RESUMEN
The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to induce the extraembryonic lineage downstream of BMP4 and other mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to ß-catenin (CTNNB1) activation, switching cell fate from trophoblast to mesoderm lineages. This study establishes PKC as a signaling boundary directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications.
Asunto(s)
Células Madre Pluripotentes , Proteína Quinasa C , Humanos , Proteína Quinasa C/metabolismo , Células Madre Embrionarias/metabolismo , Diferenciación Celular , Células Madre Pluripotentes/metabolismo , Mesodermo/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Proteína Morfogenética Ósea 4/metabolismoRESUMEN
The time course of smooth muscle contraction can be divided into two phases, the initial phase is associated with force development, whereas the sustained phase is associated with force maintenance. Cumulative evidence suggests that the two phases are regulated by different signaling pathways and that ρ-kinase (ROCK) and protein kinase C (PKC) play an important role in regulating isometric force in sustained contractions. Since the maintenance of sustained force is critical to the function of vascular smooth muscle, unraveling the complex mechanism of force maintenance is crucial for understanding the cell biology of the muscle. The present study examined the effects of ROCK and PKC on the level of phosphorylation of the 20-kD myosin light chain (MLC20) and isometric force during a sustained contraction. We used partial activation and inhibition of ROCK and PKC to reduce the isometric force by 50% of the maximal isometric force in fully activated muscle, Fmax. We then examined the level of MLC20 phosphorylation in each case. We found that in partially activated muscle the level of MLC20 phosphorylation required to maintain 50% Fmax was much lower than that required in muscles where 50% reduction in Fmax was achieved by partial inhibition of ROCK and PKC. The results can be explained by a model containing a contractile apparatus and a cytoskeletal scaffold where force generated by the contractile apparatus is transmitted to the extracellular domain through the cytoskeleton. The results indicate that ROCK and PKC play an important role in force transmission through the cytoskeleton.NEW & NOTEWORTHY The study supports a model that the maintenance of sustained force during a contraction of arterial smooth muscle is dependent on the intracellular transmission of force through the cytoskeleton and that ρ-kinase and protein kinase C plays an important role in the regulation of cytoskeletal integrity and its efficiency in force transmission.
Asunto(s)
Proteína Quinasa C , Quinasas Asociadas a rho , Animales , Ovinos , Proteína Quinasa C/metabolismo , Quinasas Asociadas a rho/metabolismo , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , Arterias Carótidas/metabolismo , FosforilaciónRESUMEN
Primary cilia, regulated via distinct signal transduction pathways, play crucial roles in various cellular behaviors. However, the full regulatory mechanism involved in primary cilia development during cellular differentiation is not fully understood, particularly for the sensory hair cells of the mammalian cochlea. In this study, we investigated the effects of the Rho-kinase inhibitor Y27632 and PKCα inhibitor GF109203X on primary cilia-related cell behavior in undifferentiated and differentiated temperature-sensitive mouse cochlear precursor hair cells (the conditionally immortalized US/VOT-E36 cell line). Our results indicate that treatment with Y27632 or GF109203X induced primary cilia elongation and tubulin acetylation in both differentiated and undifferentiated cells. Concomitant with cilia elongation, Y27632 treatment also increased Hook2 and cyclinD1 expression, while only Hook2 expression was increased after treatment with GF109203X. In the undifferentiated cells, we observed an increase in the number of S and G2/M stage cells and a decrease of G1 cells after treatment with Y27632, while the opposite was observed after treatment with GF109203X. Finally, while both treatments decreased oxidative stress, only treatment with Y27632, not GF109203X, induced cell cycle-dependent cell proliferation and cell migration.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Cóclea/citología , Proteína Quinasa C-alfa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Temperatura , Quinasas Asociadas a rho/antagonistas & inhibidores , Amidas/farmacología , Animales , Línea Celular , Cilios/metabolismo , Cóclea/efectos de los fármacos , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Ratones , Piridinas/farmacologíaRESUMEN
The mechanism that leads to a decrease in ß1-adrenergic receptor (ß1AR) expression in the failing heart remains uncertain. This study shows that cardiomyocyte ß1AR expression and isoproterenol responsiveness decrease in response to oxidative stress. Studies of mechanisms show that the redox-dependent decrease in ß1AR expression is uniquely prevented by carvedilol and not other ßAR ligands. Carvedilol also promotes the accumulation of N-terminally truncated ß1ARs that confer protection against doxorubicin-induced apoptosis in association with activation of protein kinase B. The redox-induced molecular controls for cardiomyocyte ß1ARs and pharmacologic properties of carvedilol identified in this study have important clinical and therapeutic implications.
RESUMEN
Streptozotocin (STZ)-induced chronic hyperglycemia has a detrimental effect on neurovascular coupling, linked to increased PKC-mediated phosphorylation and PKC isoform expression changes. Here, we sought to determine whether: 1) selective PKC-α/ß/γ inhibitor, GF109203X, could reverse the effects of chronic hyperglycemia on cerebrovascular reactivity; 2) pancreatic islet transplantation could prevent the development of cerebrovascular impairment seen in a rat model of Type 1 Diabetes. We studied the effect of GF109203X in diabetic (DM), non-diabetic (ND), and transplanted (TR) Lewis rats during either sciatic nerve stimulation (SNS) or the topical applications of the large-conductance Ca2+-operated K+(BKCa) channel opener, NS1619, or the K+ inward rectifier (Kir) channel agonist, KCl. Pial arteriole diameter changes were monitored using a closed cranial window in vivo microscopy technique. The pial arteriole dilatory response associated with SNS was decreased by ~45%, when comparing DM vs either ND or TR rats. Also, pial arteriolar dilations to topical KCl and NS1619 were largely attenuated in DM rats, but not in ND or TR animals. These responses were completely restored by the acute application of GF109203X to the brain surface. The PKC inhibitor had no effect on vascular responses in normoglycemic and TR animals. In conclusion, DM-associated chronic impairment of neurovascular coupling may be readily reversed by a PKC-α/ß/γ inhibitor or prevented via pancreatic islet transplantation. We believe that specific PCK isoforms (α/ß/γ) are mechanistically linked to the neurovascular uncoupling seen with hyperglycemia.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Trasplante de Islotes Pancreáticos , Acoplamiento Neurovascular , Proteína Quinasa C/antagonistas & inhibidores , Animales , Arteriolas/efectos de los fármacos , Arteriolas/fisiopatología , Bencimidazoles/farmacología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes/farmacología , Indoles/farmacología , Masculino , Maleimidas/farmacología , Neurotransmisores/farmacología , Acoplamiento Neurovascular/fisiología , Piamadre/efectos de los fármacos , Piamadre/fisiopatología , Cloruro de Potasio/farmacología , Proteína Quinasa C/metabolismo , Ratas Endogámicas Lew , Receptores KIR/agonistas , Receptores KIR/metabolismo , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiopatologíaRESUMEN
The goal of this in vitro study was to investigate the effect of mepivacaine on vasodilation induced by the ATP-sensitive potassium (KATP) channel opener levcromakalim in isolated endothelium-denuded rat aortas. The effects of mepivacaine and the KATP channel inhibitor glibenclamide, alone or in combination, on levcromakalim-induced vasodilation were assessed in the isolated aortas. The effects of mepivacaine or combined treatment with a protein kinase C (PKC) inhibitor, GF109203X, and mepivacaine on this vasodilation were also investigated. Levcromakalim concentration-response curves were generated for isolated aortas precontracted with phenylephrine or a PKC activator, phorbol 12,13-dibutyrate (PDBu). Further, the effects of mepivacaine and glibenclamide on levcromakalim-induced hyperpolarization were assessed in rat aortic vascular smooth muscle cells. Mepivacaine attenuated levcromakalim-induced vasodilation, whereas it had no effect on this vasodilation in isolated aortas pretreated with glibenclamide. Combined treatment with GF109203X and mepivacaine enhanced levcromakalim-induced vasodilation compared with pretreatment with mepivacaine alone. This vasodilation was attenuated in aortas precontracted with PDBu compared with those precontracted with phenylephrine. Mepivacaine and glibenclamide, alone or in combination, attenuated levcromakalim-induced membrane hyperpolarization. Taken together, these results suggest that mepivacaine attenuates vasodilation induced by KATP channels, which appears to be partly mediated by PKC.
RESUMEN
Spermine and spermidine are natural polyamines that are produced mainly via decarboxylation of l-ornithine and the sequential transfer of aminopropyl groups from S-adenosylmethionine to putrescine by spermidine synthase and spermine synthase. Spermine and spermidine interact with intracellular and extracellular acidic residues of different nature, including nucleic acids, phospholipids, acidic proteins, carboxyl- and sulfate-containing polysaccharides. Therefore, multiple actions have been suggested for these polycations, including modulation of the activity of ionic channels, protein synthesis, protein kinases, and cell proliferation/death, within others. In this review we summarize these neurochemical/neurophysiological/morphological findings, particularly those that have been implicated in the improving and deleterious effects of spermine and spermidine on learning and memory of naïve animals in shock-motivated and nonshock-motivated tasks, from a historical perspective. The interaction with the opioid system, the facilitation and disruption of morphine-induced reward and the effect of polyamines and putative polyamine antagonists on animal models of cognitive diseases, such as Alzheimer's, Huntington, acute neuroinflammation and brain trauma are also reviewed and discussed. The increased production of polyamines in Alzheimer's disease and the biphasic nature of the effects of polyamines on memory and on the NMDA receptor are also considered. In light of the current literature on polyamines, which include the description of an inborn error of the metabolism characterized by mild-to moderate mental retardation and polyamine metabolism alterations in suicide completers, we can anticipate that polyamine targets may be important for the development of novel strategies and approaches for understanding the etiopathogenesis of important central disorders and their pharmacological treatment.
Asunto(s)
Aprendizaje , Memoria , Espermidina/química , Espermina/química , Animales , Sitios de Unión , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/fisiopatología , Humanos , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Putrescina/química , Putrescina/metabolismo , Ratas , Espermidina/metabolismo , Espermina/metabolismoRESUMEN
Airway smooth muscle (ASM) in vivo is constantly subjected to oscillatory strain due to tidal breathing and deep inspirations. ASM contractility is known to be adversely affected by strains, especially those of large amplitudes. Based on the cross-bridge model of contraction, it is likely that strain impairs force generation by disrupting actomyosin cross-bridge interaction. There is also evidence that strain modulates muscle stiffness and force through induction of cytoskeletal remodeling. However, the molecular mechanism by which strain alters smooth muscle function is not entirely clear. Here, we examine the response of ASM to iso-velocity stretches to probe the components within the muscle preparation that give rise to different features in the force response. We found in ASM that force response to a ramp stretch showed a biphasic feature, with the initial phase associated with greater muscle stiffness compared with that in the later phase, and that the transition between the phases occurred at a critical strain of â¼3.3%. Only strains with amplitudes greater than the critical strain could lead to reduction in force and stiffness of the muscle in the subsequent stretches. The initial-phase stiffness was found to be linearly related to the degree of muscle activation, suggesting that the stiffness stems mainly from attached cross bridges. Both phases were affected by the degree of muscle activation and by inhibitors of myosin light-chain kinase, PKC, and Rho-kinase. Different responses due to different interventions suggest that cross-bridge and cytoskeletal stiffness is regulated differently by the kinases.
Asunto(s)
Contracción Muscular/fisiología , Fuerza Muscular/fisiología , Músculo Liso/fisiología , Mecánica Respiratoria/fisiología , Sistema Respiratorio , Animales , Citoesqueleto/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteína Quinasa C/metabolismo , Ovinos , Quinasas Asociadas a rho/metabolismoRESUMEN
Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca(2+)]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca(2+)]i-tension curve was higher than that of the KCl-induced [Ca(2+)]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca(2+)]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways.
Asunto(s)
Anestésicos Locales/farmacología , Aorta Torácica/efectos de los fármacos , Mepivacaína/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteína Quinasa C/fisiología , Quinasas Asociadas a rho/fisiología , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Calcio/fisiología , Endotelio Vascular/fisiología , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.
Asunto(s)
AMP Cíclico/metabolismo , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/genética , Hipófisis/efectos de los fármacos , Transducción de Señal , Somatostatina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Colforsina/farmacología , GMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Perciformes , Hipófisis/citología , Hipófisis/metabolismo , Cultivo Primario de Células , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Somatostatina/genética , Somatostatina/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Cholinergic interneurons, which provide the main source of acetylcholine (ACh) in the striatum, control the striatal local circuits and deeply involve in the pathogenesis of neurodegenerative diseases. Glycogen synthase kinase-3 (GSK-3) is a crucial kinase with diverse fundamental functions and accepted that deregulation of GSK-3 activity also plays important roles in diverse neurodegenerative diseases. However, up to now, there is no direct proof indicating whether GSK-3 activation is responsible for cholinergic dysfunction. In the present study, with combined intracerebroventricular injection of Wortmannin and GF-109203X, we activated GSK-3 and demonstrated the increased phosphorylation level of microtubule-associated protein tau and neurofilaments (NFs) in the rat striatum. The activated GSK-3 consequently decreased ACh level in the striatum as a result of the reduction of choline acetyltransferase (ChAT) activity. The alteration of ChAT activity was due to impaired ChAT distribution rather than its expression. Furthermore, we proved that cellular ChAT distribution was dependent on low phosphorylation level of NFs. Nevertheless, the cholinergic dysfunction in the striatum failed to induce significant neuronal number reduction. In summary, our data demonstrates the link between GSK-3 activation and cholinergic dysfunction in the striatum and provided beneficial evidence for the pathogenesis study of relevant neurodegenerative diseases.
Asunto(s)
Acetilcolina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Cuerpo Estriado/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Interneuronas/metabolismo , Animales , Western Blotting , Activación Enzimática , Inmunohistoquímica , Masculino , Microscopía Confocal , Enfermedades Neurodegenerativas/metabolismo , Fosforilación , Ratas , Ratas WistarRESUMEN
Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies.