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5.
MMW Fortschr Med ; 149 Suppl 4: 172-81, 2008 Jan 17.
Artigo em Alemão | MEDLINE | ID: mdl-18402243

RESUMO

BACKGROUND: The treatment of arterial hypertension in Germany is, compared to international control rates, not adequate. In particular patients having additional cardiac risk factors like diabetes mellitus, dyslipidemia, increased waist circumference as well as concomitant diseases like myocardial infarction, heart- as well as kidney failure would benefit from an effective antihypertensive therapy. Aim of the present study was therefore to investigate in more detail blood pressure control in patients receiving a fixed combination of 16 mg Candesartan and 12.5 mg hydrochlorothiazide (HCTZ). METHODS: The present studywas performed as a non-interventional observational study. Included were patients with previously uncontrolled hypertension with at least one further risk factor. Primary variable was the blood pressure reduction over a time period of 8 weeks; secondary variables were the achievement of blood pressure targets and the tolerability of Candesartan/HCTZ. RESULTS: Between August 2006 and February 2007 3,787 patients in 893 physicians' offices in Germany were included. Patients were 62.2 +/- 11.3 years old, 48.1% were female, 97.5% had at least one additional risk factor, 29.8% a cardiovascular event. The risk to die from cardiovascular disease within the next 10 years was 7.4% according to the SCORE Score. By prescribing patients a fixed combination of 16 mg Candesartan/12.5 mg HCTZ a mean blood pressure reduction of -27.2/-13.4 mmHg was achieved (p < 0.001), pulse pressure was reduced by 13.8 mmHg (p <0.001) - both compared to previous therapy. A mean of 83.1% of patients achieved the guideline defined blood pressure targets (140/90 mmHg), 42.9% of patients even 130/80 mmHg. Serious adverse events were extremely rare (0.5%; hypertensive crisis, transitory ischemic attack). CONCLUSION: Prescribing patients with previously uncontrolled hypertension a combination of 16 mg Candesartan/12.5 mg HCTZ was tolerable and effective for blood pressure reduction in patients with arterial hypertension and additional risk factors like diabetes mellitus, dyslipidemia and the metabolic syndrome.


Assuntos
Benzimidazóis/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/prevenção & controle , Hidroclorotiazida/uso terapêutico , Hipertensão/tratamento farmacológico , Vigilância de Produtos Comercializados , Tetrazóis/uso terapêutico , Idoso , Benzimidazóis/efeitos adversos , Compostos de Bifenilo/efeitos adversos , Combinação de Medicamentos , Feminino , Humanos , Hidroclorotiazida/efeitos adversos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Tetrazóis/efeitos adversos , Resultado do Tratamento
7.
Eur Respir J ; 20(1): 165-9, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12166565

RESUMO

Obliterative bronchiolitis is commonly interpreted as chronic rejection and involves the bronchial and bronchiolar epithelium. Upregulation of major histocompatibility complex (MHC) II on bronchial epithelial cells (BEC) had been hypothesised to be an important trigger of a bronchus directed rejection response. More recently, the additional expression of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on antigen presenting cells were found to play an important role in the activation of T-lymphocytes in transplant rejection. The role of the expression of these molecules by BEC is unclear. BEC obtained by bronchial brushing and bronchoalveolar lavage fluid (BALF) cells from lung transplant recipients were studied and evaluated for messenger ribonucleic acid (mRNA) expression of B7-1 and B7-2 by semi-quantitative reverse transcriptase-polymerase chain reaction. Significantly elevated B7-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios were found in BEC from patients examined during the first 3 months after lung transplantation. Interestingly, in a small group of patients with bronchiolitis obliterans syndrome the B7-1/GAPDH and B7-2/GAPDH ratios were significantly elevated for BEC, whereas no differences were found for the BALF cells. In summary, B7 messenger ribonucleic acid expression by bronchial epithelial cells may play a role in (chronic) lung allograft rejection.


Assuntos
Antígenos CD/análise , Antígenos CD/genética , Antígeno B7-1/análise , Antígeno B7-1/genética , Brônquios/patologia , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/genética , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/patologia , Expressão Gênica/genética , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/genética , Transplante de Pulmão/efeitos adversos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Adulto , Antígeno B7-2 , Bronquiolite Obliterante/patologia , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/análise , Gliceraldeído-3-Fosfato Desidrogenases/genética , Rejeição de Enxerto/patologia , Humanos , Transplante de Pulmão/patologia , Masculino , Pessoa de Meia-Idade
8.
Histochem Cell Biol ; 116(5): 411-26, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11735005

RESUMO

Endothelin is an endothelium-derived peptide with potent vasoconstrictor and mitogenic properties. Since studies concerning the immunocytochemical localization of endothelin are often inconsistent we tried to clear up some of these discrepancies by comparing specificity and labeling patterns of different endothelin antibodies. Monoclonal and polyclonal endothelin antibodies ( n=7) were examined concerning their reactivity with endothelins and heart tissues by immunoblotting. Using immunofluorescence microscopy reactivities with human non-failing hearts, failing hearts, and cultured endothelial cells were examined. Specificity of labelings was assessed by absorption controls and functional controls using endothelial cells conditioned to produce different amounts of endothelin. As shown by immunoblotting five out of seven endothelin antibodies revealed both specific endothelin reactivity and negligible non-specific reactivities with heart proteins. Immunocytochemistry showed vascular reactivity of N-terminal endothelin antibodies to be associated with alpha-smooth muscle actin expression. One N-terminal antibody showed additional nuclear reactivity, and one C-terminal antibody vimentin-like labeling patterns. Although these reactivities were abolished in absorption controls these labelings had to be graded non-specific because functional specificity of endothelin antibodies could not be proven. The remaining antibodies revealed no endothelial reactivity even after tyramide signal amplification. Cellular endothelin concentrations ranged around 2,000-fold below the detection limit of immunocytochemical methods. Discrepancies in endothelin immunocytochemistry may originate from false-positive results and from expression levels of endothelin below the detection limit of immunocytochemical methods.


Assuntos
Anticorpos Monoclonais , Endotelinas/imunologia , Endotélio Vascular/imunologia , Insuficiência Cardíaca/imunologia , Ventrículos do Coração/imunologia , Miocárdio/imunologia , Especificidade de Anticorpos , Células Cultivadas , Endotelinas/química , Reações Falso-Positivas , Imunofluorescência/métodos , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Humanos , Immunoblotting , Miocárdio/patologia , Valor Preditivo dos Testes , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes
9.
J Cardiovasc Pharmacol ; 36(6): 776-84, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11117379

RESUMO

Immunosuppression may have an important impact on early graft coronary endothelial injury. We investigated functional and morphologic coronary alterations, myocardial expression, and cardiac release of possible mediators of allograft vasculopathy within 6 months after cardiac transplantation with respect to different immunosuppressive regimens. Epicardial and microvascular endothelium-dependent and endothelium-independent vasomotor function and epicardial intimal thickening were measured in 8 transplant recipients treated with cyclosporin A (CyA), azathioprine, and prednisone (group 1), 9 transplant recipients treated with tacrolimus (TKL), azathioprine, and prednisone (group 2), and 14 patients treated with TKL, mycophenolate mofetil (MMF), and prednisone (group 3). The gene expressions of inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1, prostacyclinsynthase, and thromboxansynthase were analyzed in endomyocardial biopsy specimens using semiquantitative reverse transcription polymerase chain reaction. Transcardiac cytokine release, endothelin-1, and nitrate-release were determined from plasma samples. Epicardial endothelial dysfunction (vasoconstriction to acetylcholine > 10%) and microvascular smooth muscle cell dysfunction (flow velocity increase to adenosine and nifedipine < 2.0) were enhanced in heart transplant recipients immunosuppressed with TKL, azathioprine, and prednisone. The prevalence of epicardial dysfunction was 78% in group 2 versus 44% and 46% in group 1 and 3 (p < 0.05), respectively. The prevalence of microvascular dysfunction was 56% in group 2 versus 13% and 7% in group 1 and 3 (p < 0.02), respectively. Coronary vasomotor dysfunction was associated with increased myocardial iNOS expression (p < 0.05), decreased eNOS expression (p < 0.05), and enhanced cardiac immunoreactive interleukin-6 (p < 0.01). Coronary intimal thickening was not different between the groups. The combination of TKL and MMF appears to be superior to TKL and azathioprine (and comparable to CyA and azathioprine) concerning preservation of early coronary vasomotor function, eNOS expression, iNOS suppression as well as cardiac interleukin-6 release. This may have an important impact on subsequent development of transplant coronary atherosclerosis.


Assuntos
Doença das Coronárias/fisiopatologia , Transplante de Coração/fisiologia , Imunossupressores/efeitos adversos , Adulto , Cateterismo Cardíaco , Citocinas/metabolismo , Citocinas/farmacologia , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Endotelina-1/metabolismo , Endotélio Vascular/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Músculo Liso Vascular/fisiopatologia , Nitratos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ultrassonografia Doppler
10.
J Heart Lung Transplant ; 18(11): 1071-9, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10598730

RESUMO

BACKGROUND: Cytokines and growth factors released as part of the immune response to alloantigenic stimuli are capable of regulating endothelin-1 expression in the allograft. Endothelin plays a significant role as a modulator of coronary vascular reactivity in the early stages of atherosclerosis and may be important as a participant in and marker for cardiac allograft vasculopathy. METHODS: We characterized a possible relationship between morphological and functional coronary changes, transcardiac plasma endothelin level and myocardial endothelin-mRNA expression in 33 cardiac transplant recipients in the early, stable phase 5+/-3 months after orthotopic heart transplantation. Coronary microvascular function was determined as endothelium-dependent with acetylcholine and endothelium-independent with adenosine using intracoronary Doppler-FloWire. The percentage of the epicardial diameter changes was measured using quantitative coronary angiography. Intravascular ultrasound was performed to quantify intimal hyperplasia. Cardiac endothelin uptake or release was determined by measuring plasma endothelin levels in the coronary sinus and aorta. Myocardial endothelin-gene expression was determined using semiquantitative RT-PCR. RESULTS: The aortic endothelin levels were significantly increased in transplant recipients compared to nontransplanted patients (11.8+/-2.2 vs 7.2+/-0.9 fmol/mL; P < 0.001). Endothelin uptake was noticed in the majority of patients, and the amount of endothelin uptake was correlated to microvascular (r = 0.37; P < 0.05) and epicardial (r = 0.41; P < 0.03) endothelium-dependent vasodilatation. High mRNA signal intensity was associated with significantly reduced coronary flow response to acetylcholine compared to patients with low myocardial gene expression (coronary flow reserve 2.4+/-0.9 vs 3.4+/-0.8, respectively; P < 0.005). Morphological coronary changes early after transplantation were not correlated to endothelin plasma levels or myocardial gene expression. CONCLUSION: Coronary endothelial vasomotor dysfunction after cardiac transplantation is associated with an increased myocardial endothelin mRNA expression and decreased endothelin-uptake by the heart. We postulate that early activation in the endothelin system may have a pivotal role in the acceleration of the atherosclerotic process in transplant patients.


Assuntos
Vasos Coronários/metabolismo , Endotelinas/metabolismo , Endotélio Vascular/metabolismo , Transplante de Coração , Adulto , Biomarcadores , Biópsia , Velocidade do Fluxo Sanguíneo , Cateterismo Cardíaco , Angiografia Coronária , Circulação Coronária , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/fisiopatologia , Ecocardiografia Doppler em Cores , Endotelinas/genética , Endotélio Vascular/fisiopatologia , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/patologia , Reação em Cadeia da Polimerase , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Radioimunoensaio , Ultrassonografia de Intervenção , Vasodilatação
11.
J Mol Cell Cardiol ; 30(6): 1149-62, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9689589

RESUMO

The cellular and sub-cellular localization of the G-protein subunit, G(o) alpha, in rat heart was determined by immunofluorescence and immunoelectron microscopy. Using antibodies directed against purified G(o) alpha and an antiserum raised against the C-terminal decapeptide of G(o) alpha, strong immunoreactivity was found in the conducting system of the heart, neurons, and atrial cardiomyocytes. Labeling of ventricles was weak compared to that of atria. In neurons, immunoelectron microscopy revealed G(o) alpha was localized along the inner surface of axolemma and on axoplasmal vesicles. G(o) alpha immunoreactivity in atrial and ventricular myocytes was not restricted to sarcolemma, but was also found on sub-sarcolemmal vesicles with characteristic caveolar morphology. At the level of intercalated discs, labeling was spread over the periphery of intercalated discs avoiding its membrane structures. Additionally, in atrial endocrine cardiomyocytes, approximately 60% of secretory granules revealed G(o) alpha-labeling on the cytoplasmic surface. A small number of these granules stood out due to particularly intense labeling. The observation that these granules were found most-frequently in sub-sarcolemmal areas suggests that they may be mature granules undergoing exocytosis. Therefore, G(o) alpha found on secretory granules of endocrine cardiomyocytes may have a function in regulated exocytosis of cardiac hormones. Sarcolemmal localization of G(o) alpha in atrial and ventricular cardiomyocytes supports the role of G(o) alpha in transmembrane signal transduction. Furthermore, caveolar localization of G(o) alpha may provide a compartmental basis for integrating G(o)-mediated signaling events.


Assuntos
Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , Coelhos , Ratos , Ratos Wistar , Sarcolema/metabolismo
12.
J Cell Biochem ; 70(1): 49-59, 1998 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-9632107

RESUMO

The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca2+ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca2+ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F2alpha (PGF2alpha). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5'-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone.


Assuntos
Adenosina/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Vasos Coronários/fisiologia , Epoprostenol/fisiologia , Óxido Nítrico/fisiologia , Animais , Artérias/metabolismo , Artérias/fisiologia , Vasos Coronários/metabolismo , Técnicas In Vitro , Relaxamento Muscular/fisiologia , Tono Muscular , Cadeias Leves de Miosina/metabolismo , Fosforilação , Suínos
13.
Biochem Biophys Res Commun ; 227(2): 328-33, 1996 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-8878517

RESUMO

The contribution of increased inducible nitric oxide synthase (iNOS) activity to the development of left ventricular dysfunction after acute myocardial infarction (MI) was investigated New Zealand rabbits (n = 24) were randomly treated with either saline, S-methylisothiourea sulfate (SMT; selective iNOS inhibitor) or N-omega-nitro-L-arginine (NOLA; non-isoform selective NOS inhibitor). Left ventricular hemodynamics and myocardial blood flow were measured before coronary occlusion and on postoperative day 3 (POD 3). MI resulted in left ventricular dysfunction and increased myocardial iNOS activity. SMT and NOLA significantly inhibited iNOS activity; SMT, but not NOLA, significantly improved left ventricular maximum +dP/dt and decreased LVEDP; myocardial blood flow in the remote myocardium significantly increased after SMT. Induction of myocardial iNOS after MI on POD 3 contributes to the development of left ventricular dysfunction; modulation of iNOS activity by SMT improves left ventricular performance and may be beneficial after acute MI.


Assuntos
Inibidores Enzimáticos/farmacologia , Isotiurônio/análogos & derivados , Infarto do Miocárdio/fisiopatologia , Miocárdio/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Circulação Coronária/efeitos dos fármacos , Vasos Coronários , Indução Enzimática , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isotiurônio/farmacologia , Masculino , Infarto do Miocárdio/enzimologia , Óxido Nítrico Sintase/biossíntese , Nitroarginina/farmacologia , Coelhos , Resistência Vascular/efeitos dos fármacos , Função Ventricular Esquerda/fisiologia
14.
Mol Cell Biochem ; 160-161: 41-6, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8901453

RESUMO

Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells. Exposure of the cells up to 6 micrograms/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (Bmax) to 46.0 +/- 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca+2 channel by N-glycosylation is an important determinant for the binding of Ca+2 antagonists of the DHP-type to the alpha 1 subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded.


Assuntos
Miocárdio/metabolismo , Proteínas/metabolismo , Amidoidrolases/metabolismo , Animais , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Di-Hidropiridinas/metabolismo , Glicosilação , Cinética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Tunicamicina/farmacologia
15.
Proc Assoc Am Physicians ; 108(2): 173-8, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8705737

RESUMO

Nitric oxide (NO), an unstable radical, is synthesized from L-arginine by the constitutive (cNOS) and inducible (iNOS) forms of NOS. cNOS is present mainly in endothelial cells and plays a role in the regulation of blood flow. iNOS, the dominant enzyme in heart muscle during myocardial infarction, allograft rejection, and cardiomyopathy, is activated in macrophages. We recently described a significant increase of iNOS activity in macrophages of infarcted rabbit myocardium 24 hours after coronary occlusion, with peak activity occurring 3 days following coronary artery ligation. Inhibitors of NOS are L-arginine derivatives that inhibit both cNOS and iNOS; S-methylisothiourea (SMT) and aminoguanidine (AMG) are specific inhibitors of iNOS. Cyclosporin A and dexamethasone inhibit by interfering with protein synthesis. iNOS inhibition by SMT, NG-nitro-L-arginine (L-NNA), AMG, cyclosporin A and dexamethasone was examined in homogenates of normal, risk and infarcted myocardium. Three days after coronary artery ligation, the heart was excised and divided into normal, risk and infarcted regions. The inhibitory effect was calculated as IC50. Results shows that SMT was the most potent inhibitor with the lowest IC50; its effect, as well as the effects of L-NNA and AMG, depended on the location in the myocardium. Inhibition for SMT and AMG was greater in the normal area than in the risk and infarcted regions. AMG induced an initial rise of iNOS followed by gradual decline in the area of risk and infarction. No inhibitory effects in cyclosporin A and dexamethasone were noted.


Assuntos
Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/enzimologia , Miocárdio/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Ciclosporina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Guanidinas/farmacologia , Isotiurônio/análogos & derivados , Isotiurônio/farmacologia , Masculino , Nitroarginina , Coelhos
16.
Mol Cell Biochem ; 147(1-2): 161-8, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7494545

RESUMO

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that: 1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts. 2. Gia was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. Goa were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures. 3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.


Assuntos
Adenilil Ciclases/análise , Proteínas de Ligação ao GTP/metabolismo , Miocárdio/citologia , Receptores Muscarínicos/análise , Animais , Imunofluorescência , Proteínas de Ligação ao GTP/química , Cobaias , Imuno-Histoquímica , Membranas Intracelulares/química , Microscopia Eletrônica , Miocárdio/ultraestrutura , Ratos , Sarcolema/química , Transdução de Sinais/fisiologia
17.
J Histochem Cytochem ; 42(3): 337-43, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8308250

RESUMO

We produced antibodies against a synthetic peptide corresponding to amino acids 168-192 of the second extracellular loop of the M2 human muscarinic receptor in rabbits. In immunoblot, affinity-purified antibodies specifically recognized a major band of rat ventricular muscarinic receptor protein with a molecular weight of about 80 KD. This recognition could be blocked by pre-incubation with peptide. Moreover, with both light (LM) and electron microscopic (EM) immunocytochemistry techniques, muscarinic receptors were detected on sarcolemma and T-tubules of rat cardiomyocytes. In addition, immunoreactions were localized in membranes of capillaries. Likewise, these reactivities were abolished by pre-incubation with peptide. These results suggest that the antibodies against the second extracellular loop of human M2 muscarinic receptor could specifically recognize rat ventricular muscarinic receptor protein and could be a powerful tool to study the fate of this receptor under different pathological or physiological conditions.


Assuntos
Imunofluorescência , Ventrículos do Coração/química , Receptores Muscarínicos/análise , Acetilcolina/química , Sequência de Aminoácidos , Animais , Anticorpos , Ventrículos do Coração/ultraestrutura , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia Imunoeletrônica/métodos , Dados de Sequência Molecular , Peptídeos , Coelhos , Ratos , Receptores Muscarínicos/química , Receptores Muscarínicos/imunologia
18.
Acta Histochem ; 96(1): 87-95, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8030385

RESUMO

Localization of G proteins in the rat heart tissue was investigated using primary affinity-purified antibodies against synthetic peptides with amino acid sequences corresponding to alpha-subunits (alpha i common and alpha i 1, 2) of G proteins. Detection of immunoreactivity was performed with the peroxidase-anti-peroxidase complex (PAP), avidin-biotin complex (ABC) and fluorescein-labelled secondary antibodies for light microscopy and the protein A-gold technique for electron microscopy. In ventricles and atria, immunostaining for G proteins was detected in the sarcolemma and perinuclear space of cardiomyocytes. In endotheliocytes and fibroblasts, immunoreactivity was present also in the endoplasmic reticulum. All four immunocytochemical methods permit to demonstrate the same localization of G proteins in heart tissue. The ABC method and fluorescein labelled secondary antibodies technique showed the same sensitivity which is higher than that of the PAP method. Nomarski contrast microscopy enhanced the visualization of the final reaction product formed by the peroxidase reaction developed with diaminobenzidine in the ABC method. The results are discussed in terms of the role of G proteins in signal transduction via plasma membrane and membranes of the intracysternal space of the cell.


Assuntos
Proteínas de Ligação ao GTP/análise , Miocárdio/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Secções Congeladas , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Microscopia Eletrônica , Dados de Sequência Molecular , Ratos , Ratos Wistar , Transdução de Sinais , Inclusão do Tecido
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