RESUMO
Computer modelling has emerged as a particularly useful tool in understanding the physiology and pathophysiology of cardiac tissues. Models of ventricular, atrial and nodal tissue have evolved and include detailed ion channel kinetics and intercellular Ca(2+) handling. Purkinje fibre cells play a central role in the electrophysiology of the heart and in the genesis of cardiac arrhythmias. In this study, a new computational model has been constructed that incorporates the major membrane currents that have been isolated in recent experiments using Purkinje fibre cells. The model, which integrates mathematical models of human ion channels based on detailed biophysical studies of their kinetic and voltage-dependent properties, recapitulates distinct electrophysiological characteristics unique to Purkinje fibre cells compared to neighbouring ventricular myocytes. These characteristics include automaticity, hyperpolarized voltage range of the action potential plateau potential, and prolonged action potential duration. Simulations of selective ion channel blockade reproduce responses to pharmacological challenges characteristic of isolated Purkinje fibres in vitro, and importantly, the model predicts that Purkinje fibre cells are prone to severe arrhythmogenic activity in patients harbouring long QT syndrome 3 but much less so for other common forms of long QT. This new Purkinje cellular model can be a useful tool to study tissue-specific drug interactions and the effects of disease-related ion channel dysfunction on the cardiac conduction system.
Assuntos
Simulação por Computador , Síndrome do QT Longo/fisiopatologia , Modelos Neurológicos , Ramos Subendocárdicos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas/fisiopatologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Moduladores de Transporte de Membrana/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ramos Subendocárdicos/efeitos dos fármacosRESUMO
Hypereosinophilia may be associated with any of several underlying diseases. Atopy or allergic drug reactions are the most common causes, but infections with bacteria and parasites should also be considered in the differential diagnosis. When thorough evaluation of a patient with chronic hypereosinophilia fails to reveal an underlying disease, the diagnosis of idiopathic hypereosinophilic syndrome (HES) should be considered. We report a patient with unexplained persistent hypereosinophilia associated with a chronic pruritic rash and an underlying diagnosis of HES (lymphocytic variant).
Assuntos
Síndrome Hipereosinofílica/patologia , Ciclosporina/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Quimioterapia Combinada , Humanos , Síndrome Hipereosinofílica/tratamento farmacológico , Linfócitos , Masculino , Pessoa de Meia-Idade , Esteroides/uso terapêutico , Fatores de Tempo , Resultado do TratamentoRESUMO
Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na(+) channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca(2+))-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized Delta KPQ LQT-3 mutation but the link to altered Ca(2+) cycling related to mutant Na(+) channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca(2+) load and induction of spontaneous diastolic inward current (I(TI)) in myocytes expressing Delta KPQ Na(+) channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca(2+), and measurement of SR Ca(2+) content using a caffeine dump methodology. We compared the Ca(2+) content of Delta KPQ(+/-) myocytes displaying I(TI) to those without spontaneous diastolic activity and found that I(TI) induction correlates with higher sarcoplasmic reticulum (SR) Ca(2+). Both spontaneous diastolic I(TI) and underlying Ca(2+) waves are inhibited by ranolazine at concentrations that preferentially target I(NaL) during prolonged depolarization. Furthermore, ranolazine I(TI) inhibition is accompanied by a small but significant decrease in SR Ca(2+) content. Our results provide the first direct evidence that induction of diastolic transient inward current (I(TI)) in Delta KPQ(+/-) myocytes occurs under conditions of elevated SR Ca(2+) load.
Assuntos
Acetanilidas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Diástole/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Síndrome do QT Longo/fisiopatologia , Piperazinas/farmacologia , Animais , Relação Dose-Resposta a Droga , Camundongos , Mutação/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RanolazinaRESUMO
Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non-muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation-contraction coupling in the heart.
Assuntos
Canais de Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miocárdio/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares , Animais , Especificidade de Anticorpos , Expressão Gênica , Técnicas Imunoenzimáticas , Hibridização In Situ , Receptores de Inositol 1,4,5-Trifosfato , Músculo Liso Vascular/fisiologia , RNA Antissenso , RNA Mensageiro/genética , Ratos , Receptores de Superfície Celular/genética , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Transdução de SinaisRESUMO
The ryanodine receptor/junctional channel complex (JCC) forms the calcium release channel and foot structures of the sarcoplasmic reticulum. The JCC and the dihydropyridine (DHP) receptor in the transverse tubule are two of the major components involved in excitation-contraction (E-C) coupling in skeletal muscle. The DHP receptor is believed to serve as the voltage sensor in E-C coupling. Both the JCC and DHP receptor, as well as many skeletal muscle-specific contractile protein genes, are expressed in the BC3H1 muscle cell line. In the present study, we find that during differentiation of BC3H1 cells, induced by mitogen withdrawal, induction of the JCC and DHP receptor mRNAs is temporally similar to that of the skeletal muscle contractile protein genes alpha-tropomyosin and alpha-actin. Our data suggest that there is coordinate regulation of both the contractile protein genes (which have been studied in detail previously) and the genes encoding the calcium channels involved in E-C coupling. Induction of both calcium channels is accompanied by profound changes in BC3H1 cell morphology including the development of many components of mature skeletal muscle cells, despite lack of myoblast fusion. Visualized by electron microscopy, the JCC appears as "foot structures" located in the dyad junction between the plasmalemma and the sarcoplasmic reticulum of the BC3H1 cells. Development of foot structures is concomitant with JCC mRNA expression. Expression of the JCC and DHP receptor mRNAs and formation of the foot structures are inhibited specifically by fibroblast growth factor.
Assuntos
Canais de Cálcio/fisiologia , Substâncias de Crescimento/farmacologia , Junções Intercelulares/fisiologia , Músculos/citologia , Receptores Colinérgicos/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Linhagem Celular , Fatores de Crescimento de Fibroblastos/farmacologia , Junções Intercelulares/efeitos dos fármacos , Camundongos , Microscopia Eletrônica , Contração Muscular/efeitos dos fármacos , Músculos/metabolismo , Músculos/fisiologia , Músculos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Colinérgicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte , Microtúbulos/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo L , ATPases Transportadoras de Cálcio/isolamento & purificação , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , Imunofluorescência , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Proteínas Musculares/isolamento & purificação , Músculos/citologia , Músculos/embriologia , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/ultraestruturaRESUMO
Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.
Assuntos
Zíper de Leucina/fisiologia , Miocárdio/enzimologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Isoleucina/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/fisiologia , Fosfoproteínas Fosfatases/metabolismo , FosforilaçãoRESUMO
Excitation-contraction coupling in skeletal muscle requires the release of intracellular calcium ions (Ca2+) through ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. Half of the RyR1 channels are activated by voltage-dependent Ca2+ channels in the plasma membrane. In planar lipid bilayers, RyR1 channels exhibited simultaneous openings and closings, termed "coupled gating." Addition of the channel accessory protein FKBP12 induced coupled gating, and removal of FKBP12 uncoupled channels. Coupled gating provides a mechanism by which RyR1 channels that are not associated with voltage-dependent Ca2+ channels can be regulated.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Bicamadas Lipídicas , Músculo Esquelético/metabolismo , Polienos/farmacologia , Probabilidade , Coelhos , Proteínas Recombinantes/metabolismo , Rianodina/metabolismo , Sirolimo , Spodoptera , Proteínas de Ligação a TacrolimoRESUMO
Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn-/- mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i.
Assuntos
Canais de Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfotirosina/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Bicamadas Lipídicas , Ativação Linfocitária , Camundongos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-fyn , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
S100 protein is a calcium-binding protein found predominantly in the vertebrate nervous system. Genomic and complementary DNA probes were used in conjunction with a panel of rodent-human somatic cell hybrids to assign the gene for the beta subunit of S100 protein to the distal half of the long arm of human chromosome 21. This gene was identified as a candidate sequence which, when expressed in the trisomic state, may underlie the neurologic disturbances in Down syndrome.
Assuntos
Cromossomos Humanos Par 21 , Síndrome de Down/genética , Proteínas S100/genética , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Substâncias Macromoleculares , Hibridização de Ácido NucleicoRESUMO
Prenatal exposure to alcohol causes a wide range of deficits known as fetal alcohol spectrum disorders (FASDs). Many factors determine vulnerability to developmental alcohol exposure including timing and pattern of exposure, nutrition and genetics. Here, we characterized how a prevalent single nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene (val66met) modulates FASDs severity. This polymorphism disrupts BDNF's intracellular trafficking and activity-dependent secretion, and has been linked to increased incidence of neuropsychiatric disorders such as depression and anxiety. We hypothesized that developmental ethanol (EtOH) exposure more severely affects mice carrying this polymorphism. We used transgenic mice homozygous for either valine (BDNFval/val ) or methionine (BDNFmet/met ) in residue 68, equivalent to residue 66 in humans. To model EtOH exposure during the second and third trimesters of human pregnancy, we exposed mice to EtOH in vapor chambers during gestational days 12 to 19 and postnatal days 2 to 9. We found that EtOH exposure reduces cell layer volume in the dentate gyrus and the CA1 hippocampal regions of BDNFmet/met but not BDNFval/val mice during the juvenile period (postnatal day 15). During adulthood, EtOH exposure reduced anxiety-like behavior and disrupted trace fear conditioning in BDNFmet/met mice, with most effects observed in males. EtOH exposure reduced adult neurogenesis only in the ventral hippocampus of BDNFval/val male mice. These studies show that the BDNF val66met polymorphism modulates, in a complex manner, the effects of developmental EtOH exposure, and identify a novel genetic risk factor that may regulate FASDs severity in humans.
Assuntos
Ansiedade/genética , Fator Neurotrófico Derivado do Encéfalo/genética , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Hipocampo/efeitos dos fármacos , Mutação de Sentido Incorreto , Efeitos Tardios da Exposição Pré-Natal/genética , Animais , Condicionamento Clássico , Medo , Feminino , Hipocampo/crescimento & desenvolvimento , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
Human ovarian cancer, the leading cause of death from gynecologic malignancy, tends to remain localized to the peritoneal cavity until late in the disease. In established disease, ascitic fluid accumulates in the peritoneal cavity. We have previously demonstrated that this ascitic fluid is a potent source of in vitro mitogenic activity including at least one unique growth factor. We now report that the human ovarian adenocarcinoma line, HEY, can be induced to grow intraperitoneally in immunodeficient nude mice in the presence (23/28 mice), but not absence (0/21 mice) of ascitic fluid from ovarian cancer patients. Ascitic fluid from patients with benign disease did not have similar effects on intraperitoneal growth of HEY cells (1/15 mice). Once tumors were established by injections of exogenous ascitic fluid, they could progress in the absence of additional injections of ascitic fluid. The mice eventually developed ascitic fluid which contained potent growth factor activity, suggesting that the tumors eventually produced autologous growth factors. This nude mouse model provides a system to study the action of ovarian cancer growth factors on tumor growth in vivo and to evaluate preclinically, therapeutic approaches designed to counteract the activity of these growth factors.
Assuntos
Cistadenocarcinoma/fisiopatologia , Substâncias de Crescimento/fisiologia , Neoplasias Ovarianas/fisiopatologia , Animais , Anticorpos Monoclonais/imunologia , Ascite/patologia , Cistadenocarcinoma/patologia , Feminino , Substâncias de Crescimento/isolamento & purificação , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , Cavidade Peritoneal/patologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Imunossupressores/farmacologia , Músculo Liso/fisiologia , Polienos/farmacologia , Administração Oral , Sequência de Aminoácidos , Animais , Aorta/citologia , Sequência de Bases , Northern Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/fisiologia , Humanos , Immunoblotting , Imunossupressores/administração & dosagem , Dados de Sequência Molecular , Músculo Liso/citologia , Fases de Leitura Aberta , Fator de Crescimento Derivado de Plaquetas/fisiologia , Polienos/administração & dosagem , RNA/análise , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sirolimo , Suínos , Tacrolimo/farmacologia , Proteínas de Ligação a TacrolimoRESUMO
The molecular basis of human heart failure is unknown. Alterations in calcium homeostasis have been observed in failing human heart muscles. Intracellular calcium-release channels regulate the calcium flux required for muscle contraction. Two forms of intracellular calcium-release channels are expressed in the heart: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we showed that these two cardiac intracellular calcium release channels were regulated in opposite directions in failing human hearts. In the left ventricle, RyR mRNA levels were decreased by 31% (P < 0.025) whereas IP3R mRNA levels were increased by 123% (P < 0.005). In situ hybridization localized both RyR and IP3R mRNAs to human cardiac myocytes. The relative amounts of IP3 binding sites increased approximately 40% compared with ryanodine binding sites in the failing heart. RyR down-regulation could contribute to impaired contractility; IP3R up regulation may be a compensatory response providing an alternative pathway for mobilizing intracellular calcium release, possibly contributing to the increased diastolic tone associated with heart failure and the hypertrophic response of failing myocardium.
Assuntos
Canais de Cálcio/biossíntese , Cardiomiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas Musculares/biossíntese , Miocárdio/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Adolescente , Adulto , Northern Blotting , Canais de Cálcio/análise , Canais de Cálcio/metabolismo , Células Cultivadas , Sondas de DNA , Feminino , Expressão Gênica , Transplante de Coração , Homeostase , Humanos , Hibridização In Situ , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/metabolismo , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
Cytoplasmic free calcium ions (Ca2+) play a central role in excitation-contraction coupling of cardiac muscle. Abnormal Ca2+ handling has been implicated in systolic and diastolic dysfunction in patients with end-stage heart failure. The current study tests the hypothesis that expression of genes encoding proteins regulating myocardial Ca2+ homeostasis is altered in human heart failure. We analyzed RNA isolated from the left ventricular (LV) myocardium of 30 cardiac transplant recipients with end-stage heart failure (HF) and five organ donors (normal control), using cDNA probes specific for the cardiac dihydropyridine (DHP) receptor (the alpha 1 subunit of the DHP-sensitive Ca2+ channel) and cardiac calsequestrin of sarcoplasmic reticulum (SR). In addition, abundance of DHP binding sites was assessed by ligand binding techniques (n = 6 each for the patients and normal controls). There was no difference in the level of cardiac calsequestrin mRNA between the HF patients and normal controls. In contrast, the level of mRNA encoding the DHP receptor was decreased by 47% (P less than 0.001) in the LV myocardium from the patients with HF compared to the normal controls. The number of DHP binding sites was decreased by 35-48%. As reported previously, expression of the SR Ca(2+)-ATPase mRNA was also diminished by 50% (P less than 0.001) in the HF group. These data suggest that expression of the genes encoding the cardiac DHP receptor and SR Ca(2+)-ATPase is reduced in the LV myocardium from patients with HF. Altered expression of these genes may be related to abnormal Ca2+ handling in the failing myocardium, contributing to LV systolic and diastolic dysfunction in patients with end-stage heart failure.
Assuntos
Calsequestrina/genética , Expressão Gênica , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Receptores Nicotínicos/genética , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Canais de Cálcio , ATPases Transportadoras de Cálcio/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/análiseRESUMO
Expression of the dihydropyridine (DHP) receptor (alpha 1 subunit of L-type calcium channel) in heart is regulated by differentiation and innervation and is altered in congestive heart failure. We examined the transmembrane signaling pathways by which norepinephrine regulates DHP receptor expression in cultured neonatal rat ventricular myocytes. Using a 1.3-kb rat cardiac DHP receptor probe, and Northern analysis quantified by laser densitometry, we found that norepinephrine exposure produced a 2.2-fold increase in DHP receptor mRNA levels at 2 h followed by a decline to 50% of control at 4-48 h (P < 0.02). The alpha-adrenergic agonist phenylephrine and a phorbol ester produced a decline in mRNA levels (8-48 h). The beta-adrenergic agonist isoproterenol and 8-bromo-cAMP produced a transient increase in mRNA levels. After 24 h of exposure to isoproterenol, 3H-(+)PN200-110 binding sites increased from 410 +/- 8 to 539 +/- 39 fmol/mg (P < 0.05). The number of functional calcium channels, estimated by whole-cell voltage clamp experiments, was also increased after 24 h of exposure to isoproterenol. Peak current density (recordings performed in absence of isoproterenol) increased from -10.8 +/- 0.8 (n = 23) to -13.9 +/- 1.0 pA/pF (n = 27) (P < 0.01). Other characteristics of the calcium current (voltage for peak current, activation, and inactivation) were unchanged. Exposure for 48 h to phenylephrine produced a significant decline in peak current density (P < 0.01). We conclude that beta -adrenergic transmembrane signaling increases DHP receptor mRNA and number of functional calcium channels and that alpha - adrenergic transmembrane signaling produces a reciprocal effect. Regulation of cardiac calcium channel expression by adrenergic pathways may have physiological and pathophysiological importance.
Assuntos
Canais de Cálcio/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/metabolismo , Proteínas Musculares/biossíntese , Miocárdio/metabolismo , Receptores Colinérgicos/biossíntese , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Animais Recém-Nascidos , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Catecolaminas/farmacologia , Células Cultivadas , Eletrofisiologia , Ventrículos do Coração/citologia , Proteínas Musculares/genética , Miocárdio/citologia , Norepinefrina/farmacologia , Fenilefrina/farmacologia , RNA Mensageiro/análise , Ratos , Receptores Colinérgicos/genéticaRESUMO
We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Cardiomegalia/metabolismo , Infarto do Miocárdio/metabolismo , Fatores de Crescimento Neural/biossíntese , Norepinefrina/farmacologia , Proteínas S100 , Actinas/biossíntese , Animais , Fator Natriurético Atrial/biossíntese , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Células Cultivadas , Ecocardiografia , Regulação da Expressão Gênica , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Cadeias Pesadas de Miosina/biossíntese , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/isolamento & purificação , Receptores Adrenérgicos alfa 1/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Distribuição TecidualRESUMO
The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis.
Assuntos
Apoptose , Canais de Cálcio/deficiência , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos e Nucleares/deficiência , Linfócitos T/fisiologia , Cálcio/metabolismo , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Células Jurkat , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/química , Tapsigargina/farmacologia , Receptor fas/metabolismoRESUMO
Proliferation and cell cycle progression in response to growth factors require de novo protein synthesis. It has been proposed that binding of the eukaryotic translation initiation factor 4E (eIF-4E) to the inhibitory protein 4BP-1 blocks translation by preventing access of eIF-4G to the 5' cap of the mRNA. The signal for translation initiation is thought to involve phosphorylation of 4BP-1, which causes it to dissociate from eIF-4E and allows eIF-4G to localize to the 5' cap. It has been suggested that the ability of the macrolide antibiotic rapamycin to inhibit 4BP-1 phosphorylation is responsible for the potent antiproliferative property of this drug. We now show that rapamycin-resistant cells exhibited normal proliferation despite dephosphorylation of 4BP-1 that allows it to bind to eIF-4E. Moreover, despite rapamycin-induced dephosphorylation of 4BP-1, eIF-4E-eIF-4G complexes (eIF-4F) were still detected. In contrast, amino acid withdrawal, which caused a similar degree of 4BP-1 dephosphorylation, resulted in dissociation of the eIF-4E-eIF-4G complex. Thus, 4BP-1 dephosphorylation is not equivalent to eIF-4E inactivation and does not explain the antiproliferative property of rapamycin.
Assuntos
Proteínas de Transporte , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Animais , Células CHO , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Fator de Iniciação 4E em Eucariotos , Fator de Iniciação Eucariótico 4G , Fatores de Iniciação em Eucariotos , Substâncias Macromoleculares , Camundongos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Sirolimo/farmacologiaRESUMO
The potent antiproliferative activity of the macrolide antibiotic rapamycin is known to involve binding of the drug to its cytosolic receptor, FKBP12, and subsequent interaction with targets of rapamycin, resulting in inhibition of p70 S6 kinase (p70S6K). However, the downstream events that lead to inhibition of cell cycle progression remain to be elucidated. The antiproliferative effects of rapamycin are associated with prevention of mitogen-induced downregulation of the cyclin-dependent kinase inhibitor p27Kip1, suggesting that the latter may play an important role in the growth pathway targeted by rapamycin. Murine BC3H1 cells, selected for resistance to growth inhibition by rapamycin, exhibited an intact p70S6K pathway but had abnormally low p27 levels that were no longer responsive to mitogens or rapamycin. Fibroblasts and T lymphocytes from mice with a targeted disruption of the p27Kip1 gene had impaired growth-inhibitory responses to rapamycin. These results suggest that the ability to regulate p27Kip1 levels is important for rapamycin to exert its antiproliferative effects.