RESUMEN
BACKGROUND: In cell culture experiments, flupirtine maleate (FLU), a triaminopyridine compound, was able to protect neuronal cells from apoptotic cell death induced by prion protein fragments and beta-amyloid peptides. As FLU is a clinically safe drug, the authors started a double-blind placebo-controlled study in patients with Creutzfeldt-Jakob disease (CJD). METHODS: Twenty-eight patients with CJD were randomized to an oral treatment with either FLU (n = 13) or matching placebo (PLA; n = 15). For inclusion and continuing the study, the patients had to achieve at least 50% in two of the subscales of the dementia tests employed. A battery of standardized questionnaires was employed to monitor the progression of the disease. The main outcome variable was the cognitive part of the Alzheimer's Disease Assessment Scale (ADAS-Cog); the difference between baseline and the best score under treatment was defined as the primary efficacy variable for hypothesis testing. RESULTS: CJD types were homogeneously distributed among the treatment groups. Patients treated with FLU showed significantly less deterioration in the dementia tests than patients treated with PLA. The mean change in ADAS-Cog (baseline to best) was +8.4 (+/-15.3) in the FLU group and +20.6 (+/-15.1) in the PLA group (p = 0.02, one-sided t-test). CONCLUSIONS: FLU has beneficial effects on cognitive function in patients with CJD. These positive results also may suggest a treatment potential of FLU in other neurodegenerative disorders. However, further studies are necessary.
Asunto(s)
Aminopiridinas/uso terapéutico , Cognición/efectos de los fármacos , Síndrome de Creutzfeldt-Jakob/tratamiento farmacológico , Adulto , Anciano , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas NeuropsicológicasRESUMEN
Attempts to identify the Galpha subtypes in the two compartments of the olfactory system from Xenopus, which are supposed to be specialized for detecting aquatic and volatile odorous compounds, revealed that a Galpha(o1) subtype is characteristic for the "water nose," the lateral diverticulum, whereas a novel Galpha(s) subtype predominates in the "air nose," the medial diverticulum. The newly identified Galpha(s)-type is more closely related to Galpha(olf) of rat and human than to the known Galpha(s)-isoform of Xenopus; it is therefore considered the first identified nonmammalian Galpha(olf) subtype. Sequence comparison of Galpha(olf) from amphibia and mammals revealed a particular conservation within the alpha-helical domains, which are supposed to control the GDP/GTP-exchange rate. The selective expression of different Galpha subtypes in the two anatomically separated and functionally specialized nasal compartments parallels the expression of distinct classes of olfactory receptors. Moreover, biochemical analysis revealed that stimulation with appropriate odorous compounds elicits the formation of inositol trisphosphate in the lateral diverticulum. In contrast, cyclic adenosine monophosphate signals were induced in the medial diverticulum, and this response appears to be mediated by the novel Galpha(olf) subtype. The data indicate that olfactory sensory neurons in each of the nasal cavities are equipped not only with defined sets of receptor types but also with a distinct molecular machinery for the chemo-electrical transduction process.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/fisiología , Odorantes , Neuronas Receptoras Olfatorias/fisiología , Transducción de Señal/fisiología , Xenopus laevis/fisiología , Secuencia de Aminoácidos , Animales , AMP Cíclico/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP , Inositol 1,4,5-Trifosfato/biosíntesis , Datos de Secuencia Molecular , Mucosa Olfatoria/fisiología , FilogeniaRESUMEN
G protein betagamma (Gbetagamma) complexes are considered to play an important role in second messenger signaling of phospholipase C (PLC). Monitoring the inositol 1,4,5-trisphosphate (IP(3)) response in circumvallate tissue homogenates upon stimulation with denatonium benzoate, it was demonstrated that a glutathione S-transferase-GRK3ct fusion protein-a Gbetagamma scavenger-attenuates the bitter tastant-induced second messenger reaction. Towards an identification of the Gbetagamma complex involved in rat bitter taste transduction, it was found that the G protein beta(3) subtype is specifically expressed in taste receptor cells of circumvallate papillae. Gbeta(3)-specific antibodies blocked the denatonium benzoate-induced IP(3) formation in a dose-dependent manner; the inhibitory effect was reversed by preincubation with the antigenic peptide. A less pronounced inhibition was observed using Gbeta(1)-specific antibodies. Analyzing individual taste cells by single cell reverse transcriptase-polymerase chain reaction approaches, overlapping expression patterns for PLCbeta(2), Galpha(gust), Gbeta(3) and Ggamma(3) could be demonstrated. Furthermore, the co-expression of all profiled signal transduction components in individual taste receptor cells could be detected. These data support the concept that the denatonium benzoate-induced IP(3) response is mediated by an activation of PLCbeta(2) via a Gbetagamma complex, possibly composed of Gbeta(3) as the predominant beta subunit and Ggamma(3), and imply that multiple second messenger pathways may exist in individual taste receptor cells.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Transducción de Señal/fisiología , Papilas Gustativas/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al GTP/metabolismo , Inmunohistoquímica , Inositol 1,4,5-Trifosfato/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Papilas Gustativas/citología , Papilas Gustativas/metabolismoRESUMEN
The second messengers 3'-5'-cyclic-monophosphate (cAMP) and inositol 1,4,5-trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U-73122 led to an augmentation of odor-induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor-induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor-induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN-(2-phenylcyclopentyl)azacylotridec-1-en-2-amine (MDL-12,330 A) results in potentiation of odor-induced InsP3 formation. Concerning the molecular mechanism involved in cross-interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor-induced InsP3 signaling by cAMP may be mediated by a PKA-controlled reaction.
Asunto(s)
Vías Olfatorias/química , Vías Olfatorias/fisiología , Sistemas de Mensajero Secundario/fisiología , Inhibidores de Adenilato Ciclasa , Animales , Cilios/efectos de los fármacos , Cilios/enzimología , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Iminas/farmacología , Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Inositol 1,4,5-Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Vías Olfatorias/efectos de los fármacos , Péptidos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistemas de Mensajero Secundario/efectos de los fármacos , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
The present study indicates that male rat urinary components in female rat vomeronasal organ microvillar preparations not only induce a rapid and transient IP(3) signal, but in addition, the level of cAMP decreases with a delayed and sustained time course. This decrease seems to be a consequence of the preceding activation of the phosphoinositol pathway rather than the result of an enhanced phosphodiesterase activity or an inhibition of adenylyl cyclase (AC) via Galpha(i) or Galpha(o). This notion is supported by the finding that activation of the endogenous protein kinase C suppresses basal as well as forskolin-induced cAMP formation. Furthermore, it was observed that elevated levels of calcium inhibit cAMP formation in rat VNO microvillar preparations. These properties of cAMP signaling in the VNO of rats may be mediated by a calcium- and protein kinase C-inhibited AC VI subtype, which is localized in microvillar preparations of the VNO.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Transducción de Señal , Órgano Vomeronasal/metabolismo , Animales , Femenino , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Órgano Vomeronasal/enzimologíaRESUMEN
Fast kinetics and sensitivity of olfactory signaling raise the question of whether the participating proteins may be associated in supramolecular transduction complexes. We found evidence that caveolin proteins could play an important role in organizing signaling elements in olfactory sensory neurons. Western blot analysis indicated that caveolins are highly enriched in olfactory sensory membranes, where they co-localize in detergent-insoluble complexes with key components of the signaling pathways. Furthermore, the results of immunoprecipitation experiments suggest that G proteins and effector enzyme form preassembled subcellular complexes with caveolins. Since anti-caveolin antibodies and synthetic peptides derived from the scaffolding domains of caveolin-1 and caveolin-2 effectively attenuated second messenger responses in sensory cilia preparations in a characteristic manner, the data led to the suggestion that caveolins could mediate the assembly of signaling complexes within specialized membrane microdomains of olfactory sensory neurons.
Asunto(s)
Caveolinas , Proteínas de la Membrana/metabolismo , Mucosa Olfatoria/fisiología , Órgano Vomeronasal/fisiología , Animales , Caveolina 1 , Caveolina 2 , Fraccionamiento Celular , Cilios , Colforsina/farmacología , AMP Cíclico/metabolismo , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Proteínas de Unión al GTP Heterotriméricas/aislamiento & purificación , Masculino , Microvellosidades , Odorantes , Fragmentos de Péptidos/farmacología , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptores Odorantes/metabolismo , Sistemas de Mensajero Secundario , Transducción de Señal/efectos de los fármacosRESUMEN
An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.
Asunto(s)
Calcio/farmacología , AMP Cíclico/metabolismo , Nephropidae , Neuronas Receptoras Olfatorias/metabolismo , Transducción de Señal/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Canales de Calcio/fisiología , Membrana Celular/metabolismo , Colforsina/farmacología , Dendritas/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Activación del Canal Iónico/efectos de los fármacos , Odorantes , Neuronas Receptoras Olfatorias/ultraestructuraRESUMEN
Cyclic nucleotide-gated (CNG) channels play central roles in visual and olfactory signal transduction. In the retina, rod photoreceptors express the subunits CNCalpha1 and CNCbeta1a. In cone photoreceptors, only CNCalpha2 expression has been demonstrated so far. Rat olfactory sensory neurons (OSNs) express two homologous subunits, here designated CNCalpha3 and CNCalpha4. This paper describes the characterization of CNCbeta1b, a third subunit expressed in OSNs and establishes it as a component of the native channel. CNCbeta1b is an alternate splice form of the rod photoreceptor CNCbeta1a subunit. Analysis of mRNA and protein expression together suggest co-expression of all three subunits in sensory cilia of OSNs. From single-channel analyses of native rat olfactory channels and of channels expressed heterologously from all possible combinations of the CNCalpha3, -alpha4, and -beta1b subunits, we conclude that the native CNG channel in OSNs is composed of all three subunits. Thus, CNG channels in both rod photoreceptors and olfactory sensory neurons result from coassembly of specific alpha subunits with various forms of an alternatively spliced beta subunit.
Asunto(s)
Canales Iónicos/metabolismo , Neuronas Aferentes/metabolismo , Bulbo Olfatorio/citología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Línea Celular , Cilios/metabolismo , Clonación Molecular , AMP Cíclico/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Epitelio/metabolismo , Expresión Génica , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/química , Canales Iónicos/genética , Canales Iónicos/fisiología , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Potasio/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismoRESUMEN
Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Galpha-subunits, activation dependently labeled with [alpha-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by alpha2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gi may be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature.
Asunto(s)
Proteínas de Unión al GTP/efectos de los fármacos , Feromonas/farmacología , Órgano Vomeronasal/metabolismo , alfa-Globulinas/orina , Secuencia de Aminoácidos , Animales , Femenino , Proteínas de Unión al GTP/metabolismo , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Feromonas/orina , Etiquetas de Fotoafinidad , Ratas , Ratas Sprague-Dawley , Órgano Vomeronasal/fisiologíaRESUMEN
Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.
Asunto(s)
Cilios/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Odorantes , Mucosa Olfatoria/efectos de los fármacos , Acetatos/farmacología , Aldehídos/farmacología , Secuencia de Aminoácidos , Animales , Benzaldehídos/farmacología , Cilios/metabolismo , AMP Cíclico/biosíntesis , Ciclohexenos , Ciclopentanos/farmacología , Eugenol/farmacología , Inositol 1,4,5-Trifosfato/biosíntesis , Datos de Secuencia Molecular , Nitrilos/farmacología , Mucosa Olfatoria/metabolismo , Oxilipinas , Etiquetas de Fotoafinidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin characterized by loss of upper and lower motor neurons and concomitant astrogliosis. We have investigated the S100 beta protein levels in serum as a marker for astroglia of patients with ALS (n = 41) in comparison to a control group (n = 32). Additionally we have investigated 12 patients at different follow-up time points (minimum 6 months). We could not observe a significant difference of S100 beta protein in patients with ALS in comparison to our control group (P = 0.11) but we could clearly see a decrease of S100 beta levels in the further course of the disease. As S100 beta is also seen as a protein with nerve growth factor activity we assume that the fall of serum levels may reflect the loss of nerve growth stimulation in patients with ALS and suppose that repetitive measurements of S100 beta in serum can be used as an objective marker for disease progression.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Proteínas S100/sangre , Anciano , Biomarcadores/sangre , Proteínas de Unión al Calcio/sangre , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Inmunoensayo , Mediciones Luminiscentes , Masculino , Persona de Mediana Edad , Factores de Crecimiento Nervioso , Juego de Reactivos para Diagnóstico , Subunidad beta de la Proteína de Unión al Calcio S100RESUMEN
G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores Odorantes/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Regulación hacia Abajo , Quinasa 3 del Receptor Acoplado a Proteína-G , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Regulación hacia Arriba , Quinasas de Receptores Adrenérgicos betaRESUMEN
The chemo-electrical transduction process in olfactory neurons is accompanied by a rapid and transient increase in intracellular calcium concentrations. The notion that Na+/Ca2+ exchanger activities may play a major role in extruding calcium ions out of the cell and maintaining Ca2+ homeostasis in olfactory receptor cells was assessed by means of laser scanning confocal microscopy in combination with the fluorescent indicators Fluo-3 and Fura-Red. The data indicate that high exchanger activity, which was inhibited by amiloride derivatives, is located in the dendritic knob and probably in the olfactory cilia. This result was supported by experiments using specific antiserum raised against retinal Na+/Ca2+ exchanger protein which labelled an immunoreactive protein of 230 kDa in Western blots from olfactory tissue and strongly stained the ciliary layer of the olfactory epithelium.
Asunto(s)
Proteínas Portadoras/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Cilios/metabolismo , Epitelio/metabolismo , Colorantes Fluorescentes , Homeostasis , Inmunohistoquímica , Microscopía Confocal , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-CalcioRESUMEN
Visinin-like-protein (VILIP), a member of the neuronal subfamily of EF-hand calcium-sensor proteins is shown to be expressed in olfactory sensory cells of the rat nasal epithelium. Its prominent localization in cilia and dendritic knobs-the chemosensory compartments of olfactory neurons-suggests that the calcium-binding protein could be involved in olfactory signal transduction. Consistent with this assumption, it was found that recombinant VILIP attenuates in a calcium-dependent manner odorant-induced cAMP formation in olfactory cilia preparations. Kinetic data indicate that VILIP does not interfere with odorant-induced receptor desensitization. Since VILIP inhibits the forskolin-induced formation of cAMP, it is conceivable that VILIP may directly affect the olfactory adenylyl cyclase. Thus, VILIP may play a role in adaptation of olfactory neurons.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Receptores Sensibles al Calcio , Sistemas de Mensajero Secundario/fisiología , Animales , Calcio/metabolismo , Calcio/farmacología , Proteínas de Unión al Calcio/farmacología , Colforsina/farmacología , AMP Cíclico/biosíntesis , Inmunohistoquímica , Cinética , Proteínas del Tejido Nervioso/farmacología , Neurocalcina , Odorantes , Neuronas Receptoras Olfatorias/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
Phosducin, which tightly binds betagamma-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424). The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorant-induced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the Gbetagamma-subunit, thereby making Gbetagamma available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 --> Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 --> Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of Gbetagamma-dependent GRKs.
Asunto(s)
Proteínas del Ojo/metabolismo , Vías Olfatorias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Animales , Bovinos , AMP Cíclico/metabolismo , Proteínas del Ojo/genética , Quinasa 3 del Receptor Acoplado a Proteína-G , Reguladores de Proteínas de Unión al GTP , Humanos , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Fosfoproteínas/genética , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
The odorant-induced second messenger response in olfactory cilia was monitored in the presence of phosphatase modulators. Okadaic acid, a phosphatase inhibitor, attenuated the odorant-induced cAMP-response in a dose-dependent manner, half maximal inhibition was obtained at 1.5 nM okadaic acid indicating that phosphatase 2A may be involved. Protamine, a selective activator of phosphatase 2A, led to significantly stronger cAMP-responses. Western blot and immunohistochemical analysis employing specific antibodies revealed that phosphatase 2A is present in olfactory tissues in particular in olfactory cilia. The results suggest that phosphatase 2A may play a regulatory role in governing the responsiveness of olfactory neurons.
Asunto(s)
Cilios/enzimología , Odorantes , Neuronas Receptoras Olfatorias/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal/fisiología , Animales , Anticuerpos/inmunología , Western Blotting , Cilios/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Éteres Cíclicos/farmacología , Inmunohistoquímica , Ácido Ocadaico , Neuronas Receptoras Olfatorias/efectos de los fármacos , Fosforilación , Protaminas/farmacología , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
The increase in intracellular calcium concentration elicited by odorant stimulation seems to be involved in down-regulating the responsiveness of olfactory neurons to subsequent stimuli. The present study suggests that this regulatory effect may be due to a calcium-dependent attenuation of the olfactory signalling cascade; the odor-induced cyclic adenosine monophosphate (cAMP) response in olfactory cilia is diminished by calcium in a dose-dependent manner. This reduced cAMP signal is not due to an activation of phosphodiesterases by elevated calcium levels, but rather seems to be mediated by the inhibition of adenylate cyclase by calcium ions.
Asunto(s)
Calcio/farmacología , AMP Cíclico/metabolismo , Vías Olfatorias/metabolismo , Transducción de Señal/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/metabolismo , Animales , Cilios/efectos de los fármacos , Cilios/metabolismo , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Tabique Nasal/efectos de los fármacos , Tabique Nasal/metabolismo , Vías Olfatorias/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario , Transducción de Señal/fisiologíaRESUMEN
Strong odor stimuli elicit a slow and sustained increase of the cGMP concentration in isolated rat olfactory cilia. Elevated cGMP levels appear to attenuate the primary response to odorant stimulation. Incubating cilia with membrane-permeable cGMP derivates caused a significantly reduced cAMP signal in response to odorant stimulation. This inhibitory effect was mimicked by 8-(4-chlorophenlythio)-cGMP, a selective activator of cGMP-activated protein kinases; in contrast, a selective inhibitor, [8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate] of cGMP kinases enhanced the reactivity to odorant stimulation. The data suggest that the responsiveness of olfactory sensory cells is governed by a cGMP-dependent protein kinase. Western-blot analysis using subtype-specific antibodies indicated that cytosolic type-I cGMP kinase, but not the membrane-associated type-II cGMP kinase, is expressed in olfactory sensory neurons.
Asunto(s)
Células Quimiorreceptoras/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Mucosa Olfatoria/fisiología , Olfato/fisiología , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Guanosina Trifosfato/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Sistemas de Mensajero Secundario , Sacarosa/análogos & derivados , Gusto/fisiología , Lengua/fisiología , Animales , Femenino , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Especificidad de la Especie , Sacarosa/farmacología , Tionucleótidos/farmacología , Factores de TiempoRESUMEN
Quench-flow measurements are used to determine the subsecond kinetics of odor-induced changes in second messenger concentrations in lobster olfactory receptor neurons. Individual odors transiently and differentially increase the production of both adenosine cAMP and inositol 1,4,5-trisphosphate (IP3) within 50 msec of odor stimulation. The ability of two different odors to stimulate cAMP and IP3 correlates with the odors' ability to excite and inhibit receptor cells physiologically. These results strengthen the proposition, heretofore based largely on evidence from cultured cells, that dual second messenger pathways mediate excitatory and inhibitory input to lobster olfactory receptor cells.