RESUMEN
Melanophores, brown to black pigment cells from, for example, Xenopus laevis, contain mobile melanin filled organelles, and are well suited for studies on organelle movement. The intracellular regulation of the movement seems to be controlled by serine and threonine phosphorylations and dephosphorylations. Melatonin induces aggregation of the melanosomes to the cell centre through a G(i/o)-protein-coupled receptor, Mel1c, which leads to an inhibition of PKA and a stimulation of PP2A. However, this study shows that the melatonin-induced aggregation of melanosomes is also accompanied by tyrosine phosphorylation of a protein with a molecular weight of approximately 280 kDa. Cells pre-incubated with genistein, an inhibitor of tyrosine phosphorylations, showed inhibited melanosome movement after melatonin stimulation, and a lower degree of tyrosine phosphorylation of the approximately 280 kDa protein. The adenylyl cyclase activator forskolin, and the G(i/o) protein inhibitor pertussis toxin, also inhibited tyrosine phosphorylation of the approximately 280 kDa protein. The results indicate that melatonin stimulation generates tyrosine phosphorylation of a high molecular weight protein, an event that seems to be essential for melanosome aggregation.
Asunto(s)
Melanóforos/metabolismo , Melatonina/metabolismo , Tirosina/metabolismo , Toxina de Adenilato Ciclasa , Animales , Western Blotting , Agregación Celular , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Genisteína/farmacología , Cinética , Toxina del Pertussis , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Melatonina , Serina/metabolismo , Transducción de Señal , Treonina/metabolismo , Factores de Tiempo , Factores de Virulencia de Bordetella/farmacología , XenopusRESUMEN
1. Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an alpha 2-adrenoceptor pharmacology. We now report the cloning of the alpha 2-F, a fish skin alpha 2-receptor from the cuckoo wrasse (Labrus ossifagus). 2. Degenerate oligonucleotides corresponding to conserved regions of the human alpha 2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human alpha 2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3. A clone (pTB17BS) consisting of approximately 5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and approximately 2 kb of 5'untranslated sequence. The deduced amino acid sequence of this protein showed 47-57% identity with the human alpha 2-adrenoceptors and thus appeared to encode a fish alpha 2-adrenoceptor. 4. In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the alpha 2-F might be regulated by cyclic AMP, calcium and/or steroids. 5. The alpha 2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 +/- 0.1 nM and a Bmax of 5.7 +/- 1.0 pmol mg-1 of protein.6. Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine >p-aminoclonidine> B-HT 920> (- )-noradrenaline;for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin.7. These results show that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenoceptor subtype.
Asunto(s)
Peces/metabolismo , Receptores Adrenérgicos alfa 2/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Southern Blotting , Línea Celular , Clonación Molecular , Melanóforos/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ADN Polimerasa Dirigida por ARN/metabolismo , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/genética , Piel/metabolismoRESUMEN
Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.
Asunto(s)
Genes Reporteros , Receptores Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Benzazepinas/farmacología , Unión Competitiva , Clonación Molecular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Expresión Génica , Cobayas , Datos de Secuencia Molecular , Quinolizinas/farmacología , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa/química , Receptores Adrenérgicos alfa/efectos de los fármacos , Alineación de SecuenciaRESUMEN
The majority of studies investigating the proliferative effect of Gi/o-protein-coupled receptor agonists are performed in recombinant receptor systems or cell lines. In these systems the relative stoichiometry of receptors compared to other cell components might be changed, which may lead to anomalies in cellular responses in contrast to natural occurring systems. In the present study, we have used primary cultures of smooth muscle cells (SMCs) isolated from human myometrium to characterize the proliferative effects of agonists binding to two different G protein-coupled receptors. Treatment of quiescent SMCs with lysophosphatidic acid (LPA) and noradrenaline resulted in significant increases in [3H]thymidine incorporation. However, LPA was almost four times more effective than noradrenaline in this respect. The proliferative effects of the agonists could be completely blocked by pertussis toxin, indicating that the response are mediated through Gi/o-proteins. The selective alpha 2-adrenergic receptor (alpha 2-AR) antagonist yohimbine dose-dependently reduced the effect of noradrenaline suggesting that the proliferative response was mediated through alpha 2-ARs. The proliferative effects induced by LPA and noradrenaline was markedly reduced in SMCs treated with the tyrosine kinase inhibitor genistein and the cAMP elevating compound forskolin. However, LPA but not noradrenaline induced rapid rises in the cytosolic free Ca2+ concentration [Ca2+]i. The ability to increase Ca2+ might be one explanation why LPA produce a more pronounced proliferative response than noradrenaline in primary cultures of human myometrial SMCs.
Asunto(s)
Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Lisofosfolípidos/farmacología , Miometrio/citología , Norepinefrina/farmacología , Receptores Acoplados a Proteínas G , Adrenérgicos/metabolismo , Adrenérgicos/farmacología , Unión Competitiva , División Celular/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Colforsina/farmacología , ADN/biosíntesis , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Genisteína/farmacología , Humanos , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Norepinefrina/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Embarazo , Receptores Adrenérgicos alfa/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Ácido LisofosfatídicoRESUMEN
We demonstrate the expression of the co-activator CREB-binding protein (CBP) in the nuclei of a large number of neurons and glial structures in the rat brain and spinal cord. Immunoblotting of nuclear extracts revealed a single band at 265 kDa, the size of CBP. We found that CBP immunoreactivity was localized to cholecystokinin mRNA-expressing neurons in the hippocampus and the thalamus, suggesting that CBP may be involved in long-term memory and modulation of cortical activity. However, CBP-labeling was not ubiquitous, and many brain regions, including several mesencephalic and diencephalic nuclei, showed sparse labeling. Further, the number of neurons displaying intense CBP-labeling varied across animals in some regions, e.g., the hippocampus and the amygdala. Since competition for limited amounts of CBP and CBP-related molecules has been shown to be important for the integration of intracellular signaling pathways with transcriptional regulation, the present results suggest that varying endogenous levels of CBP in post-mitotic neurons is an important parameter in neuronal transcriptional regulation.
Asunto(s)
Acetiltransferasas/análisis , Proteínas del Tejido Nervioso/análisis , Proteínas Nucleares/análisis , Prosencéfalo/química , Transactivadores/análisis , Animales , Proteína de Unión a CREB , Colecistoquinina/genética , Inmunohistoquímica , Hibridación in Situ , Masculino , Neuronas/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that acts as an intracellular signalling molecule after receptor activation by several cytokines, e.g., interleukin-6, leptin and ciliary neurotrophic factor. We have investigated the localization of STAT3 in the rat central nervous system and dorsal root ganglia. Light microscopic immunohistochemistry showed that STAT3-like immunoreactivity (STAT3-LI) was present in the nucleus and cytoplasm of neurons. STAT3-LI was seen both in cell bodies and in proximal and distal dendrites. Many structures involved in motor functions, such as the ventral horn of the spinal cord, the motor cranial nerve nuclei, the red nucleus and the Purkinje cells of the cerebellum showed STAT3-LI. STAT3-LI was also present in many regions involved in autonomic regulation, such as the intermediolateral cell column of the spinal cord, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus nerve, the area postrema, the locus coeruleus, the Barrington's nucleus and the arcuate, the lateral, the dorsomedial, the ventromedial, and the paraventricular hypothalamic nuclei. Other structures showing STAT3-LI were the dorsal root ganglia, the thalamus (the anterodorsal and paraventricular nucleus), the cerebral neocortex (layer 5) and the olfactory bulb. The wide distribution of STAT3-LI in the nervous system is consistent with reports of cytokine actions in the brain, but the present findings further suggest novel roles for STAT3 in mediating influences of cytokines on specific neuronal circuits regulating motor, sensory and autonomic functions.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al ADN/metabolismo , Ganglios Espinales/metabolismo , Médula Espinal/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Especificidad de Anticuerpos , Cerebelo/metabolismo , Inmunohistoquímica , Líquido Intracelular/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3 , Transducción de Señal , Telencéfalo/metabolismoRESUMEN
We present a model for the activity of protein clusters based on a simultaneous desorption of an activator (agonist, substrate molecule, etc.) and an inactivator (antagonist, inhibitor, etc.) caused by the collision or interaction between two effector molecules (e.g. receptors, enzymes). This model gives rise to switch-like dose-response curves, which are difficult to explain by ordinary co-operativity. It fits with recent experimental results obtained on single cells. Some other interesting aspects of the model are also pointed out. The model is similar to the model used to explain steep 'dose-response curves' in heterogeneous catalysis, caused by the reaction between two different molecules or atoms on the surface of the catalyst.
Asunto(s)
Modelos Biológicos , Catálisis , Relación Dosis-Respuesta a DrogaRESUMEN
The effect of pertussis toxin (PT) on the aggregation of pigment granules in melanophores from cuckoo wrasse (Labrus ossifagus L.) was studied. The results indicate the presence of a PT resistant alpha 2-adrenoceptor signal transduction mechanism.
Asunto(s)
Peces/fisiología , Melanóforos/efectos de los fármacos , Toxina del Pertussis , Receptores Adrenérgicos alfa/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , Animales , Resistencia a Medicamentos , Agregación de Receptores/efectos de los fármacos , Transducción de Señal , Yohimbina/farmacologíaRESUMEN
It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.
Asunto(s)
AMP Cíclico/metabolismo , Hormonas Hipotalámicas , Melaninas/farmacología , Melanóforos/fisiología , Pigmentos Biológicos/metabolismo , Hormonas Hipofisarias/farmacología , Animales , Colforsina/farmacología , Peces , Líquido Intracelular/metabolismo , Melanóforos/efectos de los fármacos , Melanóforos/metabolismo , Transducción de Señal/efectos de los fármacos , Yohimbina/farmacologíaRESUMEN
Melanophores of isolated fish scales survive for weeks in a culture medium. During this isolation period a progressive increase in sensitivity to noradrenaline (NA) takes place. In the present study, a 100-fold increase in sensitivity to NA was found after 9 days. However, at the same time, a 12-fold decrease in sensitivity to MCH was detected.
Asunto(s)
Peces/fisiología , Hormonas Hipotalámicas , Melaninas/fisiología , Melanóforos/fisiología , Norepinefrina/fisiología , Hormonas Hipofisarias/fisiología , Animales , Técnicas de Cultivo , DesnervaciónRESUMEN
Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Melanóforos/enzimología , Melanosomas/enzimología , Proteínas Motoras Moleculares/fisiología , Óxido Nítrico/metabolismo , Orgánulos/fisiología , Transporte de Proteínas/fisiología , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 1 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanóforos/efectos de los fármacos , Melanosomas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Orgánulos/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Xenopus laevisRESUMEN
Uterine leiomyoma is a very common benign tumour with unclear pathophysiology in adult women. In the present study we have investigated the expression level of alpha(2)- and beta(2)-adrenoceptors, and the adenylyl cyclase and phosphodiesterase activity in leiomyoma tissue compared with adjacent myometrium. Our results show that the alpha(2)/beta(2)-adrenoceptor ratio is increased in leiomyoma, due to a significant decrease in beta(2)-adrenoceptor expression. These changes were not due to an increased innervation, as the tumour tissue was completely devoid of nerve fibres. Moreover, the adenylyl cyclase activity of leiomyoma membranes was found to be approximately 50% lower, whereas the phosphodiesterase activity was significantly increased (by approximately 100%). We found that stimulating an increase in intracellular cyclic AMP, by adenylyl cyclase activity through beta(2)-adrenoceptors (isoprenaline), by direct enzyme activation (forskolin), or by inhibition of phosphodiesterase activity (papaverine), potently blocked both protein and DNA synthesis in cultured leiomyoma smooth muscle cells. Our results imply the adrenoceptors might be involved in, or a consequence of, leiomyoma growth. The results also suggest a new interesting approach for leiomyoma pharmacotherapy.
Asunto(s)
Adenilil Ciclasas/metabolismo , Leiomioma/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores Adrenérgicos beta 2/biosíntesis , Neoplasias Uterinas/metabolismo , Adulto , Femenino , Humanos , Leiomioma/patología , Metionina/metabolismo , Persona de Mediana Edad , Miometrio/inervación , Miometrio/metabolismo , Miometrio/patología , Receptores Adrenérgicos alfa 2/biosíntesis , Radioisótopos de Azufre/metabolismo , Timidina/metabolismo , Tritio/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.) The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 microns 2. Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor-mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.
Asunto(s)
Peces/anatomía & histología , Melanóforos/ultraestructura , Animales , Estimulación Eléctrica , Luz , Norepinefrina/farmacologíaRESUMEN
In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 +/- 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.
Asunto(s)
Ansiolíticos/metabolismo , Gránulos Citoplasmáticos/metabolismo , Flunitrazepam/metabolismo , Melaninas/metabolismo , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Cinética , Moluscos , TritioRESUMEN
Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G-protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three-dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P < 0.05, n=200). The average ellipsoidal volume was 0.14+/-0.01 (aggregated) and 0.17+/-0.01 microm3 (dispersed), a difference of 18%. The average major diameter was 810+/-20 and 880+/-20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non-destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.
Asunto(s)
Melanosomas/ultraestructura , Piel/ultraestructura , Xenopus laevis/anatomía & histología , Animales , Agregación Celular/fisiología , Tamaño de la Célula/fisiología , Melanosomas/metabolismo , Microscopía de Fuerza Atómica , Proteínas Motoras Moleculares/metabolismo , Proteínas Motoras Moleculares/ultraestructura , Piel/metabolismo , Xenopus laevis/metabolismoRESUMEN
Pigment organelles in Xenopus laevis melanophores are used by the animal to change skin color, and they provide a good model for studying intracellular organelle transport. Movement of organelles and vesicles along the cytoskeleton is essential for many processes, such as axonal transport, endocytosis, and intercompartmental trafficking. Nitric oxide (NO) is a signaling molecule that plays a role in, among other things, relaxation of blood vessels, sperm motility, and polymerization of actin. Our study focused on the effect NO exerts on cytoskeleton-mediated transport, which has previously received little attention. We found that an inhibitor of NO synthesis, N-nitro-L-arginine methyl ester (L-NAME), reduced the melatonin-induced aggregation of the pigment organelles, melanosomes. Preaggregated melanosomes dispersed after treatment with L-NAME but not after exposure to the inactive stereoisomer (D-NAME) or the substrate for NO synthesis (L-arginine). Signal transduction by NO can be mediated through the activation of soluble guanylate cyclase (sGC), which leads to increased production of cGMP and activation of cGMP-dependent kinases (PKG). We found that both the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the cGMP analogue 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) reduced melanosome aggregation, whereas the PKG inhibitor KT582 did not. Our results demonstrate that melanosome aggregation depends on synthesis of NO, and NO deprivation causes dispersion. It seems, thus, as if NO and cGMP are essential and can regulate melanosome translocation.
Asunto(s)
Carbazoles , GMP Cíclico/análogos & derivados , Indoles , Melanóforos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiología , Alcaloides/farmacología , Animales , Arginina/farmacología , Transporte Biológico , Western Blotting , Células Cultivadas , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Guanilato Ciclasa/metabolismo , Melanóforos/citología , Microscopía Fluorescente , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Transducción de Señal , Factores de Tiempo , Xenopus laevisRESUMEN
The aim of the present investigation was to determine which subtypes of the alpha2-adrenoceptors are being expressed in the human pregnant myometrium at term pregnancy. In radioligand binding studies, the specific binding of [3H]rauwolscine to human myometrial membranes was specific and of high affinity with Kd of 2.8 +/- 0.6 nMand Bmax of 95 +/- 5 fmol/mg protein. Results from competition for the binding of [3H]rauwolscine using subtype-selective ligands, oxymetazoline (alpha2A-subptype), chlorpromazine (alpha2B-subtype) and prazosin (alpha2B-alpha2C-subtype), suggested that the alpha2A- and alpha2B-subtypes are being co-expressed. In order to examine if also the alpha2C-subtype is being expressed we used an optimal concentration of oxymetazoline or chlorpromazine which would block the high-affinity site, equivalent to the alpha2A- and alpha2B-subtype respectively. Competition curves of both oxymetazoline and chlorpromazine still showed a significantly better fit using a two-site model, suggesting that the alpha2C-subtype also is being expressed. The expression of alpha2C-subtype mRNA was confirmed using reverse transcription-polymerase chain reaction on mRNA isolated from myometrial biopsies. In conclusion, our results suggest that all three subtypes of alpha2-adrenoceptors are being coexpressed in the human myometrium at term pregnancy and that alpha2-expression is dominated by the alpha2A-subtype.
Asunto(s)
Miometrio/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Unión Competitiva , Membrana Celular/metabolismo , Clorpromazina/metabolismo , Femenino , Humanos , Oximetazolina/metabolismo , Reacción en Cadena de la Polimerasa , Prazosina/metabolismo , Embarazo , ADN Polimerasa Dirigida por ARN , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/clasificación , Tritio , Yohimbina/metabolismoRESUMEN
It has previously been shown that alpha 2-adrenoceptors (alpha 2-ARs) mediate pigment granule (melanosome) aggregation in melanophores of the teleost fish Labrus ossifagus. The present investigation scrutinized the signalling mechanisms of melanosome aggregation induced by sympathetic nerve stimulation or by exogenous addition of alpha-AR agonists and cAMP analogues. The following was observed: i) nerve-induced melanosome aggregation was associated with a rapid decrease in the cAMP level; ii) noradrenaline or medetomidine (an alpha 2-AR agonist) caused melanosome aggregation and reduced the cAMP content; iii) RP-8-Cl-cAMP, a membrane-permeating inhibitor of protein kinase A induced melanosome aggregation; and iv) B-HT 920 (an alpha 2-AR agonist) and methoxamine (an alpha 1-AR agonist) induced melanosome aggregation, although they did not reduce cAMP. It has been suggested that in some teleost species alpha 1-ARs mediate melanosome aggregation by increasing the level of intracellular calcium. However, we found that the effect of methoxamine in melanophores from Labrus ossifagus could be blocked by yohimbine (an alpha 2-AR antagonist) but not by equimolar concentration of prazosin (an alpha 1-AR antagonist). Furthermore, 1 microM ionomycin (a calcium ionophore) did not induce melanosome aggregation. Our findings therefore do not indicate that alpha 1-ARs and/or an increase in intracellular calcium mediate melanosome aggregation in Labrus ossifagus. Our results suggest that alpha 2-AR-mediated melanosome aggregation is induced by multiple signalling pathways. One of these involves a reduction in cAMP, but none involves an increase in intracellular calcium.
Asunto(s)
Melanocitos/fisiología , Melanóforos/fisiología , Receptores Adrenérgicos alfa 2/fisiología , Transducción de Señal , Animales , Estimulación Eléctrica , Peces , Hormonas Hipotalámicas/farmacología , Ionomicina/farmacología , Melaninas/farmacología , Melanocitos/efectos de los fármacos , Melanóforos/efectos de los fármacos , Hormonas Hipofisarias/farmacología , Prazosina/farmacología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sistema Nervioso Simpático/fisiologíaRESUMEN
We report here the cloning and characterization of a 5'-end alternatively spliced human cholecystokinin-B (CCK-B) receptor mRNA. The 5'-end of this CCK-B receptor transcript (termed CCK-BRtx) consisted of exon Ia, present in the ordinary full-length CCK-B receptor mRNA (CCK-BRwt), and exon Ib, present in a previously described 5'-end alternatively spliced CCK-B receptor mRNA (CCK-BRt). A short open reading frame preceded the AUG translation initiation codon of the CCK-BRtx. Transfection of COS-7 cells with the CCK-BRtx or CCK-BRt cDNAs did not lead to the appearance of peptidergic and non-peptidergic binding sites. Cell free in vitro translation yielded proteins of approximately 44 kDa (CCK-B receptor) and 40 kDa (CCK-BRt receptor) whereas no 40 kDa product was detected from the cloned CCK-BRtx cDNA. Instead, a protein product of approximately 9 kDa was visualized, the size corresponding to the predicted protein encoded by the short open reading frame. The alternatively spliced CCK-B receptor transcripts were concomitantly expressed with the ordinary full-length CCK-B receptor mRNA in the brain, pancreas, and stomach. The possibility that such transcripts are translated in vivo into truncated CCK-B receptors is discussed.