Biomedical subjects

Joël Nargeot

Publications and source records attributed to Joël Nargeot.

At least 19 recordsLinked to original sources

PubMed2006

Subunit-specific modulation of T-type calcium channels by zinc.

Zinc (Zn2+) functions as a signalling molecule in the nervous system and modulates many ionic channels. In this study, we have explored the effects of Zn2+ on recombinant T-type calcium channels (CaV3.1, CaV3.2 and CaV3.3). Using tsA-201 cells, we demonstrate that CaV3.2 current (IC50, 0.8 microm) is significantly more sensitive to Zn2+ than are CaV3.1 and CaV3.3 currents (IC50, 80 microm and approximately 160 microm, respectively). This inhibition of CaV3 currents is associated with a shift to more negative membrane potentials of both steady-state inactivation for CaV3.1, CaV3.2 and CaV3.3 and steady-state activation for CaV3.1 and CaV3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for CaV3.1 and CaV3.3, but not for CaV3.2 currents. Notably, deactivation kinetics are significantly slowed for CaV3.3 current (approximately 100-fold), but not for CaV3.1 and CaV3.2 currents. Consequently, application of Zn2+ results in a significant increase in CaV3.3 current in action potential clamp experiments, while CaV3.1 and CaV3.2 currents are significantly reduced. In neuroblastoma NG 108-15 cells, the duration of CaV3.3-mediated action potentials is increased upon Zn2+ application, indicating further that Zn2+ behaves as a CaV3.3 channel opener. These results demonstrate that Zn2+ exhibits differential modulatory effects on T-type calcium channels, which may partly explain the complex features of Zn2+ modulation of the neuronal excitability in normal and disease states.

Action Potentials↗

PubMed2006

Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels.

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for CaV3.1/alpha1G T-type calcium channels (cacna1g) abolishes T-type calcium current (I(Ca,T)) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (I(Ca,L)). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no I(Ca,T) was detected in right atrium myocytes in both wild-type and CaV3.1(-/-) mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that CaV3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

Animals↗

PubMed2006

Properties and role of voltage-dependent calcium channels during mouse skeletal muscle differentiation.

Skeletal muscle differentiation depends on calcium ions, but it is yet unclear whether calcium entry through voltage-dependent calcium channels (VDCCs) contributes to the myoblast fusion process. In this study, we investigate whether calcium influx through functional T-type VDCCs precedes and affects mouse satellite cell fusion. We report here on the properties and the role of the VDCCs expressed in differentiating mouse muscular cells using both the C2C12 cell line and primary cultures of satellite cells. We present electrophysiological and biochemical evidence demonstrating that T-type and L-type VDCCs are not present in C2C12 and primary cultures of mouse satellite cells prior to the fusion stage. Although mRNA for the T-type Ca(V)3.2 subunit was detected in differentiated C2C12 cells, no T-type calcium currents could be recorded, while both T-type and L-type calcium currents were detected after the fusion process in primary cultures. In addition, chronic application of 30 microM nickel, known to inhibit T-type Ca(V)3.2 channels, did not alter the fusion of C2C12 cells and mouse satellite cells in primary culture. Overall, the data indicate that, unlike in humans, Ca(V)3.2 T-type calcium channels play no role in mouse satellite cell fusion.

Animals↗

PubMed2006

T-type calcium channels are inhibited by fluoxetine and its metabolite norfluoxetine.

Fluoxetine, a widely used antidepressant that primarily acts as a selective serotonin reuptake inhibitor, also inhibits various neuronal ion channels. Using the whole-cell patch-clamp technique, we have examined the effects of fluoxetine and norfluoxetine, its major active metabolite, on cloned low-voltage-activated T-type calcium channels (T channels) expressed in tsA 201 cells. Fluoxetine inhibited the three T channels Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 in a concentration-dependent manner (IC(50) = 14, 16, and 30 microM, respectively). Norfluoxetine was a more potent inhibitor than fluoxetine, especially on the Ca(V)3.3 T current (IC(50) = 5 microM). The fluoxetine block of T channels was voltage-dependent because it was significantly enhanced for T channels in the inactivated state. Fluoxetine caused a hyperpolarizing shift in steady-state inactivation, with a slower rate of recovery from the inactivated state. These results indicated a tighter binding of fluoxetine to the inactivated state than to the resting state of T channels, suggesting a more potent inhibition of T channels at physiological resting membrane potential. Indeed, fluoxetine and norfluoxetine at 1 microM strongly inhibited cloned T currents (approximately 50 and approximately 75%, respectively) in action potential clamp experiments performed with firing activities of thalamocortical relay neurons. Altogether, these data demonstrate that clinically relevant concentrations of fluoxetine exert a voltage-dependent block of T channels that may contribute to this antidepressant's pharmacological effects.

Calcium Channel Blockers↗

PubMed2006

Differentiated rabbit prostatic stromal cells in primary culture display functional alpha1A-adrenoceptors.

AIMS: BPH is characterized by uncontrolled proliferation and increased contractility of prostatic smooth muscle cells. The activation of alpha1-adrenoceptors (alpha1-AR) seems involved in the latter event, but the lack of in vitro models expressing these receptors has hampered a more specific characterization of their role. In order to do so, we attempted to develop a new model of rabbit cultured prostatic stromal cells (PSC) in a non-proliferative and differentiated state. METHODS: The expression of cytoskeletal and stromal markers was confirmed by immunohistochemistry on primary cultured PSC. Alpha1-AR subtype expression was assessed by RT-PCR, while receptor coupling to the ERK1/ERK2 and calcium pathways was studied by Western Blot and Fura-2 calcium imaging, respectively. RESULTS: Cells grown under non-proliferative conditions displayed a differentiated phenotype, with expression of contractile cytoskeletal and stromal proteins. Furthermore, the alpha1A-AR was shown to activate ERK1/ERK2 as well as calcium signaling. CONCLUSION: These results emphasize the interest of this model for the characterization of PSC adrenergic regulation, in particular through the little-known alpha1A-AR.

Animals↗

PubMed2005

Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes.

OBJECTIVE: Although aldosterone has been implicated in the pathogenesis of cardiac hypertrophy and heart failure, its cellular mechanism of action on cardiomyocyte function is not yet completely elucidated. This study was designed to investigate the effect of aldosterone on calcium channel expression and cardiomyocyte contraction frequency. METHODS: Cultured neonatal rat ventricular cardiomyocytes were stimulated in vitro with 1 micromol/L aldosterone for 24 h. Calcium currents were then measured with the patch clamp technique, while calcium channel expression was assessed by real-time RT-PCR. RESULTS: In the present study, we show that aldosterone increases Ca2+ currents by inducing channel expression. Indeed, aldosterone led to a substantial increase of L- and T-type Ca2+ current amplitudes, and we found a concomitant 55% increase of the mRNA coding for alpha1C and beta2 subunits of cardiac L channels. Although T-type currents were relatively small under control conditions, they increased 4-fold and T channel alpha1H isoform expression rose in the same proportion after aldosterone treatment. Because T channels have been implicated in the modulation of membrane electrical activity, we investigated whether aldosterone affects the beating frequency of isolated cardiomyocytes. In fact, aldosterone dose-dependently increased the spontaneous beating frequency more than 4-fold. This effect of aldosterone was prevented by actinomycin D and spironolactone and reduced by RU486, suggesting a mixed mineralocorticoid/glucocorticoid receptor-dependent transcriptional mechanism. Moreover, inhibition of T currents with Ni2+ or mibefradil significantly reduced beating frequency towards control values, while conditions affecting L-type currents completely blocked contractions. CONCLUSION: Aldosterone modulates the expression of cardiac voltage-operated Ca2+ channels and accelerates beating in cultured neonatal rat ventricular myocytes. This chronotropic action of aldosterone appears to be linked to increased T channel activity and could contribute to the deleterious effect of an excess of this steroid in vivo on cardiac function.

Aldosterone↗

PubMed2005

Voltage-dependent calcium channels and cardiac pacemaker activity: from ionic currents to genes.

The spontaneous activity of pacemaker cells in the sino-atrial node controls the heart rhythm and rate under physiological conditions. Compared to working myocardial cells, pacemaker cells express a specific array of ionic channels. The functional importance of different ionic channels in the generation and regulation of cardiac automaticity is currently subject of an extensive research effort and has long been controversial. Among families of ionic channels, Ca(2+) channels have been proposed to substantially contribute to pacemaking. Indeed, Ca(2+) channels are robustly expressed in pacemaker cells, and influence the cell beating rate. Furthermore, they are regulated by the activity of the autonomic nervous system in both a positive and negative way. In this manuscript, we will first discuss how the concept of the involvement of Ca(2+) channels in cardiac pacemaking has been proposed and then subsequently developed by the recent advent in the domain of cardiac physiology of gene-targeting techniques. Secondly, we will indicate how the specific profile of Ca(2+) channels expression in pacemaker tissue can help design drugs which selectively regulate the heart rhythm in the absence of concomitant negative inotropism. Finally, we will indicate how the new possibility to assign a specific gene activity to a given ionic channel involved in cardiac pacemaking could implement the current postgenomic research effort in the construction of the cardiac Physiome.

Animals↗

PubMed2004

Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception.

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (CaV3.1, CaV3.2, and CaV3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting CaV3.2 induced a knockdown of the CaV3.2 mRNA and protein expression as well as a large reduction of 'CaV3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that CaV3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking CaV3.2 T-type channels to pain perception and suggest that CaV3.2 may offer a specific molecular target for the treatment of pain.

Animals↗

PubMed2004

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart.

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (C(t)) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Nav beta 1, Nav beta 3, Cav1.3, Cav3.1 and Cav alpha 2 delta 2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cav beta 2 and Cav gamma 4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvbeta1, MinK and Cav gamma 7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.

Animals↗

PubMed2004

Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes.

Morphine has cardioprotective effects against ischemic-reperfusion injuries. This study investigates whether morphine could mimic the antiapoptotic effect of preconditioning using a model of cultured neonatal rat cardiomyocytes subjected to metabolic inhibition (MI). To quantify MI-induced apoptosis, DNA fragmentation and mitochondrial cytochrome c release levels were measured by ELISA. MI-dependent DNA fragmentation was prevented by both Z-VAD-fmk (20 microM), a pan-caspase inhibitor, and cyclosporine A (CsA; 5 microM), a mitochondrial pore transition blocker, added during MI (36% and 54% decrease, respectively). MI-dependent cytochrome c release was not blocked by Z-VAD-fmk but was decreased (38%) by CsA during MI. Metabolic preconditioning (MIP) and preconditioning with morphine (1 microM) were also assessed. MI-dependent DNA fragmentation and cytochrome c release were prevented by MIP (40% and 45% decrease, respectively) and morphine (34% and 45%, respectively). The antiapoptotic effect of morphine was abolished by naloxone (10 nM), a nonselective opioid receptor antagonist, or xestospongin C (XeC, 400 nM), an inhibitor of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)]-mediated Ca(2+) release. Ca(2+) preconditioning, induced by increasing extracellular Ca(2+) from 1.8 to 3.3 mM, mimicked the antiapoptotic effect of morphine on DNA fragmentation (24% decrease) and cytochrome c release (57% decrease). This effect mediated by extracellular Ca(2+) was also abolished by XeC. Measurements of intracellular Ca(2+) concentration using fura-2 microspectrofluorimetry showed that morphine induces Ins(1,4,5)P(3)-dependent Ca(2+) transients abolished by 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable Ins(1,4,5)P(3) antagonist. These results suggest that morphine preconditioning prevents simulated ischemia-reperfusion-induced apoptosis via an Ins(1,4,5)P(3) signaling pathway in rat ventricular myocytes.

Animals↗

PubMed2004

Architectural and functional asymmetry of the His-Purkinje system of the murine heart.

OBJECTIVE: The aim of this work was to target a vital reporter gene in the mouse cardiac conduction system (CS) to distinguish this tissue from the surrounding myocardium in the adult heart. METHODS: A transgenic mouse line has been created in which EGFP is expressed under the control of the Cx40 gene. Correlative investigations associating EGFP imaging and electrophysiological techniques were carried out on the adult heart and isolated cardiomyocytes. RESULTS: In the heart of the Cx40(EGFP/+) mice, EGFP signal was seen in the coronary arteries, the atria, the atrioventricular (AV) node and the His-Purkinje system. The latter was found to be structurally and functionally asymmetrical. The anatomical asymmetry was apparent in both the number of strands or fasciculi making up the His bundle branches (BBs) (1 strand on the right, 20 or so on the left), and the density (low on the right, high on the left) of the network of Purkinje fibers (PFs) that extends over the ventricular wall surfaces. The profiles of the electrical activation patterns recorded on the right and left flanks of the septum were also asymmetrical, mirroring the architecture of the branches. EGFP made it easy to identify the Purkinje cells in populations of dissociated cardiomyocytes and they were investigated using the patch-clamp technique. The hyperpolarization-activated current (If) was recorded in all spontaneously active Purkinje cells. CONCLUSIONS: This investigation provides positive evidence of the asymmetry of the His-Purkinje system of the adult mouse, and the first patch-clamp recording data on murine cardiac Purkinje cells. This mouse model opens up new perspectives for investigating the contribution of specific genes to the morphology and function of the His-Purkinje system.

Action Potentials↗

PubMed2004

Plasma membrane expression of T-type calcium channel alpha(1) subunits is modulated by high voltage-activated auxiliary subunits.

It has been suggested that the auxiliary subunits of high voltage-activated (HVA) calcium channels modulate T-type, low voltage-activated (LVA) calcium channels. Such a regulation has yet to be documented, especially because there has been no biochemical characterization of T-channels. To monitor total protein levels and plasma membrane expression of T-channels in living cells, external epitopes (hemagglutinin, FLAG) were introduced into human recombinant Ca(V)3 channels that were also N-terminally fused to green fluorescent protein. Utilizing Western blot techniques, fluorescence flow cytometry, immunofluorescence, luminometry, and electrophysiology, we describe here that beta(1b) and alpha(2)-delta(1) subunits enhance the level of Ca(V)3 proteins as well as their plasma membrane expression in various expression systems. We also report that, in both Xenopus oocytes and mammalian cells, the alpha(2)-delta(1) subunits increase by at least and beta(1b) 2-fold the current density of Ca(V)3 channels with no change in the electrophysiological properties. Altogether, these data indicate that HVA auxiliary subunits modulate Ca(V)3 channel surface expression, suggesting that the membrane targeting of HVA and LVA alpha(1) subunits is regulated dynamically through the expression of a common set of regulatory subunits.

Animals↗

PubMed2004

Ca(v)3.2 calcium channels control an autocrine mechanism that promotes neuroblastoma cell differentiation.

Calcium influx via low-voltage activated alpha(1H) (Ca(v)3.2) T-currents participates in the morphological and electrical differentiation of neuroblastoma NG108-15 cells. We investigated whether an autocrine mechanism could contribute to this differentiation process. The presence of factors secreted by NG108-15 cells was identified through the use of conditioned media (CM) obtained from differentiated cells. These CM significantly increased neuritogenesis with no change in the HVA calcium channel expression. CM-induced neuritogenesis persists during alpha(1H) current block, whereas CM obtained from cells transfected with an alpha(1H) antisense did not induce neuritogenesis. These data indicate that morphological differentiation of NG108-15 cells depends on an autocrine mechanism, which is controlled by alpha(1H) currents. Such a mechanism is likely to play a role in the various differentiation processes that imply alpha(1H) T-type Ca(2+) channels.

Animals↗

PubMed2004

Dissecting the functional role of different isoforms of the L-type Ca2+ channel.

There currently exist a great number of different mouse lines in which the activity of a particular gene of interest has been inactivated or enhanced. However, it is also possible to insert specific mutations in a gene so that the pharmacological sensitivity of the gene product is altered. An example of such an approach shows how the abolition of the sensitivity of an L-type Ca(2+) channel isoform to dihydropyridines allows the investigation of the physiological role of these channels in different tissues.

Affect↗

PubMed2003

Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity.

The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for alpha(1D) (Ca(v)1.3) Ca(2+) channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (I(Ca,L)) activating at negative voltages. Pharmacological analysis of I(Ca,L) demonstrates that Ca(v)1.3 gene inactivation specifically abolishes I(Ca,L) in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Ca(v)1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.

Action Potentials↗

PubMed2003

Effects of amiodarone and dronedarone on voltage-dependent sodium current in human cardiomyocytes.

UNLABELLED: Effect of Dronedarone on Cardiac Na Current. INTRODUCTION: Amiodarone (AM) is a highly effective antiarrhythmic agent used in the management of both atrial and ventricular arrhythmias. Its noniodinated analogue dronedarone (SR) may have fewer side effects than AM. In this study, we compared the effects of AM and SR on the sodium current I(Na) in human atrial myocytes. METHODS AND RESULTS: INa was studied with the whole-cell, patch clamp technique. Both AM and SR induced a dose-dependent inhibition of I(Na) recorded at -40 mV from a holding potential of -100 mV. AM inhibited I(Na) by 41%+/- 11% (n = 4) at 3 microM, and by 80%+/- 7% (n = 5) at 30 microM. SR produced more potent block, inhibiting INa significantly at only 0.3 microM (23%+/- 10%, n = 4) and completely (97%+/- 4%, n = 4) at 3 microM. Both AM and SR had only moderate effects on voltage-dependent properties of I(Na) (current-voltage relationship, availability for activation) and had no effect on the current decay kinetics. CONCLUSION: Both AM and SR inhibit I(Na) significantly in single human atrial cells, showing that the two drugs have Class I antiarrhythmic properties. The acute effects of SR are more potent than those of AM. The study supports the idea that the iodinated form of the molecule has no part in the acute effect of AM on Na+ channels.

Adult↗

PubMed2002

Neuronal T-type alpha 1H calcium channels induce neuritogenesis and expression of high-voltage-activated calcium channels in the NG108-15 cell line.

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108-15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the alpha1H T-channel subunit. Furthermore, we describe that inhibition of alpha1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

Animals↗

PubMed2002

Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79.

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.

A Kinase Anchor Proteins↗