RESUMEN
The tumor suppressor vitamin D(3) up-regulated protein 1 (VDUP1) is expressed throughout the developing and mature Drosophila nervous system, but its regulatory pathways are not well understood. Within the developing Drosophila visual system, down-regulation of VDUP1 in lamina precursor cells (LPCs) coincided with the arrival of retinal axons into the lamina target field, suggesting VDUP1 regulation by an axonally transmitted signal. Hedgehog (Hh) is a signal well known to coordinate LPC proliferation and differentiation in response to retinal axon innervation, and analysis of orthologous dvdup1 promoters identified an evolutionarily conserved binding site for the Hh-dependent transcription factor cubitus interruptus (Ci). Hh-dependent regulation of VDUP1 in the developing lamina was confirmed in Hh loss-of-function backgrounds where VDUP1 expression was maintained in LPCs, inhibiting both cell proliferation and lamina neurogenesis. This putative coupling of VDUP1 to the Hh signaling pathway may provide novel insights into the mechanisms controlling brain growth and development.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Animales , Animales Modificados Genéticamente , Axones/fisiología , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/fisiología , Secuencia Conservada , Regulación hacia Abajo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Modelos Neurológicos , Datos de Secuencia Molecular , Neurogénesis/fisiología , Neuronas/fisiología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Lóbulo Óptico de Animales no Mamíferos/fisiología , Neuronas Retinianas/fisiología , Células Madre/fisiología , Vías Visuales/crecimiento & desarrollo , Vías Visuales/fisiologíaRESUMEN
Originally characterized as a cell-cycle inhibitor induced by vitamin D(3), the tumor suppressor vitamin-D(3) upregulated protein 1 (VDUP1) has increasingly been shown to play major physiological roles in cell differentiation and glucose metabolism. Here we show evolutionarily conserved expression patterns of VDUP1 in Drosophila and rat nervous systems, including subcellular localization--cytoplasmic enrichment in neurons and nuclear expression in glia. These anatomical correlates suggested conservation of VDUP1 regulation, which was investigated both functionally and through promoter studies. Characterization of orthologous vdup1 cis-regulatory regions identified evolutionarily conserved sequence blocks (CSBs) with similarities to neural enhancers, including basic helix-loop-helix (bHLH) transcription factor Neurogenin/Math/atonal and Mash/achaete-scute family members. E-boxes (CANNTG), the binding sites for bHLH proteins, were associated with these CSBs as well, including E-boxes known to mediate glucose-dependent upregulation of VDUP1 in nonneuronal cells. Hyperglycemia-induced upregulation of VDUP1 was observed in brain tumor cells and in the Drosophila nervous system, which resulted in developmental arrest. Taken together, these data demonstrate evolutionary conservation of VDUP1 regulation and function, and suggest an expanding role for VDUP1 in nervous system development.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Glucemia/metabolismo , Encéfalo/citología , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada , ADN/análisis , Drosophila , Proteínas de Drosophila/genética , Metabolismo Energético/fisiología , Evolución Molecular , Perfilación de la Expresión Génica , Glioma , Humanos , Inmunohistoquímica , Larva/genética , Larva/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The tumor suppressor, vitamin D(3) up-regulated protein 1 (VDUP1), regulates cell cycle progression by suppressing AP-1-dependent transcription. Loss of VDUP1 activity is associated with tumorigenesis but little is known about VDUP1 regulatory controls or developmental roles. Here we show that the Drosophila homolog of human VDUP1 (dVDUP1) is expressed throughout the nervous system at all stages of development, the first in vivo analysis of VDUP1 expression patterns in the brain. During neurogenesis dVDUP1 expression is transiently down-regulated coincident with neuroblast delamination. Subsequent to expression of the neuronal marker elav, dVDUP1 is up-regulated to varying degrees in developing neurons. In contrast, dVDUP1 expression is both robust and sustained during gliogenesis, and the cis-regulatory region of the dvdup1 gene contains consensus binding sites for the glial fate gene reversed polarity (repo). Expression of dVDUP1 in presumptive glia is lost in embryos deficient for the glial fate genes glial cells missing (gcm) and repo. Conversely, ectopic expression of gcm or repo was sufficient to induce dVDUP1 expression in the nervous system. Taken together, these data suggest a novel role for the dVDUP1 tumor suppressor during nervous system development as a regulatory target for REPO during gliogenesis.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Neuroglía/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Línea Celular , Colecalciferol/metabolismo , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Mutación , Sistema Nervioso/citología , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Severe blood loss lowers arterial pressure through a central mechanism that is thought to include opioid neurons. In this study, we investigated whether hemorrhage activates proopiomelanocortin (POMC) neurons by measuring Fos immunoreactivity and POMC mRNA levels in the medial basal hypothalamus. Hemorrhage (2.2 ml/100 g body weight over 20 min) increased the number of Fos immunoreactive neurons throughout the rostral-caudal extent of the arcuate nucleus, the retrochiasmatic area and the peri-arcuate region lateral to the arcuate nucleus where POMC neurons are located. Double label immunohistochemistry revealed that hemorrhage increased Fos expression by beta-endorphin immunoreactive neurons significantly. The proportion of beta-endorphin immunoreactive neurons that expressed Fos immunoreactivity increased approximately four-fold, from 11.7+/-1.4% in sham-operated control animals to 42.0+/-5.2% in hemorrhaged animals. Hemorrhage also increased POMC mRNA levels in the medial basal hypothalamus significantly, consistent with the hypothesis that blood loss activates POMC neurons. To test whether activation of arcuate neurons contributes to the fall in arterial pressure evoked by hemorrhage, we inhibited neuronal activity in the caudal arcuate nucleus by microinjecting the local anesthetic lidocaine (2%; 0.1 or 0.3 microl) bilaterally 2 min before hemorrhage was initiated. Lidocaine injection inhibited hemorrhagic hypotension and bradycardia significantly although it did not influence arterial pressure or heart rate in non-hemorrhaged rats. These results demonstrate that hemorrhage activates POMC neurons and provide evidence that activation of neurons in the arcuate nucleus plays an important role in the hemodynamic response to hemorrhage.
Asunto(s)
Hemorragia/fisiopatología , Hipotálamo/fisiopatología , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Bradicardia/etiología , Bradicardia/prevención & control , Hemorragia/complicaciones , Hemorragia/metabolismo , Hipotensión/etiología , Hipotensión/prevención & control , Hipotálamo/metabolismo , Hipotálamo Medio/metabolismo , Inmunohistoquímica , Inyecciones , Lidocaína/administración & dosificación , Lidocaína/farmacología , Masculino , Proopiomelanocortina/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , betaendorfina/metabolismoRESUMEN
Glycyl-glutamine (Gly-Gln) is an inhibitory dipeptide synthesized from beta-endorphin(1-31). Previously, we showed that Gly-Gln inhibits morphine conditioned place preference, tolerance, dependence and withdrawal. In this study, we tested whether Gly-Gln's inhibitory activity extends to other rewarding drugs, specifically nicotine. Rats were conditioned with nicotine (0.6 mg/kg, s.c.) for four days and tested on day five. Glycyl-glutamine (100 nmol i.c.v.) inhibited acquisition and expression of a nicotine place preference significantly. Cyclo(Gly-Gln) (100 nmol i.c.v. or 25 mg/kg i.p.), a cyclic Gly-Gln derivative, blocked expression of nicotine place preference but Gly-d-Gln (100 nmol i.c.v.) was ineffective. To study nicotine withdrawal, rats were treated with nicotine (9 mg/kg/day) for seven days and conditioned place aversion was induced with mecamylamine (1 mg/kg, s.c.). Glycyl-glutamine blocked acquisition of place aversion to mecamylamine but not U50,488, a kappa opioid receptor agonist. Glycyl-glutamine thus inhibits the rewarding effects of nicotine and attenuates withdrawal in nicotine dependent rats.
Asunto(s)
Condicionamiento Psicológico/efectos de los fármacos , Dipéptidos/farmacología , Nicotina/farmacología , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Animales , Reacción de Prevención/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inyecciones Intraventriculares , Inyecciones Subcutáneas , Masculino , Mecamilamina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/agonistas , Síndrome de Abstinencia a Sustancias/prevención & control , Factores de TiempoRESUMEN
This study investigated whether the lateral hypothalamus (LH) contributes to the depressor response evoked by somato-visceral nociception. Lidocaine (2%; 0.1, 0.3 or 1.0 microl) or muscimol (0.34 nmol; 0.5 microl) was microinjected into the rostral LH of halothane-anesthetized rats bilaterally and somato-visceral nociception was induced 2 min later by injecting 5% acetic acid (0.5 ml) intraperitoneally. Lidocaine and muscimol inhibited the hypotension and bradycardia caused by somato-visceral nociception significantly without affecting cardiovascular function in normotensive animals.
Asunto(s)
Bradicardia/fisiopatología , Agonistas del GABA/administración & dosificación , Homocisteína/análogos & derivados , Hipotensión/fisiopatología , Área Hipotalámica Lateral/efectos de los fármacos , Muscimol/administración & dosificación , Animales , Bradicardia/etiología , Fenómenos Fisiológicos Cardiovasculares , Homocisteína/administración & dosificación , Hipotensión/etiología , Masculino , Microinyecciones , Dolor/complicaciones , Dolor/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
This study tested the hypothesis that morphine and other opiates cause urinary retention by activating mu opioid receptors in the midbrain periaqueductal gray (PAG) region. Selective mu, delta and kappa receptor agonists were microinjected into the PAG of urethane-anesthetized rats and the amplitude and incidence of bladder contractions were recorded during continuous saline infusion. Arterial pressure was monitored through a femoral artery catheter. Microinjection of the mu receptor agonist DAMGO into the ventrolateral PAG (vlPAG) suppressed volume-evoked bladder contractions completely. Bladder contractions ceased within 5 min of DAMGO injection and remained essentially undetectable for the rest of the 20 min recording period. Microinjection of the delta receptor agonist DPDPE into the vlPAG did not significantly affect either the amplitude of bladder contractions or the time interval separating contractions. The kappa receptor agonist U-69593 caused no discernible change in amplitude but increased the interval between bladder contractions significantly. Microinjection of DAMGO, DPDPE or U-69593 into the lateral or dorsolateral PAG columns was ineffective. DAMGO injection into the vlPAG increased arterial pressure whereas DPDPE and U-69593 produced a small but significant depressor response. These data support the hypothesis that mu and kappa receptors in the vlPAG participate in the micturition reflex.
Asunto(s)
Sustancia Gris Periacueductal/metabolismo , Receptores Opioides mu/metabolismo , Reflejo/fisiología , Micción/fisiología , Administración Intravesical , Anestésicos/farmacología , Animales , Bencenoacetamidas/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Encefalina D-Penicilamina (2,5)/farmacología , Femenino , Microinyecciones , Morfina/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Narcóticos/farmacología , Sustancia Gris Periacueductal/efectos de los fármacos , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/efectos de los fármacos , Reflejo/efectos de los fármacos , Cloruro de Sodio/administración & dosificación , Vejiga Urinaria/inervación , Vejiga Urinaria/fisiopatología , Micción/efectos de los fármacosRESUMEN
PURPOSE: We investigated the effects of the combination of bladder outlet obstruction and diabetes mellitus on in vitro rat bladder body strip function. MATERIALS AND METHODS: Longitudinal strips were removed from ventral and dorsal detrusor of age matched control, 2-week diabetic, 2-week obstructed and 2-week obstructed diabetic rats. Contractile responses to electrical field stimulation, carbachol, adenosine triphosphate, KCl and phenylephrine, and relaxations in response to norepinephrine and isoproterenol were measured. RESULTS: Bladders from diabetic, obstructed and obstructed diabetic rats were 1.6-fold, 2.6-fold and 3.6-fold heavier than those from controls. Responses of bladder strips from diabetics to all stimuli were similar to those of controls. Strips from obstructed rats were significantly less responsive to norepinephrine than those from controls or diabetics and strips from obstructed diabetics were significantly less responsive to norepinephrine and isoproterenol than those from all other groups. Strips from obstructed diabetics had significantly decreased responses to field stimulation, while responses to carbachol were decreased to a lesser extent. Responses of strips from obstructed rats to field stimulation were also decreased compared with controls but were significantly greater than those of the obstructed diabetic group. Responses to adenosine triphosphate, KCl and phenylephrine were similar in all groups. CONCLUSIONS: The combination of outlet obstruction and diabetes mellitus causes significant increases in bladder mass compared with either diabetes or obstruction alone. Bladder strips from obstructed diabetics show characteristics of denervation accompanied by alterations in beta-adrenergic function, suggesting that the coexistence of outlet obstruction and diabetes increases the rate of development of bladder decompensation.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Obstrucción del Cuello de la Vejiga Urinaria/fisiopatología , Vejiga Urinaria/fisiopatología , Animales , Diabetes Mellitus Experimental/complicaciones , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Vejiga Urinaria/efectos de los fármacos , Obstrucción del Cuello de la Vejiga Urinaria/complicacionesRESUMEN
PURPOSE: We investigated the relationship of bladder mass to responses to electrical field stimulation and adrenergic agonists in diabetic rat bladders. MATERIALS AND METHODS: Longitudinal strips were removed from the ventral and dorsal detrusor of age matched control, 2-month diabetic and sucrose drinking rats. Contractile responses to electrical field stimulation, KCl and phenylephrine, and relaxation in response to norepinephrine and isoproterenol were measured. RESULTS: Bladders from sucrose drinking and diabetic rats weighed significantly more than those of controls. Diabetic rats were divided into 2 groups with the bladder weighing less than or greater than 265 mg. Strips from small diabetic bladders were generally more responsive to field stimulation and norepinephrine than those from control or sucrose drinking rats. Conversely decreased function was especially apparent in dorsal strips from large diabetic bladders. Ventral strips were significantly more sensitive to the relaxant actions of norepinephrine and isoproterenol than dorsal strips. CONCLUSIONS: Our results suggest that the responsiveness of diabetic rat bladder to electrical field stimulation and adrenergic agonists is related to bladder mass, analogous to observations after partial outlet obstruction. Decreased function was particularly apparent in dorsal strips from diabetic rats with a large bladder.