finally, treatment with ISO significantly improved motor function in our test model. Conclusion: In conclusion, activation of the BK channel during acute SCI may be a novel therapeutic target for acute SCI.
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Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. To gain deeper insights into the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared with proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date, which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques.
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Espectrometria de Massas/métodos , Placa Aterosclerótica/química , Proteoma/análise , Proteínas do Citoesqueleto/metabolismo , Endarterectomia das Carótidas , Proteínas da Matriz Extracelular/metabolismo , Humanos , Masculino , Miócitos de Músculo Liso , Acidente Vascular Cerebral/prevenção & controle , Remodelação VascularRESUMO
The fabricated small intestine submucosa (SIS) - hydroxyapatite (HAp) sponges can act as biomimetic scaffolds to be utilized in tissue engineering and regeneration. Here we developed SIS-HAp sponges and investigated their mechanical, physical and chemical characteristics using scanning electron microscopy, Fourier transformed infrared spectroscopy, uniaxial compression, porosity, and swelling testing techniques. The results demonstrated mechanical properties superior to comparable bone substitutes fabricated with similar methods. SIS-HAp scaffolds possess an interconnected macroporosity, similar to that of trabecular bone, hence presenting a novel biomaterial that may serve as a superior bone substitute and tissue scaffold.
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Materiais Biocompatíveis/química , Durapatita/química , Mucosa Intestinal/química , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Força Compressiva , Mucosa Intestinal/fisiologia , Microscopia Eletrônica de Varredura , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier , Suínos , Engenharia TecidualRESUMO
Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.
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Aciltransferases/metabolismo , Biopolímeros/biossíntese , Bryopsida/enzimologia , Carotenoides/biossíntese , Parede Celular/enzimologia , Proteínas de Plantas/metabolismo , Esporos/fisiologia , Briófitas/enzimologia , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Reação em Cadeia da PolimeraseRESUMO
Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophages with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient's LDL cholesterol (R2 = 0.39, Significance F = 0.05). Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. The abundance of iron in symptomatic plaques is associated with the source patient's LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin.
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Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Ferro/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Idoso , Doenças das Artérias Carótidas/diagnóstico , Doenças das Artérias Carótidas/cirurgia , Diagnóstico por Imagem/métodos , Endarterectomia das Carótidas , Feminino , Humanos , Imuno-Histoquímica , Masculino , SíncrotronsRESUMO
The adrenal medulla plays a key role in the physiological responses of developing and mature mammals by releasing catecholamines (CAT) during stress. In rodents and humans, the innervation of CAT-producing, adrenomedullary chromaffin cells (AMCs) is immature or absent during early postnatal life, when these cells possess 'direct' hypoxia- and CO2/H(+)-chemosensing mechanisms. During asphyxial stressors at birth, these mechanisms contribute to a CAT surge that is critical for adaptation to extra-uterine life. These direct chemosensing mechanisms regress postnatally, in parallel with maturation of splanchnic innervation. Here, we review the evidence that neurotransmitters released from the splanchnic nerve during innervation activate signaling cascades that ultimately cause regression of direct AMC chemosensitivity to hypoxia and hypercapnia. In particular, we consider the roles of cholinergic and opioid receptor signaling, given that splanchnic nerves release acetylcholine and opiate peptides onto their respective postsynaptic nicotinic and opioid receptors on AMCs. Recent in vivo and in vitro studies in the rat suggest that interactions involving α7 nicotinic acetylcholine receptors (nAChRs), the hypoxia inducible factor (HIF)-2α signaling pathway, protein kinases and ATP-sensitive K(+) (KATP) channels contribute to the selective suppression of hypoxic chemosensitivity. In contrast, interactions involving µ- and/or δ-opiod receptor signaling pathways contribute to the suppression of both hypoxic and hypercapnic chemosensitivity, via regulation of the expression of KATP channels and carbonic anhydrase (CA I and II), respectively. These data suggest that the ontogeny of O2 and CO2/H(+) chemosensitivity in chromaffin cells can be regulated by the tonic release of presynaptic neurotransmitters.
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Medula Suprarrenal/inervação , Medula Suprarrenal/fisiologia , Células Cromafins/fisiologia , Neurotransmissores/metabolismo , Nervos Esplâncnicos/metabolismo , Medula Suprarrenal/embriologia , Animais , Dióxido de Carbono/metabolismo , Hipóxia Celular , Humanos , Oxigênio/metabolismo , Transdução de Sinais , Nervos Esplâncnicos/embriologiaRESUMO
Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.
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Modelos Animais de Doenças , Fármacos Neuroprotetores/administração & dosagem , Riluzol/administração & dosagem , Traumatismos da Medula Espinal/prevenção & controle , Traumatismos da Medula Espinal/fisiopatologia , Animais , Vértebras Cervicais , Avaliação Pré-Clínica de Medicamentos/métodos , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Potenciais Somatossensoriais Evocados/fisiologia , Feminino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/patologia , Fatores de TempoRESUMO
Cell-based therapies using neural stem cells (NSCs) have shown positive outcomes in various models of neurological injury and disease. Induced pluripotent stem cells (iPSCs) address many problems associated with NSCs from various sources, including the immune response and cell availability. However, due to inherent differences between embryonic stem cells (ESCs) and iPSCs, detailed characterization of the iPS-derived NSCs will be required before translational experiments can be performed. Murine piggyBac transposon iPSCs were clonally expanded in floating sphere colonies to generate primitive NSCs initially with serum-free media (SFM) containing the leukemia inhibitory factor and followed by SFM with the fibroblast growth factor-2 (FGF2) to form colonies of definitive NSCs (dNSCs). Primitive and definitive clonally derived neurospheres were successfully generated using the default conditions from iPSCs and ESCs. However, the iPSC-dNSCs expressed significantly higher levels of pluripotency and nonectoderm lineage genes compared to equivalent ESC-dNSCs. The addition of the bone morphogenetic proteins antagonist, Noggin, to the media significantly increased primary neurosphere generation from the iPSC lines, but did not affect the dNSC sphere colonies generated. The induction of the NOTCH pathway by the Delta-like ligand 4 (DLL4) improved the generation and quality of dNSCs, as demonstrated by a reduction in pluripotency and nonectodermal markers, while maintaining NSC-specific gene expression. The iPS-dNSCs (+DLL4) showed functional neural differentiation by immuncytochemical staining and electrophysiology. This study suggests the intrinsic differences between ESCs and iPSCs in their ability to acquire a dNSC fate that can be overcome by inducing the NOTCH pathway.
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Diferenciação Celular , Elementos de DNA Transponíveis/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/fisiologia , Células Cultivadas , Dipeptídeos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Potenciais da Membrana , Proteínas de Membrana/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/fisiologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Repressoras , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/fisiologia , TranscriptomaRESUMO
At birth, rat adrenomedullary chromaffin cells (AMCs) respond directly to asphyxial stressors such as hypoxia and hypercapnia by triggering catecholamine secretion, which is critical for proper transition to extrauterine life. These non-neurogenic responses are suppressed postnatally in parallel with the development of splanchnic innervation, and reappear following denervation of the adult adrenal gland. To test whether neural factors released from the splanchnic nerve may regulate AMC chemosensitivity, we previously showed that nicotinic agonists in utero and in vitro suppressed hypoxia, but not hypercapnia, sensitivity. Here, we considered the potential role of opiate peptides which are also released from the splanchnic nerve and act via postsynaptic µ-, δ- and -opioid receptors. Treatment of neonatal rat AMC cultures for â¼1 week with µ- and/or δ- (but not ) opioid agonists (2 µm) led to a marked suppression of both hypoxia and hypercapnia sensitivity, as measured by K(+) current inhibition and membrane depolarization; co-incubation with naloxone prevented the effects of combined opioids. The suppression of hypoxia sensitivity was attributable to upregulation of K(ATP) current density and the K(ATP) channel subunit Kir6.2, and was reversed by the K(ATP) channel blocker, glibenclamide. By contrast, suppression of hypercapnia sensitivity was associated with down-regulation of two key mediators of CO(2) sensing, i.e. carbonic anhydrase I and II. Collectively, these studies point to a novel role for opioid receptor signalling in the developmental regulation of chromaffin cell chemosensitivity, and suggest that prenatal exposure to opioid drugs could lead to impaired arousal responses in the neonate.
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Glândulas Suprarrenais/citologia , Analgésicos Opioides/farmacologia , Células Cromafins/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Glândulas Suprarrenais/inervação , Animais , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Hipóxia Celular , Células Cultivadas , Células Cromafins/metabolismo , Expressão Gênica/efeitos dos fármacos , Glibureto/farmacologia , Canais KATP/antagonistas & inibidores , Canais KATP/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Ratos , Ratos Wistar , Receptores Opioides/agonistas , Receptores Opioides/genética , Receptores Opioides/metabolismo , Nervos Esplâncnicos/fisiologiaRESUMO
Pulmonary neuroepithelial bodies (NEBs), composed of clusters of amine [serotonin (5-HT)] and peptide-producing cells, are widely distributed within the airway mucosa of human and animal lungs. NEBs are thought to function as airway O(2)-sensors, since they are extensively innervated and release 5-HT upon hypoxia exposure. The small cell lung carcinoma cell line (H146) provides a useful model for native NEBs, since they contain (and secrete) 5-HT and share the expression of a membrane-delimited O(2) sensor [classical NADPH oxidase (NOX2) coupled to an O(2)-sensitive K(+) channel]. In addition, both native NEBs and H146 cells express different NADPH oxidase homologs (NOX1, NOX4) and its subunits together with a variety of O(2)-sensitive voltage-dependent K(+) channel proteins (K(v)) and tandem pore acid-sensing K(+) channels (TASK). Here we used H146 cells to investigate the role and interactions of various NADPH oxidase components in O(2)-sensing using a combination of coimmunoprecipitation, Western blot analysis (quantum dot labeling), and electrophysiology (patchclamp, amperometry) methods. Coimmunoprecipitation studies demonstrated formation of molecular complexes between NOX2 and K(v)3.3 and K(v)4.3 ion channels but not with TASK1 ion channels, while NOX4 associated with TASK1 but not with K(v) channel proteins. Downregulation of mRNA for NOX2, but not for NOX4, suppressed hypoxia-sensitive outward current and significantly reduced hypoxia -induced 5-HT release. Collectively, our studies suggest that NOX2/K(v) complexes are the predominant O(2) sensor in H146 cells and, by inference, in native NEBs. Present findings favor a NEB cell-specific plasma membrane model of O(2)-sensing and suggest that unique NOX/K(+) channel combinations may serve diverse physiological functions.
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Células Quimiorreceptoras/fisiologia , Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Corpos Neuroepiteliais/fisiologia , Oxigênio/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Glicoproteínas de Membrana/química , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Serotonina/metabolismo , Canais de Potássio Shal/química , Canais de Potássio Shal/fisiologia , Canais de Potássio Shaw/química , Canais de Potássio Shaw/fisiologiaRESUMO
Spinal cord injury (SCI) causes neuronal death, demyelination of surviving axons, and altered ion channel functioning, resulting in impaired axonal conduction. The large-conductance, voltage and Ca(2+)-activated K(+) (BK or Maxi K(+)) channels contribute to the repolarization phase of action potentials. Therefore, they may play a significant role in regulating axonal conduction in SCI. In this paper, using combined electrophysiological and molecular approaches, we tested the hypothesis that the deficit in axonal conduction in chronic SCI is partially due to the activation of axonal BK channels. BK channels were found to be expressed in spinal cord white matter axons. These channels are not sensitive to BK channel blocker iberiotoxin in uninjured cords, likely reflecting their juxtaparanodal localization. After chronic injury, BK channels were exposed due to axonal demyelination at the injured site and their activation was found to depend on calcium influx, likely through N-type voltage-dependent calcium channels. Activation of BK channels introduced a reduction in the size of the compound action potentials (CAPs) and in axonal response to high frequency stimulation (HFS). Administration of BK channel blocker iberiotoxin significantly enhanced axonal conduction in the injured cords. Thus, pharmacological targeting of axonal BK channels may provide a therapeutic strategy for the treatment of chronic SCI, by restoring conduction to the remaining functional axons.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Fibras Nervosas Mielinizadas/metabolismo , Traumatismos da Medula Espinal/metabolismo , Potenciais de Ação/fisiologia , Animais , Feminino , Regulação da Expressão Gênica , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
The reduction of molecular oxygen in individual cells during the process of oxidative phosphorylation is central to oxidative metabolism and bioenergetic homeostasis. As such, any insufficiency in molecular oxygen availability represents a severe threat to sustained life. Thus, as with other similar multicellular organisms, the human body has evolved various peripheral chemosensory pathways that play a key role in sampling arterial PO2 values and initiating corrective reflex responses so as to maintain homeostasis. Research on these peripheral chemosensors can trace their origins to the cross circulation studies of Corneille Heymans in the early 20th century. Since then, it has become increasingly apparent that defects in these chemosensory pathways play a key role in various pathological conditions, e.g. Sudden Infant Death Syndrome (SIDS), and therefore an understanding of the underlying mechanisms is of critical importance. This review aims to discuss the advantages and disadvantages of the various experimental models employed in studying the mechanisms by which acute peripheral chemosensing occurs.
Assuntos
Fosforilação Oxidativa , Oxigênio/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cães , Técnicas Eletroquímicas , Fenômenos Eletrofisiológicos , Humanos , Camundongos , Modelos Animais , Oxigênio/análise , Técnicas de Patch-Clamp , Coelhos , RatosRESUMO
Changes in intracellular Ca(2+) play a key role in regulating gene expression and developmental changes in oligodendroglial precursor cells (OPCs). However, the mechanisms by which Ca(2+) influx in OPCs is controlled remains incompletely understood. Although there are several mechanisms that modulate Ca(2+) influx, in many systems the large-conductance, voltage- and Ca(2+) -activated K(+) channel (BK channel) plays an important role in regulating both membrane excitability and intracellular Ca(2+) levels. To date, the role of the BK channel in the regulation of intracellular Ca(2+) in oligodendroglial lineage cells is unknown. Here we investigated whether cells of the oligodendroglial lineage express BK channels and what potential role they play in regulation of Ca(2+) influx in these cells. In oligodendrocytes derived from differentiated adult neural precursor cells (NPCs, obtained from C57bl6 mice) we observed outward currents that were sensitive to the BK channel blocker iberiotoxin (IbTx). Further confirmation of the expression of the BK channel was obtained utilizing other blockers of the BK channel and by confocal immunofluoresence labelling of the BK channel on oligodendroglia. Using Fura-2AM to monitor intracellular Ca(2+) , it was observed that inhibition of the BK channel during glutamate-induced depolarization led to an additive increase in intracellular Ca(2+) levels. Electrophysiological difference currents demonstrated that the expression levels of the BK channel decrease with developmental age. This latter finding was further corroborated via RT-PCR and Western blot analysis. We conclude that the BK channel is involved in regulating Ca(2+) influx in OPCs, and may potentially play a role during differentiation of oligodendroglial lineage cells.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/biossíntese , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Western Blotting , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The fetus and neonate experience variable patterns of low P(O)2(hypoxia) ranging from acute, sustained, and intermittent. Adaptation to hypoxia involves activation of key transcription factors, known as hypoxia-inducible factors (e.g. HIF-1α, HIF-2α), which regulate a number of genes in different cell types. This review focuses on the signaling pathways that mediate proper physiological responses of perinatal adrenomedullary chromaffin cells (AMC) to varying patterns of hypoxic challenges, and particularly on the controversial role of reactive oxygen species (ROS). At birth, acute hypoxia (seconds to minutes) directly stimulates catecholamine release from AMC via K+ channel inhibition, mediated by a decrease in mitochondrial-derived ROS. By contrast, exposure to chronic sustained hypoxia (CSH) induces HIF-2α in a fetal-derived chromaffin cell line independently of changes in ROS. Exposure to chronic intermittent hypoxia (CIH) activates antioxidant responses via the regulator Nrf-2, in association with an increase in ROS and the induction of HIF-1α. We propose that the physiological responses of perinatal AMC to hypoxia and the ensuing directional changes in ROS are dependent on the pattern and duration of the hypoxic exposure.