RESUMO
Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated ß1 adrenergic receptor (ß1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the ß1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. ß1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the ß1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.
Assuntos
Tamanho Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Catecolaminas/farmacologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Proteína 1 Homóloga a Discs-Large , Transferência Ressonante de Energia de Fluorescência , Coração/efeitos dos fármacos , Coração/fisiologia , Técnicas In Vitro , Isoproterenol/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Adrenérgicos beta 1/genética , Rolipram/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Beta-adrenergic receptor induces cAMP/Protein kinase A (PKA) activation to regulate cardiac contraction. Using real-time fluorescence resonance energy transfer imaging for highly sensitive detection of cAMP and PKA activities, we show two distinct phases in isoproterenol dose-dependent responses in cardiomyocytes: a transient and dose-dependent increase in cAMP and PKA activities at lower concentrations from 10(-12) to 10(-8) M; and a saturated initial increases at higher concentrations from 10(-8) to 10(-5) M followed by a rapid decrease to different levels that were later sustained in a dose-dependent manner. The dose-dependent temporal responses are patterned by equilibrium between receptor-activated adenylyl cyclase (AC) and phosphodiesterase (PDE). At lower concentrations, cAMP is produced in an agonist dose-dependent manner with AC as a rate-limiting factor. However, the cAMP activities are confined within local domains for phosphorylation of PDE isoforms in the receptor complex but not for phosphorylation of phospholamban and troponin I. At higher concentrations, isoproterenol promotes a dose-dependent selective dissociation of PDE4D but not ACVI from the receptor complex, which shifts the equilibrium between AC and PDE. This shifted balance leads to sustained cAMP accumulation and diffusion for PKA phosphorylation of phospholamban and troponin I, and for myocyte contraction. Pharmacological inhibition or overexpression of either ACVI or PDE4D8 disrupts the balance and shapes the temporal responses in cAMP accumulation. Together, our data reveal a new paradigm for adrenergic agonist dose-dependent cAMP/PKA activities for substrate-specific phosphorylation dictated by dual regulation of AC and PDE in cardiomyocytes.
Assuntos
Adenilil Ciclases/metabolismo , Miócitos Cardíacos/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Agonistas Adrenérgicos/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Contração Miocárdica/efeitos dos fármacos , Fosforilação , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild-type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real-time changes in intracellular ([Ca(2+)](i)) and protein kinase A (PKA) activity were measured by fura-2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTR(inh)-172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyocytes activated L-type Ca(2+) channels, leading to a transient increase of [Ca(2+)](i) and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) and Ca(2+)-activated Cl(-) channels (CaCC) because simultaneous addition of myristoylated-autocamtide-2-related inhibitory peptide or niflumic acid and CFTR(inh)-172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Transferência Ressonante de Energia de Fluorescência , Frequência Cardíaca/fisiologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , FosforilaçãoRESUMO
Progressive decrease in neuronal function is an established feature of Alzheimer's disease (AD). Previous studies have shown that amyloid beta (Abeta) peptide induces acute increase in spontaneous synaptic activity accompanied by neurotoxicity, and Abeta induces excitotoxic neuronal death by increasing calcium influx mediated by hyperactive alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. An in vivo study has revealed subpopulations of hyperactive neurons near Abeta plaques in mutant amyloid precursor protein (APP)-transgenic animal model of Alzheimer's disease (AD) that can be normalized by an AMPA receptor antagonist. In the present study, we aim to determine whether soluble Abeta acutely induces hyperactivity of AMPA receptors by a mechanism involving beta(2) adrenergic receptor (beta(2)AR). We found that the soluble Abeta binds to beta(2)AR, and the extracellular N terminus of beta(2)AR is critical for the binding. The binding is required to induce G-protein/cAMP/protein kinase A (PKA) signaling, which controls PKA-dependent phosphorylation of GluR1 and beta(2)AR, and AMPA receptor-mediated excitatory postsynaptic currents (EPSCs). beta(2)AR and GluR1 also form a complex comprising postsynaptic density protein 95 (PSD95), PKA and its anchor AKAP150, and protein phosphotase 2A (PP2A). Both the third intracellular (i3) loop and C terminus of beta(2)AR are required for the beta(2)AR/AMPA receptor complex. Abeta acutely induces PKA phosphorylation of GluR1 in the complex without affecting the association between two receptors. The present study reveals that non-neurotransmitter Abeta has a binding capacity to beta(2)AR and induces PKA-dependent hyperactivity in AMPA receptors.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de AMPA/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Linhagem Celular , Células Cultivadas , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoprecipitação , Camundongos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteína Fosfatase 2/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon beta-adrenergic stimulation under stress. Both beta(1)- and beta(2)-adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that beta(2)ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the beta(2)AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the beta(2)AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the beta(2)AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of beta(2)AR. Our data underscore the complex regulation of intracellular cAMP by beta(2)AR-associated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Miócitos Cardíacos/citologia , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/fisiologia , Animais , Animais Recém-Nascidos , Sequência de Bases , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas , RatosRESUMO
Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G(s) and G(i) proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G(i) inhibitor pertussis toxin (PTX). Further studies revealed that activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/G(i) coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (> or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR blocked receptor internalization and coupling to G(i) proteins, probably by preventing the receptor's transportation to access G(i) proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G(i)-specific inhibitor, G(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Camundongos , Células Musculares/metabolismo , Mutagênese Sítio-Dirigida , Toxina Pertussis/farmacologia , Fosforilação , Ligação ProteicaRESUMO
cAMP/protein kinase (PK)A activation represents a key signaling mechanism for neurohormonal stimulation of diversified physiological processes. Using real-time, fluorescence resonance energy transfer-based imaging of PKA activity in neonatal cardiac myocytes, we report that sustained activation of PKA induced by beta-adrenoceptor (betaAR) dictates signaling propagation for substrate phosphorylation and myocyte contraction. Activation of betaARs in wild-type myocytes induces strong and sustained PKA activities, which are rapidly attenuated on washing away agonist or adding antagonist to the cells. The sustained PKA activities promote signaling propagation to the sarcoplasmic reticulum for phosphorylation of phospholamban and increases in myocyte contraction. Addition of antagonist after betaAR stimulation significantly attenuates PKA phosphorylation of phospholamban and rapidly reduces contraction rate increases. Moreover, stimulation of beta(1)AR subtype induces PKA activities similar to those in wild-type cells. In contrast, stimulation of beta(2)AR subtype induces strong initial activation of PKA similar to those induced by beta(1)AR; however, the activities are rapidly decreased to baseline levels. The transient PKA activities are sufficient for phosphorylation of the overexpressed beta(2)ARs under agonist stimulation, but not phospholamban. Further analysis reveals that phosphodiesterase 4 is the major family that shapes PKA activities under betaAR stimulation. Inhibition of phosphodiesterase 4 extends beta(2)AR-induced PKA activities, promotes PKA phosphorylation of phospholamban, and ultimately enhances myocyte contraction responses. Together, our data have revealed insights into kinetics of PKA activities in signaling propagation under neurohormonal stimulation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/enzimologia , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/fisiologia , Antagonistas Adrenérgicos/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas de Receptores Adrenérgicos beta 2 , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Activation of the beta adrenergic receptor (betaAR) induces a tightly controlled cAMP/protein kinase A (PKA) activity to ensure an agonist dose-dependent and saturable contraction response in animal heart. We have found that stimulation of beta(1)AR by isoproterenol induces maximal contraction responses at the dose of 1 microM in cardiac myocytes; however, cAMP accumulation continues to increase with higher agonist concentrations. Dose-dependent cAMP accumulation is tightly controlled by negative regulator phosphodiesterase 4 (PDE4) that hydrolyzes cAMP. At 1 nM isoproterenol, cAMP accumulation is minimal because of the hydrolysis of cAMP by PDE4, which leads to a small increase in PKA phosphorylation of phospholamban and troponin I (TnI), and contraction responses. Inhibition of PDE4 activity with rolipram enhances cAMP accumulation, yields maximal PKA phosphorylation of phospholamban and TnI, and myocyte contraction responses. In contrast, at 10 microM isoproterenol, despite the negative effect of PDE4, cAMP accumulation is sufficient for maximal PKA phosphorylation of phospholamban and TnI. Inhibition of PDE4 with rolipram enhances cAMP accumulation, but not PKA phosphorylation and contraction responses. It is interesting that activities of both PKA and protein phosphatase 2A (PP2A) are enhanced under beta(1)AR activation with 10 microM isoproterenol, and PP2A is recruited to PKA/A kinase-anchoring protein complex. Inhibition of PP2A with okadaic acid further enhances the phosphorylation of phospholamban and TnI as well as contraction responses induced by 10 microM isoproterenol. Therefore, PP2A plays a key role in limiting PKA phosphorylation of phospholamban and TnI for myocyte contraction responses under beta(1)AR stimulation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Miocárdio/metabolismo , Proteína Fosfatase 2/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais , Animais , Western Blotting , AMP Cíclico/metabolismo , Ativação Enzimática , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Miocárdio/citologia , Miocárdio/enzimologia , Inibidores de Fosfodiesterase/farmacologia , FosforilaçãoRESUMO
Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous beta(2)AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated beta(2)AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated beta(2)AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated beta(2)AR coupling to G(s) proteins, only Epi-activated receptors were capable of coupling to G(i) proteins. Subsequent studies showed that the Epi-activated beta(2)AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and G(i) coupling. In contrast, the NE-activated beta(2)ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to G(i). Moreover, inhibiting beta(2)AR phosphorylation by betaARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and G(i) coupling. Together, our data revealed that distinct temporal phosphorylation of beta(2)AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent beta(2)AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.
Assuntos
Epinefrina/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Miócitos Cardíacos/metabolismo , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Agonistas alfa-Adrenérgicos , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Epinefrina/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Norepinefrina/metabolismo , Ácido Okadáico/farmacologia , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.
Assuntos
Actinas/química , Citoesqueleto/metabolismo , Músculos/citologia , Fosfolipase D/fisiologia , 1-Butanol/química , Actinas/metabolismo , Animais , Arginina Vasopressina/química , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Regulação para Baixo , Immunoblotting , Microscopia de Fluorescência , Músculo Esquelético/citologia , Miogenina/metabolismo , Faloidina/farmacologia , Fenótipo , Ácidos Fosfatídicos/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase D/metabolismo , Plasmídeos/metabolismo , Isoformas de Proteínas , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We have previously shown that myogenesis induction by Arg8-vasopressin (AVP) in L6 rat myoblasts involves a sustained stimulation of type 4 cAMP-phosphodiesterase. In this model, we observed that a transient cAMP generation occurs in the minutes following AVP addition. Evidence suggests that cAMP generation is due to the prostaglandins produced in response to AVP binding to V1a receptors and subsequent activation of phospholipase A2. The early cAMP increase was effective in activating cAMP-dependent protein kinase (PKA) and increasing phosphorylation of CREB transcription factor. Inhibition of PKA by compound H89 prior to AVP addition led to a significant reduction of expression of the differentiation marker creatine kinase, whereas H89 added 1-5 h after AVP had no significant effect. Furthermore, PKA inhibition 24 h after the beginning of AVP treatment potentiated differentiation. This shows that both an early activation and a later down-regulation of the cAMP pathway are required for AVP induction of myogenesis. Because phosphodiesterase PDE4D3 overexpressed in L6 cells lost its ability to potentiate AVP-induced differentiation when mutated and rendered insensitive to PKA phosphorylation and activation, we hypothesize that the early cAMP increase is required to trigger the down-regulation of cAMP pathway through stimulation of phosphodiesterase.
Assuntos
Diferenciação Celular , AMP Cíclico/metabolismo , Mioblastos/citologia , Sulfonamidas , Animais , Arginina Vasopressina/farmacologia , Sequência de Bases , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA , Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Mioblastos/enzimologia , Mioblastos/metabolismo , Ratos , Transdução de SinaisRESUMO
Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Compartimento Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura Livres de Soro , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Isoenzimas/genética , Isoenzimas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosforilação , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca(2+) concentration ([Ca(2+)](o)) produced membrane hyperpolarization and a concentration-dependent increase of [Ca(2+)](i) due to Ca(2+) influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca(2+)-sensing receptor. [Ca(2+)](o)-induced [Ca(2+)](i) increase was inhibited by Ca(2+) channel inhibitors and appeared to be modulated by several kinase activities. [Ca(2+)](i) increase was potentiated by depletion of intracellular Ca(2+) stores and depressed by inactivation of the Na(+)/Ca(2+) exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca(2+)](i) increase in L6-C5 cells, was not modified by high [Ca(2+)](o). On the contrary, AVP potentiated the [Ca(2+)](i) increase in the presence of elevated [Ca(2+)](o). Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca(2+)](o), and the amplitude of the responses was correlated with the myogenic potential of the cells.