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The local anesthetics lidocaine and articaine are among the most widely used drugs in the dentist's arsenal, relieving pain by blocking voltage-dependent Na+ channels and thus preventing transmission of the pain signal. Given reports of infrequent but prolonged paresthesias with 4% articaine, we compared its neurotoxicity and functional impairment by screening cultured neural SH-SY5Y cells with formulations used in patients (2% lidocaine + 1:100,000 epinephrine or 4% articaine + 1:100,000 epinephrine) and with pure formulations of the drugs. Voltage-dependent sodium channels Na(v)1.2 and Na(v)1.7 were expressed in SH-SY5Y cells. To test the effects on viability, cells were exposed to drugs for 5 minutes, and after washing, cells were treated with the ratiometric Live/Dead assay. Articaine had no effect on the survival of SH-SY5Y cells, while lidocaine produced a significant reduction only when used as pure powder. To determine reversibility of blockage, wells were exposed to drugs for 5 minutes and returned for medium for 30 minutes, and the calcium elevation induced by depolarizing cells with a high-potassium solution was measured using the calcium indicator Fura-2. High potassium raised calcium in control SH-SY5Y cells and those treated with articaine, but lidocaine treatment significantly reduced the response. In conclusion, articaine does not damage neural cells more than lidocaine in this in vitro model. While this does not question the safety of lidocaine used clinically, it does suggest that articaine is no more neurotoxic, at least in the in vitro setting.
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Anestésicos Locales/farmacología , Carticaína/farmacología , Lidocaína/farmacología , Neuronas/efectos de los fármacos , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Anestésicos Locales/toxicidad , Señalización del Calcio/efectos de los fármacos , Carticaína/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Lidocaína/toxicidad , Canal de Sodio Activado por Voltaje NAV1.2/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Neuronas/metabolismo , Neuronas/patología , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Medición de Riesgo , Factores de Tiempo , Bloqueadores del Canal de Sodio Activado por Voltaje/toxicidadRESUMEN
The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.
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Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.
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Canales de Calcio/fisiología , Activación del Canal Iónico/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores Purinérgicos/fisiología , Percepción del Gusto/fisiología , Gusto/fisiología , Animales , Canales de Calcio/análisis , Femenino , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/análisis , Receptores Purinérgicos/análisis , Transmisión Sináptica/fisiología , XenopusRESUMEN
Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1-/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.
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Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Células Epiteliales/metabolismo , Lisosomas/metabolismo , Receptor Toll-Like 3/agonistas , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Autofagia , Biomarcadores , Calcio/metabolismo , Células Cultivadas , Concentración de Iones de Hidrógeno , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
The transient receptor potential cation channel mucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calcium in RPE cells and whether such release is affected by lysosomal accumulations. The TRPML agonist ML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-l-phenylalanine-ß-naphthylamide. Treatment with desipramine to inhibit acid sphingomyelinase or YM201636 to inhibit PIKfyve also reduced the cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML1-/- mice showed no response to ML-SA1. Cotreatment with chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomal Ca2+ release. Lysosomal Ca2+ release was also impaired in RPE cells from the ATP-binding cassette, subfamily A, member 4-/- mouse model of Stargardt's retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models, suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calcium levels in RPE cells, but this response is reduced by lysosomal accumulation.-Gómez, N. M., Lu, W. Lim, J. C., Kiselyov, K., Campagno, K. E., Grishchuk, Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.
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Señalización del Calcio , Metabolismo de los Lípidos , Lisosomas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Lisosomas/patología , Degeneración Macular/congénito , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Ratones Noqueados , Ftalimidas/farmacología , Quinolinas/farmacología , Epitelio Pigmentado de la Retina/patología , Enfermedad de Stargardt , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Inflammatory responses play a key role in many neural pathologies, with localized signaling from the non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. The NLRP3 inflammasome requires two stages to contribute: priming and activation. The priming stage involves upregulation of inflammasome components while the activation stage results in the assembly and activation of the inflammasome complex. The priming step can be rate limiting and can connect insult to chronic inflammation, but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammation is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure increased mRNA for inflammasome components IL-1ß, NLRP3, ASC, and CASP1 in rat and mouse retinas. The elevation was greater 1 day after the insult, with the rise in IL-1ß most pronounced. The P2X7 receptor was implicated in the mechanosensitive priming of IL-1ß mRNA in vivo, as the antagonist Brilliant Blue G (BBG) blocked the increased expression, the agonist BzATP mimicked the pressure-dependent rise in IL-1ß, and the rise was absent in P2X7 knockout mice. In vitro measurements from optic nerve head astrocytes demonstrated an increased expression of IL-1ß following stretch or swelling. This increase in IL-1ß was eliminated by degradation of extracellular ATP with apyrase, or by the block of pannexin hemichannels with carbenoxolone, probenecid, or 10panx1 peptide. The rise in IL-1ß expression was also blocked by P2X7 receptor antagonists BBG, A839977 or A740003. The rise in IL-1ß was prevented by blocking transcription factor NFκB with Bay 11-7082, while the swelling-dependent fall in NFκB inhibitor IκB-α was reduced by A839977 and in P2X7 knockout mice. In summary, mechanical trauma to the retina primed NLRP3 inflammasome components, but only if there was ATP release through pannexin hemichannels, and autostimulation of the P2X7 receptor. As the P2X7 receptor can also trigger stage two of inflammasome assembly and activation, the P2X7 receptor may have a central role in linking mechanical strain to neuroinflammation.
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Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.
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Diabetes Mellitus Experimental/metabolismo , Curación de Fractura/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antígenos CD34/sangre , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/fisiopatología , Factor VIII/metabolismo , Curación de Fractura/inmunología , Hiperglucemia/sangre , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Hiperglucemia/fisiopatología , Inflamación/sangre , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/fisiopatología , Masculino , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/sangre , Polietilenglicoles/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response.
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Astrocitos/metabolismo , Interleucina-6/fisiología , Neuronas/metabolismo , Receptores Purinérgicos P2X7/fisiología , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Inyecciones , Interleucina-6/genética , Presión Intraocular , Ratones , Ratones Noqueados , Nervio Óptico/patología , Agonistas del Receptor Purinérgico P2X/administración & dosificación , Agonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7/genética , Células Ganglionares de la Retina/efectos de los fármacos , Regulación hacia Arriba/genética , Cuerpo VítreoRESUMEN
Mechanical strain due to increased pressure or swelling activates inflammatory responses in many neural systems. As cytokines and chemokine messengers lead to both pro-inflammatory and neuroprotective actions, understanding the signaling patterns triggered by mechanical stress may help improve overall outcomes. While cytokine signaling in neural systems is often associated with glial cells like astrocytes and microglia, the contribution of neurons themselves to the cytokine response is underappreciated and has bearing on any balanced response. Mechanical stretch of isolated neurons was previously shown to trigger ATP release through pannexin hemichannels and autostimulation of P2X7 receptors (P2X7Rs) on the neural membrane. Given that P2X7Rs are linked to cytokine activation in other cells, this study investigates the link between neuronal stretch and cytokine release through a P2X7-dependent pathway. Cytokine assays showed application of a 4% strain to isolated rat retinal ganglion cells (RGCs) released multiple cytokines. The P2X7R agonist BzATP also released multiple cytokines; Interleukin 3 (IL-3), TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 were released by both stimuli, with the release of IL-3 greatest with either stimuli. Stretch-dependent IL-3 release was confirmed with ELISA and blocked by P2X7R antagonists A438079 and Brilliant Blue G (BBG), implicating autostimulation of the P2X7R in stretch-dependent IL-3 release. Neuronal IL-3 release triggered by BzATP required extracellular calcium. The IL-3Rα receptor was expressed on RGCs but not astrocytes, and both IL-3Rα and IL-3 itself were predominantly expressed in the retinal ganglion cell layer of adult retinal sections, implying autostimulation of receptors by released IL-3. While the number of surviving ganglion cells decreased with time in culture, the addition of IL-3 protected against this loss of neurons. Expression of mRNA for IL-3 and IL-3Rα increased in rat retinas stretched with moderate intraocular pressure (IOP) elevation; BBG blocked the rise in IL-3, implicating a role for the P2X7R in transcriptional regulation in vivo. In summary, mechanical stretch triggers release of cytokines from neurons that can convey neuroprotection. The enhancement of these signals in vivo implicates P2X7R-mediated IL-3 signaling as an endogenous pathway that could minimize damage following neuronal exposure to chronic mechanical strain.
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Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD(+)/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.
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4-Aminobutirato Transaminasa/metabolismo , Metabolismo Energético , Homeostasis , Sueño , 4-Aminobutirato Transaminasa/genética , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Encéfalo/fisiología , Drosophila melanogaster , Ácido Glutámico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutación , Estrés OxidativoRESUMEN
Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer's disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimer's disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body.
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Células Epiteliales/fisiología , Lisosomas/fisiología , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/citología , Animales , Autofagia/fisiología , Humanos , Concentración de Iones de Hidrógeno , Epitelio Pigmentado de la Retina/patologíaRESUMEN
As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.
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Adenosina Trifosfato/metabolismo , Astrocitos/fisiología , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Disco Óptico/fisiología , Estrés Mecánico , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Conexinas/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Glaucoma/fisiopatología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas de Transporte de Nucleótidos/metabolismo , Disco Óptico/efectos de los fármacos , Presión Osmótica/fisiología , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas Long-EvansRESUMEN
OBJECTIVES: Timely augmentation of the physiological events of dentoalveolar repair is a prerequisite for the optimization of the outcome of regeneration. This study aimed to develop a treatment strategy to promote dentoalveolar regeneration by the combined delivery of the early mitogenic factor platelet-derived growth factor (PDGF) and the late osteogenic differentiation factor simvastatin. MATERIALS AND METHODS: By using the coaxial electrohydrodynamic atomization technique, PDGF and simvastatin were encapsulated in a double-walled poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) (PDLLA-PLGA) microspheres in five different modes: microspheres encapsulating bovine serum albumin (BB), PDGF alone (XP), simvastatin alone (SB), PDGF-in-core and simvastatin-in-shell (PS), and simvastatin-in-core and PDGF-in-shell (SP). The microspheres were characterized using scanning electronic microscopy, and the in vitro release profile was evaluated. Microspheres were delivered to fill large osteotomy sites on rat maxillae for 14 and 28 days, and the outcome of regeneration was evaluated by microcomputed tomography and histological assessments. RESULTS: Uniform 20-µm controlled release microspheres were successfully fabricated. Parallel PDGF-simvastatin release was noted in the PS group, and the fast release of PDGF followed by the slow release of simvastatin was noted in the SP group. The promotion of osteogenesis was observed in XP, PS, and SP groups at day 14, whereas the SP group demonstrated the greatest bone fill, trabecular numbers, and thickest trabeculae. Bone bridging was evident in the PS and SP group, with significantly increased osteoblasts in the SP group, and osteoclastic cell recruitment was promoted in all bioactive molecule-treated groups. At day 28, osteogenesis was promoted in all bioactive molecule-treated groups. Initial corticalization was noted in the XP, PS, and SP groups. Osteoblasts appeared to be decreased in all groups, and significantly, a greater osteoclastic cell recruitment was noted in the SB and SP groups. CONCLUSIONS: Both PDGF and simvastatin facilitate dentoalveolar regeneration, and sequential PDGF-simvastatin release (SP group) further accelerated the regeneration process through the enhancement of osteoblastogenesis and the promotion of bone maturation.
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Proceso Alveolar/efectos de los fármacos , Proceso Alveolar/fisiología , Factor de Crecimiento Derivado de Plaquetas/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regeneración/efectos de los fármacos , Simvastatina/administración & dosificación , Simvastatina/farmacología , Proceso Alveolar/diagnóstico por imagen , Animales , Humanos , Ácido Láctico/química , Masculino , Microesferas , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Sprague-Dawley , Microtomografía por Rayos XRESUMEN
Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.
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Metabolismo de los Lípidos , Lisosomas/metabolismo , Fagosomas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Bovinos , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Transcripción GenéticaRESUMEN
Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of retinal pigmented epithelial cells from elderly ABCA4(-/-) mice, a model of recessive Stargardt's retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.
Asunto(s)
Agonistas de Dopamina/farmacología , Células Epiteliales/metabolismo , Lisosomas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de Dopamina D5/agonistas , Epitelio Pigmentado de la Retina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Western Blotting , Catepsina D/metabolismo , Bovinos , Línea Celular , Células Epiteliales/efectos de los fármacos , Citometría de Flujo , Fluorescencia , Silenciador del Gen , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ratones , Ratones Noqueados , Pepstatinas , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , ARN Interferente Pequeño , Receptores de Dopamina D1/genética , Receptores de Dopamina D5/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.
Asunto(s)
Adenosina Trifosfato/fisiología , Receptores Purinérgicos P2X7/fisiología , Células Ganglionares de la Retina/fisiología , Animales , Conexinas/fisiología , Proteínas del Tejido Nervioso/fisiología , Ratas , Ratas Long-Evans , Estrés MecánicoRESUMEN
BACKGROUND: Diabetes is known to impair wound healing and deteriorate the periodontal condition. There is limited information about the patterns and events associated with periodontal wound repair. In this study, we evaluate the dynamics of periodontal wound repair using micro-computed tomography (microCT) and immunohistochemistry. METHODS: Thirty-six male rats were used, and diabetes was induced by streptozotocin. The maxillary first molars were extracted, and a tooth-associated osseous defect was created in the extraction area. Animals were sacrificed after 7, 14, and 21 days. Volumetry and distribution of bone trabeculae were evaluated by microCT imaging. The patterns of healing and collagen alignment were evaluated by histology. Advanced glycation end-product (AGE) deposition and expression of the receptor for AGEs (RAGE), tartrate-resistant acid phosphatase, and proliferating cell nuclear antigen were evaluated by histochemical and immunohistochemical staining. RESULTS: Diabetic animals demonstrated a significantly reduced bone volume and trabecular number as well as thinner trabeculae and more trabecular separation in osseous defects. The early stage was characterized by significantly reduced cellular proliferation and prolonged active inflammation without evident bone resorption, whereas delayed recovery of collagen realignment, matrix deposition, and bone turnover was noted in later stages. Although AGEs and RAGE were present during healing in diabetes and controls, a stronger and more persistent level of expression was observed in the group with diabetes CONCLUSIONS: Diabetes significantly delayed osseous defect healing by augmenting inflammation, impairing proliferation, and delaying bone resorption. The AGE-RAGE axis can be activated under metabolic disturbance and inflammation.