RESUMO
Hematological cancer survivors are highly vulnerable to cardiometabolic complications impacting long-term health status, quality of life and survival. Elevated risk of diabetes and cardiovascular disease arises not only from the effects of the cancers themselves, but also from the toxic effects of cancer therapies, and deconditioning arising from reduced physical activity levels. Regular physical activity can circumvent or reverse adverse effects on the heart, skeletal muscle, vasculature and blood cells, through a combination of systemic and molecular mechanisms. We review the link between hematological cancers and cardiometabolic risk with a focus on adult survivors, including the contributing mechanisms and discuss the potential for physical activity interventions, which may act to oppose the negative effects of both physical deconditioning and therapies (conventional and targeted) on metabolic and growth signaling (kinase) pathways in the heart and beyond. In this context, we focus particularly on strategies targeting reducing and breaking up sedentary time and provide recommendations for future research.
Assuntos
Cardiopatias/etiologia , Cardiopatias/fisiopatologia , Neoplasias Hematológicas/complicações , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Animais , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Gerenciamento Clínico , Metabolismo Energético , Testes de Função Cardíaca , Neoplasias Hematológicas/terapia , HumanosRESUMO
The physiological functions and cellular signaling of Class II phosphoinositide 3-kinases (PI3Ks) remain largely unknown. Platelets express two Class II PI3Ks: PI3KC2α and PI3KC2ß. PI3KC2α deficiency was recently reported to cause disruption of the internal membrane reserve structure of platelets (open canalicular system, OCS) that results in dysregulated platelet adhesion and impaired arterial thrombosis in vivo. Notably, these effects on platelets occurred despite normal agonist-induced 3-phosphorylated phosphoinositide (3-PPI) production and cellular activation in PI3KC2α-deficient platelets. However, the potential compensatory actions of PI3KC2ß in platelets have not yet been investigated. Here, we report the first mice deficient in both PI3KC2α and PI3KC2ß (no Class II PI3Ks in platelets) and reveal a nonredundant role for PI3KC2α in mouse platelet structure and function. Specifically, we show that the disrupted OCS and impaired thrombus stability observed in PI3KC2α-deficient platelets does not occur in PI3KC2ß-deficient platelets and is not exaggerated in platelets taken from mice deficient in both enzymes. Furthermore, detailed examination of 3-PPI production in platelets from this series of mice revealed no changes in either unactivated or activated platelets, including those with a complete lack of Class II PI3Ks. These findings indicate a nonredundant role for PI3KC2α in regulating platelet structure and function, and suggest that Class II PI3Ks do not significantly contribute to the acute agonist-induced production of 3-PPIs in these cells.
Assuntos
Plaquetas/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/deficiência , Trombose/sangue , Trombose/genética , Animais , Plaquetas/ultraestrutura , Classe II de Fosfatidilinositol 3-Quinases/genética , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Adesividade Plaquetária , Contagem de Plaquetas , Testes de Função PlaquetáriaRESUMO
The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by a-disintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10-mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 µg/mL), collagen (10 µg/mL), and convulxin (0.1 µg/mL) to shear stress (1000-10000 s(-1), 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Plaquetas/citologia , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína ADAM10 , Coagulação Sanguínea , Plaquetas/metabolismo , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Venenos de Crotalídeos/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Peptídeos/metabolismo , Trombose/metabolismoRESUMO
BACKGROUND: Current abacavir exposure has been reported to be associated with cardiovascular disease. Changes in platelet reactivity could plausibly explain the clinically observed pattern of association. OBJECTIVE: To determine if platelet reactivity changed following abacavir exposure and whether this effect was reversible on cessation of the drug. METHODS: In an open-label, interventional study abacavir, 600 mg daily, was added to a suppressive antiretroviral regimen in 20 adult HIV-positive men. Platelet function, estimated by the phosphorylated vasodilator-stimulated phosphoprotein (P-VASP) assay and through measurement of the expression and shedding of platelet-specific receptors, was assessed at baseline, following 15 days of abacavir and at completion of a 28-day washout period. RESULTS: The VASP-index decreased significantly from 79.1% [interquartile range (IQR) 47.8-87.6] to 32.6% (IQR -11.5-51.0) following 15 days of abacavir administration (P = 0.010), and returned to baseline levels following the washout period (day 43 =76.3%; IQR 40.7-92.3). There was no change in resting (prostaglandin E1 alone) P-VASP but a slight increase in P-VASP within stimulated platelets (prostaglandin E1 and adenosine diphosphate). Integrin ß3 levels decreased significantly [208.5 ng/ml (IQR 177.0-231.1) to 177.5 ng/ml (IQR 151.7-205) Pâ<â0.001] and there was a nonsignificant trend towards decreased soluble glycoprotein VI levels [baseline; 72.5 ng/ml (95% CI 58.3-81.5) vs. day 15; 45.0 ng/ml (95% CI 33.0-98.2) Pâ=â0.79]. CONCLUSION: Abacavir led to reversible changes in platelet function and structure. The clinical implications of these changes are uncertain; they may represent negative feedback mechanisms in response to an abacavir-associated prothrombotic state.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/efeitos adversos , Didesoxinucleosídeos/administração & dosagem , Didesoxinucleosídeos/efeitos adversos , Infecções por HIV/tratamento farmacológico , Ativação Plaquetária/efeitos dos fármacos , Adulto , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
In addition to playing a central role in normal haemostasis, platelets make important contributions to host inflammatory and immune responses to injury or infection. Under pathophysiological conditions where platelet function is not tightly controlled, platelets also play critical roles in pathogenic processes underlying cardiovascular disease, uncontrolled inflammation, coagulopathy and in tumour metastasis. Neutrophil extracellular traps (NETs) are webs of histone-modified nuclear material extruded from activated neutrophils during inflammatory responses and these degranulation events can be directly triggered by platelet/neutrophil engagement. Emerging research describes how NETs influence platelet function, particularly in the setting of infection and inflammation. Especially intriguing is the potential for platelet-driven coagulation to be modulated by NETs in plasma and interstitial spaces. These findings also reveal new perspectives related to improved therapy for venous thrombosis.
Assuntos
Plaquetas/fisiologia , Armadilhas Extracelulares/fisiologia , Infecções/sangue , Ativação de Neutrófilo/fisiologia , Neutrófilos/fisiologia , Animais , Coagulação Sanguínea , Plaquetas/metabolismo , Núcleo Celular/metabolismo , Hemostasia , Histonas/química , Humanos , Inflamação , Leucócitos/metabolismo , Neutrófilos/metabolismo , Trombose Venosa/metabolismoRESUMO
In addition to playing a central role in normal haemostasis, platelets make important contributions to host inflammatory and immune responses to injury or infection. Under pathophysiological conditions where platelet function is not tightly controlled, platelets also play critical roles in pathogenic processes underlying cardiovascular disease, uncontrolled inflammation, coagulopathy and in tumour metastasis. Neutrophil extracellular traps (NETs) are webs of histone-modified nuclear material extruded from activated neutrophils during inflammatory responses and these degranulation events can be directly triggered by platelet/neutrophil engagement. Emerging research describes how NETs influence platelet function, particularly in the setting of infection and inflammation. Especially intriguing is the potential for platelet-driven coagulation to be modulated by NETs in plasma and interstitial spaces. These findings also reveal new perspectives related to improved therapy for venous thrombosis.
RESUMO
INTRODUCTION: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. AIM: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. METHODS: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. RESULTS: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an â¼40-kDa Syk fragment. CONCLUSIONS: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.
Assuntos
Síndromes Mielodisplásicas/fisiopatologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Colágeno/fisiologia , Transdução de Sinais/fisiologia , Idoso , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Proteínas Tirosina Quinases/metabolismo , Quinase SykRESUMO
Apocynin, or a (myelo)peroxidase-derived product thereof, is a powerful inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Apocynin has also been shown to prevent aggregation of platelets in response to agonists such as collagen and thrombin. The aims of this study were to establish whether NADPH oxidase activity is required for aggregation of murine platelets to collagen and other agonists and whether the anti-aggregatory effects of apocynin are due to an inhibitory action against this enzyme. Washed platelets were isolated from male C57BL6 (wild-type), Nox2-deficient (Nox2(-/y )), and p47phox-deficient (p47phox(-/-)) mice for assessment of aggregation and NADPH oxidase subunit (Nox2, p47phox) expression. Collagen and U46619 elicited aggregation of murine platelets, and these responses were inhibited by apocynin at concentrations ≥100 µM. By contrast, aggregations to a direct protein kinase C activator, phorbol-12,13-dibutyrate, were insensitive to apocynin. Immunoblotting of platelet protein homogenates from wild-type mice with anti-Nox2 or p47phox antibodies revealed strong bands at 58 and 50 kDa, respectively. While expression of these immunoreactive bands was greatly diminished in platelets from Nox2(-/y ) and p47phox(-/-) mice, collagen still elicited aggregations that were similar to those observed in platelets from wild-types. Moreover, apocynin was an equally effective inhibitor of aggregation in platelets from all three mouse strains. In conclusion, these data suggest that NADPH oxidase-derived reactive oxygen species play no role in the aggregation response of washed murine platelets to collagen. Thus, our observation that apocynin is a powerful inhibitor of platelet aggregation raises further questions about the selectivity of this drug as an NADPH oxidase inhibitor.
Assuntos
Acetofenonas/farmacologia , Plaquetas/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Células Cultivadas , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genéticaRESUMO
Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.
Assuntos
Plaquetas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Fator de von Willebrand/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Inibidores Enzimáticos/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Peptídeos/genética , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas/genética , Estrutura Terciária de Proteína , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
Several recent findings point to an important role for redox regulation of platelet responses to collagen involving the receptor, glycoprotein (GP)VI. First, the antioxidant dietary compound, quercetin, was shown to inhibit GPVI-dependent platelet activation and signaling responses to collagen. Second, collagen increased platelet production of the oxygen radical, superoxide anion (O2-), mediated by the multi-subunit enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase. In that case, O2- was implicated in regulating not initial aggregation, but collagen-induced thrombus stabilization involving release of ADP. Third, our laboratory showed that an unpaired thiol in the GPVI cytoplasmic tail undergoes rapid oxidation to form GPVI homodimers following ligand binding, preceding GPVI signaling and ectodomain metalloproteolysis, and indicating formation of an oxidative submembranous environment in activated platelets. This review examines receptor/redox regulation in other cells, and relevance to the pathophysiological function of GPVI and other platelet receptors initiating thrombus formation in haemostasis or thrombotic diseases such as heart attack and stroke.
Assuntos
Plaquetas/metabolismo , Antígenos CD36/metabolismo , NADPH Oxidases/metabolismo , Transdução de Sinais , Superóxidos/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Colágeno/metabolismo , Citoplasma/metabolismo , Dimerização , Cardiopatias/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Oxirredução , Agregação Plaquetária , Estrutura Terciária de Proteína , Quercetina/metabolismo , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Trombose/metabolismoRESUMO
Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.
Assuntos
Plaquetas/metabolismo , Membrana Celular/metabolismo , Adesividade Plaquetária , Glicoproteínas da Membrana de Plaquetas/agonistas , Transdução de Sinais , Trombose/metabolismo , Substituição de Aminoácidos , Vasos Sanguíneos/lesões , Vasos Sanguíneos/metabolismo , Membrana Celular/genética , Colágeno/genética , Colágeno/metabolismo , Venenos de Crotalídeos/farmacologia , Dimerização , Dipeptídeos/farmacologia , Dissulfetos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C , Ligantes , Metaloproteases/antagonistas & inibidores , Metaloproteases/genética , Metaloproteases/metabolismo , Mutação de Sentido Incorreto , Oxirredução/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinase Syk , Trombose/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/fisiologiaRESUMO
P2Y1 [P2 (purinergic type-2)-receptor 1] is a G-protein-coupled ADP receptor that regulates platelet activation and ADP-induced Ca2+ signalling. Studies using P2Y1-knockout mice, G(q)-deficient mice or P2Y1-selective inhibitors have previously identified a key role for P2Y1 in pathophysiological thrombus formation at high shear stress. We provide evidence that a positively charged juxtamembrane sequence within the cytoplasmic C-terminal tail of P2Y1 can bind directly to the cytosolic regulatory protein calmodulin. Deletion by mutagenesis of the calmodulin-binding domain of P2Y1 inhibits intracellular Ca2+ flux in transfected cells. These results suggest that the interaction of calmodulin with the P2Y1 C-terminal tail may regulate P2Y1-dependent platelet aggregation.
Assuntos
Plaquetas/metabolismo , Calmodulina/metabolismo , Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Sinalização do Cálcio , Calmodulina/antagonistas & inibidores , Humanos , Ativação Plaquetária , Agregação Plaquetária/fisiologia , Ligação Proteica , Receptores Purinérgicos P2Y1 , Sulfonamidas/farmacologiaRESUMO
Expression of the wild type alpha subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active G alpha q subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac G alpha q overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose , Tamanho Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Addition of ATP to neonatal rat cardiomyocytes has been reported to inhibit hypertrophic growth responses, even though G(q)-coupled receptors are activated. In the current study, we investigated hypertrophic responses to activation of G(q)-coupled-purinergic receptors on cardiomyocytes using UTP as an alternative agonist to ATP. UTP (100 microM) activated phospholipase C via G(q) similarly to ATP, and responses to the two agonists were not additive. Similarly, UTP and ATP both induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), while having little effect on p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase. However, addition of UTP (100 microM) to cardiomyocytes caused hypertrophic growth indicated by increased protein content without DNA synthesis. ATP (100 microM) caused no increase in protein. We conclude that activation of purinergic receptors on neonatal cardiomyocytes initiates hypertrophic signaling pathways, but that prolonged exposure to ATP, but not UTP, has growth-inhibitory effects.
Assuntos
Trifosfato de Adenosina/farmacologia , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , Uridina Trifosfato/farmacologia , Animais , Animais Recém-Nascidos , Hipertrofia , Isoenzimas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfolipase C beta , Fosfolipase C gama , Antagonistas de Receptores Purinérgicos P1 , Antagonistas do Receptor Purinérgico P2 , Ratos , Ratos Sprague-Dawley , Fosfolipases Tipo C/metabolismoRESUMO
Activation of G(q)-coupled alpha(1)-adrenergic receptors leads to hypertrophic growth of neonatal rat ventricular cardiomyocytes that is associated with increased expression of hypertrophy-related genes, including atrial natriuretic peptide (ANP) and myosin light chain-2 (MLC), as well as increased ribosome synthesis. The role of inositol phosphates in signaling pathways involved in these changes in gene expression was examined by overexpressing inositol phosphate-metabolizing enzymes and determining effects on ANP, MLC, and 45 S ribosomal gene expression following co-transfection of appropriate reporter gene constructs. Overexpression of enzymes that metabolize inositol 1,4,5-trisphosphate did not reduce ANP or MLC responses, but overexpression of the enzyme primarily responsible for metabolism of inositol 4,5-bisphosphate (Ins(1,4)P(2)), inositol polyphosphate 1-phosphatase (INPP), reduced ANP and MLC responses associated with alpha(1)-adrenergic receptor-mediated hypertrophy. Similarly overexpressed INPP reduced ANP and MLC responses associated with contraction-induced hypertrophy. In addition, overexpression of INPP reduced the increase in ribosomal DNA transcription associated with both hypertrophic models. Hypertrophied cells from both cell models as well as ventricular tissue from mouse hearts hypertrophied by pressure overload in vivo contained heightened levels of Ins(1,4)P(2), suggesting reduced INPP activity in three different models of hypertrophy. These studies provide evidence for an involvement of Ins(1,4)P(2) in hypertrophic signaling pathways in ventricular myocytes.