RESUMEN
The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.
Asunto(s)
Carcinoma/genética , Carcinoma/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción Genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Paclitaxel/farmacología , Fosforilación , Proteómica , Interferencia de ARN , Factor de Transcripción Sp1/metabolismoRESUMEN
RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as 'pocket proteins', because they share a 'pocket' domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the 'viral hypothesis' of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.
Asunto(s)
Virus ADN/genética , Proteínas Oncogénicas/fisiología , Proteína de Retinoblastoma/fisiología , Adenoviridae/genética , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/fisiología , Humanos , Papillomaviridae/genéticaRESUMEN
Nm23-NDPKs besides contributing to the maintenance of the cellular nucleoside triphosphate pool, exert regulatory properties in a variety of cellular events including proliferation, invasiveness, development, differentiation, and gene regulation. This review focuses on recently discovered protein-protein interactions involving the Nm23 proteins. The findings herein summarized provide new and intriguing suggestions for a more extensive understanding of the biological functions of the Nm23 proteins.
Asunto(s)
Antígenos Virales/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas Nucleares/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Telómero/metabolismo , Activación Transcripcional/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos Virales/química , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/clasificación , Diferenciación Celular/fisiología , División Celular , Antígenos Nucleares del Virus de Epstein-Barr , Humanos , Proteínas Nucleares/química , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/clasificación , Monoéster Fosfórico Hidrolasas , Unión Proteica , Proteínas Proto-Oncogénicas/química , Relación Estructura-Actividad , Telómero/química , Técnicas del Sistema de Dos HíbridosRESUMEN
The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Apoptosis/fisiología , División Celular/fisiología , Péptidos/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas E1A de Adenovirus/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica , Receptores de Cinasa C Activada , Células Tumorales CultivadasRESUMEN
A cDNA coding for the human neuronal nicotinic alpha7 receptor subunit with Leu-248 mutated to threonine was expressed in Xenopus oocytes. When activated by acetylcholine (AcCho), the receptors expressed generated currents that had low desensitization, linear current-voltage relation, and high apparent affinity for both AcCho and nicotine. These characteristics are similar to those already described for the chick threonine-for-leucine-247 alpha7 nicotinic AcCho receptor (nAcChoR) mutant (L247Talpha7). These properties were all substantially maintained when the human L248Talpha7 mutant was transiently expressed in human Bosc 23 cells. Simultaneous whole-cell clamp and fluorescence measurements with the Ca(2+) indicator dye Fura-2 showed that nicotine induced a Ca(2+) influx in standard 2 mM Ca(2+) solution. The average fractional Ca(2+) current flowing through L248Talpha7 nAcChoRs was 6.7%, which is larger than that flowing through muscle alpha(beta)epsilon(delta) nAcChoRs (4.1%). The relative Ca(2+) permeability, determined in oocytes in the absence of Cl(-), was measured from the shift in reversal potential caused by increasing the external Ca(2+) concentration from 1 to 10 mM. The human wild-type alpha7 nAcChoR was found to be more permeable than the L248Talpha7 mutant to Ca(2+). Our findings indicate that the Ca(2+) permeability of the homomeric alpha7 nAcChoR is larger than that of the heteromeric neuronal nicotinic receptors studied to date and is possibly similar to that of the N-methyl-D-aspartate subtype of brain glutamate receptors.
Asunto(s)
Calcio/metabolismo , Leucina/genética , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Treonina/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Transporte Iónico , Mutagénesis Sitio-Dirigida , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Espectrometría de FluorescenciaRESUMEN
The growth-related gene product beta (GRObeta) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GRObeta receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GRObeta receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GRObeta activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GRObeta-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.
Asunto(s)
Cerebelo/metabolismo , Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Esfingomielinas/metabolismo , Animales , Factores Quimiotácticos/genética , Activación Enzimática , Regulación de la Expresión Génica , Sustancias de Crecimiento/genética , Hidrólisis , Proteínas Quinasas JNK Activadas por Mitógenos , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
We report here that, in cultured cerebellar granule cells, the CXC chemokine GRObeta stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous postsynaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRObeta-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRObeta modulates neurotransmitter release in the cerebellum through the activation of the extracellular signal-regulated kinases pathway.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Inhibidores de Crecimiento/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Quinasas Activadas por Mitógenos , Neurotransmisores/metabolismo , Células de Purkinje/enzimología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Quimiocina CXCL1 , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Flavonoides/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células de Purkinje/citología , Ratas , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacologíaRESUMEN
1. We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the gamma subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5. 2. We studied unitary ACh-evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6). 3. At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24-26 degrees C. 4. On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the gammas subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period.
Asunto(s)
Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Receptores Colinérgicos/metabolismo , Animales , Células Cultivadas , Electrofisiología , Exones , Femenino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/inervación , Técnicas de Placa-Clamp , Embarazo , ARN Mensajero/biosíntesis , Ribonucleasas/metabolismoRESUMEN
We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/farmacología , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/farmacología , Células de Purkinje/efectos de los fármacos , Animales , Animales Recién Nacidos , Calcio/metabolismo , Quimiocina CXCL1 , Citosol/metabolismo , Conductividad Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Compuestos Orgánicos , Células de Purkinje/metabolismo , Células de Purkinje/fisiología , Ratas , Ratas Wistar , Receptores de Quimiocina/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8BRESUMEN
nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.
Asunto(s)
Acetilcolina/farmacología , Receptores Nicotínicos/fisiología , Animales , Línea Celular , Pollos , Clonación Molecular , Femenino , Hexametonio/farmacología , Humanos , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Neuronas/fisiología , Antagonistas Nicotínicos/farmacología , Oocitos/fisiología , Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/química , Proteínas Recombinantes/biosíntesis , Transfección , XenopusRESUMEN
To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat alpha 3 beta 4, chick alpha 3 beta 2, alpha 4 beta 2, alpha 7 and alpha 8), expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of alpha 8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19-23 and 32-45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric alpha 7 and alpha 8 nAChRs (6-7 ms) than for heteromeric nAChRs (1-3 ms), with the exception of alpha 3 beta 4 nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (alpha 3 beta 4 and alpha 3 beta 2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChR channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.
Asunto(s)
Acetilcolina/farmacología , Neuronas/efectos de los fármacos , Receptores Nicotínicos/fisiología , Animales , Pollos , Humanos , Técnicas de Placa-Clamp , Ratas , Receptores Nicotínicos/efectos de los fármacos , TransfecciónRESUMEN
A large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR gamma subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short gamma subunit [gamma(s)] with alpha, beta and delta subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the gamma by the gamma(s) subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that alpha beta gamma(s) delta AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the gamma subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/metabolismo , Receptores Colinérgicos/metabolismo , Acetilcolina/farmacología , Animales , Línea Celular , Conductividad Eléctrica , Humanos , Cinética , Ratones , Receptores Colinérgicos/química , Receptores Colinérgicos/fisiología , Relación Estructura-ActividadRESUMEN
The action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the potent stimulator of protein kinase C (PKC), on acetylcholine-activated currents (I(Ach)) was investigated in voltage clamped Xenopus laevis oocytes injected with RNAs encoding murine embryonic nicotinic acetylcholine receptor (AChR) subunits. Comparable potentiation and acceleration of decay of I(ACh) were observed within minutes of phorbol ester application in oocytes injected with various RNA subunit combinations: (i) alpha beta gamma delta; (ii) alpha beta gamma; (iii) alpha beta delta; and (iv) alpha beta gamma delta(AAA), a mutant of the delta subunit with serine residues 360-361-362 mutated to alanine. Our findings indicate that the effects on I(ACh) induced by PKC stimulation are independent of both gamma and delta subunits and, accordingly, of the presence of PKC phosphorylation sites on delta subunit. It is here suggested a novel PKC-dependent modulatory mechanism of cholinergic receptor which does not involve direct phosphorylation of the AChR and requires phosphorylation of intermediate regulatory protein(s).
Asunto(s)
Mutación/efectos de los fármacos , Oocitos/efectos de los fármacos , Ésteres del Forbol/farmacología , Receptores Nicotínicos/efectos de los fármacos , Animales , XenopusRESUMEN
A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.
Asunto(s)
Agonistas Nicotínicos/química , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Serotonina/química , Animales , Pollos , Activación del Canal Iónico , Leucina/química , Potenciales de la Membrana , Neuronas/química , Oocitos , Relación Estructura-Actividad , Treonina/química , Xenopus laevisRESUMEN
M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major "classic" heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45 degrees C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)+ RNA extracted from cells treated at 45 degrees C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer.
Asunto(s)
Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/química , Animales , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/inmunología , Humanos , Peso Molecular , Conejos , Células Tumorales CultivadasRESUMEN
The rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents (IACh) under various patch-clamp configurations. We observed a progressive slowing of IACh decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when IACh decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A+)RNA extracted from C2C12 myoblasts or mature myotubes, IACh decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced IACh decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR.
Asunto(s)
Músculos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/farmacología , Animales , Línea Celular , Medios de Cultivo , Electrofisiología , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Músculos/citología , Músculos/ultraestructura , Oocitos/metabolismo , Poli A/biosíntesis , ARN Mensajero/biosíntesis , XenopusRESUMEN
The modulation of acetylcholine-activated current (IACh) by protein kinase C (PKC) was studied in Xenopus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine alpha beta gamma delta subunits of the nicotinic ACh receptor (nAChR). Voltage-clamped oocytes were treated for 90 sec with 12-O-tetradecanoylphorbol-13-acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of IACh were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20-30 min; then a slight depression of IACh developed. By this time, an initial PKC down regulation was observed. At the peak of TPA-induced potentiation, dose-response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4 alpha-phorbol 12,13-didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect IACh, while staurosporine (5-10 microM), a potent inhibitor of PKC activity, suppressed the action of TPA on IACh. In oocytes co-injected with C2C12 mRNA and with rat brain mRNA, IACh was potentiated by 5-hydroxy-tryptamine (10 microM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR-channel activity in cell-attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic-type muscle nAChRs.
Asunto(s)
Acetilcolina/farmacología , Proteína Quinasa C/farmacología , Receptores Nicotínicos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Electrofisiología , Ratones , Ratones Endogámicos , Oocitos , Receptores Nicotínicos/fisiología , Serotonina/farmacología , Factores de Tiempo , XenopusRESUMEN
Nicotinic acetylcholine receptors (nAcChoRs) of skeletal muscle are heterosubunit ligand-gated channels that mediate signal transmission from motor nerves to muscle. While cloning murine nAcChoR subunits, to gain an insight into the receptor diversity across species, we detected two forms of gamma subunits in the myogenic C2C12 cell line. Both forms are functional when expressed in Xenopus oocytes. One gamma subunit [long gamma (gamma 1)] was almost identical to that previously cloned in the murine BC3H-1 tumor cell line. The second form of gamma subunit [short gamma (gamma s)] lacked 156 bp (52 amino acids) in the extracellular N terminus, adjoining the hydrophobic segment M1, which corresponds to the fifth exon of the gamma-subunit gene. The two forms of gamma subunit coexist during myogenesis in vitro and in 17-day embryonic and denervated adult muscle fibers in vivo. However, the gamma s variant was the only form of gamma subunit in newborn muscle. In dissociated muscle fibers of newborn mice, AcCho-evoked channel openings were more prolonged when compared with C2C12 myotubes or denervated adult muscle fibers. The gamma s subunit may, thus, contribute to the structural and functional diversity of nAcChoRs in muscle cells.
Asunto(s)
Empalme Alternativo , Músculo Esquelético/metabolismo , Receptores Muscarínicos/biosíntesis , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Embrión de Mamíferos , Embrión no Mamífero , Exones , Femenino , Expresión Génica , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Conformación Proteica , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Receptores Muscarínicos/química , Transcripción Genética , Xenopus laevisRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine secreted by activated astrocytes and is known to alter evoked synaptic activity in slices of adult rat hippocampus. In this paper we show that TNF-alpha increases the frequency of spontaneous miniature synaptic currents in cultured hippocampal neurons, acting at nanomolar concentrations. In addition, we show that the mRNA for the 55 kDa TNF-alpha receptor (TNF-R1) is detected in embryonic rat hippocampal cultures, as well as in acutely dissected embryonic and adult rat hippocampi. Possible transduction pathways mediating the TNF-alpha effect are discussed.
Asunto(s)
Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Calcio/metabolismo , Citosol/metabolismo , Conductividad Eléctrica , Hipocampo/citología , Hipocampo/fisiología , Neuronas/fisiología , Concentración Osmolar , Reacción en Cadena de la Polimerasa , Ratas/embriología , Ratas Sprague-Dawley , Receptores de Citocinas/metabolismo , Sinapsis/fisiología , Transcripción GenéticaRESUMEN
The action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10(-5) to 10(-3) M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond -10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10(-6) M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule.