RESUMEN
Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at â¼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. ß-Adrenergic receptor kinase 1 (ßARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.
Asunto(s)
Vasos Sanguíneos/fisiología , Luz , Opsinas de Bastones/metabolismo , Transducción de Señal/fisiología , Vasodilatación/fisiología , Animales , Vasos Sanguíneos/metabolismo , Western Blotting , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Flujometría por Láser-Doppler , Ratones , Miografía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vasodilatación/efectos de la radiaciónRESUMEN
In autonomic ganglia, acetylcholine (ACh) is released from preganglionic nerve terminals and binds to nicotinic ACh receptors (nAChRs) on postganglionic neurons, resulting in a brief, short-lived synaptic potential (fast excitatory postsynaptic potential [fEPSP]). Although nerve growth factor (NGF) is known to affect sensory and sympathetic nerves, especially during development, little is known regarding its effect on parasympathetic nerves, especially on adult neurons. Elevated levels of NGF and NGF-mediated neural plasticity may have a role in airway diseases, such as asthma and chronic obstructive pulmonary disease. In this study, we characterize the composition and response of nAChRs in parasympathetic neurons located in lower airways of mice, and note the effects of NGF on fEPSPs and on nicotinic currents. Based on immunohistochemical staining, nAChRs are made up of α-3 and ß-4 subunits; in addition, tropomyosin-related kinase A, the receptor for NGF, is also expressed by the neurons. Vagus nerve evoked fEPSPs and inward currents evoked by a nicotinic receptor agonist (1,1-dimethyl-4-phenylpiperazinium) were increased by NGF. NGF also affected the action potential after hyperpolarization. These studies were done in mice, which are routinely used to study airway diseases, such as asthma, where the allergen-induced contraction of airway smooth muscle has a well-defined parasympathetic cholinergic component.
Asunto(s)
Potenciales Postsinápticos Excitadores , Factor de Crecimiento Nervioso/fisiología , Nervio Vago/fisiopatología , Potenciales de Acción , Animales , Asma/fisiopatología , Masculino , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Receptores Nicotínicos/metabolismo , Transmisión SinápticaRESUMEN
Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.
Asunto(s)
Alérgenos , Esofagitis Eosinofílica/metabolismo , Esófago/inervación , Fibras Nerviosas Amielínicas/metabolismo , Ovalbúmina , Células Receptoras Sensoriales/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Nervio Vago/metabolismo , Potenciales de Acción , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Esofagitis Eosinofílica/inmunología , Esofagitis Eosinofílica/fisiopatología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Cobayas , Isotiocianatos/farmacología , Masculino , Mastocitos/inmunología , Mastocitos/metabolismo , Fibras Nerviosas Amielínicas/efectos de los fármacos , Fibras Nerviosas Amielínicas/inmunología , Ganglio Nudoso/efectos de los fármacos , Ganglio Nudoso/inmunología , Ganglio Nudoso/metabolismo , Sensación , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/inmunología , Factores de Tiempo , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/inmunología , Nervio Vago/efectos de los fármacos , Nervio Vago/inmunología , Nervio Vago/fisiopatologíaRESUMEN
Chronic debilitating pruritus is a cardinal feature of atopic dermatitis (AD). Little is known about the underlying mechanisms. Antihistamines lack efficacy in treating itch in AD, suggesting the existence of histamine-independent itch pathways in AD. Transient receptor potential ankyrin 1 (TRPA1) is essential in the signaling pathways that promote histamine-independent itch. In this study, we tested the hypothesis that TRPA1-dependent neural pathways play a key role in chronic itch in AD using an IL-13-transgenic mouse model of AD. In these mice, IL-13 causes chronic AD characterized by intensive chronic itch associated with markedly enhanced growth of dermal neuropeptide-secreting afferent nerve fibers and enhanced expression of TRPA1 in dermal sensory nerve fibers, their dorsal root ganglia, and mast cells. Inhibition of TRPA1 with a specific antagonist in these mice selectively attenuated itch-evoked scratching. Genetic deletion of mast cells in these mice led to significantly diminished itch-scratching behaviors and reduced TRPA1 expression in dermal neuropeptide containing afferents in the AD skin. Interestingly, IL-13 strongly stimulates TRPA1 expression, which is functional in calcium mobilization in mast cells. In accordance with these observations in the AD mice, TRPA1 expression was highly enhanced in the dermal afferent nerves, mast cells, and the epidermis in the lesional skin biopsies from patients with AD, but not in the skin from healthy subjects. These studies demonstrate a novel neural mechanism underlying chronic itch in AD and highlight the complex interactions among TRPA1(+) dermal afferent nerves and TRPA1(+) mast cells in a Th2-dominated inflammatory environment.
Asunto(s)
Canales de Calcio/metabolismo , Dermatitis Atópica/inmunología , Mastocitos/fisiología , Fibras Nerviosas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Prurito/inmunología , Canales de Potencial de Receptor Transitorio/metabolismo , Acetanilidas/administración & dosificación , Animales , Canales de Calcio/genética , Células Cultivadas , Enfermedad Crónica , Citocinas/inmunología , Dermatitis Atópica/tratamiento farmacológico , Modelos Animales de Enfermedad , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuropéptidos/metabolismo , Prurito/prevención & control , Purinas/administración & dosificación , Canal Catiónico TRPA1 , Balance Th1 - Th2 , Canales de Potencial de Receptor Transitorio/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.
Asunto(s)
Digoxina/farmacología , Hipertensión Pulmonar/prevención & control , Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/complicaciones , Activación Transcripcional/fisiología , Análisis de Varianza , Animales , Presión Sanguínea/efectos de los fármacos , Calcio/metabolismo , Digoxina/sangre , Hipertensión Pulmonar/etiología , Hipertrofia Ventricular Derecha/prevención & control , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Ratones , Microscopía Confocal , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacosRESUMEN
Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.
Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/irrigación sanguínea , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Permeabilidad Capilar , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Poli(ADP-Ribosa) PolimerasasRESUMEN
RATIONALE: Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES: To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS: ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS: In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS: HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.
Asunto(s)
Angiopoyetinas/metabolismo , Aterosclerosis/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/fisiopatología , Apnea Obstructiva del Sueño/fisiopatología , Grasa Subcutánea/fisiopatología , Adipocitos/metabolismo , Adulto , Anciano , Proteína 4 Similar a la Angiopoyetina , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Femenino , Humanos , Hipoxia/metabolismo , Lipoproteína Lipasa/antagonistas & inhibidores , Ratones , Ratones Endogámicos SENCAR , Persona de Mediana Edad , Obesidad/metabolismo , Obesidad/fisiopatología , Apnea Obstructiva del Sueño/metabolismo , Grasa Subcutánea/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.
Asunto(s)
Citocinas/fisiología , Dermatitis Atópica/patología , Fibrosis/etiología , Interleucina-13/fisiología , Animales , Progresión de la Enfermedad , Humanos , Interleucina-13/genética , Ratones , Ratones Transgénicos , Transgenes , Linfopoyetina del Estroma TímicoRESUMEN
We addressed the hypothesis that allergic inflammation in guinea pig airways leads to a phenotypic switch in vagal tracheal cough-causing, low-threshold mechanosensitive Aδ neurons, such that they begin expressing functional transient receptor potential vanilloid (TRPV1) channels. Guinea pigs were actively sensitized to ovalbumin (OVA) and beginning 21 days later exposed via aerosol to OVA daily for 3 days. Tracheal-specific neurons were identified in the nodose ganglion using retrograde tracing techniques. Tracheal specific neurons were isolated, and mRNA expression was evaluated at the single-neuron level using RT-PCR analysis. Electrophysiological studies have revealed that the vast majority of vagal nodose afferent nerves innervating the trachea are capsaicin-insensitive Aδ-fibers. Consistent with this, we found <20% of these neurons express TRPV1 mRNA or respond to capsaicin in a calcium assay. Allergen exposure induced de novo TRPV1 mRNA in a majority of the tracheal-specific nodose neurons (P < 0.05). The allergen-induced TRPV1 induction was mimicked by applying either brain-derived neurotrophic factor (BDNF) or glial-derived neurotrophic factor (GDNF) to the tracheal lumen. The BDNF-induced phenotypic change observed at the level of mRNA expression was mimicked using a calcium assay to assess functional TRPV1 ion channels. Finally, OVA exposure induced BDNF and GDNF production in the tracheal epithelium, the immediate vicinity of the nodose Aδ -fibers terminations. The induction of TRPV1 in nodose tracheal Aδ -fibers would substantively expand the nature of stimuli capable of activating these cough-causing nerves.
Asunto(s)
Alérgenos/inmunología , Mecanorreceptores/metabolismo , Ganglio Nudoso/patología , Ovalbúmina/inmunología , Canales Catiónicos TRPV/metabolismo , Tráquea/inervación , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Señalización del Calcio , Células Cultivadas , Expresión Génica , Perfilación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Cobayas , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Factor de Crecimiento Nervioso/fisiología , Neuronas/metabolismo , Ganglio Nudoso/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de la Célula Individual , Canales Catiónicos TRPV/genética , Tráquea/inmunología , Tráquea/patologíaRESUMEN
Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.
Asunto(s)
Acuaporina 1/genética , Calcio/metabolismo , Movimiento Celular , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Arteria Pulmonar/patología , Regulación hacia Arriba/genética , Animales , Aorta/patología , Acuaporina 1/metabolismo , Hipoxia de la Célula , Proliferación Celular , Espacio Intracelular/metabolismo , Masculino , Músculo Liso Vascular/patología , Ratas , Ratas WistarRESUMEN
Hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 and resistin-like molecule α, belongs to a novel class of cysteine-rich secreted proteins. It exhibits mitogenic and chemotactic properties during pulmonary hypertension-associated vascular remodeling, as well as fibrogenic properties during pulmonary fibrosis. HIMF expression in the lung was reported to be regulated by Th2 cytokines (IL-4 and IL-13) via the transcription factor STAT6 pathway in a bleomycin-induced pulmonary fibrosis model. However, in this study, we found that in the hypoxia-induced pulmonary hypertension model, lung HIMF expression is increased in IL-4 and STAT6 knockout (KO) mice to the same degree as in wild-type (WT) mice, suggesting that induction of HIMF expression does not require Th2 regulation in this model. We also found that HIMF-induced proliferative activity, hypertrophy, collagen, and extracellular matrix deposition in the pulmonary arteries are significantly less in IL-4 KO mice than in WT mice. In addition, HIMF-induced production of angiogenic factors/chemokines, such as vascular endothelial growth factor, MCP-1, and stromal-derived factor-1, in the lung resident cells, as well as macrophage infiltration, were significantly suppressed in the lungs of IL-4 KO mice. We also show that IL-4 was significantly increased in the lungs of HIMF-treated WT mice. Our in vitro studies using pulmonary microvascular endothelial cells revealed that HIMF stimulated cell proliferation, vascular endothelial growth factor expression, and MCP-1 production in a manner that is dependent on the IL-4/IL-4Rα system. These findings suggest that IL-4 signaling may play a significant role in HIMF-induced lung inflammation and vascular remodeling.
Asunto(s)
Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-4/metabolismo , Neumonía/metabolismo , Transducción de Señal/inmunología , Animales , Movimiento Celular , Proliferación Celular , Células Endoteliales/inmunología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Hipertensión Pulmonar/metabolismo , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interleucina-4/inmunología , Pulmón/irrigación sanguínea , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Neumonía/inmunología , Neumonía/patología , Fibrosis Pulmonar/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lung transplantation has become the standard of care for particular individuals with advanced lung disease. However, this surgical procedure involves interruption of the lower vagal nerve fibers which leads to loss of the protective cough reflex. Injury of the neural pathways involved with the sensory limb of the cough reflex is associated with an increased risk of complications involving the allograft. While loss of the cough reflex was once considered permanent, recent evidence indicates functional and structural restoration is a time-dependent process that occurs 6-12 months after lung transplantation. The implication that the cough reflex may be reestablished in lung transplant recipients provides insight into the dynamic response to airway neural injury that may lead to improvements in allograft tissue repair.
Asunto(s)
Tos/fisiopatología , Trasplante de Pulmón/fisiología , Reflejo , Animales , Humanos , Trasplante de Pulmón/efectos adversos , Sistema Respiratorio/inervación , Traumatismos del Nervio Vago/etiologíaRESUMEN
Sialic acid-binding immunoglobulin-like lectin (Siglec)-F, an inhibitory receptor on mouse eosinophils, preferentially recognizes the glycan ligand 6'-sulfated sialyl Lewis X, but little is known about the requirements for its lung expression. RT-PCR and immunohistochemistry were used to detect and localize the sulfotransferase keratin sulfate galactose 6-O sulfotransferase (KSGal6ST, also known as carbohydrate sulfotransferase 1; gene name, Chst1) that is putatively required for 6'-sulfated Sialyl Lewis X synthesis. RT-PCR detected the greatest constitutive expression of Chst1 in lung, liver, and spleen tissue. Immunohistochemistry localized the expression of KSGal6ST in lung tissue primarily to airway epithelium. Siglec-F-Ig fusion protein selectively bound in a similar pattern, and was unaffected in lung tissue treated with methanol or deficient in Type 2 α2,3 sialyltransferase (St3gal2), but was eliminated by proteinase K or sialidase, and was absent in tissue deficient in the Type 3 α2,3 sialyltransferase (St3gal3). Binding of the Siglec-F-Ig fusion protein was similar in pattern to, and completely blocked by, a plant lectin recognizing α2,3-linked sialic acid. Thus, α2,3-linked sialic acid-containing glycoprotein Siglec-F ligands and the enzymes required for their synthesis are constitutively expressed in murine lungs, especially by airway epithelium. St3gal3, but not St3gal2, is required for constitutive Siglec-F ligand synthesis. The survival of eosinophils entering the lung may be shortened by encountering these Siglec-F sialoside ligands.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Polisacáridos/inmunología , Polisacáridos/metabolismo , Animales , Anticuerpos Antiidiotipos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Omalizumab , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Sialiltransferasas/deficiencia , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , beta-Galactosida alfa-2,3-Sialiltransferasa , Carbohidrato SulfotransferasasRESUMEN
We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aß-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as â¼50% of the nodose neurons. GFRα2 was expressed in â¼50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.
Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Pulmón/inervación , Ganglio Nudoso/fisiología , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Tráquea/inervación , Animales , Cobayas , Masculino , Receptor trkA/biosíntesis , Receptor trkB/biosíntesis , Receptor trkC/biosíntesis , Células Receptoras Sensoriales , Canales Catiónicos TRPV/biosíntesisRESUMEN
Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality, which have been attributed to intermittent hypoxia (IH). The effects of IH on lung structure and function are unknown. We used a mouse model of chronic IH, which mimics the O(2) profile in patients with OSA. We exposed adult C57BL/6J mice to 3 mo of IH with a fraction of inspired oxygen (F(I)(O(2))) nadir of 5% 60 times/h during the 12-h light phase. Control mice were exposed to room air. Lung volumes were measured by quasistatic pressure-volume (PV) curves under anesthesia and by water displacement postmortem. Lungs were processed for morphometry, and the mean airspace chord length (Lm) and alveolar surface area were determined. Lung tissue was stained for markers of proliferation (proliferating cell nuclear antigen), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling), and type II alveolar epithelial cells (surfactant protein C). Gene microarrays were performed, and results were validated by real-time PCR. IH increased lung volumes by both PV curves (air vs. IH, 1.16 vs. 1.44 ml, P < 0.0001) and water displacement (P < 0.01) without changes in Lm, suggesting that IH increased the alveolar surface area. IH induced a 60% increase in cellular proliferation, but the number of proliferating type II alveolocytes tripled. There was no increase in apoptosis. IH upregulated pathways of cellular movement and cellular growth and development, including key developmental genes vascular endothelial growth factor A and platelet-derived growth factor B. We conclude that IH increases alveolar surface area by stimulating lung growth in adult mice.
Asunto(s)
Hipoxia/patología , Pulmón/patología , Animales , Secuencia de Bases , Enfermedad Crónica , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Hipoxia/etiología , Hipoxia/genética , Hipoxia/fisiopatología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Derivado de Plaquetas/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Apnea Obstructiva del Sueño/complicaciones , Apnea Obstructiva del Sueño/patología , Apnea Obstructiva del Sueño/fisiopatología , Factor de Crecimiento Transformador beta/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. METHODS AND RESULTS: Immunoblot showed increased α(1)-, α(8)- and α(v)-integrins, and decreased α(5)-integrin levels in PAs of both models. ß(1)- and ß(3)-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α(1)- and α(5)-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α(1)-ligand collagen IV. Moreover, α(1)-integrin binding hexapeptide GRGDTP elicited an enhanced Ca(2+) response, whereas the response to α(5)-integrin binding peptide GRGDNP was reduced in CH-PASMCs. CONCLUSION: Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression.
Asunto(s)
Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , Integrinas/metabolismo , Músculo Liso Vascular/metabolismo , Arteria Pulmonar/metabolismo , Animales , Señalización del Calcio/fisiología , Enfermedad Crónica , Modelos Animales de Enfermedad , Quinasa 1 de Adhesión Focal/metabolismo , Hipertensión Pulmonar/inducido químicamente , Masculino , Monocrotalina/farmacología , Fosforilación/fisiología , Ratas , Ratas WistarRESUMEN
We have identified a distinct subtype of airway vagal afferent nerve that plays an essential role in regulating the cough reflex. These afferents are exquisitely sensitive to punctate mechanical stimuli, acid, and decreases in extracellular chloride concentrations, but are insensitive to capsaicin, bradykinin, histamine, adenosine, serotonin, or changes in airway intraluminal pressures. In this study we used intravital imaging, retrograde neuronal tracing, and electrophysiological analyses to characterize the structural basis for their peculiar mechanical sensitivity and to further characterize the regulation of their excitability. In completing these experiments, we uncovered evidence for an essential role of an isozyme of Na(+)-K(+) ATPase in regulating cough. These vagal sensory neurons arise bilaterally from the nodose ganglia and are selectively and brilliantly stained intravitally with the styryl dye FM2-10. Cough receptor terminations are confined and adherent to the extracellular matrix separating the airway epithelium and smooth muscle layers, a site of extensive remodeling in asthma and chronic obstructive pulmonary disease. The cough receptor terminals uniquely express the alpha(3) subunit of Na(+)-K(+) ATPase. Intravital staining of cough receptors by FM2-10, cough receptor excitability in vitro, and coughing in vivo are potently and selectively inhibited by the sodium pump inhibitor ouabain. These data provide the first detailed morphological description of the peripheral terminals of the sensory nerves regulating cough and identify a selective molecular target for their modulation.
Asunto(s)
Tos/fisiopatología , Reflejo/fisiología , Células Receptoras Sensoriales/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Nervio Vago/fisiología , Animales , Adhesión Celular , Tos/enzimología , Epitelio/anatomía & histología , Matriz Extracelular/fisiología , Cobayas , Isoenzimas/metabolismo , Masculino , Músculo Liso/anatomía & histología , Ganglio Nudoso/anatomía & histología , Ganglio Nudoso/citología , Ganglio Nudoso/fisiología , Ratas , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/enzimología , Tráquea/anatomía & histología , Tráquea/citología , Tráquea/fisiología , Nervio Vago/anatomía & histología , Nervio Vago/citologíaRESUMEN
Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1ß had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.
Asunto(s)
Alérgenos/inmunología , Pulmón/inmunología , Pulmón/inervación , Neuronas/metabolismo , Neurotransmisores/metabolismo , Neurotrofina 3/metabolismo , Sistema Nervioso Parasimpático/metabolismo , Animales , Esófago/inervación , Esófago/patología , Técnica del Anticuerpo Fluorescente , Secciones por Congelación , Cobayas , Masculino , Neuronas/inmunología , Neuronas/patología , Sistema Nervioso Parasimpático/inmunología , Sistema Nervioso Parasimpático/patología , Neumonía/inmunología , Neumonía/patologíaRESUMEN
Two major types of nociceptors have been described in dorsal root ganglia (DRGs). In comparison, little is known about the vagal nociceptor subtypes. The vagus nerves provide much of the capsaicin-sensitive nociceptive innervation to visceral tissues, and are likely to contribute to the overall pathophysiology of visceral inflammatory diseases. The cell bodies of these afferent nerves are located in the vagal sensory ganglia referred to as nodose and jugular ganglia. Neurons of the nodose ganglion are derived from the epibranchial placodes, whereas jugular ganglion neurons are derived from the neural crest. In the adult mouse, however, there is often only a single ganglionic structure situated alone in the vagus nerve. By employing Wnt1Cre/R26R mice, which express ß-galactosidase only in neural crest derived neurons, we found that this single vagal sensory ganglion is a fused ganglion consisting of both neural crest neurons in the rostral portion and non-neural crest (nodose) neurons in the more central and caudal portions of the structure. Based on their activation and gene expression profiles, we identified two major vagal capsaicin-sensitive nociceptor phenotypes, which innervated a defined target, namely the lung in adult mice. One subtype is non-peptidergic, placodal in origin, expresses P2X2 and P2X3 receptors, responds to α,ß-methylene ATP, and expresses TRKB, GFRα1 and RET. The other phenotype is derived from the cranial neural crest and does not express P2X2 receptors and fails to respond to α,ß-methylene ATP. This population can be further subdivided into two phenotypes, a peptidergic TRKA(+) and GFRα3(+) subpopulation, and a non-peptidergic TRKB(+) and GFRα1(+) subpopulation. Consistent with their similar embryonic origin, the TRPV1 expressing neurons in the rostral dorsal root ganglia were more similar to jugular than nodose vagal neurons. The data support the hypothesis that vagal nociceptors innervating visceral tissues comprise at least two major subtypes. Due to distinctions in their gene expression profile, each type will respond to noxious or inflammatory conditions in their own unique manner.
Asunto(s)
Pulmón/inervación , Cresta Neural/citología , Ganglio Nudoso/citología , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Calcio/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Cobayas , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
The pathophysiology of airway diseases, such as asthma, is increasingly studied using transgenic mice and other mouse models of airway inflammation where allergen-induced changes in airway smooth muscle tone and mucous secretion is due, in part, to activation of preganglionic airway parasympathetic nerves. Ganglionic parasympathetic neurons located in the airways in several species, including humans, have anatomical and electrophysiological properties that limit transmission of preganglionic synaptic input. In this study, intracellular recordings were made from neurons in parasympathetic ganglia located on the trachea and bronchi of adult mice to determine electrophysiological properties associated with regulation of transmission of preganglionic input. Ganglionic neurons were characterized as having either tonic or phasic action potential accommodation patterns. Tonic neurons responded with repetitive action potentials sustained throughout a depolarizing current step, whereas phasic neurons generated one or a burst of action potential(s) and accommodated. A small subset displayed both patterns. Phasic neurons could be further differentiated as usually having either short- or long-duration afterhyperpolarizing potential following single and multiple action potentials. In most cells, stimulation of preganglionic nerves elicited one population of nicotinic fast excitatory postsynaptic potentials that were graded in amplitude, usually suprathreshold for action potential generation, and did not decrease in amplitude during higher frequency stimulation. Dye injection into the neurons revealed that dendrites were either absent or very short. These results provide evidence that in contrast to the characteristics of airway parasympathetic neurons reported in other species, including human, the electrophysiological and synaptic properties, and anatomical characteristics of mouse lower airway ganglionic neurons, are less associated with integration of presynaptic input.