RESUMO
Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Neurônios/fisiologia , Síndromes Neurotóxicas/metabolismo , Septinas/metabolismo , Animais , Toxinas Botulínicas Tipo A/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação/genética , Neurônios/microbiologia , Síndromes Neurotóxicas/microbiologia , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Septinas/genéticaRESUMO
Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.
Assuntos
Toxinas Botulínicas/metabolismo , Paralisia/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxina Tetânica/metabolismo , Animais , Western Blotting , Toxinas Botulínicas/genética , Toxinas Botulínicas/farmacocinética , Cerebelo/metabolismo , Camundongos , Mutação , Doenças Neuromusculares/metabolismo , Neurônios/metabolismo , Neurotoxinas/genética , Neurotoxinas/metabolismo , Neurotoxinas/farmacocinética , Peptídeo Hidrolases/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Nervo Isquiático/fisiopatologia , Nervo Isquiático/cirurgia , Medula Espinal/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Toxina Tetânica/genética , Toxina Tetânica/farmacocinéticaRESUMO
As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Ceratoconjuntivite Seca/imunologia , Ativação Linfocitária/imunologia , Transferência Adotiva , Animais , Autoantígenos/imunologia , Separação Celular , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
cAMP mediates diverse cellular signals including prostaglandin (PG) E(2)-mediated intraocular pressure (IOP)-lowering activity in human ocular ciliary smooth muscle cells (hCSM). We have identified gene regulatory networks and key genes upon activation of the cAMP pathway in hCSM, using novel agonists highly selective for PGE(2) receptor subtypes EP2 or EP4, which are G protein-coupled receptors well known to activate cAMP signaling. Here we describe a novel, EP2/EP4-induced, primate-specific gene of hitherto unknown function, also known as C6orf176 (chromosome 6 open reading frame 176) and recently reclassified as noncoding RNA in NCBI's database. Its expression, as determined by quantitative real-time RT-PCR (qRT-PCR), is dramatically upregulated (>2,000-fold) subsequent to transduction of EP2/EP4/Gs/cAMP signaling not only in hCSM, but also in HEK cells overexpressing the recombinant receptors. Moreover, activation of other IOP lowering, Gs-coupled prostanoid receptors, such as DP1 and IP, as well as a direct activator of adenylyl cyclase, forskolin, also substantially upregulated C6orf176 in hCSM, while FP and TP, which are Gq-coupled prostanoid receptor subtypes, did not. Novel transcript variants carrying open reading frames, derived from an at least 67 kb genomic locus on chromosome 6q27 with putative alternative transcription start sites, were identified. Transcriptional upregulation of transcript variants as well as of two genes expressed in antisense orientation that partially overlap the transcribed C6orf176 region was observed, to varying degrees, subsequent to induction of cAMP signaling using various agonists. Small interfering RNA-mediated C6orf176 gene silencing experiments showed modulation of several cAMP-responsive genes. These transcriptional activities identify C6orf176 as a potential biomarker and/or therapeutic target in context with diseases linked to deregulated cAMP signaling. Also, the cAMP-inducible C6orf176 gene locus could be useful as a model system for studying transcriptional regulation by chromatin and RNA polymerase II.
Assuntos
AMP Cíclico/metabolismo , Expressão Gênica , RNA não Traduzido/genética , Cromatina/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Células HEK293 , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transcrição Gênica , Regulação para CimaRESUMO
Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E-)BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Cerebelo/metabolismo , Leucina , Fármacos Neuromusculares/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Células Receptoras Sensoriais/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Toxinas Botulínicas Tipo A/genética , Calcitonina/genética , Calcitonina/metabolismo , Cerebelo/citologia , Feminino , Masculino , Camundongos , Mutação , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Células Receptoras Sensoriais/citologia , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.
Assuntos
Corpo Ciliar/metabolismo , Regulação da Expressão Gênica , Miócitos de Músculo Liso/metabolismo , Receptores de Prostaglandina E Subtipo EP2/fisiologia , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Células Cultivadas , Corpo Ciliar/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Análise em Microsséries , Miócitos de Músculo Liso/efeitos dos fármacos , Prostaglandinas Sintéticas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos de Validação como AssuntoRESUMO
Prodrugs of 5-aminosalicylic acid (5-ASA), such as sulfasalazine, have been the mainstay for the treatment and maintenance of inflammatory bowel disease (IBD) for decades, which is attributable to their antiadaptive immune activity. However, 5-ASA compromises regeneration of intestinal epithelia and induces apoptosis. The majority of patients eventually undergo colectomy. Agonists for the prostaglandin E(2) subtype 4 (EP4) receptor have been shown to protect epithelial barrier against colitis-inducing agents and could be valuable alternatives for sulfasalazine. Here, we compared sulfasalazine and a novel EP4 agonist for their abilities to prevent colitis induction and relieve symptoms of established colitis in a dextran sulfate sodium-indomethacin mouse model. The EP4 agonist dose-dependently alleviated weight loss in colitis mice. Compared with sulfasalazine at 100 mg/kg on the colitis induction model, the EP4 agonist at 0.2 mg/kg was superior in reducing colitis symptoms, preventing increase of innate immune cells, and ameliorating inflammation in colon. In mice with established colitis, sulfasalazine quickly reversed weight loss but with fading efficacy. The EP4 agonist, in contrast, had slow but sustained effects on body weight gain and was more efficacious in epithelial regeneration. Such temporal differences between sulfasalazine and the EP4 agonist actions seemingly led to no additive effect in combination therapy. In conclusion, the EP4 agonist would be more efficacious in the maintenance of remission because of both anti-innate immune responses and epithelial regeneration activity, whereas sulfasalazine would be more suitable for induction of remission because of its rapid onset of antiadaptive inflammation action.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Colite/tratamento farmacológico , Colite/prevenção & controle , Dinoprostona/análogos & derivados , Dinoprostona/uso terapêutico , Receptores de Prostaglandina E Subtipo EP4/agonistas , Sulfassalazina/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colite/sangue , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/farmacologia , Diarreia/prevenção & controle , Dinoprostona/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Contagem de Eritrócitos , Feminino , Hematócrito , Hemoglobinas/metabolismo , Indometacina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Sulfassalazina/farmacologiaRESUMO
PURPOSE: To develop a focal photoreceptor degeneration model by blue light-emitting diode (LED)-induced phototoxicity (LIP) and investigate the protective effects of topical brimonidine (BMD) or intravitreal brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (bFGF). METHODS: In anesthetized, dark-adapted, adult female Swiss mice, the left eye was dilated and exposed to blue light (10 seconds, 200 lux). After LIP, full-field electroretinograms (ERG) and spectral-domain optical coherence tomography (SD-OCT) were obtained longitudinally, and reactive-Iba-1+monocytic cells, TUNEL+ cells and S-opsin+ cone outer segments were examined up to 7 days. Left eyes were treated topically with BMD (1%) or vehicle, before or right after LIP, or intravitreally with BDNF (2.5 µg), CNTF (0.2 µg), bFGF (0.5 µg), or corresponding vehicle right after LIP. At 7 days, S-opsin+ cone outer segments were counted within predetermined fixed-size areas (PFA) centered on the lesion in both flattened retinas. RESULTS: SD-OCT showed a circular region in the superior-temporal left retina with progressive thinning (207.9 ± 5.6 µm to 160.7 ± 6.8 µm [7 days], n = 8), increasing TUNEL+ cells (peak at 3 days), decreasing S-opsin+ cone outer segments, and strong microglia activation. ERGs were normal by 3 days. Total S-opsin+ cones in the PFA for LIP-treated and fellow-retinas were 2330 ± 262 and 5601 ± 583 (n = 8), respectively. All neuroprotectants (n = 7-11), including topical BMD pre- or post-LIP, or intravitreal BDNF, CNTF, and bFGF, showed significantly greater S-opsin+ cone survival than their corresponding vehicle-treated groups. CONCLUSIONS: LIP is a reliable, quantifiable focal photoreceptor degeneration model. Topical BMD or intravitreal BDNF, CNTF, or bFGF protect against LIP-induced cone-photoreceptor loss. TRANSLATIONAL RELEVANCE: Topical BMD or intravitreal BDNF, CNTF, or bFGF protect cones against phototoxicity.
RESUMO
PURPOSE: Compelling evidence suggests that alpha2 agonists, such as brimonidine, protect retinal ganglion cells (RGCs) from injury in a wide range of animal models. However, the mechanism of action for this protection and the physiological role of the alpha2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the alpha2 adrenergic system in the modulation of cytosolic Ca(2+) signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place. METHODS: Functional Ca(2+) imaging at the inner plexiform layer (IPL) and outer plexiform layer (OPL) of living rat retinal slices was conducted with a high-speed confocal system. The relative changes of cytosolic free Ca(2+) were monitored with the fluorescent Ca(2+) dye fluo-4. The Ca(2+) signal was elicited by membrane depolarization produced by a high K(+) (40 mM) Ringer solution that was delivered rapidly and briefly to the test regions of the retinal slice by a custom-made multichannel local perfusion system. RESULTS: A brief application (8 seconds) of high K(+) Ringer elicited a robust cytosolic Ca(2+) increase at the IPL and OPL. In both cases, this Ca(2+) signal was eliminated by nimodipine, a selective L-type voltage-gated Ca(2+)-channel blocker, or when the extracellular Ca(2+) in the Ringer was replaced with equal molar EGTA. At IPL, the Ca(2+) signal was also suppressed in a dose-dependent manner by brimonidine and other alpha2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic alpha2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the alpha2 receptor agonists had no effect on the high K(+) Ringer-elicited cytosolic Ca(2+) signal at OPL. Blocking the N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K(+)-elicited Ca(2+) signal by approximately 20% at IPL. D-AP5 had no effect on the Ca(2+) signal at OPL. CONCLUSIONS: These findings provide the first direct evidence of alpha2 receptor-mediated modulation of L-type Ca(2+) channel activity in the CNS (the retina is part of the CNS). This alpha2 modulation appears to occur at the IPL but not at the OPL of the retina. These findings suggest that a physiological function of the retinal alpha2 system is the regulation of synaptic transmission at IPL and that brimonidine and other alpha2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca(2+) either in RGCs, in their presynaptic cells, or in both.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Adrenérgicos alfa 2/fisiologia , Células Ganglionares da Retina/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Compostos de Anilina , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Microscopia Confocal , Nimodipina/farmacologia , Ratos , Ratos Endogâmicos BN , Transmissão Sináptica/fisiologia , XantenosRESUMO
PURPOSE: Despite structural similarity with prostaglandin F(2 alpha), the ocular hypotensive agent bimatoprost (Lumigan; Allergan, Inc., Irvine, CA) shows unique pharmacology in vitro and functional activity in vivo. Unfortunately, the precise mechanisms that underlie bimatoprost's distinctive impact on aqueous humor dynamics are unclear. The purpose of the present study was to investigate the effects of bimatoprost and a novel prostamide-selective antagonist AGN 211334 on human conventional drainage. METHODS: Two model systems were used to test the consequences of bimatoprost and/or AGN 211334 treatment on conventional drainage. Human anterior segments in organ culture were perfused at a constant flow rate of 2.5 microL/min while pressure was recorded continuously. After stable baseline facilities were established, segments were treated with drug(s), and pressure was monitored for an additional 3 days. In parallel, the drugs' effects on hydraulic conductivity of human trabecular meshwork (TM) cell monolayers were evaluated. Pharmacological properties of AGN 211334 were characterized in isolated feline iris preparations in organ culture and heterologously expressed G-protein-coupled receptors were examined in vitro. RESULTS: Bimatoprost increased outflow facility by an average of 40% +/- 10% within 48 hours of treatment (n = 10, P < 0.001). Preincubation or coincubation with AGN 211334 significantly blunted bimatoprost's effects by 95% or 43%, respectively. Similar results were obtained in cell culture experiments in which bimatoprost increased hydraulic conductivity of TM cell monolayers by 78% +/- 25%. Pretreatment with AGN 211334 completely blocked bimatoprost's effects, while coincubation decreased its effects on average by 74%. In both models, AGN 211334 alone significantly decreased fluid flux across trabecular tissues and cells. CONCLUSIONS: The findings indicate that bimatoprost interacts with a prostamide receptor in the trabecular meshwork to increase outflow facility.
Assuntos
Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Cloprostenol/análogos & derivados , Lipídeos/farmacologia , Malha Trabecular/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Amidas/antagonistas & inibidores , Animais , Humor Aquoso/metabolismo , Bimatoprost , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Gatos , Técnicas de Cultura de Células , Cloprostenol/antagonistas & inibidores , Cloprostenol/farmacologia , Dinoprosta/farmacologia , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Feminino , Humanos , Pressão Intraocular/fisiologia , Iris/efeitos dos fármacos , Lipídeos/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Oxazóis/farmacologia , Receptores de Tromboxanos/antagonistas & inibidores , Receptores de Tromboxanos/metabolismo , Proteínas Recombinantes , Malha Trabecular/metabolismoRESUMO
AIM: To determine the aqueous humour concentration of the acid hydrolysis products of bimatoprost and latanoprost after a single topical dose of bimatoprost 0.03% or latanoprost 0.005% in humans. METHODS: Randomised, controlled, double-masked, prospective study. 48 eyes of 48 patients scheduled for routine cataract surgery were randomised in an 8:2:2 ratio to treatment with a single 30 mul drop of bimatoprost 0.03%, latanoprost 0.005% or placebo at 1, 3, 6 or 12 h before the scheduled cataract surgery. Aqueous humour samples were withdrawn at the beginning of the surgical procedure and analysed using high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Bimatoprost acid (17-phenyl trinor prostaglandin F2alpha) was detected in aqueous samples at a mean concentration of 5.0 nM at hour 1, 6.7 nM at hour 3 and 1.9 nM at hour 6 after bimatoprost treatment. After latanoprost treatment, the mean concentration of latanoprost acid (13,14-dihydro-17-phenyl trinor prostaglandin F2alpha) in aqueous samples was 29.1 nM at hour 1, 41.3 nM at hour 3 and 2.5 nM at hour 6. Acid metabolites were below the limit of quantitation in all samples taken 12 h after dosing and in all samples from placebo-treated patients. None of the samples from latanoprost-treated patients contained quantifiable levels of non-metabolised latanoprost. Non-metabolised bimatoprost was detected in aqueous samples at a mean concentration of 6.6 nM at hour 1 and 2.4 nM at hour 3 after bimatoprost treatment. CONCLUSIONS: Low levels of bimatoprost acid were detected in aqueous humour samples from patients with cataract treated with a single dose of bimatoprost. Latanoprost acid concentrations in samples from patients treated with latanoprost were at least sixfold higher. These results suggest that bimatoprost acid in the aqueous humour does not sufficiently account for the ocular hypotensive efficacy of bimatoprost.
Assuntos
Amidas/metabolismo , Anti-Hipertensivos/metabolismo , Humor Aquoso/metabolismo , Catarata/metabolismo , Cloprostenol/análogos & derivados , Bimatoprost , Extração de Catarata , Cloprostenol/metabolismo , Método Duplo-Cego , Humanos , Lipídeos , Estudos ProspectivosRESUMO
PURPOSE: The aim of this study was to identify the changes in the primate visual system after a single session of photodynamic therapy (PDT) in an intact nonhuman primate retina. METHODS: As part of a larger study, PDT (wavelength 689 nm, 50 J/cm2, 600 mW/cm2, 83 seconds, 4-mm spot size) with verteporfin (6 mg/m2 intravenous infusion) was performed in one eye each of two cynomolgus monkeys. Fundus photography, fluorescein angiography (FA), indocyanine green angiography (ICG), optical coherence tomography (OCT), and multifocal electroretinography (mfERG) were performed at baseline and 12 time points (1-283 days) after PDT. In addition, retinal histopathologic findings were evaluated at 9 months. RESULTS: Various morphologic changes, including whitening of the treated area, RPE proliferation, closure of the choroidal vasculature, and subretinal edema (followed by foveolar thinning) were observed. Most of the changes persisted and were detectable in histopathologic evaluation at 9 months. Reductions of the mfERG amplitude, followed by varying degrees of recovery from the treated and the border regions, were observed. This was accompanied by progressive delay of P1 peak time up to 3 months after treatment, followed by complete recovery at 9 months. In addition, the nontreated area showed amplitude and timing mfERG deficits, which underwent gradual (but not complete) recovery. CONCLUSIONS: In a primate model, under standard clinical parameters, a single PDT treatment resulted in various dynamic morphologic and functional retinal changes detectable for up to 9 months after treatment. The significance of the observed changes and possible ways of pharmacologic interference with PDT adverse effects are discussed.
Assuntos
Arteriopatias Oclusivas/induzido quimicamente , Doenças da Coroide/induzido quimicamente , Corioide/irrigação sanguínea , Edema Macular/induzido quimicamente , Fotoquimioterapia/efeitos adversos , Retina/efeitos dos fármacos , Animais , Arteriopatias Oclusivas/diagnóstico , Corioide/patologia , Doenças da Coroide/diagnóstico , Eletrorretinografia/efeitos dos fármacos , Feminino , Angiofluoresceinografia , Seguimentos , Verde de Indocianina , Macaca fascicularis , Edema Macular/diagnóstico , Fármacos Fotossensibilizantes/efeitos adversos , Porfirinas/efeitos adversos , Retina/patologia , Tomografia de Coerência Óptica , VerteporfinaRESUMO
Acute or chronic glaucoma is often associated with an increase in intraocular pressure (IOP). In many patients, however, therapeutic pressure reduction does not halt disease progression. Neuroprotection has been proposed as a complementary therapeutic approach. We previously demonstrated effective T-cell-based neuroprotection in experimental animals vaccinated with the synthetic copolymer glatiramer acetate (copolymer-1, Cop-1), a weak agonist of self-antigens. This study was undertaken to test different routes and modes of vaccination with Cop-1 as treatment modalities for protection against retinal ganglion cell (RGC) death caused by chronic elevation of IOP in rats, and to determine whether anatomical neuroprotection is accompanied by functional neuroprotection. In a chronic model of unilaterally high IOP, Cop-1 vaccination, with or without an adjuvant, protected rats against IOP-induced loss of RGCs by eliciting a systemic T-cell-mediated response capable of cross-reacting with self-antigens residing in the eye. In rats deprived of T cells, Cop-1 (unlike treatment with alpha2-adrenoreceptor agonists) was not protective of RGCs, substantiating the contention that its beneficial effect is not conferred directly but is T-cell-mediated. Pattern electroretinography provided evidence of functional protection. Thus, vaccination with adjuvant-free Cop-1 can protect RGCs from the consequences of elevated IOP in rats. This protection is manifested both morphologically and functionally. These findings can be readily implemented for the development of a therapeutic vaccination to arrest the progression of glaucoma.
Assuntos
Modelos Animais de Doenças , Pressão Intraocular/efeitos dos fármacos , Proteínas Repressoras/uso terapêutico , Células Ganglionares da Retina/efeitos dos fármacos , Linfócitos T/imunologia , Vacinação , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Apoptose , Proteínas de Sinalização Intercelular CCN , Citoproteção/imunologia , Eletrorretinografia , Pressão Intraocular/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Proteínas Repressoras/administração & dosagem , Células Ganglionares da Retina/imunologia , Células Ganglionares da Retina/patologia , Fatores de TempoRESUMO
PURPOSE: To determine the effect of bimatoprost on intraocular pressure in the prostaglandin FP receptor knockout mouse. METHODS: The IOP response to a single 1.2-microg (4 microL) dose of bimatoprost was measured in the treated and untreated fellow eyes of homozygote (FP+/+, n = 9) and heterozygote (FP+/-, n = 10) FP-knockout mice, as well as in wild-type C57BL/6 mice (FP+/+, n = 20). Serial IOP measurements were also performed after topical bimatoprost in a separate generation of homozygous FP-knockout mice and wild-type littermate control animals (n = 4 per group). Aqueous humor protein concentrations were measured to establish the state of the blood-aqueous barrier. Tissue, aqueous humor and vitreous concentrations of bimatoprost, latanoprost, and their C-1 free acids were determined by liquid chromatography and tandem mass spectrometry. RESULTS: A significant reduction in IOP was observed in the bimatoprost-treated eye of wild-type mice at 2 hours, with a mean difference and 95% confidence interval (CI) of the difference in means of -1.33 mm Hg (-0.81 to -1.84). Bimatoprost did not lead to a significant reduction in IOP in either the heterozygous knockout -0.36 mm Hg (-0.82 to +0.09) or homozygous FP-knockout mice 0.25 mm Hg (-0.38 to +0.89). The lack of an IOP response in the FP-knockout mice was not a consequence of blood-aqueous barrier breakdown, as there was no significant difference in aqueous humor protein concentration between treated and fellow eyes. Tissue and aqueous humor concentrations of bimatoprost, latanoprost, and their C-1 free acids indicate that latanoprost, but not bimatoprost, is hydrolyzed in the mouse eye after topical administration. CONCLUSIONS: An intact FP receptor gene is critical to the IOP response to bimatoprost in the mouse eye.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Intraocular/efeitos dos fármacos , Lipídeos/farmacologia , Receptores de Prostaglandina/fisiologia , Amidas , Animais , Anti-Hipertensivos/farmacocinética , Humor Aquoso/metabolismo , Bimatoprost , Barreira Hematoaquosa/fisiologia , Cloprostenol/análogos & derivados , Proteínas do Olho/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Lipídeos/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/farmacologia , Receptores de Prostaglandina/deficiência , Tonometria OcularRESUMO
1. The pharmacology of bimatoprost, a synthetic prostaglandin-amide, was examined in prostaglandin F(2alpha) (PGF(2alpha))-sensitive preparations. Bimatoprost potently contracted the rabbit isolated uterus (pEC(50)=7.92+/-0.16). In contrast, bimatoprost exhibited weak excitatory activity in human myometrium from pregnant and nonpregnant donors, mouse uterus, rat uterus, and endothelium-intact rabbit jugular veins, and did not stimulate DNA synthesis in mouse fibroblasts. 2. The possibility that the effects of bimatoprost may reflect partial agonism at prostanoid FP receptors was examined and the contractile effects of full agonists, 17-phenyl PGF(2alpha) (FP) and U-46619 (TP, a control), were determined in the absence and presence of 1 muM bimatoprost on the mouse uterus. Analyses of the agonist-agonist functional studies showed no antagonism, indicating that bimatoprost is not a partial agonist. 3. Bioassay metabolism studies of bimatoprost and latanoprost (FP receptor agonist prodrug) in the rabbit uterus were conducted using recipient mouse uterus. Results indicated that the potent responses to bimatoprost in the rabbit uterus are produced by the intact molecule and not by its putative free acid metabolite, 17-phenyl PGF(2alpha). Some hydrolysis of latanoprost to latanoprost free acid appears to have occurred in the rabbit uterus, according to biological detection. 4. The pharmacology of bimatoprost could not be explained by its interaction with known prostanoid FP receptors and was independent of species-, tissue-, or preparation-related factors. The potent contractile effects of bimatoprost in the rabbit uterus provide further pharmacological evidence for the presence of a novel receptor population that preferentially recognises bimatoprost.
Assuntos
Lipídeos/farmacologia , Receptores de Prostaglandina/agonistas , Contração Uterina/efeitos dos fármacos , Útero/efeitos dos fármacos , Células 3T3 , Amidas , Animais , Bimatoprost , Cloprostenol/análogos & derivados , DNA/biossíntese , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Veias Jugulares/efeitos dos fármacos , Veias Jugulares/metabolismo , Veias Jugulares/fisiologia , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Miométrio/fisiologia , Gravidez , Coelhos , Ratos , Ratos Sprague-Dawley , Útero/metabolismo , Útero/fisiologiaRESUMO
PURPOSE: To determine, using electrophysiological measures of visual system function, whether oral daily dosing of memantine is both safe and effective to reduce the injury associated with experimental glaucoma in primates. METHODS: Argon laser treatment of the anterior chamber angle was used to induce chronic ocular hypertension (COHT) in the right eye of 18 macaque monkeys. Nine animals were orally dosed daily with 4 mg/kg memantine while the other nine animals received an oral dose of vehicle only. Using both conventional and multifocal methods, recordings of the electroretinogram (ERG) were made at approximately 3, 5, and 16 months after elevation of the intraocular pressure (IOP). Recordings of the visually-evoked cortical potential (VECP) were also made at the 16-month time point. RESULTS: Chronic ocular hypertension was associated with a reduction in the amplitude of components of the multifocal ERG response and visually-evoked cortical potential. Memantine-treated animals suffered less amplitude reduction for these measures than did vehicle-treated animals, though this treatment effect on the ERG measures was observed only at the early time points (3 and 5 months post IOP elevation). Memantine treatment was not associated with an effect on either the kinetics or amplitude of ERG or VECP response measures obtained from the normotensive eyes. CONCLUSIONS: Systemic treatment with memantine, a compound which does not lower intraocular pressure, was both safe and effective for reduction of functional loss associated with experimental glaucoma.
Assuntos
Eletrorretinografia/efeitos dos fármacos , Potenciais Evocados Visuais/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glaucoma/tratamento farmacológico , Memantina/uso terapêutico , Administração Oral , Animais , Contagem de Células , Doença Crônica , Modelos Animais de Doenças , Potenciais Evocados Visuais/fisiologia , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacocinética , Glaucoma/fisiopatologia , Ácido Glutâmico/metabolismo , Pressão Intraocular , Macaca fascicularis , Memantina/administração & dosagem , Memantina/farmacocinética , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/fisiopatologia , Doenças do Nervo Óptico/tratamento farmacológico , Doenças do Nervo Óptico/fisiopatologia , Retina/efeitos dos fármacos , Retina/fisiopatologia , Células Ganglionares da Retina/patologia , Segurança , Resultado do Tratamento , Corpo Vítreo/metabolismoRESUMO
PURPOSE: To determine, using anatomic measurements, whether daily oral dosing with memantine is both safe and effective to reduce the injury associated with experimental glaucoma in primates. METHODS: Argon laser treatment of the anterior chamber angle was used to induce chronic ocular hypertension (COHT) in the right eyes of 18 macaque monkeys. Nine animals were daily orally dosed with 4 mg/kg memantine while the other nine animals received vehicle only. Measurements of intraocular pressure (IOP) from both eyes of all animals were made at regular intervals. Appearance of the optic nerve head, retinal vessels, and surrounding retina was documented with stereo fundus photographs obtained at multiple time points throughout the study. Measurements of optic nerve head topography were obtained from confocal laser scans made from animals with the highest IOPs at approximately 3, 5, and 10 months after elevation of IOP. At approximately 16 months after IOP elevation, animals were killed and histologic counts of cells in the retinal ganglion cell (RGC) layer were made. RESULTS: Histologic measurements showed that, for animals with moderate elevation of IOP, memantine treatment was associated with an enhanced survival of RGCs in the inferior retina. Measurements of optic nerve head topography showed less IOP-induced change in memantine-treated animals. This effect was seen in measurements of both the cup and the neuroretinal rim. A comparison of these same histologic and morphologic measurements in normotensive eyes from the two treatment groups showed that memantine treatment was not associated with any significant effects on these eyes. CONCLUSIONS: Histologic measurements of RGC survival as well as tomographic measurements of nerve head topography show that systemic treatment with memantine, a compound which does not lower intraocular pressure, is both safe and effective to reduce changes associated with experimental glaucoma.
Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glaucoma/tratamento farmacológico , Memantina/uso terapêutico , Disco Óptico/patologia , Retina/patologia , Células Ganglionares da Retina/patologia , Administração Oral , Animais , Contagem de Células , Sobrevivência Celular , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Glaucoma/patologia , Pressão Intraocular , Macaca fascicularis , Memantina/administração & dosagem , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/patologia , Doenças do Nervo Óptico/tratamento farmacológico , Doenças do Nervo Óptico/patologia , Segurança , Resultado do TratamentoRESUMO
Four criteria are used to evaluate the potential usefulness of an agent for neuroprotection in glaucoma: 1) the agent must have a target in the retina; 2) it must be neuroprotective in animal models; 3) it must reach neuroprotective concentrations in the posterior segment after clinical dosing; and finally, 4) it must be shown to be neuroprotective in clinical trials. The alpha-2 adrenergic agonist brimonidine has met the first three criteria and clinical trials to establish the fulfillment of the fourth criterion are ongoing. The effects of brimonidine are mediated by its interaction with alpha-2 adrenergic receptors that are present in the retina. Activation of alpha-2 receptors by brimonidine has been shown to effectively promote the survival and function of retinal ganglion cells in a variety of animal models of optic injury relevant to glaucoma such as the chronic ocular hypertensive rat and rat optic nerve crush. Brimonidine has also been shown to be neuroprotective in the rat ischemia reperfusion model that evaluates general hypoxic damage to the whole retina. Clinical dosing of the topical formulation of brimonidine results in brimonidine concentrations in the posterior segment that are sufficient for both pharmacological activity at alpha-2 adrenergic receptors and neuroprotection. Finally, clinical trials are in progress to investigate the ability of brimonidine to protect human retinal ganglion cells and the visual field in glaucoma-related disease.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/uso terapêutico , Glaucoma/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Quinoxalinas/uso terapêutico , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Tartarato de Brimonidina , Humanos , Receptores Adrenérgicos alfa 2/metabolismoRESUMO
Recent studies have suggested glaucomatous loss of retinal ganglion cells and their axons in Alzheimer's disease. Amyloid beta peptides and phosphorylated tau protein have been implicated in the selective regional neuronal loss and protein accumulations characteristic of Alzheimer's disease. Similar protein accumulations are not present on glaucomatous retinal ganglion cells. Neurons die in both Alzheimer's disease and glaucoma by apoptosis, although the signaling pathways for neuronal degradation appear to differ in the two diseases. Alzheimer's disease features a loss of locus ceruleus noradrenergic neurons, which send axon terminals to the brain regions suffering neuronal apoptosis and results in reductions in noradrenaline in those regions. Activation of alpha-2 adrenergic receptors reduces neuronal apoptosis, in part through a protein kinase B (Akt)-dependent signaling pathway. Loss of noradrenaline innervation facilitates neuronal apoptosis in Alzheimer's disease models and may act similarly in glaucoma. Alpha-2 adrenergic receptor agonists offer the potential to slow the neuronal loss in both diseases by compensating for lost noradrenaline innervation.
Assuntos
Agonistas alfa-Adrenérgicos/uso terapêutico , Doença de Alzheimer/prevenção & controle , Apoptose/efeitos dos fármacos , Glaucoma/prevenção & controle , Receptores Adrenérgicos alfa 2/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Citoproteção , Glaucoma/metabolismo , Humanos , Fármacos Neuroprotetores/uso terapêutico , Células Ganglionares da Retina/metabolismoRESUMO
PURPOSE: To evaluate the neuroprotective effect of memantine, an NMDA receptor channel blocker, in two retinal ganglion cell (RGC) injury models in rats. METHODS: Neuroprotective effect of memantine was tested in partial optic nerve injury and chronic ocular hypertensive models. In the optic nerve injury model, memantine (0.1 - 30 mg/kg) was injected intraperitoneally immediately after injury. Two weeks later, optic nerve function was determined by measuring compound action potential and surviving RGC was determined by retrograde labeling with dextran tetramethyl rhodamine. Chronic ocular hypertension was attained by laser photocoagulation of episcleral and limbal veins. Memantine (5 or 10 mg/kg) was administered continuously each day with an osmotic pump, either immediately after or 10 days after first laser photocoagulation, for 3 weeks, after which RGC survival was determined. RESULTS: Two weeks after partial optic nerve injury, there was approximately 80% reduction in RGC number. Memantine (5 mg/kg) caused a twofold increase in compound action potential amplitude and a 1.7-fold increase in survival of RGCs, respectively. In the chronic ocular hypertension model there was 37% decrease in RGCs after 3 weeks of elevated intraocular pressure. Memantine (10 mg/kg daily) reduced ganglion cell loss to 12% when applied immediately after first laser photocoagulation, and prevented any further loss when applied 10 days after first laser photocoagulation. CONCLUSION: The protective effect of memantine suggests that excessive stimulation of NMDA receptors by glutamate is involved in causing cell damage in these RGC injury models.