RESUMEN
OBJECTIVES: To explore what it is like to be a pregnant smoker in Sunderland and to inform the development of a Smoking Cessation Programme; to use qualitative techniques to develop a cessation programme tailored to pregnant smokers. STUDY DESIGN: The intervention follows years of social marketing research and development in Sunderland, Wearside, UK. METHODS: Information derived from nine focus groups (mainly with women from deprived areas, social class C2D and E) provided insights into the issues facing smoking pregnant women. This information was then used to overcome barriers to smoking cessation using the principles of social marketing. The number of women recruited into a specially designed smoking cessation support initiative was compared with women recruited into comparable groups in the North East. RESULTS: Recruitment of pregnant (and non-pregnant) smokers to the new NHS smoking cessation programme in Sunderland has increased during the intervention phase compared with neighbouring Primary Care Trust areas (in which different smoking cessation interventions targeted at pregnant women were being undertaken). CONCLUSIONS: This innovative intervention has been successful in generating ideas, guiding development of a customer-friendly service and encouraging women to come forward for smoking cessation support during their pregnancy. The target population have welcomed the approach, and health professionals have enjoyed and benefited from the role play with professional actors.
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Complicaciones del Embarazo , Derivación y Consulta/tendencias , Cese del Hábito de Fumar , Prevención del Hábito de Fumar , Mercadeo Social , Femenino , Promoción de la Salud/métodos , Humanos , Embarazo , Salud Pública , Clase Social , Reino UnidoRESUMEN
To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.
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Proteínas Morfogenéticas Óseas/fisiología , Corazón/embriología , Miocardio/citología , Proteínas de Xenopus , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Sistema Digestivo/embriología , Regulación hacia Abajo , Endodermo/metabolismo , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Regulación del Desarrollo de la Expresión Génica , Marcadores Genéticos , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/biosíntesis , Hibridación in Situ , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteína smad6 , Transactivadores/metabolismo , Factores de Transcripción/biosíntesis , Transcripción Genética , XenopusRESUMEN
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Embrión no Mamífero/fisiología , Células Precursoras Eritroides/citología , Regulación del Desarrollo de la Expresión Génica , Secuencia de Aminoácidos , Animales , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Supervivencia Celular/fisiología , Embrión no Mamífero/citología , Eritrocitos/citología , Células Precursoras Eritroides/fisiología , Eritropoyesis , Regulación Enzimológica de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Morfogénesis , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción Genética , Xenopus laevisRESUMEN
We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.
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Arrestinas/fisiología , Bencenoacetamidas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores Opioides kappa/fisiología , Secuencia de Aminoácidos , Animales , Encefalina D-Penicilamina (2,5) , Encefalinas/farmacología , Femenino , Quinasa 3 del Receptor Acoplado a Proteína-G , Datos de Secuencia Molecular , Pirrolidinas/farmacología , Ratas , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/química , Relación Estructura-Actividad , Arrestina beta 2 , beta-ArrestinasRESUMEN
Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Here, we report that extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. Interestingly, the Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. This may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Calcio/fisiología , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Transcripción Genética/fisiología , Animales , Transporte Biológico/fisiología , Señalización del Calcio , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Neuronas/fisiología , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Transducción de Señal/fisiologíaRESUMEN
The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.
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Calcio/metabolismo , Activación del Canal Iónico , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Animales , Sitios de Unión , Calmodulina/genética , Calmodulina/metabolismo , Electrofisiología , Glutatión Transferasa/metabolismo , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , XenopusRESUMEN
The type 9 adenylyl cyclase (AC9) is a widely distributed adenylyl cyclase that was originally cloned from a mouse cDNA library. Here we report the cloning, chromosomal mapping, and regulatory properties of human AC9 (HGMW-approved symbol ADCY9). Although the human AC9 sequence shows 86% homology with mouse AC9, divergence at the C2a/C2b junction results in an alternative C2b amino acid sequence. In situ hybridization localized the human AC9 gene to both human and mouse chromosomes 16. AC9 mRNA is present in all tissues examined, with the highest levels found in skeletal muscle, heart, and brain. To characterize the regulatory properties of human AC9 in vivo, the enzyme was expressed in HEK-293 cells. Human AC9 is stimulated by beta-adrenergic receptor activation but is insensitive to forskolin, Ca2+ and somatostatin. In contrast to mouse AC9, the activity of human AC9 is unaffected by inhibitors of calcineurin. These data emphasize the importance of determining the regulatory properties of human adenylyl cyclases.
Asunto(s)
Adenilil Ciclasas/genética , Adenilil Ciclasas/fisiología , Mapeo Cromosómico , Inhibidores de Adenilato Ciclasa , Secuencia de Aminoácidos , Animales , Northern Blotting , Calcineurina/farmacología , Línea Celular , Cromosomas Humanos Par 16/genética , Clonación Molecular , Colforsina/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/fisiología , Riñón/embriología , Ratones , Datos de Secuencia MolecularRESUMEN
We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a Kd in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.
Asunto(s)
Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico , Secuencia de Bases , Sitios de Unión , Encéfalo/enzimología , Células COS , Cinética , Quinasas de Proteína Quinasa Activadas por Mitógenos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Plásmidos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TransfecciónRESUMEN
The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which was due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site was identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Animales , Células COS , Colforsina/farmacología , Fosforilación , SpodopteraRESUMEN
The CD5 receptor on T lymphocytes is involved in T cell activation and T-B cell interactions. In the present study, we have characterized the signaling pathways induced by anti-CD5 stimulation in human T lymphocytes. In T lymphocytes, anti-CD5 co-stimulation enhances the phytohemagglutinin/anti-CD28-induced interleukin-2 (IL-2) mRNA accumulation 1.6-fold and IL-2 protein secretion 2. 2-fold, whereby the up-regulation is mediated at both the transcriptional and post-transcriptional level. The CD5 signaling pathway up-regulates the IL-2 gene expression by increasing the DNA binding and transactivation activity of activator protein 1 but affects none of the other transcription factors like nuclear factor of activated T cells, nuclear factor kappaB, Oct, and CD28-responsive complex/nuclear factor of mitogen-activated T cells involved in the regulation of the IL-2 promoter activity. The CD5-induced increase of the activator protein 1 activity is mediated through the activation of calcium/calmodulin-dependent (CaM) kinase type IV, and is independent of the activation of mitogen-activated protein kinases Jun N-terminal kinase, extracellular signal-regulated kinase, and p38/Mpk2, and calcium/calmodul-independent kinase type II. The expression of a dominant negative mutant of CaM kinase IV in T lymphocytes transfected with an IL-2 promoter-driven reporter construct completely abrogates the response to CD5 stimulation, indicating that CaM kinase IV is essential to the CD5 signaling pathway. In addition, it is demonstrated that calcium/calmodulin-dependent kinase type IV is also involved in the stabilization of the IL-2 transcripts, which is observed after co-stimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with anti-CD5.
Asunto(s)
Antígenos CD5/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Transducción de Señal , Linfocitos T/citología , Factor de Transcripción AP-1/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Sitios de Unión , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Expresión Génica , Humanos , Técnicas In Vitro , Interleucina-2/genética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Linfocitos T/enzimología , Activación TranscripcionalRESUMEN
Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/deficiencia , Alanina , Animales , Sitios de Unión , Calcio/farmacología , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Humanos , Riñón , Cinética , Potenciación a Largo Plazo , Memoria , Ratones , Ratones Mutantes Neurológicos , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , SerinaRESUMEN
Inhibition of type III adenylyl cyclase (III-AC) by intracellular Ca2+ in vivo provides a mechanism for attenuation of hormone-stimulated cAMP signals in olfactory epithelium, heart, and other tissues (Wayman, G. A., Impey, S., and Storm, D. R. (1995) J. Biol. Chem. 270, 21480-21486). Although the mechanism for Ca2+ inhibition of III-AC in vivo has not been defined, inhibition is not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, Ca2+ inhibition of III-AC is antagonized by KN-62, a CaM-dependent kinase inhibitor. In addition, constitutively activated CaM kinase II inhibits the enzyme. These data suggest that CaM kinase II regulates the activity of III-AC by direct phosphorylation or by an indirect mechanism involving phosphorylation of a protein that inhibits III-AC. Here we report that III-AC is phosphorylated in vivo when intracellular Ca2+ is increased and that phosphorylation is prevented by CaM-dependent kinase inhibitors. Site-directed mutagenesis of a CaM kinase II consensus site (Ser-1076 to Ala-1076) in III-AC greatly reduced Ca2+-stimulated phosphorylation and inhibition of III-AC in vivo. These data support the hypothesis that Ca2+ inhibition of III-AC is due to direct phosphorylation of the enzyme by CaM kinase II in vivo.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores de Adenilato Ciclasa , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN Complementario/genética , Humanos , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfoserina/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Various forms of cross-talk between the Ca2+ and cAMP signal transduction systems can occur in animal cells depending upon the types of adenylyl cyclases present. Here, we report that Ca2+ oscillations can be generated by hormone stimulation of type III adenylyl cyclase expressed in HEK-293 cells. These Ca2+ oscillations are apparently due to the unique regulatory features of type III adenylyl cyclase, which is stimulated by hormones and inhibited by elevated Ca2+ in vivo. Ca2+ oscillations were generated by glucagon, isoproterenol, or forskolin stimulation of type III adenylyl cyclase and were dependent upon the activity of cAMP- and calmodulin-dependent protein kinases. Ca2+ oscillations were not solely dependent upon cAMP increases since dibutyryl cAMP or (Sp)-cAMP did not stimulate Ca2+ oscillations. We hypothesize that stimulation of type III adenylyl cyclase leads to increased cAMP, activation of inositol 1,4,5-trisphosphate receptors, and elevation of intracellular Ca2+. As free Ca2+ increases, type III adenylyl cyclase activity is attenuated by CaM kinase(s) and intracellular cAMP levels decrease. When cAMP levels drop below a threshold level, the inositol 1,4,5-trisphosphate receptor is dephosphorylated and Ca2+ is resequestered. This cycle is repeated if type III adenylyl cyclase is chronically exposed to an activator. This unique mechanism for generation of Ca2+ oscillations in cells is distinct from others documented in the literature.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Colforsina/farmacología , Glucagón/farmacología , Isoproterenol/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Canales de Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Carbacol/farmacología , Línea Celular , AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Isoquinolinas/farmacología , Riñón , Cinética , Modelos Biológicos , Oscilometría , Fosfatidilinositoles/metabolismo , Piperazinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Glucagón/efectos de los fármacos , Receptores de Glucagón/fisiología , Transducción de Señal , Factores de TiempoRESUMEN
Type III adenylyl cyclase is stimulated by beta-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca2+ and calmodulin. However, the enzyme is stimulated by Ca2+ and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein Gs (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by Gs-coupled receptors and intracellular Ca2+ in vivo. Surprisingly, intracellular Ca2+ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca2+, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca2+ was not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca2+ inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca2+ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Adenilil Ciclasas/metabolismo , Calcio/farmacología , Glucagón/farmacología , Isoenzimas/metabolismo , Isoproterenol/farmacología , Receptores de Glucagón/fisiología , Inhibidores de Adenilato Ciclasa , Animales , Encéfalo/enzimología , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Bovinos , Línea Celular , Membrana Celular/enzimología , Embrión de Mamíferos , Proteínas de Unión al GTP/metabolismo , Biblioteca de Genes , Guanilil Imidodifosfato/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoquinolinas/farmacología , Riñón , Cinética , Piperazinas/farmacología , Receptores de Glucagón/biosíntesis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Lisofosfolípidos , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Esfingosina/análogos & derivados , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Calcio/metabolismo , Calcio/fisiología , Adhesión Celular , División Celular/efectos de los fármacos , Membrana Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Fosfatidilinositoles/metabolismo , Transducción de Señal , Esfingosina/farmacologíaRESUMEN
Catalytic subunits of mammalian adenylyl cyclases have been proposed to contain 12 transmembrane domains, a property shared with some voltage-sensitive ion channels. Here we report that adenylyl cyclase activity in cerebellar neurons is synergistically stimulated by depolarizing agents and beta-adrenergic receptor activation. This phenomenon is Ca(2+)-independent and not attributable to Ca(2+)-stimulated adenylyl cyclase activity. Cholera toxin and forskolin also synergistically stimulate adenylyl cyclase activity in combination with depolarizing agents. We hypothesize that conformational changes in the catalytic subunit of the enzymes caused by changes in the membrane potential may enhance stimulation of adenylyl cyclases by the guanylyl nucleotide stimulatory protein. This novel mechanism for regulation of adenylyl cyclases generates robust cAMP signals that may contribute to various neuromodulatory events including some forms of neuroplasticity.
Asunto(s)
Adenilil Ciclasas/metabolismo , Neuronas/enzimología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/enzimología , Toxina del Cólera/farmacología , Colforsina/farmacología , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Activación Enzimática , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Activación del Canal Iónico , Isoproterenol/farmacología , Ratones , Neuronas/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , Veratridina/farmacologíaRESUMEN
Studies carried out with mammals and invertebrates suggest that Ca(2+)-sensitive adenylyl cyclases may be important for neuroplasticity. Long-term potentiation in the hippocampus requires increases in intracellular Ca2+ which are accompanied by elevated cyclic AMP (cAMP). Furthermore, activation of cAMP-dependent protein kinase is required for the late stage of long-term potentiation in the CA1 region of the hippocampus, which is also sensitive to inhibitors of transcription. Therefore, some forms of synaptic plasticity may require coordinate regulation of transcription by Ca2+ and cAMP. In this study, we demonstrate that the expression of type I adenylyl cyclase in HEK-293 cells allows Ca2+ to stimulate reporter gene activity mediated through the cAMP response element. Furthermore, simultaneous activation by Ca2+ and isoproterenol caused synergistic stimulation of transcription in HEK-293 cells and cultured neurons. We propose that Ca2+ and neurotransmitter stimulation of type I adenylyl cyclase may play a role in synaptic plasticity by generating optimal cAMP signals for regulation of transcription.
Asunto(s)
Adenilil Ciclasas/fisiología , Calcio/administración & dosificación , AMP Cíclico/fisiología , Elementos de Facilitación Genéticos , Isoproterenol/administración & dosificación , Receptores de AMP Cíclico/fisiología , Transducción de Señal , Transcripción Genética , Línea Celular , Cerebelo/fisiología , Corteza Cerebral/fisiología , Sinergismo Farmacológico , Regulación de la Expresión Génica , Humanos , Neuronas/fisiología , Sistemas de Mensajero SecundarioRESUMEN
The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vivo (Choi, E. J., Wong, S. T., Hinds, T. R. and Storm, D. R. (1992) J. Biol. Chem. 267, 12440-12442; Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268, 23766-23768). In this study, we examined the sensitivity of the type I adenylyl cyclase expressed in HEK-293 cells to beta-adrenergic agonists or glucagon when intracellular Ca2+ was elevated by Ca2+ ionophore or carbachol. Although previous studies have shown that this enzyme can be directly stimulated by activated Gs in vitro, we demonstrate that it is not stimulated by Gs-coupled receptors in vivo. However, the enzyme was stimulated by Gs-coupled receptors in vivo when it was activated by intracellular Ca2+. For example, the Ca2+ ionophore A23187 stimulated the enzyme 3 +/- 0.5-fold (n = 9) and isoproterenol alone did not stimulate the enzyme, but the combination of the two stimulated type I adenylyl cyclase 13 +/- 2-fold (n = 9) in vivo. Similarly, 500 nM glucagon alone did not stimulate the enzyme but the combination of A23187 and glucagon activated the enzyme 90 +/- 8-fold (n = 4). Synergistic stimulation of type I adenylyl cyclase activity was also obtained with combinations of carbachol and isoproterenol or glucagon. This phenomenon was not observed with a mutant enzyme that is insensitive to Ca2+ and calmodulin, suggesting that conformational changes caused by binding of calmodulin to the type I adenylyl cyclase enhance binding or coupling to activated Gs. These data illustrate that this adenylyl cyclase can couple Ca2+ and neurotransmitter signals to generate optimal cAMP levels, a property of the enzyme that may be important for its role in learning and memory in mammals.