RESUMEN
Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Canales de Potasio de Tipo Rectificador Tardío/antagonistas & inhibidores , Ganglios Sensoriales/efectos de los fármacos , Gentamicinas/toxicidad , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/toxicidad , Nervio Vestibular/efectos de los fármacos , Potenciales de Acción , Animales , Células Cultivadas , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Femenino , Ganglios Sensoriales/enzimología , Masculino , Ratones Endogámicos ICR , Neuronas/enzimología , Fosforilación , Transducción de Señal , Nervio Vestibular/enzimologíaRESUMEN
Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.
Asunto(s)
Diabetes Mellitus Tipo 2/terapia , Retinopatía Diabética/prevención & control , Disbiosis/terapia , Ayuno , Microbioma Gastrointestinal , Retina/patología , Vasos Retinianos/patología , Animales , Bacteroidetes/crecimiento & desarrollo , Bacteroidetes/inmunología , Bacteroidetes/aislamiento & purificación , Ácidos y Sales Biliares/uso terapéutico , Colon/efectos de los fármacos , Colon/inmunología , Colon/metabolismo , Colon/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/microbiología , Diabetes Mellitus Tipo 2/patología , Retinopatía Diabética/complicaciones , Retinopatía Diabética/inmunología , Retinopatía Diabética/patología , Disbiosis/complicaciones , Disbiosis/microbiología , Disbiosis/patología , Heces/microbiología , Firmicutes/crecimiento & desarrollo , Firmicutes/inmunología , Firmicutes/aislamiento & purificación , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/inmunología , Ganglios Sensoriales/metabolismo , Ganglios Sensoriales/patología , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/inmunología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/inmunología , Células Caliciformes/metabolismo , Células Caliciformes/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/patología , Masculino , Ratones Endogámicos DBA , Ratones Mutantes , Microvasos/efectos de los fármacos , Microvasos/inmunología , Microvasos/metabolismo , Microvasos/patología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Retina/efectos de los fármacos , Retina/inmunologíaRESUMEN
BNN27, a C17-spiroepoxy derivative of DHEA, was shown to have antiapoptotic properties via mechanisms involving the nerve growth factor receptors (tropomyosin-related kinase A [TrkA]/neurotrophin receptor p75 [p75NTR]). In this study, we examined the effects of BNN27 on neural/glial cell function, apoptosis, and inflammation in the experimental rat streptozotocin (STZ) model of diabetic retinopathy (DR). The ability of BNN27 to activate the TrkA receptor and regulate p75NTR expression was investigated. BNN27 (2,10, and 50 mg/kg i.p. for 7 days) administration 4 weeks post-STZ injection (paradigm A) reversed the diabetes-induced glial activation and loss of function of amacrine cells (brain nitric oxide synthetase/tyrosine hydroxylase expression) and ganglion cell axons via a TrkA receptor (TrkAR)-dependent mechanism. BNN27 activated/phosphorylated the TrkAY490 residue in the absence but not the presence of TrkAR inhibitor and abolished the diabetes-induced increase in p75NTR expression. However, it had no effect on retinal cell death (TUNEL+ cells). A similar result was observed when BNN27 (10 mg/kg i.p.) was administered at the onset of diabetes, every other day for 4 weeks (paradigm B). However, BNN27 decreased the activation of caspase-3 in both paradigms. Finally, BNN27 reduced the proinflammatory (TNFα and IL-1ß) and increased the anti-inflammatory (IL-10 and IL-4) cytokine levels. These findings suggest that BNN27 has the pharmacological profile of a therapeutic for DR, since it targets both the neurodegenerative and inflammatory components of the disease.
Asunto(s)
Células Amacrinas/efectos de los fármacos , Antiinflamatorios/uso terapéutico , Deshidroepiandrosterona/uso terapéutico , Retinopatía Diabética/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Receptor trkA/agonistas , Retina/efectos de los fármacos , Células Amacrinas/inmunología , Células Amacrinas/metabolismo , Células Amacrinas/patología , Animales , Antiinflamatorios/administración & dosificación , Axones/efectos de los fármacos , Axones/inmunología , Axones/metabolismo , Axones/patología , Deshidroepiandrosterona/administración & dosificación , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/inmunología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Relación Dosis-Respuesta a Droga , Proteínas del Ojo/agonistas , Proteínas del Ojo/metabolismo , Femenino , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/inmunología , Ganglios Sensoriales/metabolismo , Ganglios Sensoriales/patología , Masculino , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroglía/patología , Fármacos Neuroprotectores/administración & dosificación , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/agonistas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Retina/inmunología , Retina/patología , Retina/fisiopatología , EstreptozocinaRESUMEN
Neurons of the Grueneberg ganglion (GG) in the anterior nasal region of mice respond to a small set of odorous compounds, including given dimethylpyrazines present in mouse urine. Consequently, mouse pups living in murine colonies are presumably commonly exposed to such GG-activating substances. Since stimulation of the GG elicits alarm and stress reactions in mice, the question arises whether such a GG activation potentially inducing stress could be reduced when pups might rather feel secure in the presence of their mother. Being together with their warmth-giving dam, mouse pups experience a nest temperature of â¼35⯰C. Therefore, we hypothesized that such a warm temperature may attenuate the responses of GG neurons to dimethylpyrazines. Monitoring the expression of the activity marker c-Fos, GG responses to dimethylpyrazines were significantly lower in pups exposed to these substances at 35⯰C compared to exposure at 30⯰C. By contrast, dimethylpyrazine-induced responses of neurons in the main olfactory epithelium were not diminished at 35⯰C in comparison to 30⯰C. The attenuated chemosensory responses of GG neurons at 35⯰C coincided with a reduced dimethylpyrazine-evoked activation of the glomeruli in the olfactory bulb innervated by GG neurons. The reduction in dimethylpyrazine-evoked GG responses by warm temperatures was positively correlated with exposure time, suggesting that warm temperatures might enhance desensitization processes in GG neurons. In summary, the findings indicate that warm temperatures similar to those in mouse nests in the presence of the dam attenuate GG activation by colony-derived odorants.
Asunto(s)
Bulbo Olfatorio/metabolismo , Mucosa Olfatoria/metabolismo , Pirazinas/administración & dosificación , Células Receptoras Sensoriales/metabolismo , Animales , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/metabolismo , Calor , Ratones Endogámicos C57BL , Odorantes , Bulbo Olfatorio/efectos de los fármacos , Mucosa Olfatoria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Transient receptor potential vanilloid (TRPV) 4 is a nonselective cation channel expressed in sensory neurons such as those in the dorsal root and trigeminal ganglia, kidney, and inner ear. TRPV4 is activated by mechanical stress, heat, low osmotic pressure, low pH, and phorbol derivatives such as 4α-phorbol 12,13-didecanoate (4α-PDD). We investigated the expression of TRPV4 in rat vestibular ganglion (VG) neurons. The TRPV4 gene was successfully amplified from VG neuron mRNA using reverse-transcription polymerase chain reaction. Furthermore, immunoblotting showed positive expression of TRPV4 protein in VG neurons. Immunohistochemistry indicated that TRPV4 was localized predominantly on the plasma membrane of VG neurons. Calcium (Ca2+) imaging of VG neurons showed that 4α-PDD and/or hypotonic stimuli caused an increase in intracellular Ca2+ concentration ([Ca2+]i) that was almost completely inhibited by ruthenium red, a selective antagonist of TRPV channels. Interestingly, a [Ca2+]i increase was evoked by both hypotonic stimuli and 4α-PDD in approximately 38% of VG neurons. These data indicate that TRPV4 is functionally expressed in VG neurons as an ion channel and that TRPV4 likely participates in VG neurons for vestibular neurotransmission as an osmoreceptor and/or mechanoreceptor.
Asunto(s)
Ganglios Sensoriales/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Canales Catiónicos TRPV/genética , Nervio Vestibular/metabolismo , Animales , Calcio/metabolismo , Ganglios Sensoriales/efectos de los fármacos , Neuronas/efectos de los fármacos , Forboles/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPV/metabolismo , Nervio Vestibular/efectos de los fármacosRESUMEN
P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,ß-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Ganglios Sensoriales/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Ganglios Sensoriales/efectos de los fármacos , Ratones Endogámicos C57BL , Fenoles/farmacología , Compuestos Policíclicos/farmacología , Receptores Purinérgicos P2X3/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ganglio del Trigémino/citología , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Mas-related genes (Mrgs) belong to a large family of G protein-coupled receptor genes found in rodents. Human MRGX proteins are G protein-coupled 7-transmembrane proteins sharing 41-52% amino acid identity with each other, but have no orthologs in rodents. MrgX2 is a member of the MrgX family. MRGX2 is expressed in the small neurons of sensory ganglia and mast cells. It can interact with a series of factors and genes such as the peptides substance P, vasoactive intestinal peptide, cortistatin (CST), proadrenomedullin N-terminal peptide (PAMP), LL-37, PMX-53 and ß-defensins. MRGX2 is related to nociception, adrenal gland secretion and mast cell degranulation. Recent research on MrgX2 provides insights into its role in nociception and anti-microbial activities. This article reviewed the origin, expression and function of MrgX2, and discussed possible future research focus.
Asunto(s)
Ganglios Sensoriales/fisiología , Proteínas del Tejido Nervioso/metabolismo , Nocicepción/fisiología , Dolor/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Adrenomedulina/farmacología , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Dextrorfano/farmacología , Evolución Molecular , Ganglios Sensoriales/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Mastocitos/citología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuropéptidos/farmacología , Nocicepción/efectos de los fármacos , Dolor/genética , Dolor/fisiopatología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Transducción de Señal , Sustancia P/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
The primary vestibular neurons convey afferent information from hair cells in the inner ear to the vestibular nuclei and the cerebellum. The intrinsic firing properties of vestibular ganglion cells (VGCs) are heterogeneous to sustained membrane depolarization, and undergo marked developmental changes from phasic to tonic types during the early postnatal period. Previous studies have shown that low-voltage-activated potassium channels, Kv1 and Kv7, play a critical role in determining the firing pattern of VGCs. In the present study, we explored the developmental changes in the properties of hyperpolarization-activated current (Ih) in rat VGCs and the role played by Ih in determining the firing properties of VGCs. Tonic firing VGCs showed a larger current density of Ih as compared to phasic firing VGCs, and tonic firing VGCs became phasic firing in the presence of ZD7288, an Ih channel blocker, indicating that Ih contributes to control the firing pattern of VGCs. The amplitude of Ih increased and the activation kinetics of Ih became faster during the developmental period. Analysis of developmental changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels revealed that expression of HCN1 protein and its mRNA increased during the developmental period, whereas expression of HCN2-4 protein and its mRNA did not change. Our results suggest that HCN1 channels as well as Kv1 channels are critical in determining the firing pattern of rat VGCs and that developmental up-regulation of HCN1 transforms VGCs from phasic to tonic firing phenotypes.
Asunto(s)
Potenciales de Acción/fisiología , Ganglios Sensoriales/crecimiento & desarrollo , Neuronas/fisiología , Nervio Vestibular/crecimiento & desarrollo , Potenciales de Acción/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/fisiología , Regulación del Desarrollo de la Expresión Génica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Nervio Vestibular/efectos de los fármacos , Nervio Vestibular/fisiologíaRESUMEN
Previous studies indicated that intracerebroventricular administration of nerve growth factor (NGF) leads to massive Schwann cell hyperplasia surrounding the medulla oblongata and spinal cord. This study was designed to characterize the proliferation of peripheral glial cells, that is, Schwann and satellite cells, in the trigeminal ganglia and dorsal root ganglia (DRG) of adult rats during two weeks of NGF infusion using bromodeoxyuridine (BrdU) to label dividing cells. The trigeminal ganglia as well as the cervical and lumbar DRG were analyzed. Along the entire neuraxis a small number of dividing cells were observed within these regions under physiological condition. NGF infusion has dramatically increased the generation of new cells in the neuronal soma and axonal compartments of sensory ganglia and along the dorsal root and the dorsal root entry zone. Quantification of BrdU positive cells within sensory ganglia revealed a 2.3- to 3-fold increase in glial cells compared to controls with a similar response to NGF for the different peripheral ganglia examined. Immunofluorescent labeling with S100ß revealed that Schwann and satellite cells underwent mitosis after NGF administration. These data indicate that intracerebroventricular NGF infusion significantly induces gliogenesis in trigeminal ganglia and the spinal sensory ganglia and along the dorsal root entry zone as well as the dorsal root.
Asunto(s)
Ganglios Sensoriales/crecimiento & desarrollo , Factor de Crecimiento Nervioso/administración & dosificación , Neuroglía/fisiología , Raíces Nerviosas Espinales/crecimiento & desarrollo , Animales , Axones/efectos de los fármacos , Ganglios Sensoriales/efectos de los fármacos , Infusiones Intraventriculares , Neuroglía/efectos de los fármacos , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/crecimiento & desarrollo , Raíces Nerviosas Espinales/efectos de los fármacosRESUMEN
The 5-hydroxytryptamine type 3 (5-HT3) receptor is a ligand-gated ion channel and a member of the Cys-loop family of receptors. Previous studies have shown 5-HT3 receptor expression in various neural cells of the central and peripheral nervous systems. Although the function and distribution of the 5-HT3 receptor has been well established, its role in the inner ear is still poorly understood. Moreover, no study has yet determined its localization and function in the peripheral vestibular nervous system. In the present study, we reveal mRNA expression of both 5-HT3A and 5-HT3B receptor subunits in the mouse vestibular ganglion (VG) by RT-PCR and in situ hybridization (ISH). We also show by ISH that 5-HT3 receptor mRNA is only expressed in the VG (superior and inferior division) in the peripheral vestibular nervous system. Moreover, we performed Ca(2+) imaging to determine whether functional 5-HT3 receptors are present in the mouse VG, using a selective 5-HT3 receptor agonist, SR57227A. In wild mice, 32% of VG neurons responded to the agonist, whereas there was no response in 5-HT3A receptor knockout mice. These results indicate that VG cells express functional 5-HT3 receptor channels and might play a modulatory role in the peripheral vestibular nervous system.
Asunto(s)
Ganglios Sensoriales/metabolismo , Neuronas/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Animales , Calcio/metabolismo , Tamaño de la Célula , Células Cultivadas , Ganglios Sensoriales/efectos de los fármacos , Hibridación Fluorescente in Situ , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Imagen Óptica , Piperidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Serotonina 5-HT3/genética , Agonistas del Receptor de Serotonina 5-HT3/farmacología , Ganglio Espiral de la Cóclea/efectos de los fármacos , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
BACKGROUND: Theophylline has been used in the treatment of asthma and chronic obstructive pulmonary disease for more than 80 years. In addition to bronchodilator and anti-inflammatory activity, clinical studies have suggested that theophylline acts as an antitussive agent. Cough is the most frequent reason for consultation with a family doctor, and treatment options are limited. Determining how theophylline inhibits cough might lead to the development of optimized compounds. OBJECTIVE: We sought to investigate the inhibitory activity of theophylline on vagal sensory nerve activity and the cough reflex. METHODS: Using a range of techniques, we investigated the effect of theophylline on human and guinea pig vagal sensory nerve activity in vitro and on the cough reflex in guinea pig challenge models. RESULTS: Theophylline was antitussive in a guinea pig model, inhibited activation of single C-fiber afferents in vivo and depolarization of human and guinea pig vagus in vitro, and inhibited calcium influx in airway-specific neurons in vitro. A sequence of pharmacological studies on the isolated vagus and patch clamp and single-channel inside-out experiments showed that the effect of theophylline was due to an increase in the open probability of calcium-activated potassium channels. Finally, we demonstrated the antitussive activity of theophylline in a cigarette smoke exposure model that exhibited enhanced tussive responses to capsaicin. CONCLUSION: Theophylline inhibits capsaicin-induced cough under both normal and "disease" conditions by decreasing the excitability of sensory nerves through activation of small- and intermediate-conductance calcium-activated potassium channels. These findings could lead to the development of optimized antitussive compounds with a reduced side effect potential.
Asunto(s)
Antitusígenos/farmacología , Tos/etiología , Reflejo/efectos de los fármacos , Reflejo/fisiología , Teofilina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Antitusígenos/administración & dosificación , Calcio/metabolismo , Capsaicina/farmacología , Tos/tratamiento farmacológico , Modelos Animales de Enfermedad , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/metabolismo , Cobayas , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Fibras Nerviosas Amielínicas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Calcio-Activados/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Teofilina/administración & dosificación , Nervio Vago/efectos de los fármacos , Nervio Vago/fisiologíaRESUMEN
Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Ganglios Sensoriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Animales , Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Bortezomib , Núcleo Celular/fisiología , Reparación del ADN/efectos de los fármacos , Ganglios Sensoriales/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Masculino , Microscopía Confocal , Cuerpos de Nissl/efectos de los fármacos , Cuerpos de Nissl/fisiología , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Pirazinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The aim of the present work was to investigate the effect of ouabagenin on the growth and proliferation of cells in organotypic culture. The objects of study were explants of nerve, cardiac, retina and liver tissue of 10-12 day old chicken embryos. Inhibitor of Na+,K(+)-ATPase ouabagenin was investigated in a wide range of concentrations (0.1 nM-1 mM). It has been found that the ouabagenin controls cell growth and proliferation in a dose-dependent manner and tissue-unspecific. The data obtained show that ouabagenin regulates only the pumping function of Na+,K(+)-ATPase.
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Inhibidores Enzimáticos/farmacología , Ganglios Sensoriales/efectos de los fármacos , Hígado/efectos de los fármacos , Ouabaína/análogos & derivados , Retina/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Ganglios Sensoriales/citología , Ganglios Sensoriales/enzimología , Hígado/citología , Hígado/enzimología , Miocardio/citología , Miocardio/enzimología , Ouabaína/farmacología , Retina/citología , Retina/enzimología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (-)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, - five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.
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Ganglios Sensoriales/efectos de los fármacos , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Tráquea/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Ganglios Sensoriales/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotina/administración & dosificación , Receptores Nicotínicos/genética , Células Receptoras Sensoriales/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/genética , Tráquea/metabolismo , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Immunohistochemical studies of sympathetic ganglia have indicated that the normal rat superior cervical ganglion contains both SP-IR and CGRP-IR fibres, and CGRP- and SP-immunoreactivity coexist in some fibres. In rat sympathetic ganglia decentralization by preganglionic denervation leads to intraganglionic increase of peptidergic fibres immunoreactive (IR) for substance P (SP) and calcitonin gene-related peptide. We explored the sources of SP- and CGRP-IR fibres in normal and in chronically decentralized rat SCGs. The distribution of immunoreactivities for CGRP and SP was determined in SCGs of normal rats and of rats following preganglionic denervation followed by sensory denervation. Ganglia were studied after short-term (2-5 days) sensory denervation, and long-term (7-16 months) sympathetic denervation followed by short-term (2 days) sensory denervation. To explore for the production of SP and CGRP by intrinsic neurones within the ganglion, normal and chronically decentralized SCGs were examined following pretreatment by local in vivo application of colchicine. Normal and chronically decentralized ganglia were also injected with fluorescent tracer Fluorogold for retrograde tracing of extrinsic fibres back to their neurones of origin. The observations suggest that in normal SCG in the rat the SP-IR and CGRP-IR nerve fibres are derived via direct links from vagus and glossopharyngeal nerves and the cervical plexus, or from nerve fibres running along the cervical sympathetic trunk, and the external carotid and the internal carotid nerves. Sensory nerve inputs to the rat SCG following decentralization may contribute to the low levels of ganglionic activation observable in the autonomic failure of multiple system atrophy in man.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Atrofia de Múltiples Sistemas/metabolismo , Fibras Nerviosas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas , Sustancia P/metabolismo , Ganglio Cervical Superior/cirugía , Animales , Biomarcadores/metabolismo , Colchicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/metabolismo , Ganglios Sensoriales/patología , Ganglios Sensoriales/cirugía , Inmunohistoquímica , Masculino , Atrofia de Múltiples Sistemas/patología , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/patología , Ratas , Ratas Wistar , Ganglio Cervical Superior/efectos de los fármacos , Ganglio Cervical Superior/metabolismo , Ganglio Cervical Superior/patología , Simpatectomía , Factores de Tiempo , Moduladores de Tubulina/farmacologíaRESUMEN
Venoms often target vital processes to cause paralysis or death, but many types of venom also elicit notoriously intense pain. While these pain-producing effects can result as a byproduct of generalized tissue trauma, there are now multiple examples of venom-derived toxins that target somatosensory nerve terminals in order to activate nociceptive (pain-sensing) neural pathways. Intriguingly, investigation of the venom components that are responsible for evoking pain has revealed novel roles and/or configurations of well-studied toxin motifs. This review serves to highlight pain-producing toxins that target the capsaicin receptor, TRPV1, or members of the acid-sensing ion channel family, and to discuss the utility of venom-derived multivalent and multimeric complexes.
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Dolor Agudo/inducido químicamente , Proteínas del Tejido Nervioso/agonistas , Nociceptores/efectos de los fármacos , Proteínas/toxicidad , Agonistas de los Canales de Sodio , Canales Catiónicos TRPV/agonistas , Ponzoñas/toxicidad , Canales Iónicos Sensibles al Ácido , Dolor Agudo/etiología , Dolor Agudo/metabolismo , Animales , Mordeduras y Picaduras/metabolismo , Mordeduras y Picaduras/fisiopatología , Ganglios Sensoriales/efectos de los fármacos , Ganglios Sensoriales/metabolismo , Humanos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Nociceptores/metabolismo , Conformación Proteica , Subunidades de Proteína/análisis , Subunidades de Proteína/química , Subunidades de Proteína/toxicidad , Proteínas/análisis , Proteínas/química , Canales de Sodio/química , Canales de Sodio/metabolismo , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/metabolismo , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismo , Ponzoñas/química , Ponzoñas/enzimologíaRESUMEN
BACKGROUND: Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase. RESULTS: We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents. CONCLUSIONS: Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.
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Ganglios Sensoriales/efectos de los fármacos , Eliminación de Gen , Proteínas de Microfilamentos/genética , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Tamoxifeno/farmacología , Animales , Células Cultivadas , Ganglios Sensoriales/metabolismo , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Células Receptoras Sensoriales/metabolismoAsunto(s)
Cloropirifos/toxicidad , Citoplasma/efectos de los fármacos , Ganglios Sensoriales/efectos de los fármacos , Ganglios Espinales/patología , Neuronas/patología , Células Receptoras Sensoriales/efectos de los fármacos , Tritolilfosfatos/toxicidad , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Animales , MasculinoRESUMEN
An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 µg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.
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Biomarcadores/metabolismo , Ganglios Sensoriales/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Octopodiformes/genética , Nervio Óptico/efectos de los fármacos , Proteoma/análisis , Proteómica/métodos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Actinas/genética , Actinas/metabolismo , Acil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasas/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Animales , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Ganglios Sensoriales/metabolismo , Redes Reguladoras de Genes , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Metanol/efectos adversos , Octopodiformes/efectos de los fármacos , Nervio Óptico/metabolismo , Mapeo Peptídico , Proteoma/genética , Máquina de Vectores de Soporte , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain. RESULTS: Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Ca(v)2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation. CONCLUSIONS: The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.