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1.
Br J Pharmacol ; 172(17): 4430-41, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26103136

RESUMEN

BACKGROUND AND PURPOSE: Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4ß2 nAChRs regulates receptor recovery from desensitization. EXPERIMENTAL APPROACH: Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4ß2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce(-/-) and wild-type mice. KEY RESULTS: Inhibiting PKCε impaired the magnitude of α4ß2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce(-/-) mice. Sazetidine-A-induced hypothermia, which is mediated by α4ß2 nAChR desensitization, was more severe and prolonged in Prkce(-/-) than in wild-type mice. CONCLUSIONS AND IMPLICATIONS: PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4ß2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.


Asunto(s)
Proteína Quinasa C-epsilon/metabolismo , Receptores Nicotínicos/metabolismo , Recuperación de la Función/fisiología , Animales , Humanos , Hipotermia/inducido químicamente , Hipotermia/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotina/farmacología , Fosforilación/fisiología , Recuperación de la Función/efectos de los fármacos
2.
Br J Pharmacol ; 172(2): 642-53, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24827408

RESUMEN

BACKGROUND AND PURPOSE: We recently found that PKCε was required for spinal analgesic synergy between two GPCRs, δ opioid receptors and α2 A adrenoceptors, co-located in the same cellular subpopulation. We sought to determine if co-delivery of µ and δ opioid receptor agonists would similarly result in synergy requiring PKCε. EXPERIMENTAL APPROACH: Combinations of µ and δ opioid receptor agonists were co-administered intrathecally by direct lumbar puncture to PKCε-wild-type (PKCε-WT) and -knockout (PKCε-KO) mice. Antinociception was assessed using the hot-water tail-flick assay. Drug interactions were evaluated by isobolographic analysis. KEY RESULTS: All agonists produced comparable antinociception in both PKCε-WT and PKCε-KO mice. Of 19 agonist combinations that produced analgesic synergy, only 3 required PKCε for a synergistic interaction. In these three combinations, one of the agonists was morphine, although not all combinations involving morphine required PKCε. Morphine + deltorphin II and morphine + deltorphin I required PKCε for synergy, whereas a similar combination, morphine + deltorphin, did not. Additionally, morphine + oxymorphindole required PKCε for synergy, whereas a similar combination, morphine + oxycodindole, did not. CONCLUSIONS AND IMPLICATIONS: We discovered biased agonism for a specific signalling pathway at the level of spinally co-delivered opioid agonists. As the bias is only revealed by an appropriate ligand combination and cannot be accounted for by a single drug, it is likely that the receptors these agonists act on are interacting with each other. Our results support the existence of µ and δ opioid receptor heteromers at the spinal level in vivo. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.


Asunto(s)
Analgésicos Opioides/farmacología , Analgésicos Opioides/uso terapéutico , Dolor/tratamiento farmacológico , Proteína Quinasa C-epsilon/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Quimioterapia Combinada , Femenino , Calor , Ligandos , Masculino , Ratones Noqueados , Morfina/farmacología , Morfina/uso terapéutico , Morfolinas/farmacología , Morfolinas/uso terapéutico , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Dolor/metabolismo , Multimerización de Proteína , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Médula Espinal/metabolismo
3.
Genes Brain Behav ; 12(2): 250-62, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22853648

RESUMEN

The CRF (corticotropin-releasing factor) system is a key mediator of the stress response. Alterations in CRF signaling have been implicated in drug craving and ethanol consumption. The development of negative reinforcement via activation of brain stress systems has been proposed as a mechanism that contributes to alcohol dependence. Here, we isolated a gain-of-function allele of seb-3, a CRF receptor-like GPCR in Caenorhabditis elegans, providing an in vivo model of a constitutively activated stress system. We also characterized a loss-of-function allele of seb-3 and showed that SEB-3 positively regulates a stress response that leads to an enhanced active state of locomotion, behavioral arousal and tremor. SEB-3 also contributed to acute tolerance to ethanol and to the development of tremor during ethanol withdrawal. Furthermore, we found that a specific CRF(1) receptor antagonist reduced acute functional tolerance to ethanol in mice. These findings demonstrate functional conservation of the CRF system in responses to stress and ethanol in vertebrates and invertebrates.


Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Etanol/toxicidad , Respuesta al Choque Térmico/genética , Locomoción/genética , Receptores de Hormona Liberadora de Corticotropina/fisiología , Receptores Acoplados a Proteínas G/fisiología , Síndrome de Abstinencia a Sustancias/genética , Alelos , Secuencia de Aminoácidos , Animales , Nivel de Alerta/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Etanol/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Estrés Fisiológico , Temblor/genética
4.
Genes Brain Behav ; 8(5): 493-9, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19243450

RESUMEN

Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCepsilon) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCepsilon signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCepsilon in the amygdala reduced ethanol consumption and preference in a limited-access paradigm. Further, mice that are heterozygous for the PKCepsilon allele consume less ethanol compared with wild-type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCepsilon in the amygdala compared with wild-type mice. We conclude that amygdala PKCepsilon is important for ethanol consumption in mice.


Asunto(s)
Trastornos del Sistema Nervioso Inducidos por Alcohol/enzimología , Alcoholismo/enzimología , Amígdala del Cerebelo/enzimología , Predisposición Genética a la Enfermedad , Proteína Quinasa C-epsilon/genética , Trastornos del Sistema Nervioso Inducidos por Alcohol/genética , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Alcoholismo/genética , Alcoholismo/fisiopatología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiopatología , Animales , Química Encefálica/genética , Depresores del Sistema Nervioso Central/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Etanol/farmacología , Frecuencia de los Genes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recompensa
5.
Genes Brain Behav ; 7(3): 323-33, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17908177

RESUMEN

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCepsilon) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCepsilon activator, psiepsilonRACK, increased levels of pro-CRF, whereas reducing PKCepsilon levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCepsilon by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCepsilon(-/-) mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCepsilon control over anxiety-like behavior through regulation of CRF in the amygdala.


Asunto(s)
Amígdala del Cerebelo/enzimología , Ansiedad/psicología , Hormona Liberadora de Corticotropina/fisiología , Proteína Quinasa C-epsilon/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Animales , Ansiedad/genética , Hormona Liberadora de Corticotropina/administración & dosificación , Hormona Liberadora de Corticotropina/farmacología , Ratones , Ratones Noqueados , Neuronas/enzimología , Neuronas/fisiología , Proteína Quinasa C-epsilon/deficiencia , Proteína Quinasa C-epsilon/genética , Interferencia de ARN , ARN Mensajero/genética
6.
Behav Neurosci ; 121(2): 439-42, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17469934

RESUMEN

Determining the intracellular signaling pathways that mediate the rewarding effects of ethanol may help identify drug targets to curb excessive alcohol consumption. Mice lacking the epsilon isoform of protein kinase C (PKCepsilon) voluntarily consumed less ethanol than wild-type mice in two-bottle choice and operant self-administration assays. Decreased consumption may reflect either increased or decreased sensitivity to the rewarding effects of ethanol. Alternatively, decreased voluntary consumption may reflect a change in sensitivity to the aversive effects of ethanol. The authors used place conditioning to determine that PKCepsilon null mice have an increased sensitivity to the aversive effects of ethanol but a decreased sensitivity to the rewarding effects of ethanol. Together these data suggest that PKCepsilon null mice voluntarily consume less ethanol because they derive less reward and are more sensitive to the aversive effects of ethanol.


Asunto(s)
Reacción de Prevención/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Proteína Quinasa C-epsilon/fisiología , Animales , Depresores del Sistema Nervioso Central/sangre , Etanol/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recompensa
7.
Genes Brain Behav ; 6(8): 776-83, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17376149

RESUMEN

Activation of adenosine receptors in the brain reduces anxiety-like behavior in animals and humans. Because nucleoside transporters regulate adenosine levels, we used mice lacking the type 1 equilibrative nucleoside transporter (ENT1) to investigate whether ENT1 contributes to anxiety-like behavior. The ENT1 null mice spent more time in the center of an open field compared with wild-type littermates. In the elevated plus maze, ENT1 null mice entered more frequently into and spent more time exploring the open arms. The ENT1 null mice also spent more time exploring the light side of a light-dark box compared with wild-type mice. Microinjection of an ENT1-specific antagonist, nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside), into the amygdala of C57BL/6J mice reduced anxiety-like behavior in the open field and elevated plus maze. These findings show that amygdala ENT1 modulates anxiety-like behavior. The ENT1 may be a drug target for the treatment of anxiety disorders.


Asunto(s)
Amígdala del Cerebelo/metabolismo , Ansiedad/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Conducta Exploratoria/fisiología , Actividad Motora/fisiología , Análisis de Varianza , Animales , Ansiedad/genética , Tranportador Equilibrativo 1 de Nucleósido/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Neostriado/metabolismo , Estadísticas no Paramétricas
8.
Neuroscience ; 145(1): 350-6, 2007 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-17204374

RESUMEN

Small-fiber painful peripheral neuropathy, a complication of chronic ethanol ingestion, is more severe in women. In the present study, we have replicated this clinical finding in the rat and evaluated for a role of estrogen and second messenger signaling pathways. The alcohol diet (6.5% ethanol volume:volume in Lieber-DeCarli formula) induced hyperalgesia with more rapid onset and severity in females. Following ovariectomy, alcohol failed to induce hyperalgesia in female rats, well past its time to onset in gonad intact males and females. Estrogen replacement reinstated alcohol neuropathy in the female rat. The protein kinase A (PKA) inhibitor (Walsh inhibitor peptide, WIPTIDE) only attenuated alcohol-induced hyperalgesia in female rats. Inhibitors of protein kinase Cepsilon (PKCepsilon-I) and extracellular-signal related kinase (ERK) 1/2 (2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene (U0126)) attenuated hyperalgesia in males and females, however the degree of attenuation produced by PKCepsilon-I was much greater in females. In conclusion, estrogen plays an important role in the expression of pain associated with alcohol neuropathy in the female rat. In contrast to inflammatory hyperalgesia, in which only the contribution of PKCepsilon signaling is sexually dimorphic, in alcohol neuropathy PKA as well as PKCepsilon signaling is highly sexually dimorphic.


Asunto(s)
Alcoholes , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Estrógenos/fisiología , Neuralgia/inducido químicamente , Proteína Quinasa C-epsilon/fisiología , Caracteres Sexuales , Análisis de Varianza , Animales , Interacciones Farmacológicas , Inhibidores Enzimáticos/administración & dosificación , Femenino , Hiperalgesia/inducido químicamente , Hiperalgesia/enzimología , Hiperalgesia/fisiopatología , Masculino , Neuralgia/enzimología , Neuralgia/fisiopatología , Ovariectomía/métodos , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Ratas , Ratas Sprague-Dawley
9.
Genes Brain Behav ; 6(4): 329-38, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16899053

RESUMEN

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.


Asunto(s)
Aprendizaje por Asociación/fisiología , Conducta Animal/fisiología , Condicionamiento Clásico/fisiología , Morfina/farmacología , Proteína Quinasa C-epsilon/genética , Animales , Aprendizaje por Asociación/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Ratones , Ratones Noqueados , Narcóticos/farmacología , Proteína Quinasa C-epsilon/metabolismo , Distribución Aleatoria , Receptores Opioides mu/fisiología , Recompensa , Autoadministración , Factores de Tiempo
10.
Pharmacol Ther ; 109(1-2): 227-37, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16102840

RESUMEN

Recent evidence indicates that ethanol modulates the function of specific intracellular signaling cascades, including those that contain cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase A (PKA), protein kinase C (PKC), the tyrosine kinase Fyn, and phospholipase D (PLD). In some cases, the specific components of these cascades appear to mediate the effects of ethanol, whereas other components indirectly modify responses to ethanol. Studies utilizing selective inhibitors and genetically modified mice have identified specific isoforms of proteins involved in responses to ethanol. The effects of ethanol on neuronal signaling appear restricted to certain brain regions, partly due to the restricted distribution of these proteins. This likely contributes specificity to ethanol's actions on behavior. This review summarizes recent work on ethanol and intracellular signal transduction, emphasizing studies that have identified specific molecular events that underlie behavioral responses to ethanol.


Asunto(s)
Conducta Animal/efectos de los fármacos , Conducta/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Transducción de Señal/fisiología , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Humanos , Fosfolipasa D/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo
12.
Am J Physiol Lung Cell Mol Physiol ; 289(6): L1083-93, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16085670

RESUMEN

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.


Asunto(s)
Hipoxia/enzimología , Pulmón/irrigación sanguínea , Pulmón/enzimología , Proteína Quinasa C-epsilon/metabolismo , Circulación Pulmonar , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipoxia/genética , Hipoxia/patología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico/administración & dosificación , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo III , Proteína Quinasa C-epsilon/deficiencia , Proteína Quinasa C-epsilon/genética , Circulación Pulmonar/efectos de los fármacos , Circulación Pulmonar/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
13.
Cell Mol Life Sci ; 62(2): 119-27, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15666084

RESUMEN

Pharmacological studies with drugs that activate or inhibit several protein kinase C (PKC) isozymes have identified the PKC family of serine-threonine kinases as important in the regulation of gamma-aminobutyric acid type A (GABA(A)) receptor function. PKC modulates GABA(A) receptor surface density, chloride conductance and receptor sensitivity to positive allosteric modulators such as neurosteroids, ethanol, benzodiazepines and barbiturates. Recent studies using PKC isozyme-selective reagents and gene-targeted mice have begun to identify critical roles for three isozymes, PKCbetaII, PKCvarepsilon and PKCgamma, in various aspects of GABA(A) receptor regulation. Progress in this field touches upon therapeutic areas that are of great clinical importance such as anxiety and addiction. Increased understanding of how PKC regulates GABA(A) receptors and which PKC isozymes are involved holds promise for development of new treatments for diverse neuropsychiatric disorders.


Asunto(s)
Proteína Quinasa C/fisiología , Receptores de GABA-A/metabolismo , Regulación Alostérica , Animales , Barbitúricos/farmacología , Benzodiazepinas/farmacología , Etanol/farmacología , Ratones
14.
J Pharmacol Exp Ther ; 305(1): 264-70, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12649378

RESUMEN

Ethanol intoxication results partly from actions of ethanol at specific ligand-gated ion channels. One such channel is the GABA(A) receptor complex, although ethanol's effects on GABA(A) receptors are variable. For example, we found that hippocampal neurons from selectively bred mice and rats with high hypnotic sensitivity to ethanol have increased GABA(A) receptor-mediated synaptic responses during acute ethanol treatment compared with mice and rats that display low behavioral sensitivity to ethanol. Here we investigate whether specific protein kinase C (PKC) isozymes modulate hypnotic and GABA(A) receptor sensitivity to ethanol. We examined acute effects of ethanol on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in mice lacking either PKCgamma (PKCgamma(-/-)) or PKCepsilon (PKCepsilon(-/-)) isozymes and compared the results to those from corresponding wild-type littermates (PKCgamma(+/+) and PKCepsilon(+/+)). GABA(A) receptor-mediated IPSCs were evoked in CA1 pyramidal neurons by electrical stimulation in stratum pyramidale, and the responses were recorded in voltage-clamp mode using whole-cell patch recording techniques. Ethanol (80 mM) enhanced the IPSC response amplitude and area in PKCgamma(+/+) mice, but not in the PKCgamma(-/-) mice. In contrast, ethanol markedly potentiated IPSCs in the PKCepsilon(-/-) mice, but not in PKCepsilon(+/+) littermates. There was a positive correlation between ethanol potentiation of IPSCs and the ethanol-induced loss of righting reflex such that mice with larger ethanol-induced increases in GABA(A) receptor-mediated IPSCs also had higher hypnotic sensitivity to ethanol. These results suggest that PKCgamma and PKCepsilon signaling pathways reciprocally modulate both ethanol enhancement of GABA(A) receptor function and hypnotic sensitivity to ethanol.


Asunto(s)
Etanol/farmacología , Hipocampo/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores de GABA-A/metabolismo , Animales , Electrofisiología , Femenino , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C-epsilon
15.
J Neurosci ; 21(17): 6933-9, 2001 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-11517280

RESUMEN

Inflammatory pain, characterized by a decrease in mechanical nociceptive threshold (hyperalgesia), arises through actions of inflammatory mediators, many of which sensitize primary afferent nociceptors via G-protein-coupled receptors. Two signaling pathways, one involving protein kinase A (PKA) and one involving the epsilon isozyme of protein kinase C (PKCepsilon), have been implicated in primary afferent nociceptor sensitization. Here we describe a third, independent pathway that involves activation of extracellular signal-regulated kinases (ERKs) 1 and 2. Epinephrine, which induces hyperalgesia by direct action at beta(2)-adrenergic receptors on primary afferent nociceptors, stimulated phosphorylation of ERK1/2 in cultured rat dorsal root ganglion cells. This was inhibited by a beta(2)-adrenergic receptor blocker and by an inhibitor of mitogen and extracellular signal-regulated kinase kinase (MEK), which phosphorylates and activates ERK1/2. Inhibitors of G(i/o)-proteins, Ras farnesyltransferases, and MEK decreased epinephrine-induced hyper-algesia. In a similar manner, phosphorylation of ERK1/2 was also decreased by these inhibitors. Local injection of dominant active MEK produced hyperalgesia that was unaffected by PKA or PKCepsilon inhibitors. Conversely, hyperalgesia produced by agents that activate PKA or PKCepsilon was unaffected by MEK inhibitors. We conclude that a Ras-MEK-ERK1/2 cascade acts independent of PKA or PKCepsilon as a novel signaling pathway for the production of inflammatory pain. This pathway may present a target for a new class of analgesic agents.


Asunto(s)
Hiperalgesia/fisiopatología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nociceptores/fisiopatología , Antagonistas de Receptores Adrenérgicos beta 2 , Animales , Células Cultivadas , Cruzamientos Genéticos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Hiperalgesia/inducido químicamente , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nociceptores/efectos de los fármacos , Dimensión del Dolor , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C-epsilon , Ratas , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas ras/metabolismo
16.
Eur J Neurosci ; 13(12): 2227-33, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11454025

RESUMEN

We have evaluated the contribution of differences in second messenger signalling to sex differences in inflammatory pain and its control by sex hormones. In normal male but not female rats, epinephrine-induced mechanical hyperalgesia was antagonized by inhibitors of protein kinase Cepsilon (PKCepsilon), protein kinase A (PKA) and nitric oxide synthetase (NOS). Similarly, in PKCepsilon knockout mice, a contribution of PKCepsilon to epinephrine-dependent mechanical hyperalgesia occurred in males only. In contrast, hyperalgesia induced by prostaglandin E2, in both females and males, was dependent on PKA and NO. In both sexes, inhibitors of mitogen-activated protein kinase/extracellular-signal related kinase kinase (MEK) inhibited epinephrine hyperalgesia. In gonadectomized females, the second messenger contributions to epinephrine hyperalgesia demonstrated the pattern seen in males. Administration of oestrogen to gonadectomized females fully reconstituted the phenotype of the normal female. These data demonstrate gender differences in PKCepsilon, PKA and NO signalling in epinephrine-induced hyperalgesia which are oestrogen dependent and appear to be exerted at the level of the beta-adrenergic receptor or the G-protein to which it is coupled.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Dolor/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Agonistas Adrenérgicos/farmacología , Animales , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiopatología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Dinoprostona/farmacología , Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Femenino , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/fisiopatología , Péptidos y Proteínas de Señalización Intercelular , Isoenzimas/efectos de los fármacos , Isoenzimas/genética , MAP Quinasa Quinasa 1 , Masculino , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico Sintasa/efectos de los fármacos , Dolor/inducido químicamente , Dolor/fisiopatología , Péptidos/farmacología , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Transducción de Señal/efectos de los fármacos , omega-N-Metilarginina/farmacología
17.
Biochem Biophys Res Commun ; 285(4): 997-1006, 2001 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-11467851

RESUMEN

Deposition of plaques containing Abeta is considered important in the pathogenesis of Alzheimer's disease. Phorbol esters that activate protein kinase C (PKC) promote alpha-secretase-mediated processing of the beta amyloid precursor protein (APP), which generally reduces formation of Abeta. To determine which PKC isozymes mediate this process, we studied CHO cells that express human APP751. Phorbol 12-myristate, 13-acetate (PMA)-stimulated APP secretion, which was reduced by a general PKC inhibitor bisindoylmaleimide I, but not by Gö 6976, which inhibits PKCalpha, beta, gamma, and mu. Since PKCdelta and epsilon were the only other PMA-sensitive isozymes present, we studied cells that express selective peptide inhibitors of these isozymes. Expression of the PKCepsilon inhibitor inhibited PMA-induced APPs secretion and suppression of Abeta production. In contrast, the PKCdelta inhibitor had no effect. These results provide evidence that PKCepsilon decreases Abeta production by promoting alpha-secretase mediated cleavage of APP.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Enfermedad de Alzheimer/etiología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas , Células CHO , Carbazoles/farmacología , Cricetinae , Regulación hacia Abajo , Activación Enzimática , Humanos , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-epsilon , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología
18.
Alcohol Clin Exp Res ; 25(5 Suppl ISBRA): 60S-66S, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-11391051

RESUMEN

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Paula L. Hoffman and Takeshi Yagi. The presentations were (1) cAMP signaling in ethanol sensitivity and tolerance, by Boris Tabakoff; (2) Synaptic signaling pathways of Fyn-tyrosine kinase, by Takeshi Yagi; (3) Ethanol drinking and sensitization in dopaminergic and serotonergic receptor knockouts, by Tamara J. Phillips; (4) ICAM-1 is involved in early alcohol-induced liver injury in the mouse given enteral alcohol, by Hiroshi Kono; and (5) Strategies for targeted and regulated knockouts, by Robert O. Messing and Doo-Sup Choi.


Asunto(s)
Consumo de Bebidas Alcohólicas/genética , AMP Cíclico/genética , Hepatopatías Alcohólicas/genética , Ratones Noqueados/genética , Ratones Transgénicos/genética , Proteínas Proto-Oncogénicas/genética , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Depresores del Sistema Nervioso Central/farmacología , AMP Cíclico/metabolismo , Etanol/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Hepatopatías Alcohólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/metabolismo , Ratones Transgénicos/metabolismo , Modelos Animales , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas c-fyn , Receptores Dopaminérgicos/deficiencia , Receptores Dopaminérgicos/genética , Receptores de Serotonina/deficiencia , Receptores de Serotonina/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
19.
Alcohol Clin Exp Res ; 25(5 Suppl ISBRA): 76S-81S, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-11391054

RESUMEN

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase Cepsilon-regulated sensitivity of gamma-aminobutyric acid type A (GABAA) receptors to allosteric agonists, by Robert O. Messing, A. M. Sanchez-Perez, C. W. Hodge, T. McMahon, D. Wang, K. K. Mehmert, S. P. Kelley, A. Haywood, and M. F. Olive; (2) Genetic and functional analysis of a GABAA receptor gamma2 subunit variant: A candidate for quantitative trait loci involved in alcohol sensitivity and withdrawal, by Kari J. Buck and Heather M. Hood; (3) Tryptophan-scanning mutagenesis in GABAA receptor subunits: Channel gating and alcohol actions, by Susumu Ueno; and (4) Can a single binding site account for actions of alcohols on GABAA and glycine receptors? by R. Adron Harris, Yuri Blednov, Geoffrey Findlay, and Maria Paola Mascia.


Asunto(s)
Conducta Animal/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Mutación/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Secuencia de Aminoácidos/efectos de los fármacos , Secuencia de Aminoácidos/genética , Animales , Conducta Animal/fisiología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Agonistas del GABA/farmacología , Humanos , Isoenzimas/efectos de los fármacos , Isoenzimas/genética , Ratones , Mutación/genética , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Receptores de GABA-A/genética , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/genética
20.
Neuroscience ; 103(1): 171-9, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11311798

RESUMEN

Withdrawal from chronic ethanol consumption can be accompanied by motor seizures, which may be a result of altered GABA(A) receptor function. Recently, we have generated and characterized mice lacking the epsilon isoform of protein kinase C as being supersensitive to the behavioral and biochemical effects of positive GABA(A) receptor allosteric modulators, including ethanol. The aim of the present study was to determine whether protein kinase C-epsilon null mutant mice display altered seizure severity during alcohol withdrawal. In addition, we used c-fos immunohistochemistry immediately following seizure assessment to identify potential brain regions involved in any observed differences in withdrawal severity. Mice were allowed to consume an ethanol-containing or control liquid diet as the sole source of food for 14 days. During the 7-h period following removal of the diet, both ethanol-fed wild-type and protein kinase C-epsilon null mutant mice displayed an overall increase in Handling-Induced Convulsion score versus control-fed mice. However, at 6 and 7h following diet removal, the Handling-Induced Convulsion score was reduced in ethanol-fed protein kinase C-epsilon null mutant mice compared to ethanol-fed wild-type mice. Ethanol-fed protein kinase C-epsilon null mutant mice also exhibited a decrease in the number of Fos-positive cells in the lateral septum, and an increase in the number of Fos-positive cells in the dentate gyrus, mediodorsal thalamus, paraventricular nuclei of the thalamus and hypothalamus, and substantia nigra compared to ethanol-fed wild-type mice. These data demonstrate that deletion of protein kinase C-epsilon results in diminished progression of ethanol withdrawal-associated seizure severity, suggesting that selective pharmacological inhibitors of protein kinase C-epsilon may be useful in the treatment of seizures during alcohol withdrawal. These data also provide insight into potential brain regions involved in generation or suppression of ethanol withdrawal seizures.


Asunto(s)
Convulsiones por Abstinencia de Alcohol/metabolismo , Convulsiones por Abstinencia de Alcohol/fisiopatología , Encéfalo/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Convulsiones por Abstinencia de Alcohol/enzimología , Animales , Temperatura Corporal , Encéfalo/enzimología , Inmunohistoquímica , Isoenzimas/deficiencia , Isoenzimas/genética , Masculino , Ratones , Ratones Mutantes , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Proteínas Proto-Oncogénicas c-fos/metabolismo
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