RESUMO
The influence of cell confluence on the ß-adrenoceptor (ß-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. ß-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the ß-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100µM) induced a similar saturating response in both LD and HD cells. A ß1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a ß2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a ß-AR ligand, identified two binding sites in HD cells, corresponding to ß1- and ß2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to ß2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10µM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1µM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the ß-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both ß1- and ß2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only ß2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the ß1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular ß-AR signalling.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta/genética , Transdução de Sinais/genética , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Colforsina/análogos & derivados , Colforsina/farmacologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Diterpenos , Transferência Ressonante de Energia de Fluorescência , Imidazóis/farmacologia , Isoproterenol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/administração & dosagem , Propanolaminas/farmacologia , Ratos , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Although cardiac cytosolic cyclic 3',5'-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3(-)) and Ca(2+), sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca(2+) entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na(+)/Ca(2+) exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3(-) rescued the sensitization of mitochondria to Ca(2+)-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies.
Assuntos
Apoptose , Cálcio/metabolismo , AMP Cíclico/farmacologia , Mitocôndrias Cardíacas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/química , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bicarbonatos/farmacologia , Camptotecina/toxicidade , Células Cultivadas , Dactinomicina/toxicidade , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Peróxido de Hidrogênio/toxicidade , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Permeabilidade , Ratos , Ratos Wistar , Troponina I/metabolismoRESUMO
BACKGROUND AND PURPOSE: This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by ß-adrenoceptors, following heart failure (HF). EXPERIMENTAL APPROACH: Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta. KEY RESULTS: HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF2α -stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF2α -precontracted arteries, ß-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries. CONCLUSIONS AND IMPLICATIONS: Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to ß-adrenoceptor stimulation.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Insuficiência Cardíaca/fisiopatologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Dinoprosta/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Quinolonas/farmacologia , RNA Mensageiro/metabolismo , Ratos Wistar , Vasoconstrição/efeitos dos fármacosRESUMO
Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.
Assuntos
Fenômenos Eletrofisiológicos , Coração/fisiologia , Disseminação de Informação/métodos , Modelos Biológicos , Projetos de Pesquisa/normas , Animais , Humanos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
PURPOSE: The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca(2+)-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl(-) channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. METHODS: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl(-) current was examined using whole-cell patch clamp analysis. RESULTS: Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibited the function of wild-type bestrophin-1 channels in a dominant negative manner. CONCLUSIONS: This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.
Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Proteínas Mutantes/genética , Idade de Início , Substituição de Aminoácidos , Bestrofinas , Linhagem Celular , Criança , Pré-Escolar , Canais de Cloreto , Cloretos/metabolismo , Análise Mutacional de DNA , Éxons/genética , Feminino , Genes Dominantes , Humanos , Transporte de Íons/genética , Rim , Degeneração Macular/diagnóstico , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Técnicas de Patch-Clamp , Linhagem , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Análise de Sequência de DNA , Relação Estrutura-Atividade , TransfecçãoRESUMO
The cAMP pathway is of cardinal importance for heart physiology and pathology. The spatial organization of the various components of the cAMP pathway is thought to allow the segregation of functional responses triggered by the different neuromediators and hormones that use this pathway. PDEs (phosphodiesterases) hydrolyse cAMP (and cGMP) and play a major role in this process by preventing cAMP diffusion to the whole cytosol and inadequate target activation. The development of olfactory cyclic nucleotide-gated channels to directly monitor cAMP beneath the plasma membrane in real time allows us to gain new insights into the molecular mechanisms responsible for cAMP homoeostasis and hormonal specificity in cardiac cells. The present review summarizes the recent results we obtained using this approach in adult rat ventricular myocytes. In particular, the role of PDEs in the maintenance of specific cAMP signals generated by beta-adrenergic receptors and other G(s)-coupled receptors will be discussed.
Assuntos
AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Canais Iônicos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.
Assuntos
Miocárdio/imunologia , Miocárdio/metabolismo , Receptores de Serotonina/química , Receptores de Serotonina/imunologia , Adenilil Ciclases/metabolismo , Animais , Especificidade de Anticorpos , Fibrilação Atrial/etiologia , Fibrilação Atrial/imunologia , Autoanticorpos , Células COS , Ativação Enzimática , Epitopos/química , Epitopos/genética , Humanos , Técnicas In Vitro , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Receptores 5-HT4 de Serotonina , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The serotonin 5-HT(4) receptor has recently gained a lot of attention for its functional roles in central processes such as memory and cognition. In this study, we show that activation of the human 5-HT(4) (h5-HT(4)) receptor stimulates the secretion of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha). 5-HT enhanced the level of secreted sAPPalpha in a time- and dose-dependent manner in Chinese hamster ovary cells stably expressing the h5-HT(4(e)) receptor isoform. The increase was inhibited by the selective 5-HT(4) receptor antagonist, GR113808. The 5-HT(4) selective agonists, prucalopride and renzapride, also increased secreted sAPPalpha in IMR32 human neuroblastoma cells. The stimulatory effect of 5-HT was mimicked by forskolin, a direct activator of adenylyl cyclase, and 8-bromo-cAMP, a membrane-permeant cAMP analogue. On the contrary, inhibition of protein kinase A (PKA) by H89 potentiated the 5-HT-induced increase in both secreted and cellular sAPPalpha. This phenomenon involves a novel PKA-independent stimulatory process that overcomes a PKA-dependent inhibitory one. Finally, activation of the h5-HT(4(e)) receptor did not modify extracellular amyloid beta-protein in Chinese hamster ovary cells transfected with the human APP695. Given the neuroprotective and enhancing memory effects of sAPPalpha, our results may open a new avenue for the treatment of Alzheimer's disease.
Assuntos
Receptores de Serotonina/metabolismo , Serotonina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Adenilil Ciclases/metabolismo , Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzamidas/farmacologia , Benzofuranos/farmacologia , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células CHO , Colforsina/farmacologia , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/farmacologia , Ligantes , Memória , Reação em Cadeia da Polimerase , Ligação Proteica , Isoformas de Proteínas , Radioimunoensaio , Receptores 5-HT4 de Serotonina , Sulfonamidas/farmacologia , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
1. The regulation of L-type Ca(2+) current (I(Ca)) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 microM to 1 mM) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 microM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2. SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 microM)-mediated stimulation of I(Ca) via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP. 3. When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I(Ca) to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP). 4. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 microM) on FSK-stimulated I(Ca) when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5. A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 microM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 microM) or SNAP (10 microM) on I(Ca) but had no effect on the distant response. 6. When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed. 7. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca(2+) channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca(2+) channels and PDE2.
Assuntos
Adenina/análogos & derivados , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Óxido Nítrico/farmacologia , Função Ventricular/efeitos dos fármacos , Adenina/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Benzoatos/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Condutividade Elétrica , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Membranas Intracelulares/metabolismo , Isoproterenol/farmacologia , Miocárdio/citologia , Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Técnicas de Patch-Clamp , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo , Rana esculenta , Distribuição TecidualRESUMO
5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.
Assuntos
Processamento Alternativo/genética , Sistema Nervoso Central/fisiologia , Receptores de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Elementos Antissenso (Genética) , Expressão Gênica , Humanos , RNA Mensageiro/análise , Receptores 5-HT4 de Serotonina , Taq PolimeraseRESUMO
1. The regulation of the L-type Ca(2+) current (I(Ca)) by intracellular cGMP was investigated in human atrial myocytes using the whole-cell patch-clamp technique. 2. Intracellular application of 0.5 microM cGMP produced a strong stimulation of basal I(Ca) (+64 +/- 5 %, n = 60), whereas a 10-fold higher cGMP concentration induced a 2-fold smaller increase (+36 +/- 8 %, n = 35). 3. The biphasic response of I(Ca) to cGMP was not mimicked by the cGMP-dependent protein kinase (PKG) activator 8-bromoguanosine 3',5' cyclic monophosphate (8-bromo-cGMP, 0.5 or 5 microM), and was not affected by the PKG inhibitor KT 5823 (100 nM). 4. In contrast, cGMP stimulation of I(Ca) was abolished by intracellular perfusion with PKI (10 microM), a selective inhibitor of the cAMP-dependent protein kinase (PKA). 5. Selective inhibition of the cGMP-inhibited phosphodiesterase (PDE3) by extracellular cilostamide (100 nM) strongly enhanced basal I(Ca) in control conditions (+78 +/- 13 %, n = 7) but had only a marginal effect in the presence of intracellular cGMP (+22 +/- 7 % in addition to 0.5 microM cGMP, n = 11; +20 +/- 22 % in addition to 5 microM cGMP, n = 7). 6. Application of erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, 30 microM), a selective inhibitor of the cGMP-stimulated phosphodiesterase (PDE2), fully reversed the secondary inhibitory effect of 5 microM cGMP on I(Ca) (+99 +/- 16 % stimulation, n = 7). 7. Altogether, these data indicate that intracellular cGMP regulates basal I(Ca) in human atrial myocytes in a similar manner to NO donors. The effect of cGMP involves modulation of the cAMP level and PKA activity via opposite actions of the nucleotide on PDE2 and PDE3.
Assuntos
Canais de Cálcio Tipo L/metabolismo , GMP Cíclico/farmacologia , Fibras Musculares Esqueléticas/enzimologia , Miocárdio/citologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Átrios do Coração/citologia , Humanos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Quinolonas/farmacologia , Estimulação QuímicaRESUMO
1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Coração/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Animais , Eletrofisiologia , Guanilato Ciclase/antagonistas & inibidores , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/fisiologia , Microdiálise , Miocárdio/citologia , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
We have previously suggested that the recognition of a cross-reactive epitope on the 5-HT4 receptor and the 52-kDa SSA/Ro protein by serotonin-antagonizing autoantibodies could explain the electrophysiological symptoms of congenital heart block in neonatal lupus. To confirm this hypothesis, we immunized female mice with four synthetic peptides corresponding to the recognized epitopes. All mice developed anti-peptide antibodies, which cross-reacted with the Ro52 and 5-HT4 receptor peptides and recognized both cognate proteins. Peptide-immune mice were mated. The pups from mice immunized with the Ro52 peptides had no symptoms of neonatal lupus apart from bradycardia. However, pups from mice immunized with the 5-HT4 receptor peptides and bradycardia, atrioventricular block of type I or II, longer QT intervals, skin rashes and neuromotor problems. The 5-HT4 receptor was detectable in the different fetal tissues affected (heart, skin and brain) by immunohistochemistry. Hearts from diseased pups were less developed and showed disorganized myocardial hyperplasia, compared to the normal littermates. These results demonstrate that the serotoninergic 5-HT4 receptor is the antigenic target of physiopathological autoantibodies in neonatal lupus.
Assuntos
Lúpus Vulgar/etiologia , Fragmentos de Peptídeos/imunologia , Receptores de Serotonina/imunologia , Animais , Animais Recém-Nascidos , Autoanticorpos/análise , Reações Cruzadas , Feminino , Imunização , Lúpus Vulgar/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores 5-HT4 de SerotoninaRESUMO
The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP]i) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole-cell L-type Ca2+ current (ICa). Ratiometric imaging was used to monitor [cAMP]i changes in response to the beta-adrenergic agonist isoprenaline (ISO) or to the direct adenylyl cyclase activator forskolin (FSK). FlCRhR fluorescence was distributed in the cytosol in a striated pattern, with high fluorescence in the I-bands and low fluorescence in the A-bands. This pattern of distribution was mimicked by fluorescein dextran, another high molecular weight fluorescent molecule, and was therefore likely to be due to anisotropic diffusion of the probe in the cytosol due to the hindrance generated by sarcomeric proteins in the A-bands. Introduction of FlCRhR into the cell induced a small approximately 70% stimulatory effect on basal ICa, attenuating about 2-fold a subsequent response of ICa to 1-10 microM ISO (from 400 to 200%). Brief (10 s) application of a saturating concentration of ISO (1-20 microM) to the cell induced a transient increase in both ICa and [cAMP]i. However, the [cAMP]i transient was approximately 2-fold shorter in duration than the ICa transient, i.e. ICa was still strongly enhanced when [cAMP]i had already returned to control level. This indicates that hydrolysis of cAMP by phosphodiesterases is not a rate limiting step in the recovery of ICa from ISO stimulation. When the application of ISO was maintained, ICa and [cAMP]i responses followed a similar time course, with a half-maximal response at approximately 60 s. This suggests that activation of Ca2+ channels by cAMP-dependent protein kinase occurs on a much faster time scale than the rise in [cAMP]i. When the cells were exposed to FSK (13 microM), both responses of ICa and [cAMP]i were approximately 2-fold slower than with ISO. This demonstrates that the slower response of ICa to FSK is due to a slower rise in [cAMP]i rather than to some inhibitory effect of FSK on ICa or to a direct or priming effect of the stimulatory G protein Gs on Ca2+ channels. Simultaneous measurements of [cAMP]i and ICa changes in intact cardiac myocytes opens the way to dissect the temporal sequence of events in the cAMP cascade mediating the response of the heart to a large number of hormones and inotropic agents.
Assuntos
Canais de Cálcio Tipo L/metabolismo , AMP Cíclico/metabolismo , Miocárdio/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Colforsina/farmacologia , Citosol/metabolismo , Corantes Fluorescentes , Proteínas de Ligação ao GTP/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Isoproterenol/farmacologia , Potenciais da Membrana/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/citologia , Técnicas de Patch-Clamp , Rana esculentaRESUMO
The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.
Assuntos
Anticorpos Antinucleares/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/complicações , Bloqueio Cardíaco/etiologia , Lúpus Eritematoso Sistêmico/complicações , Miocárdio/imunologia , RNA Citoplasmático Pequeno , Receptores de Serotonina/imunologia , Ribonucleoproteínas/imunologia , Adulto , Idoso , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Doenças Autoimunes/imunologia , Células CHO , Canais de Cálcio/metabolismo , Cricetinae , Cricetulus , Reações Cruzadas , Feminino , Bloqueio Cardíaco/congênito , Bloqueio Cardíaco/imunologia , Humanos , Imunidade Materno-Adquirida , Recém-Nascido , Ativação do Canal Iônico , Transporte de Íons , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Gravidez , Complicações na Gravidez/imunologia , Coelhos , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores 5-HT4 de Serotonina , Proteínas Recombinantes de Fusão/imunologia , TransfecçãoRESUMO
New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT(4) receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human 5-HT(4(e)) isoform stably expressed in C6 glial cells with [(3)H]GR 113808 as the radioligand. The affinity values (K(i)) depended upon the substituent on the aromatic ring. A chlorine atom produced a marked drop in activity (K(i) > 100 nM), while a m-methoxy group gave a compound with nanomolar affinity (K(i) = 3 nM). The most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, respectively). K(i) values for 9a and 9r were determined for the 5-HT(4(a)), 5-HT(4(b)), 5-HT(4(c)), and 5-HT(4(d)) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT(4(e)) receptor and shifted the 5-HT concentration-effect curve on adenylyl cyclase activity with pK(D) values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT on the L-type calcium current (I(Ca)) with a K(D) value of 0.7 nM.
Assuntos
Ácido 4-Aminobenzoico/síntese química , Piperazinas/síntese química , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/síntese química , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/farmacologia , Adenilil Ciclases/metabolismo , Animais , Células COS , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular , Humanos , Técnicas In Vitro , Miocárdio/citologia , Miocárdio/metabolismo , Neuroglia/citologia , Técnicas de Patch-Clamp , Piperazinas/química , Piperazinas/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ensaio Radioligante , Receptores de Serotonina/metabolismo , Receptores 5-HT4 de Serotonina , Antagonistas da Serotonina/química , Antagonistas da Serotonina/farmacologia , para-AminobenzoatosRESUMO
The recently identified C-terminal splice variant of the human 5-HT(4) receptor, the h5-HT(4(d)) receptor, was stably expressed in a CHO cell line at 493+/-25 fmol mg(-1) protein. We analysed its pharmacological properties by measuring binding affinities and 5-HT(4) ligand-induced cyclic AMP production. The pharmacological binding profile determined in competition studies with the specific antagonist [(3)H]-GR113808 revealed a rank order of affinity of 5-HT(4) ligands for the h5-HT(4(d)) receptor that was consistent with those previously reported for other 5-HT(4) receptor isoforms. In adenylyl cyclase functional assays, the h5-HT(4(d)) receptor displayed equipotent coupling for all 5-HT(4) agonists tested (EC(50) in the range of 1 - 6 nM). EC(50) values were lower than those previously obtained with the 5-HT(4(e)) receptor stably expressed in CHO cells indicating that the 5-HT(4(d)) receptor was more efficiently coupled to its effector than the 5-HT(4(e)) receptor isoform. Moreover, in terms of agonist efficacy (E(max)), the benzamide derivative, renzapride displayed full agonist properties at the h5-HT(4(d)) receptor (same E(max) as 5-HT) whereas it was previously shown to be a partial agonist at the h5-HT(4(e)) receptor. A constitutive activity of the h5-HT(4(d)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand. Surprisingly, two 5-HT(4) ligands, SB204070 and RS39604 which are described as highly potent antagonists in various biological models, revealed partial agonist properties at the h5-HT(4(d)) receptor. We conclude that C-terminal tails of 5-HT(4) receptor isoforms may directly influence their functional properties.
Assuntos
Receptores de Serotonina/fisiologia , Animais , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Humanos , Indóis/metabolismo , Isoformas de Proteínas , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores 5-HT4 de Serotonina , Relação Estrutura-Atividade , Sulfonamidas/metabolismoRESUMO
Among the five human 5-HT(4) (h5-HT(4)) receptor isoforms, the h5-HT(4(a)) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT(4) receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. Ligand binding or competition studies with two h5-HT(4) receptor agonists, serotonin and ML10302 and two h5-HT(4) receptor antagonists, [(3)H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. Ligand binding experiments revealed that [(3)H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT(4(a)) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [(3)H]-GR113808 and to serotonin. According to these results, we propose ligand-receptor complex models with serotonin and [(3)H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [(3)H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [(3)H]-GR113808.
Assuntos
Mutagênese Sítio-Dirigida/genética , Receptores de Serotonina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Ligação Competitiva/genética , Western Blotting , Células COS , Membrana Celular/metabolismo , AMP Cíclico/biossíntese , Humanos , Indóis/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/imunologia , Receptores 5-HT4 de Serotonina , Serotonina/metabolismo , Serotonina/farmacologia , Sulfonamidas/metabolismoRESUMO
1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.
Assuntos
Cátions/metabolismo , Coração/embriologia , Canais Iônicos/biossíntese , Miocárdio/metabolismo , Proteínas do Tecido Nervoso , Células-Tronco/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Agonistas Muscarínicos/farmacologia , Miocárdio/citologia , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Canais de Potássio , Células-Tronco/citologiaRESUMO
RT - PCR technique was used to clone the human 5-HT(4(e)) receptor (h5-HT(4(e))) from heart atrium. We showed that this h5-HT(4(e)) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT(4(e)) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg(-1) protein, respectively. Expression of h5-HT(4(e)) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [(3)H]-GR113808 of a number of 5-HT(4) ligands, was consistent with that previously reported for other 5-HT(4) receptor isoforms. Surprisingly, we found that the rank order of potencies (EC(50)) of 5-HT(4) agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (K(i)) obtained from binding assays. Furthermore, EC(50) values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT(4(e)) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca(2+) currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT(4(e)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand and two 5-HT(4) antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT(4(e)) receptor has a pharmacological profile which is close to the native h5-HT(4) receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.