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1.
NMR Biomed ; 27(9): 1085-93, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25060359

RESUMO

Cerebrovascular abnormality is frequently accompanied by cognitive dysfunctions, such as dementia. Antibodies against the α1 -adrenoceptor (α1 -AR) can be found in patients with Alzheimer's disease with cerebrovascular disease, and have been shown to affect the larger vessels of the brain in rodents. However, the impact of α1 -AR antibodies on the cerebral vasculature remains unclear. In the present study, we established a neuroimaging method to measure the relative cerebral blood volume (rCBV) in small rodents with the ultimate goal to detect changes in blood vessel density and/or vessel size induced by α1 -AR antibodies. For this purpose, mapping of R2 * and R2 was performed using MRI at 9.4 T, before and after the injection of intravascular iron oxide particles (ferumoxytol). The change in the transverse relaxation rates (ΔR2 *, ΔR2 ) showed a significant rCBV decrease in the cerebrum, cortex and hippocampus of rats (except hippocampal ΔR2 ), which was more pronounced for ΔR2 * than for ΔR2 . Immunohistological analyses confirmed that the α1 -AR antibody induced blood vessel deficiencies. Our findings support the hypothesis that α1 -AR antibodies lead to cerebral vessel damage throughout the brain, which can be monitored by MRI-derived rCBV, a non-invasive neuroimaging method. This demonstrates the value of rCBV estimation by ferumoxytol-enhanced MRI at 9.4 T, and further underlines the significance of this antibody in brain diseases involving vasculature impairments, such as dementia.


Assuntos
Autoanticorpos/imunologia , Volume Sanguíneo/imunologia , Circulação Cerebrovascular/imunologia , Óxido Ferroso-Férrico , Angiografia por Ressonância Magnética/métodos , Receptores Adrenérgicos alfa 1/imunologia , Animais , Velocidade do Fluxo Sanguíneo/imunologia , Determinação do Volume Sanguíneo/métodos , Meios de Contraste , Masculino , Microvasos/imunologia , Microvasos/patologia , Ratos , Ratos Wistar
2.
Mol Cell Biochem ; 333(1-2): 233-42, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19685172

RESUMO

Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the alpha(1)-adrenergic receptor (alpha(1)-AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (anti-alpha(1)) was generated against the second extracellular loop of the alpha(1)-AR that bound to its peptide epitope with high affinity (K (D) approximately 50 nM). We studied anti-alpha(1) effects on intracellular calcium (Ca(i)), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-alpha(1) induced pronounced but transient increases in Ca(i) in CHO cells expressing the human alpha(1)-AR (CHO-alpha(1)) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-alpha(1) on Ca(i). However, preincubation with the antibody attenuated the effect of the alpha(1)-AR antagonist prazosin. In neonatal cardiomyocytes anti-alpha(1) induced a robust phosphorylation of a 15-kDa protein that is involved in alpha(1)-AR signalling. Our data support the notion that elevation of Ca(i) is a general feature of agonistic antibodies' action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the alpha(1)-AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action.


Assuntos
Anticorpos Monoclonais/farmacologia , Sinalização do Cálcio , Miócitos Cardíacos/química , Proteínas/metabolismo , Receptores Adrenérgicos alfa 1/fisiologia , Agonistas de Receptores Adrenérgicos alfa 1 , Animais , Animais Recém-Nascidos , Autoanticorpos , Células CHO , Cricetinae , Cricetulus , Humanos , Fosforilação , Ratos , Receptores Adrenérgicos alfa 1/genética , Transfecção
3.
J Mol Med (Berl) ; 85(12): 1405-12, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17717642

RESUMO

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cardiotônicos/farmacologia , Permeabilidade da Membrana Celular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Leves de Miosina/farmacologia , Fragmentos de Peptídeos/farmacologia , Miosinas Ventriculares/farmacologia , Animais , Cardiotônicos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Estimulação Elétrica , Humanos , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos WKY , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Miosinas Ventriculares/metabolismo
4.
Front Biosci (Landmark Ed) ; 23(11): 2082-2089, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29772547

RESUMO

Agonistic autoantibodies (agAAB) for alpha-1 adrenoceptor were found in approx. 50% of patients with Alzheimer's disease. These antibodies activate the receptor and trigger the signal cascades similarly to how natural agonists do. The agAAB bond to the receptor is persistent and prolonged. This results in a non-physiological elevation of intracellular calcium. An animal model has shown that agAAB causes macrovascular and microvascular impairment in the vessels of the brain. Reduction in blood flow and the density of intact vessels was significantly demonstrated. The agAAB was removed through immunoadsorption in a small cohort of patients with Alzheimer's disease. Subsequent follow-up observations over 12-18 months noted stabilization of cognition levels.


Assuntos
Doença de Alzheimer/imunologia , Autoanticorpos/imunologia , Demência/imunologia , Receptores Adrenérgicos alfa 1/imunologia , Doença de Alzheimer/sangue , Doença de Alzheimer/metabolismo , Animais , Autoanticorpos/sangue , Encéfalo/irrigação sanguínea , Encéfalo/imunologia , Encéfalo/metabolismo , Demência/sangue , Demência/metabolismo , Modelos Animais de Doenças , Humanos
5.
FASEB J ; 20(10): 1653-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16873888

RESUMO

The causal relationship between obesity and heart failure is broadly acknowledged; however, the pathophysiological mechanisms involved remain unclear. In this study we investigated whether human adipocytes secrete cardioactive substances that may affect cardiomyocyte contractility. We cultivated adipocytes obtained from human white adipose tissue and incubated isolated rat adult cardiomyocytes with adipocyte-conditioned or control medium. This is the first report to demonstrate that human adipocytes exhibit cardiodepressant activity with a direct and acute effect on cardiomyocyte contraction. This adipocyte-derived negative inotropic activity directly depresses shortening amplitude as well as intracellular systolic peak Ca2+ in cardiomyocytes within a few minutes. The adipocyte-derived cardiodepressant activity was dose-dependent and was completely blunted by heating or by trypsin digestion. Filtration of adipocyte-conditioned medium based on molecular mass characterized the cardiodepressant activity at between 10 and 30 kDa. In summary, adipose tissue exerts highly potent activity with an acute depressant effect directly on cardiomyocytes, which may well contribute to increased heart failure risk in overweight patients.


Assuntos
Adipócitos/fisiologia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Adipócitos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/análise , Insuficiência Cardíaca/etiologia , Humanos , Peso Molecular , Obesidade/complicações , Comunicação Parácrina , Ratos
6.
FASEB J ; 20(7): 865-73, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16675844

RESUMO

In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart.


Assuntos
Coração/fisiologia , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Actinas , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica , Terapia Genética , Humanos , Masculino , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Ligação Proteica , Ratos , Ratos Endogâmicos WKY
7.
FASEB J ; 19(6): 503-11, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15791000

RESUMO

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor substances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to approximately 50%. This suggests that the Ca2+-calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the nucleus upon vasopressin stimulation. Inhibition of protein kinase C (PKC) isoforms by bisindolylmaleimide had no significant influence on human ALC-1 promoter activity. Thus, our results demonstrate a dominant role of Ca2+-calmodulin-dependent signaling pathways in the regulation of human ALC-1 expression.


Assuntos
Cálcio/farmacologia , Calmodulina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas/genética , Animais , Transporte Biológico/efeitos dos fármacos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/análise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Embrião de Mamíferos , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Genótipo , Coração , Humanos , Immunoblotting , Isoenzimas/análise , Luciferases/genética , Miócitos Cardíacos , Fatores de Transcrição NFATC/análise , Fatores de Transcrição NFATC/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Proteínas Recombinantes de Fusão , Transfecção , Vasopressinas/farmacologia
8.
Ther Apher Dial ; 20(5): 523-529, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27096216

RESUMO

Dementia has been shown to be associated with agonistic autoantibodies. The deleterious action of autoantibodies on the α1-adrenergic receptor for brain vasculature has been demonstrated in animal studies. In the current study, 169 patients with dementia were screened for the presence of agonistic autoantibodies. 47% of patients suffering from mild to moderate Alzheimer's disease and/or vascular dementia carried these autoantibodies. Eight patients positive for autoantibodies underwent immunoadsorption. Patients treated on four consecutive days were subsequently negative for autoantibodies and displayed stabilization of cognitive and mental condition during 12-18 months' follow-up. In patients treated for 2-3 days, autoantibodies were reduced by only 78%. They suffered a rebound of autoantibodies during follow-up, benefited from immunoadsorption too, but their mental parameters worsened. We provide first data on the clinical relevance of agonistic autoantibodies in dementia and show that immunoadsorption is safe and efficient in removing autoantibodies with overall benefits for patients.


Assuntos
Autoanticorpos/imunologia , Demência/terapia , Técnicas de Imunoadsorção , Receptores Adrenérgicos alfa 1/imunologia , Idoso , Demência/imunologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Resultado do Tratamento
9.
Cardiovasc Res ; 57(3): 694-703, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12618231

RESUMO

OBJECTIVE: The role of cAMP in beta(2)-adrenoceptor signaling and its functional relevance in adult rat heart has been the subject of considerable controversy. Therefore, we investigated the beta(2)-adrenoceptor pathways in both adult cardiomyocytes and in the intact hearts of Wistar rats with respect to protein kinase A (at Ser16)-, the key event in shortening of relaxation time, and CaM kinase II (at Thr17)-dependent phospholamban phosphorylation. METHODS: Contractile and cellular beta(1)/beta(2)-adrenergic responses were studied in parallel on the same perfused rat heart. (-)Isoproterenol and the beta(2)-adrenergic agonists zinterol and procaterol were used to discriminate the beta-adrenoceptor subtype-related actions. RESULTS: Beta(2)-adrenoceptor stimulation induces protein kinase A-dependent phospholamban phosphorylation in both adult cardiomyocytes and in adult hearts of rats. The beta(2)-adrenoceptor-mediated shortening of relaxation time in the heart correlates with Ser16 phosphorylation. Adenosine elicited antiadrenergic action on both beta(1)- and beta(2)-adrenergic signaling cascades by reducing the phosphorylation status of phospholamban. Only beta(1)-adrenoceptor stimulation produced significant CaM kinase II-related Thr17 phosphorylation, troponin I phosphorylation and activation of phosphorylase a. CONCLUSIONS: Our findings clearly show that beta(2)-adrenoceptor signaling is coupled to phospholamban phosphorylation and shortening of relaxation time in the adult rat heart.


Assuntos
Coração/fisiologia , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais/fisiologia , Adenosina/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Etanolaminas/farmacologia , Masculino , Células Musculares/fisiologia , Contração Muscular/efeitos dos fármacos , Fosforilase a/metabolismo , Fosforilação , Ratos , Ratos Wistar , Troponina I/metabolismo
10.
Antioxid Redox Signal ; 6(2): 345-52, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15025936

RESUMO

Potential ortho- and pathophysiological roles for nitric oxide synthases (NOS) in cardiac functions have been and are continuing to be described. However, cellular signaling mechanisms controlling nitric oxide (NO) production in the heart remain obscure. The aim of this study was to investigate signaling mechanisms involved in regulation of NOS expression and NO generation in cardiomyocytes. Using immunocytochemical methods in conjunction with western blotting, we have found that cultured neonatal rat cardiomyocytes express constitutively all three NOS isoforms targeted predominantly to the particulate component of cardiomyocytes - mitochondria and along contractile fibers, as well as along plasma membrane including T-tubules. Biochemical assay of NO generation has shown that exposure of cultured neonatal rat cardiac cells to isoproterenol (beta-adrenergic stimulation), iloprost [stable prostaglandin I(2) (PGI(2)) analogue], as well as inflammatory cytokines and dibutyryl adenosine-3',5'-monophosphate (db-cAMP), resulted in a marked up-regulation of NOS expression by cardiomyocytes. In db-cAMP-stimulated cells, inhibition of protein kinase A (PKA) and protein kinase C (PKC) reduced immunolabeling of NOS and concomitantly lowered NO production. Taken together, these data point to an involvement of beta-adrenergic mechanisms, cytokine and PGI(2) receptors, adenylyl cyclase, PKA, and PKC in the control of NO generation and expression of NOS in rat cardiomyocytes.


Assuntos
Regulação Enzimológica da Expressão Gênica , Isoenzimas/metabolismo , Miócitos Cardíacos/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Animais Recém-Nascidos , Bucladesina/farmacologia , Células Cultivadas , Isoenzimas/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Nitritos/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologia
11.
PLoS One ; 7(7): e41602, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22860001

RESUMO

BACKGROUND: Circulating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α(1)-adrenergig receptor were detected in patients suffering from widespread diseases such as hypertension and type 2 diabetes. Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α(1)-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments. METHODOLOGY/PRINCIPAL FINDINGS: Using a rat model we studied the long-term action of antibodies against the α(1)-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α(1)-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with (relative units ± standard error, n = 10) 0.839 ± 0.026 versus 0.919 ± 0.026 statistical significance (p<0.05) for peptide-immunized rats. CONCLUSION/SIGNIFICANCE: We present evidence that antibodies to the α(1)-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.


Assuntos
Autoanticorpos/imunologia , Encéfalo/irrigação sanguínea , Receptores Adrenérgicos alfa 1/imunologia , Animais , Autoanticorpos/sangue , Encéfalo/imunologia , Encéfalo/metabolismo , Circulação Cerebrovascular/imunologia , Angiografia por Ressonância Magnética , Masculino , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/metabolismo
12.
PLoS One ; 3(11): e3742, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19011682

RESUMO

BACKGROUND: Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target. METHODOLOGY/PRINCIPAL FINDINGS: To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca(2+) in cardiomyocytes and induce mesentery artery segment contraction. CONCLUSIONS/SIGNIFICANCE: Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.


Assuntos
Autoanticorpos/imunologia , Hipertensão/imunologia , Receptores Adrenérgicos alfa/imunologia , Adsorção/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Animais , Autoanticorpos/isolamento & purificação , Autoanticorpos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Ativação Enzimática/efeitos dos fármacos , Mapeamento de Epitopos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Hipertensão/fisiopatologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Fosfolipases A2/metabolismo , Proteína Quinase C/metabolismo , Estrutura Secundária de Proteína , Ratos , Receptores Adrenérgicos alfa/química
13.
J Biol Chem ; 277(24): 21306-14, 2002 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-11925434

RESUMO

Endothelial K+ and Ca2+ homeostasis plays an important role in the regulation of tissue supply and metabolism under normal and pathological conditions. However, the exact molecular mechanism of how Ca2+ is involved in the regulation of K+ homeostasis in capillary endothelial cells, especially under oxidative stress, is not clear. To reveal Ca2+-triggered pathways, which modulate K+ homeostasis, Ca2+/calmodulin-dependent protein kinase II and voltage-gated outward K+ currents were studied in rat brain capillary endothelial cells under hypoxia. Whole cell voltage-clamp measurements showed voltage-gated outward K+ current with transient and sustained components. mRNA and protein of Ca2+/calmodulin-dependent protein kinase II delta2 and two gamma isoenzymes were identified. Activation of the isoforms (autophosphorylation) was typically achieved by the Ca2+ ionophore ionomycin, which was prevented by the Ca2+/calmodulin-dependent protein kinase II-specific inhibitor KN-93. Hypoxia resulted in autophosphorylation of the delta2 and gammaB isoforms, augmented the current amplitude, increased the inactivation time constant, and decreased the extent of inactivation of the transient current. KN-93 prevented both the activation of the isoforms and the alterations in the K+ current characteristics. It is concluded that the activation of Ca2+/calmodulin-dependent protein kinase II decreases inactivation of the voltage-gated outward K+ current, thereby counteracting depolarization of the hypoxic endothelium.


Assuntos
Encéfalo/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Capilares/enzimologia , Endotélio Vascular/enzimologia , Hipóxia , Potássio/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Immunoblotting , Microscopia de Contraste de Fase , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
J Mol Cell Cardiol ; 34(3): 321-34, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11945024

RESUMO

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.


Assuntos
Calcineurina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , Adulto , Western Blotting , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calsequestrina/metabolismo , Feminino , Humanos , Isoenzimas/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Fosforilação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático
15.
J Biol Chem ; 278(36): 33809-17, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12777394

RESUMO

S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with approximately 4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after beta-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in beta-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Camundongos Transgênicos , Contração Miocárdica , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Northern Blotting , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Ecocardiografia , Isoproterenol/farmacologia , Cinética , Camundongos , Testes de Precipitina , Ligação Proteica , Receptores Adrenérgicos beta/metabolismo , Proteínas S100 , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Fatores de Tempo
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