RESUMEN
The pathology of migraine, a common neurological disease, involves sensitization and activation of trigeminal nociceptive neurons to promote hyperalgesia and allodynia during an attack. Migraineurs often exhibit characteristics of a hyperexcitable or hypervigilant nervous system. One of the primary reported risk factors for development of a hyperexcitable trigeminal system is chronic, unmanaged stress and anxiety. While primary traumatic stress is a commonly cited risk factor for many pain conditions, exposure to secondary traumatic stress early in life is also thought to be a contributing risk factor. The goal of this study was to investigate cellular changes within the spinal trigeminal nucleus and trigeminal ganglion mediated by secondary traumatic stress. Male Sprague Dawley rats (sender) were subjected to forced swim testing (primary traumatic stress) and were then housed in close visual, olfactory, and auditory proximity to the breeding male and female rats, pregnant female rats, or female rats and their nursing offspring (all receivers). In response to secondary stress, levels of calcitonin gene-related peptide, active forms of the mitogen activated protein kinases ERK, JNK, and p38, and astrocyte expression of glial fibrillary acidic protein were significantly elevated in the spinal trigeminal nucleus in day 45 offspring when compared to naïve offspring. In addition, increased nuclear expression of ERK and p38 was observed in trigeminal ganglion neurons. Our results demonstrate that secondary traumatic stress promotes cellular events associated with prolonged trigeminal sensitization in the offspring, and provides a mechanism of how early life stress may function as a risk factor for migraine.
Asunto(s)
Sensibilización del Sistema Nervioso Central/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Trastornos de Estrés Traumático/patología , Ganglio del Trigémino/patología , Núcleo Espinal del Trigémino/patología , Animales , Modelos Animales de Enfermedad , Femenino , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratas , Ratas Sprague-Dawley , Trastornos de Estrés Traumático/fisiopatología , NataciónRESUMEN
OBJECTIVES: To investigate and discuss the effects of cocoa on orofacial pain. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the University of Florida (UF). Male and female hairless rats (N=20/group) were tested. MATERIALS AND METHODS: Rats were tested using the Orofacial Pain Assessment Device (OPAD) before and after changing their food from the standard chow to a cocoa-enriched or control-equivalent diet. RESULTS: Male rats fed the cocoa diet had a significantly higher operant pain index when tested at 37°C as compared to control diet-fed animals. Female rats on the cocoa diet had a significantly higher pain index when tested at 18°C and 44°C, as compared to animals fed the control diet. Capsaicin-induced pain was inhibited, with cocoa-diet male rats having a significantly higher pain index than control-diet male rats and cocoa-diet female rats at both 37°C and 44°C. Cocoa-diet female rats had a significantly higher pain index at 44°C than control-diet females. Mechanical sensitivity was affected following capsaicin cream, with a significantly decreased tolerated bottle distance in both cocoa- and control-diet animals, but there was no difference between cocoa- and control-diet groups. CONCLUSION: Using the OPAD operant system, we demonstrated that a diet rich in cocoa was effective in inhibiting neurogenic inflammatory pain in rats. This has implications for the use of novel alternative therapies such as diet modification for pain control.
Asunto(s)
Cacao , Dieta , Dolor Facial/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Dimensión del Dolor , Ratas , Ratas sin Pelo , Ratas Sprague-DawleyRESUMEN
PURPOSE: Analog development of existing drugs and direct drug delivery to the lungs by inhalation as treatments for multiple and extensively drug resistant (MDR and XDR) tuberculosis (TB) represent new therapeutic strategies. Pyrazinamide (PZA) is critical to drug sensitive TB therapy and is included in regimens for MDR TB. However, PZA-resistant Mycobacterium tuberculosis (Mtb) strains threaten its use. Pyrazinoic acid esters (PAEs) are PZA analogs effective against Mtb in vitro, including against the most common PZA resistant strains. However, PAEs require testing for TB efficacy in animal models. METHODS: PAEs were delivered daily as aqueous dispersions from a vibrating mesh nebulizer to Mtb infected guinea pigs for 4 weeks in a regimen including orally administered first-line TB drugs. RESULTS: PAEs tested as a supplement to oral therapy significantly reduced the organ bacterial burden in comparison to infected, untreated control animals. Thus, PAE aerosol therapy is a potentially significant addition to the regimen for PZA resistant MDR-TB and XDR-TB treatment. Interestingly, low dose oral PZA treatment combined with standard therapy also reduced bacterial burden. This observation may be important for PZA susceptible disease treatment. CONCLUSION: The present study justifies further evaluation of PZA analogs and their lung delivery to treat TB.
Asunto(s)
Antituberculosos/administración & dosificación , Mycobacterium tuberculosis/efectos de los fármacos , Pirazinamida/análogos & derivados , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Administración por Inhalación , Aerosoles , Animales , Ésteres , Cobayas , Masculino , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/fisiología , Pirazinamida/administración & dosificación , Resultado del Tratamiento , Tuberculosis/tratamiento farmacológico , Tuberculosis/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/metabolismoRESUMEN
Combining the advantage of higher efficacy due to local pulmonary administration of pyrazinoic acid (POA) and potent effect of pyrazinoic acid ester (PAE) delivered as an aerosol would aid in tuberculosis therapy. A combination spray dried dry powder, composed of POA, PAE (n-propyl POA), maltodextrin and leucine, was prepared for aerosol delivery to animals. Solid-state characteristics of morphology (scanning electron microscopy) crystallinity (X-ray powder diffraction), thermal properties (thermogravimetric analysis and differential scanning calorimetry) and moisture content (Karl Fisher) were evaluated. Particle size distributions, by volume (laser diffraction) for the dispersed powder and by mass (inertial impaction) were determined. Efficient delivery of the powder to a nose only animal exposure chamber employed a novel rotating brush/micro-fan apparatus. Spherical, crystalline particles were prepared. The volume median diameter, â¼1.5µm, was smaller than the mass median aerodynamic diameter, â¼3.0µm, indicating modest aggregation. Drug content variations were observed across the particle size distribution and may be explained by PAE evaporative losses. Delivery to the nose-only exposure chamber indicated that boluses could be administered at approximately 3min intervals to avoid aerosol accumulation and effect uniform dose delivery with successive doses suitable for future pharmacokinetic and pharmacodynamic studies.
Asunto(s)
Administración Intranasal/instrumentación , Administración Intranasal/veterinaria , Inhaladores de Polvo Seco/métodos , Inhaladores de Polvo Seco/veterinaria , Polvos/uso terapéutico , Pirazinamida/análogos & derivados , Administración por Inhalación , Animales , Combinación de Medicamentos , Composición de Medicamentos/métodos , Composición de Medicamentos/veterinaria , Tamaño de la Partícula , Polvos/administración & dosificación , Pirazinamida/administración & dosificación , Pirazinamida/uso terapéuticoRESUMEN
Since the 1990s the rising incidence of multiple drug resistant TB, particularly in the context of human immunodeficiency virus co-infected patients, has threatened global TB control. At that time funding agencies began to support formal investigation of aerosol therapy which until then had been the subject of case reports of individual investigators. Over the last decade, proponents of aerosol therapy have increased in number within the TB research community as the incidence of multiple and extremely drug resistant TB has increased dramatically around the world. Aerosol therapy offers the potential to deliver drug at target concentrations directly into the lungs, use the alveolar-capillary interface to achieve systemic levels, while reducing the risk of systemic toxicity seen with parentally administered doses. In addition, there are insufficient new drugs in the pipeline to anticipate the appearance of a new regimen in time to assure future control of drug resistance. Consequently, alternative strategies are critical to achieving global TB control, and inhaled therapies should be considered as one such strategy.
Asunto(s)
Antituberculosos/administración & dosificación , Tuberculosis/tratamiento farmacológico , Administración por Inhalación , Animales , Antituberculosos/metabolismo , Predicción , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Humanos , Nebulizadores y Vaporizadores/tendencias , Resultado del Tratamiento , Tuberculosis/metabolismoRESUMEN
Tuberculosis is the most serious infectious disease caused by a single organism, Mycobacterium tuberculosis (Mtb). The standard of care is a protracted and complex drug treatment regimen made more complicated and of longer duration by the incidence of multiple and extensively drug resistant disease. Pulmonary delivery of aerosols as a supplement to the existing regimen offers the advantage of delivering high local drug doses to the initial site of infection and most prominent organ system involved in disease. Pyrazinamide is used in combination with other drugs to treat tuberculosis. It is postulated that the action of pyrazinoic acid (POA), the active moiety of pyrazinamide, may be enhanced by local pH adjustment, when presented as a salt form. POA was prepared as leucine (POA-leu) and ammonium salts (POA-NH4), spray dried, and characterized in terms of physicochemical properties (melting point, crystallinity, moisture content), aerodynamic performance (aerodynamic particle size distribution, emitted dose), and in vitro inhibitory effect on two mycobacteria (Mtb and Mycobacterium bovis). Particles were prepared in sizes suitable for inhalation (3.3 and 5.4 µm mass median aerodynamic diameter and 61 and 40% of the aerodynamic particle size distribution less than 4.46 µm, as measured by inertial impaction, for POA-leu and POA-NH4, respectively) and with properties (stoichiometric 1:1 ratio of salt to drug, melting points at â¼180 °C, with water content of <1%) that would support further development as an inhaled dosage form. In addition, POA salts demonstrated greater potency in inhibiting mycobacterial growth compared with POA alone, which is promising for therapy.
Asunto(s)
Antituberculosos/administración & dosificación , Rociadores Nasales , Pirazinamida/análogos & derivados , Tuberculosis/tratamiento farmacológico , Administración por Inhalación , Antituberculosos/química , Desecación , Inhaladores de Polvo Seco , Humanos , Nanopartículas/química , Tamaño de la Partícula , Difracción de Polvo , Pirazinamida/administración & dosificación , Pirazinamida/química , Sales (Química)/administración & dosificación , Sales (Química)/química , Difracción de Rayos XRESUMEN
Pain patients who are nicotine dependent report a significantly increased incidence and severity of pain intensity. The goal of this study was to determine the effects of prolonged nicotine administration on inflammatory proteins implicated in the development of peripheral and central sensitization of the trigeminal system. Behavioral, immunohistochemical, and microarray studies were utilized to investigate the effects of nicotine administered daily for 14 days via an Alzet® osmotic pump in Sprague Dawley rats. Systemic nicotine administration caused a significant increase in nocifensive withdrawals to mechanical stimulation of trigeminal neurons. Nicotine stimulated expression of the pro-inflammatory signal transduction proteins phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and protein kinase A (PKA) in the spinal trigeminal nucleus. Nicotine also promoted elevations in the expression of glial fibrillary acidic protein (GFAP), a biomarker of activated astrocytes, and the microglia biomarker ionized calcium-binding adapter molecule 1 (Iba1). Similarly, levels of eleven cytokines were significantly elevated with the largest increase in expression of TNF-α. Levels of PKA, p-ERK, and p-JNK in trigeminal ganglion neurons were increased by nicotine. Our findings demonstrate that prolonged systemic administration of nicotine promotes sustained behavioral and cellular changes in the expression of key proteins in the spinal trigeminal nucleus and trigeminal ganglion implicated in the development and maintenance of peripheral and central sensitization.
Asunto(s)
Sensibilización del Sistema Nervioso Central/efectos de los fármacos , Estimulantes Ganglionares/farmacología , Nicotina/farmacología , Médula Espinal/efectos de los fármacos , Ganglio del Trigémino/efectos de los fármacos , Núcleo Espinal del Trigémino/efectos de los fármacos , Animales , Sensibilización del Sistema Nervioso Central/fisiología , Cotinina/sangre , Citocinas/metabolismo , Inmunohistoquímica , Masculino , Nocicepción/efectos de los fármacos , Nocicepción/fisiología , Estimulación Física , Análisis por Matrices de Proteínas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Ganglio del Trigémino/metabolismo , Núcleo Espinal del Trigémino/metabolismoRESUMEN
Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX1R) and receptor 2 (OX2R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX1R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central neurons and nociception.
Asunto(s)
Azepinas/farmacología , Bencimidazoles/farmacología , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Nocicepción/efectos de los fármacos , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Adyuvante de Freund , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Masculino , Proteínas de Microfilamentos/metabolismo , FN-kappa B/metabolismo , Neuroglía/inmunología , Neuronas/inmunología , Nocicepción/fisiología , Receptores de Orexina/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/inmunología , Núcleo Espinal del Trigémino/efectos de los fármacos , Núcleo Espinal del Trigémino/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Intranasal noninhaled delivery of carbon dioxide (CO2) is efficacious in the symptomatic treatment of seasonal allergic rhinitis. The goal of this study was to determine whether and how 100% CO2 inhibits mast cell degranulation, thereby possibly contributing to the reduction of symptoms in seasonal allergic rhinitis. METHODS: Peritoneal mast cells isolated from rats and labelled with sulforhodamine-B (SFRM-B) were used to determine whether CO2 treatment could block mast cell degranulation and histamine release in response to 48/80. In addition, the effect of CO2 on intracellular calcium levels in unstimulated and stimulated mast cells was determined by fluorescent microscopy. RESULTS: Treatment with 48/80 caused >90% of mast cells containing SFRM-B to degranulate, resulting in a marked decrease in the fluorescent intensity within the mast cells, and simultaneously causing a significant increase in histamine release. Significantly, the stimulatory effect of 48/80 on fluorescent intensity and histamine levels was greatly inhibited (>95%) to near control levels by pretreatment with 100% CO2. Treatment with 48/80 also caused a robust transient increase in intracellular calcium, whereas pretreatment with CO2 repressed the increase in calcium (>70%) in response to 48/80. CONCLUSIONS: Results from this study provide the first evidence of a unique regulatory mechanism by which CO2 inhibits mast cell degranulation and histamine release by repressing stimulated increases in intracellular calcium. Thus, our data provide a plausible explanation for the reported therapeutic benefit of noninhaled intranasal delivery of 100% CO2 to treat allergic rhinitis.
Asunto(s)
Calcio/metabolismo , Dióxido de Carbono/farmacología , Degranulación de la Célula/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Mastocitos/efectos de los fármacos , Animales , Células Cultivadas , Formaldehído/farmacología , Liberación de Histamina/efectos de los fármacos , Inmunosupresores/farmacología , Espacio Intracelular/metabolismo , Masculino , Mastocitos/metabolismo , Ratas , Ratas Sprague-Dawley , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Migraine is a neurovascular disorder characterized by recurrent episodic headaches, and is caused by abnormal processing of sensory information due to peripheral and/or central sensitization. The exact pathophysiological mechanism underlying migraine is not fully understood; however, cortical spreading depression (CSD) is thought to provide the basis for migraine aura and may serve as a trigger of migraine pain. CSD depends on neuronal-glial cell communication, which is mediated by intercellular transfer of messengers through connexin-containing gap junctions, as well as messengers released into the extracellular space by non-junctional connexin-containing hemichannels. These processes are believed to be important in peripheral sensitization within the trigeminal ganglion and to lead to central sensitization. The novel benzopyran compound tonabersat binds selectively to a unique site in the brain. In preclinical studies, tonabersat markedly reduced CSD and CSD-associated events and inhibited gap-junction communication between neurons and satellite glial cells in the trigeminal ganglion. Together, these findings suggest that tonabersat should have clinical application in preventing migraine attacks.
Asunto(s)
Analgésicos/farmacología , Benzamidas/farmacología , Benzopiranos/farmacología , Encéfalo/efectos de los fármacos , Trastornos Migrañosos/fisiopatología , Trastornos Migrañosos/terapia , Animales , Encéfalo/fisiopatología , Depresión de Propagación Cortical/fisiología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , HumanosRESUMEN
Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague-Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP(8-37). Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neuroglía/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Ganglio del Trigémino/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Células Cultivadas , Femenino , Neuroglía/fisiología , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo II/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Péptido Relacionado con el Gen de Calcitonina/fisiología , Ganglio del Trigémino/citología , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Elevated nitric oxide (NO) and proton levels in synovial fluid are implicated in joint pathology. However, signaling pathways stimulated by these molecules that mediate inflammation and pain in the temporomandibular joint (TMJ) have not been investigated. The goal of this study was to determine the effect of NO-proton stimulation of rat trigeminal neurons on the in vivo expression of mitogen-activated protein kinases (MAPKs) and phosphatases (MKPs) in trigeminal ganglion neurons and satellite glial cells. Low levels of the active MAPKs extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), and p38 were localized in the cytosol of neurons and satellite glial cells in unstimulated animals. However, increased levels of active ERK and p38, but not JNK, were detected in the cytosol and nucleus of V3 neurons and satellite glial cells 15 min and 2 h following bilateral TMJ injections of an NO donor diluted in pH 5.5 medium. While ERK levels returned to near basal levels 24 h after stimulation, p38 levels remained significantly elevated. In contrast to MKP-2 and MKP-3 levels that were barely detectable in neurons or satellite glial cells, MKP-1 staining was readily observed in satellite glial cells in ganglia from unstimulated animals. However, neuronal and satellite glial cell staining for MKP-1, MKP-2, and MKP-3 was significantly increased in response to NO-protons. Increased active ERK and p38 levels as well as elevated MKP levels were also detected in neurons and satellite glial cells located in V2 and V1 regions of the ganglion. Our data provide evidence that NO-proton stimulation of V3 neurons results in temporal and spatial changes in expression of active ERK and p38 and MKPs in all regions of the ganglion. We propose that in trigeminal ganglia these cellular events, which are involved in peripheral sensitization as well as control of inflammatory and nociceptive responses, may play a role in TMJ pathology.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Óxido Nítrico/metabolismo , Protones , Ganglio del Trigémino/citología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Indoles , Masculino , Proteínas de Neurofilamentos/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
We have investigated the cellular mechanisms by which changes in intracellular calcium (Ca2+) can differentially regulate gene expression. Two Ca2+ paradigms, involving prolonged and transient Ca2+ increases, were used. As a starting point, we studied the slow, prolonged elevation of Ca2+ caused by activation of 5-HT1 receptors. We had previously shown that 5-HT1 agonists inhibit calcitonin gene-related peptide (CGRP) transcription and secretion. The Ca2+ ionophore, ionomycin, was used to produce a prolonged elevation of the Ca2+ signal similar to that generated by 5-HT1 receptor agonists. Ionomycin treatment of the neuronal-like CA77 cell line specifically inhibited mitogen-activated protein (MAP) kinase stimulation of the CGRP enhancer and two synthetic MAP kinase-responsive reporter genes (4- to 10-fold). We then showed that ionomycin repression of promoter activity involved selective induction of MAP kinase phosphatase-1 (MKP-1), but not MKP-2, and that overexpression of MKP-1 was sufficient to repress CGRP enhancer activity. These effects were then compared with a Ca2+ paradigm involving a transient elevation in Ca2+ as seen after depolarization. At 4 h after the transient increase in Ca2+, the CGRP enhancer and synthetic MAP kinase-responsive reporter genes were stimulated. In contrast, exposure to depolarizing stimuli overnight caused only a less than 2-fold inhibition of promoter activity. We propose that the duration of the Ca2+ signal can determine the magnitude of a negative feedback loop that leads to differential regulation of MAP kinase-responsive genes.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteínas de Ciclo Celular , Regulación de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Línea Celular , Fosfatasa 1 de Especificidad Dual , Elementos de Facilitación Genéticos , Inhibidores Enzimáticos/farmacología , Genes Reporteros/efectos de los fármacos , Proteínas Inmediatas-Precoces/biosíntesis , Ionomicina/farmacología , Ionóforos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/metabolismo , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Receptores de Serotonina/fisiología , Receptores de Serotonina 5-HT1 , Agonistas de Receptores de Serotonina/farmacología , Glándula Tiroides , Factores de TiempoRESUMEN
OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.
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Ceguera/veterinaria , Brotes de Enfermedades/veterinaria , Infecciones Virales del Ojo/veterinaria , Macropodidae/virología , Orbivirus/aislamiento & purificación , Infecciones por Reoviridae/veterinaria , Animales , Australia/epidemiología , Secuencia de Bases , Ceguera/epidemiología , Ceguera/virología , Cartilla de ADN/química , ADN Viral/química , Infecciones Virales del Ojo/epidemiología , Infecciones Virales del Ojo/virología , Femenino , Masculino , Datos de Secuencia Molecular , Orbivirus/clasificación , Orbivirus/genética , Filogenia , Reacción en Cadena de la Polimerasa , Infecciones por Reoviridae/epidemiología , Infecciones por Reoviridae/virologíaRESUMEN
We have investigated the regulation of calcitonin gene-related peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of headache. However, despite this clinical efficacy, the cellular target and mechanism of sumatriptan action are not well understood beyond the pharmacology of its recognition of the 5-HT1 class of serotonin receptors. We have used cultured trigeminal neurons to demonstrate that sumatriptan can directly repress CGRP secretion from sensory neurons. The stimulated secretion in response to depolarization or inflammatory agents was inhibited, but not the basal secretion rate. Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the classical role ascribed to the 5-HT1 receptors. Instead, activation of 5-HT1 receptors caused a slow and remarkably prolonged increase in intracellular calcium. The inhibition of CGRP secretion is attenuated by the phosphatase inhibitor okadaic acid, suggesting that sumatriptan action is mediated by calcium-recruited phosphatases. These results suggest that 5-HT1 agonists may block a deleterious feedback loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimulated neuropeptide release.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Neuronas/fisiología , Sumatriptán/farmacología , Ganglio del Trigémino/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Células HeLa , Humanos , Inflamación , Cinética , Modelos Neurológicos , Neuronas/citología , Neuronas/efectos de los fármacos , Ácido Ocadaico/farmacología , Oxadiazoles/farmacología , Cloruro de Potasio/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/biosíntesis , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/genética , Receptores de Serotonina/fisiología , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/biosíntesis , Agonistas de Receptores de Serotonina/farmacología , Transfección , Ganglio del Trigémino/citología , Triptaminas/farmacologíaRESUMEN
We have investigated the mechanisms underlying regulation of the calcitonin gene-related peptide (CGRP) cell-specific enhancer. Recently, we reported that this enhancer is inhibited by serotonin type-1 (5-HT1) agonists, similar to currently used antimigraine drugs. We have now tested whether this repression involves a mitogen-activated protein (MAP) kinase pathway. We first demonstrate that the CGRP enhancer is strongly (10-fold) activated by a constitutively active MAP kinase kinase (MEK1), yielding reporter activities 100-fold above the enhancerless control. The involvement of a MAP kinase pathway was confirmed by down-regulation of reporter activity upon cotransfection of a dominant negative Ras. Activation of the enhancer by MEK1 was blocked in a dose-dependent manner by the 5-HT1 receptor agonist CGS 12066A (CGS). Since it is not known whether the CGRP enhancer factors are immediate targets of MAP kinases, we then used EIk-1- and c-Jun-dependent reporter genes that are directly activated by the ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinase) MAP kinases. CGS treatment repressed the activation of both of these reporters, suggesting that at least two MAP kinases are the immediate targets of CGS-mediated repression. We further demonstrate that 5-HT1 agonists inactivate ERK by dephosphorylation, even in the presence of constitutively activated MEK1. This inactivation appears to be due to a marked increase in the level of MAP kinase phosphatase-1. These results have defined a novel and general mechanism by which 5-HT1 receptor agonists can repress MAP kinase activation of target genes, such as CGRP.
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Péptido Relacionado con Gen de Calcitonina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Proteínas Quinasas Activadas por Mitógenos , Fosfoproteínas Fosfatasas , Serotonina/fisiología , Factores de Transcripción , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carcinoma Medular , Fosfatasa 1 de Especificidad Dual , Genes jun/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Fosforilación , Proteínas Quinasas/genética , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Quinoxalinas/farmacología , Ratas , Agonistas de Receptores de Serotonina/farmacología , Neoplasias de la Tiroides , Transfección , Células Tumorales Cultivadas , Proteína Elk-1 con Dominio etsAsunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Enfermedades de los Bovinos/inmunología , Animales , Diarrea Mucosa Bovina Viral/epidemiología , Diarrea Mucosa Bovina Viral/inmunología , Bovinos , Enfermedades de los Bovinos/sangre , Enfermedades de los Bovinos/epidemiología , Chlamydia/inmunología , Infecciones por Chlamydia/epidemiología , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/veterinaria , Coxiella burnetii/inmunología , Virus de la Diarrea Viral Bovina/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Fiebre Efímera/epidemiología , Fiebre Efímera/inmunología , Virus de la Fiebre Efímera Bovina/inmunología , Leptospira interrogans/inmunología , Infecciones por Mycobacterium/epidemiología , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/veterinaria , Mycobacterium avium subsp. paratuberculosis/inmunología , Fiebre Q/epidemiología , Fiebre Q/inmunología , Fiebre Q/veterinaria , Respirovirus/inmunología , Infecciones por Respirovirus/epidemiología , Infecciones por Respirovirus/inmunología , Australia del Sur/epidemiología , Enfermedad de Weil/epidemiología , Enfermedad de Weil/inmunología , Enfermedad de Weil/veterinariaRESUMEN
We have investigated the control of calcitonin gene-related peptide (CGRP) expression by a serotonergic agonist that is related pharmacologically to currently used antimigraine drugs. During migraines, CGRP levels are elevated but then returned to normal by a 5-HT1 receptor agonist, sumatriptan. However, neither the molecular nor cellular targets of this drug are known. Trigeminal neurons are the major source of cerebrovascular CGRP, and thus we have used trigeminal primary cultures and the neuronal-like CA77 thyroid C-cell line as a model. We first demonstrate that sumatriptan and another 5-HT1 agonist, CGS 12066A (CGS), cause a robust and prolonged increase with oscillations in intracellular calcium in CA77 cells. CGS caused a similar increase in trigeminal cultures. We then show that CGS treatment leads to a decrease in CGRP mRNA levels in the CA77 cells. This decrease is attributable to the repression of promoter activity through two discrete elements: (1) the cAMP-responsive region, via a cAMP-independent mechanism; and (2) the cell-specific enhancer, which binds the upstream stimulatory factor helix-loop-helix protein and a cell-specific activator. These results demonstrate that activation of the endogenous 5-HT1 receptor is coupled to calcium signaling pathways and leads to inhibition of CGRP gene transcription.
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Péptido Relacionado con Gen de Calcitonina/genética , Regiones Promotoras Genéticas/fisiología , Receptores de Serotonina/genética , Animales , Calcio/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Neuronas/química , Neuronas/citología , Neuronas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Quinoxalinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Agonistas de Receptores de Serotonina/farmacología , Sumatriptán/farmacología , Neoplasias de la Tiroides , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Ganglio del Trigémino/química , Ganglio del Trigémino/citología , Células Tumorales CultivadasRESUMEN
Serotonergic neurons play key roles in modulating a wide variety of behavioral and homeostatic processes. However, there is a paucity of good model systems to study these neurons at a molecular level. In this review we will present evidence that cell lines derived from an unexpected source, thyroid parafollicular cells (PF) (also called C cells), fit the criteria for use as models for the study of serotonergic neurons. A strength of PF cell lines over other cell lines is that the parental PF cells have serotonergic properties and a neuronal potential that is consistent with their neural crest origin. Furthermore, PF cells and PF cell lines are capable of expressing the fundamental properties of serotonergic neurons, including: (1) serotonin (5-HT) biosynthesis by tryptophan hydroxylase (TPH), (2) vesicular 5-HT storage and regulated release, (3) expression of a 5-HT autoreceptor, and (4) expression of the 5-HT transporter. In this review, we will focus primarily on the serotonergic and neuronal properties of the rat CA77 PF cell line and the parental rat PF cells. The applicability of CA77 cells for molecular analyses will be described. First, their use for studies on the glucocorticoid regulation of the TPH gene will be discussed. Second, control of the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene will be discussed, with particular emphasis on the application of serotonergic drugs in treating migraine headaches. These examples highlight the versatility of thyroid PF cell lines as a system for studying the control of both serotonin biosynthesis and physiological actions.