RESUMO
Regulation of the sodium pump during normal pregnancy and its effect on the function of cardiomyocytes is poorly understood. Our objective was to evaluate the possible implication of the Na(+)-K(+)-ATPase, the sodium pump which controls cellular ionic and metabolic homeostasis, in the adaptations of cardiomyocytes to normal pregnancy. We have used Western blots and patch-clamp measurements to identify changes in the sodium pump proteins. Confocal microscopy was applied to estimate intracellular sodium concentration. Time-resolved spectroscopy was employed to measure mitochondrial NAD(P)H fluorescence and estimate oxidative metabolic state. Optical microscopy was adopted to study the contractility responses of cardiomyocytes. Cells from non-pregnant and pregnant rats (1 day prior parturition) were studied. Our results showed lower protein expression of the α1 Na(+)-K(+)-ATPase isoform in cardiomyocytes in pregnant rats, decreased sodium pump membrane current and elevated steady-state sodium concentration. In addition, ouabain, the inhibitor of the sodium pump capable of increasing cardiomyocyte contractility in non-pregnant rats in a concentration-dependent manner, failed to affect cell contractions in pregnant rats. We also noted modified responsiveness of the mitochondrial metabolic state to ouabain in cardiac cells. The gathered data confirmed that in pregnant rats, the sodium pump protein content and transmembrane flux are decreased, while the sensitivity of cardiomyocyte contractility and the sensitivity of mitochondrial metabolic redox state to ouabain are modified, pointing to regulation of the Na(+)-K(+)-ATPase during cardiac cell adaptations to normal pregnancy.
Assuntos
Adaptação Fisiológica , Miócitos Cardíacos/fisiologia , Prenhez/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Feminino , Contração Muscular/efeitos dos fármacos , NADP/metabolismo , Ouabaína/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence. Here, we describe detection and measurement of endogenous mitochondrial flavin fluorescence directly in living cardiac cells using fluorescence lifetime imaging microscopy (FLIM) after excitation with 473 nm picoseconds (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.
Assuntos
Flavinas/química , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , Animais , Metabolismo Energético , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Ratos , Espectrometria de FluorescênciaRESUMO
To better understand pH-dependence of endogenous fluorescence of algae, we employed spectroscopy and microscopy methods, including advanced time-resolved fluorescence imaging microscopy (FLIM), using green algae Chlorella sp. as a model system. Absorption spectra confirmed two peaks, at 400-420 nm and 670 nm. Emission was maximal at 680 nm, with smaller peaks between 520 and 540 nm. Acidification led to a gradual decrease in the red fluorescence intensity with the maximum at 680 nm when excited by 450 nm laser. FLIM measurements, performed using 475 nm picoseconds excitation, uncovered that this effect is accompanied by a shortening of the tau1 fluorescence lifetime. Under severe acidification, we also noted an increase in the green fluorescence with a maximum between 520-540 nm and a shift toward 690-700 nm of the red fluorescence, accompanied by prolongation of the tau2 fluorescence lifetime. Gathered data increase our knowledge on the responsiveness of algae to acidification and indicate that endogenous fluorescence derived from chlorophylls can potentially serve as a biosensing tool for monitoring pH change in its natural environment.
Assuntos
Chlorella/química , Clorofila/química , Fluorescência , Concentração de Íons de HidrogênioRESUMO
The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD(P) H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level. In the future, this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , NADP/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Técnicas In Vitro , Miócitos Cardíacos/citologia , NADP/análise , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
We present a new concept of cardiac cells based on an analogy with lasers, practical implementations of quantum resonators. In this concept, each cardiac cell comprises a network of independent nodes, characterised by a set of discrete energy levels and certain transition probabilities between them. Interaction between the nodes is given by threshold-limited energy transfer, leading to quantum-like behaviour of the whole network. We propose that in cardiomyocytes, during each excitation-contraction coupling cycle, stochastic calcium release and the unitary properties of ionic channels constitute an analogue to laser active medium prone to "population inversion" and "spontaneous emission" phenomena. This medium, when powered by an incoming threshold-reaching voltage discharge in the form of an action potential, responds to the calcium influx through L-type calcium channels by stimulated emission of Ca2+ ions in a coherent, synchronised and amplified release process known as calcium-induced calcium release. In parallel, phosphorylation-stimulated molecular amplification in protein cascades adds tuneable features to the cells. In this framework, the heart can be viewed as a coherent network of synchronously firing cardiomyocytes behaving as pulsed laser-like amplifiers, coupled to pulse-generating pacemaker master-oscillators. The concept brings a new viewpoint on cardiac diseases as possible alterations of "cell lasing" properties.
Assuntos
Miocárdio/citologia , Cálcio/metabolismo , Lasers , FosforilaçãoRESUMO
The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/biossíntese , Cálcio/fisiologia , Sulfonamidas , Zona Glomerulosa/metabolismo , Adolescente , Adulto , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Isoquinolinas/farmacologia , Cinética , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Níquel/farmacologia , Nifedipino/farmacologia , Inibidores de Proteínas Quinases , Fatores de Tempo , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacosRESUMO
OBJECTIVES: Pregnancy is an important physiological condition associated with hemodynamic and endocrine changes that affect the heart. Nevertheless, very little is known about cardiomyocyte remodeling in this condition. Here, we studied the morphological, functional and metabolic remodeling of rat left ventricular myocytes that occurs in late stages of normal pregnancy (P) and in experimental preeclampsia induced by elevated (0.9%) sodium intake (P0.9). METHODS: We applied confocal microscopy to examine the morphology and the contractility of single cells, while the patch clamp technique was used to assay ionic currents. RESULTS: Our results revealed a significant increase in the volume of single left ventricular cardiac myocytes in P, mainly resulting from cell elongation. In P0.9, further increase in the cell length led to a significant rise in the length/width ratio. Cell contractility was significantly decreased in glucose-based solutions in response to stimulation at 0.5 Hz and 6 Hz in P as well as in P0.9. The density of L-type calcium current (I(Ca)L) was not significantly altered in P or in P0.9. Metabolic substrates lactate and pyruvate, increased in the blood of P and P0.9 rats, enhanced contractility in P, without affecting I(Ca)L. The same effect, present but blunted in P0.9, was associated with a significant increase in I(Ca)L. CONCLUSION: Our results demonstrate that processes of adaptive remodeling take place in normal pregnancy, while maladaptive components are identified in experimental preeclampsia; they also reveal an adaptation in the use of energy substrates in pregnancy and its impairment by sodium supplementation.
Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Pré-Eclâmpsia/etiologia , Sódio/efeitos adversos , Animais , Contagem de Células , Tamanho Celular/efeitos dos fármacos , Ecocardiografia , Feminino , Glucose/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Microscopia Confocal , Modelos Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Técnicas de Patch-Clamp , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Ácido Pirúvico/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação VentricularRESUMO
Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca(2+)- activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.
Assuntos
Receptores de Angiotensina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Modulation of ionic Ca2+ currents by dopamine (DA) could play a pivotal role in the control of steroid secretion by the rat adrenal glomerulosa cells. In the present study, we report that DA decreases the T-type Ca2+ current amplitude in these cells. The use of pharmacological agonists and antagonists reveals that this effect is mediated by activation of the D1-like receptors. Modulation by cAMP is complex inasmuch as preincubation of the cells with 8-Br-cAMP or the specific adenylyl cyclase inhibitor, 2',3'-dideoxyadenosine, have no effect per se, but prevent the DA-induced inhibition. The inhibitory effect of DA was abolished by addition of GDPbetaS to the pipette medium but not by pertussis toxin. If a cell is dialyzed with medium containing G alpha(s)-GDP, the inhibitory effect is reduced and cannot be recovered by the addition of GTPgammaS, indicating that the alpha(s) is not involved, but rather the betagamma-subunit. Indeed, DA-induced inhibition was mimicked by G betagamma in the pipette and 8-Br-cAMP in the bath. Similarly, G betagamma release from the activation of the AT1 receptor of angiotensin II did affect the current amplitude only in the presence of 8-Br-cAMP in the bath. The mitogen-activated protein kinase cascade, which can be activated by receptors coupled to Gs, was not involved as shown by the lack of activation of p42mapk by DA and the absence of effect of the mitogen-activated protein kinase inhibitor, PD 098059, on the DA-induced inhibition. Because the binding of G betagamma-subunits to various effectors involves the motif QXXER, we therefore tested the effect of the QEHA peptide on the inhibition of the T-type Ca2+ current induced by DA. The peptide, added to the medium pipette (200 microM), abolished the effect of DA. We conclude that the presence of the G betagamma and an increase in cAMP concentration are both required to inhibit the T-type Ca2+ current in rat adrenal glomerulosa cells.
Assuntos
Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores de Dopamina D1/metabolismo , Zona Glomerulosa/metabolismo , Adenilil Ciclases/química , Adenilil Ciclases/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Fosforilação , Ratos , Sistemas do Segundo Mensageiro , Tionucleotídeos/farmacologiaRESUMO
In the present study, we report that ACTH induces a transient chloride current. The lack of correlation between ACTH-induced cAMP production and amplitude of the Cl- current, as well as the absence of stimulation by forskolin or 8Br-cAMP indicated that the ACTH-induced current was not cAMP-dependent. We explored the possibility that one or several elements of the Ras/Raf MAPK cascade were involved. Indeed, we found that ACTH at 10(-10) M induced activation of Ras. Inhibition of the current by QEHA peptide, a Gbetagamma sequestrant, demonstrated that Gbetagamma subunits transduced the message. Blockage of the Ras activation using an inhibitor of farnesyl transferase (BZA-5B) or the monoclonal antibody H-Ras(259) abrogated the current. Moreover, the addition of Ras-GTPyS in the pipette medium gave rise to the Cl- current. Treatment of the cells with BZA decreased the aldosterone secretion induced by 10(-10) M ACTH but not that induced by 10(-8) M ACTH, confirming the involvement of Ras in steroid secretion. We conclude that ACTH triggers a Cl- current through the activation of the Ras protein by Gbetagamma subunits. This current, activated at physiological ACTH concentrations (1 to 100 pM) where cAMP production is very low, could play a significant role in aldosterone production.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Canais de Cloreto/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Zona Glomerulosa/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aldosterona/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Oligopeptídeos/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Long-Evans , Zona Glomerulosa/citologiaRESUMO
The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca2+ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone. Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.
Assuntos
Hormônio Adrenocorticotrópico/fisiologia , AMP Cíclico/metabolismo , Zona Glomerulosa/fisiologia , Animais , Canais de Cloreto/metabolismo , Fosfatidilinositóis/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Ratos , Tirosina/metabolismo , Zona Glomerulosa/enzimologia , Zona Glomerulosa/metabolismoRESUMO
A new setup for time-resolved fluorescence micro-spectroscopy of cells, based on multi-dimensional time-correlated single photon counting, was designed and tested. Here we demonstrate that the spectrometer allows fast and reproducible measurements of endogenous flavin fluorescence measured directly in living cardiac cells after excitation with visible picosecond laser diodes. Two complementary approaches for the analysis of spectrally- and time-resolved autofluorescence data are presented, comprising the fluorescence decay fitting by exponential series and the time-resolved emission spectroscopy analysis. In isolated cardiac myocytes, we observed three distinct lifetime pools with characteristic lifetime values spanning from picosecond to nanosecond range and the time-dependent red shift of the autofluorescence emission spectra. We compared obtained results to in vitro recordings of free flavin adenine dinucleotide (FAD) and FAD in lipoamide dehydrogenase (LipDH). The developed setup combines the strength of both spectral and fluorescence lifetime analysis and provides a solid base for the study of complex systems with intrinsic fluorescence, such as identification of the individual flavinoprotein components in living cardiac cells. This approach therefore constitutes an important instrumental advancement towards redox fluorimetry of living cardiomyocytes, with the perspective of its applications in the investigation of oxidative metabolic state under pathophysiological conditions, such as ischemia and/or metabolic disorders.
Assuntos
Biofísica/métodos , Fótons , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Animais , Di-Hidrolipoamida Desidrogenase/química , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Fluorescência , Modelos Estatísticos , Miócitos Cardíacos/citologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We applied linear unmixing approach to reveal individual components of intrinsic flavin fluorescence signal recorded in living cardiac cells by spectrally resolved confocal microscopy. Responses of whole-cell autofluorescence to modulators of cell metabolism and respiration were used as a tool of separation of its components; their spectral profiles, estimated by principal component analysis, correspond to free FAD and FAD bound to different enzymes of electron transport chain.
Assuntos
Algoritmos , Flavinas/metabolismo , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Respiração Celular/fisiologia , Células Cultivadas , Humanos , OxirreduçãoRESUMO
Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm of [K+]e with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n = 4. Time constants ranged from 1 to 4 msec with a maximum at -25 mV. The steady-state activation curve spanned the voltage range -50 +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time constants tau 1 and tau 2 were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage range -40 to -20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of this "very slow inactivation" process (tau vs) determined for voltage steps at -60 and -50 mV were 15 and 10 sec respectively. A "facilitation process" of the peak current was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range -100 to -50 mV. Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed in relation with the membrane electrogenesis of this cell type.
Assuntos
Córtex Suprarrenal/fisiologia , Técnicas de Patch-Clamp , Zona Fasciculada/fisiologia , Animais , Bovinos , Células Cultivadas , Condutividade Elétrica , Cinética , Potássio/metabolismo , Zona Fasciculada/citologiaRESUMO
The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.
Assuntos
Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Líquido Intracelular/metabolismo , Tacrolimo/farmacologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/enzimologia , Córtex Suprarrenal/metabolismo , Animais , Inibidores de Calcineurina , Canais de Cálcio/metabolismo , Bovinos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/enzimologia , Microssomos/enzimologia , Microssomos/metabolismo , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/enzimologiaRESUMO
The effects of angiotensin II (100 nM) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10(-5) M), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of -10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl(-)-free solution, the outward current reversed at -78.5 mV, a value close to EK. It was blocked by external TEA (20 mM) and by apamin (50 nM). In K(+)-free solution, the transient outward current, sensitive to Cl- channel blocker DPC (400 microM), reversed at -52 mV, a more positive potential than ECl. Its magnitude changed in the same direction as the driving force for Cl-. The hormone-induced transient outward current was never observed when EGTA (5 mM) was added to the pipette solution. The plateau current was suppressed in nominally CA(2+)-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 microM) but not by nisoldipine (0.5-1 microM) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca(2+)-dependent K+ and Cl- currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]i originating from intra- and extracellular stores.
Assuntos
Angiotensina II/farmacologia , Cálcio/metabolismo , Canais de Cloreto/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Zona Fasciculada/efeitos dos fármacos , Animais , Cálcio/farmacologia , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Canais de Cloreto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/fisiologia , Zona Fasciculada/fisiologiaRESUMO
Adrenocorticotropic hormone (ACTH) is one of the principal activator of aldosterone secretion in rat zona glomerulosa cells, but its action on chloride currents is not well established. Here, we demonstrate that the hormone provoked a transient increase in a chloride current with a small unitary conductance estimated at 3.35 pS. Amplitude, as well as time-dependent increase of the ACTH-induced chloride current was independent of the intracellular cAMP concentration. In contrary, its decrease was sensitive to alkaline phosphatase and PKA-inhibitor H-89, indicating that protein phosphorylation, at least in part via PKA, is involved in the decline of the current.