RESUMEN
Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.
Asunto(s)
Bicarbonatos , Simportadores de Sodio-Bicarbonato , Ratas , Animales , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Bicarbonatos/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Sodio/metabolismo , Isoformas de Proteínas/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.
Asunto(s)
Feto/fisiología , Placenta/fisiología , Trofoblastos/fisiología , Animales , Educación , Epigénesis Genética/fisiología , Femenino , Feto/irrigación sanguínea , Feto/citología , Feto/inmunología , Humanos , Transporte Iónico/fisiología , Intercambio Materno-Fetal/fisiología , Placenta/irrigación sanguínea , Placenta/citología , Placenta/inmunología , Placentación/fisiología , Embarazo , Proteómica/métodos , Trofoblastos/citología , Trofoblastos/inmunologíaRESUMEN
In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.
Asunto(s)
Angiotensina II/farmacología , Cardiotónicos/farmacología , Angiotensina II/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Gatos , Colagenasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Indoles/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Microscopía Fluorescente , Miocardio/citología , Naftalenos/farmacología , Músculos Papilares/metabolismo , Técnicas de Placa-Clamp , Fosforilación , Proteína Quinasa C/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Rianodina/farmacología , Retículo Sarcoplasmático/metabolismo , Transducción de Señal , Tapsigargina/farmacología , Factores de TiempoRESUMEN
The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A-mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7+/-0.5 to 13.1+/-0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca(2+) transient (CaT), and pH(i) in indo-1 and seminaphthorhodafluor (SNARF)-1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160+/-34%) and CaT (70+/-5%), GLP-1 induced a significant decrease in contraction amplitude (-27+/-5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1-loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (DeltapH(i) -0.09+/-0.02 and -0.08+/-0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1-induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to beta-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin-sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca(2+) responsiveness probably resulting from intracellular acidification.
Asunto(s)
AMP Cíclico/metabolismo , Glucagón/farmacología , Corazón/efectos de los fármacos , Miocardio/metabolismo , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , Animales , Calcio/metabolismo , Cardiotónicos/farmacología , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Péptido 1 Similar al Glucagón , Corazón/fisiología , Concentración de Iones de Hidrógeno , Isoproterenol/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Ratas , Ratas Sprague-Dawley , Tionucleótidos/farmacología , Factores de TiempoRESUMEN
Angiotensin II (AngII) is a circulating peptide that produces a positive inotropic effect in the heart in several species, including humans. The subcellular mechanisms involved in producing this effect have been the focus of numerous studies; however, the results of these studies have generated considerable controversy. Although part of the controversy might arise from species and developmental differences, conflicting results have also been reported in the same species. To further complicate the understanding of the cardiac actions of AngII, the binding of the peptide to its transmembrane G-protein-coupled receptors has been shown to activate signalling cascades that involve numerous second messengers. Among these, inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) have been shown to have the potential to modulate either one or both of the two basic mechanisms known to increase contractility: (i) an increase in the intracellular Ca2+ concentration ([Ca2+]i); or (ii) an increase in myofilament responsiveness to Ca2+. The aim of this review is to examine the effect of AngII on the fundamental components of cardiac excitation-contraction coupling: calcium currents, Na+/Ca2+ exchange, sarcoplasmic reticulum (SR)-CaZ+ release, calcium transients and contractile proteins. An answer to the following question is sought: Is the positive inotropic effect of AngII due to an increase in [Ca2+]i, to an increase in myofilament responsiveness to Ca2+, or to both?
RESUMEN
1. Cat ventricular myocytes loaded with [Ca2+]i- and pHi-sensitive probes were used to examine the subcellular mechanism(s) of the Ang II-induced positive inotropic effect. Ang II (1 microM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 +/- 22 and 43 +/- 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0.06 +/- 0.03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+-H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. 2. Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II-induced positive inotropic effect. 3. Ang II significantly increased the L-type Ca2+ current, as assessed by using the perforated patch-clamp technique (peak current recorded at 0 mV: -1.88 +/- 0.16 pA pF-1 in control vs. -3.03 +/- 0.20 pA pF-1 after 6-8 min of administration of Ang II to the bath solution). 4. The positive inotropic effect of Ang II was not modified in the presence of either KB-R7943, a specific blocker of the Na+-Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. 5. The above results allow us to conclude that in the cat ventricle the Ang II-induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L-type Ca2+ current being the dominant mechanism underlying this increase.
Asunto(s)
Angiotensina II/farmacología , Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Gatos , Electrofisiología , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Indoles , Potenciales de la Membrana/fisiología , Miocardio/ultraestructura , Técnicas de Placa-Clamp , Retículo Sarcoplasmático/fisiología , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
1. The perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+-HCO3- cotransport in isolated rat ventricular myocytes. 2. Switching from Hepes buffer to HCO3- buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 +/- 0.4 mV (n = 9, P < 0.05). In the presence of HCO3-, the anion blocker SITS depolarized RMP by 2.6 +/- 0.5 mV (n = 5, P < 0.05). No HCO3--induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 +/- 6.1 % shorter in the presence of HCO3- than in its absence (n = 6, P < 0.05). 3. Quasi-steady-state currents were evoked by voltage-clamped ramps ranging from -130 to +30 mV, during 8 s. The development of a novel component of Na+-dependent and Cl--independent steady-state outward current was observed in the presence of HCO3-. The reversal potential (Erev) of the Na+-HCO3- cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3-:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole-cell experiments. Under these conditions, INa,Bic reversed at -96.4 +/- 1.9 mV (n = 5), being consistent with the influx of 2 HCO3- ions per Na+ ion through the Na+-HCO3- cotransporter. 4. In the presence of external HCO3-, after 10 min of depolarizing the membrane potential (Em) with 45 mM extracellular K+, a significant intracellular alkalinization was detected (0.09 +/- 0. 03 pH units; n = 5, P < 0.05). No changes in pHi were observed when the myocytes were pre-treated with the anion blocker DIDS (0.001 +/- 0.024 pH units; n = 5, n.s.), or when exposed to Na+-free solutions (0.003 +/- 0.037 pH units; n = 6, n.s.). 5. The above results allow us to conclude that the cardiac Na+-HCO3- cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Corazón/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Benzopiranos , Bicarbonatos/metabolismo , Células Cultivadas , Colorantes Fluorescentes , HEPES , Ventrículos Cardíacos , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Miocardio/citología , Miocardio/metabolismo , Técnicas de Placa-Clamp , Ratas , Sodio/metabolismo , Sodio/farmacología , Simportadores de Sodio-Bicarbonato , Espectrometría de FluorescenciaRESUMEN
The present study examines the intracellular pH (pHi) dependence of angiotensin (ANG) II-induced positive inotropic effect in cat papillary muscles contracting isometrically (0.2 Hz, 30 degrees C). Muscles were loaded with the fluorescent dye 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester for simultaneous measurement of pHi and contractility. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (n = 4), there was a temporal dissociation between the positive inotropic and the alkalinizing effects of ANG II (0.5 microM). The positive inotropic effect of ANG II peaked at 9.7 +/- 0.8 min (240 +/- 57% above control) without significant changes in pHi. The increase in pHi became significant (0.05 +/- 0.01 pH units) only after 16 min of exposure to the drug, when the positive inotropic effect of ANG II was already fading. In HCO3- buffer (n = 7), the ANG II-induced positive inotropic effect occurred without significant pHi changes. In the presence of 5 microM ethyl isopropyl amiloride (EIPA, to specifically inhibit the Na+/H+ exchanger), the alkalinizing effect of ANG II was changed to a significant decrease in pHi, despite which ANG II still increased contractility by 87 +/- 16% (n = 6). The results indicate that in HEPES buffer only a fraction of the ANG II-induced positive inotropic effect can be attributed to a pHi change, whereas in a physiological CO2-HCO3- medium the positive inotropic effect of ANG II is independent of pHi changes. Furthermore, an ANG II-induced increase in myocardial contractility was observed even when ANG II administration elicited a decrease in pHi, as occurred after Na+/H+ exchanger blockade. The results show that in feline myocardium, the increase in contractility evoked by ANG II in a physiological CO2-HCO3- medium is not due to an increase in Ca2+ myofilament sensitivity secondary to an increase in myocardial pHi.