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J Nargeot

Publications and source records attributed to J Nargeot.

At least 37 records · Page 2Linked to original sources
PubMed2000

Evidence for tetrodotoxin-sensitive sodium currents in primary cultured myocytes from human, pig and rabbit arteries.

Primary cultured human coronary myocytes express a tetrodotoxin-sensitive sodium current (I(Na)). Here, we have investigated whether I(Na) is expressed in vascular smooth muscles cells (VSMCs) isolated from other large arteries, and other mammals. VSMCs were enzymatically dissociated, kept in primary culture, and macroscopic I(Na) was recorded using the whole-cell patch-clamp technique. We found that I(Na) is expressed in VSMCs grown from human aortic (90%; n=50) and pulmonary (44%; n=19) arteries, and in the human aortic myocyte cell line HAVSMC (94%; n=27). I(Na) was also detected in pig coronary (60%; n=33), and rabbit aortic (47%; n=15), but not in rat aortic VSMCs (n=20). These different I(Na) had similar voltage thresholds for activation (approximately equal to -50 mV), and were highly sensitive to extracellularly applied tetrodotoxin. We conclude that I(Na) is expressed in VSMCs grown from various types of large arteries in humans, pig and rabbit.

Animals↗
PubMed2000

Facilitation of L-type calcium currents by diastolic depolarization in cardiac cells: impairment in heart failure.

OBJECTIVE: Decay kinetics of the voltage-gated L-type Ca(2+) current (I(CaL)) control the magnitude of Ca(2+) influx during the cardiac action potential. We investigated the influence of changes in diastolic membrane potential on I(CaL) decay kinetics in cardiac cells. METHODS: Cells were isolated enzymatically from rat ventricles, human right atrial appendages obtained during corrective heart surgery and left ventricles from end-stage failing hearts of transplant recipients. The whole-cell patch-clamp technique was used to evoke I(CaL) by a 100-ms depolarizing test pulse to -10 mV. Conditioning potentials between -80 and 0 mV were applied for 5 s prior to the test pulse. RESULTS: Depolarizing the cells between -80 and -50 mV prior to the test pulse slowed the early inactivation of I(CaL) both in rat ventricular and human atrial cells. This slowing resulted in a significant increase of Ca(2+) influx. This type of facilitation was not observed when the sarcoplasmic reticulum (SR) Ca(2+) content was depleted using ryanodine which reduced the rate of inactivation of I(CaL), or when Ba(2+) replaced Ca(2+) as the permeating ion. Facilitation was favored by intracellular cAMP-promoting agents that, in addition to increasing current peak amplitude, enhanced the fast Ca(2+)-dependent inactivation of I(CaL). Facilitation was impaired in atrial and ventricular human failing hearts. CONCLUSION: Decay kinetics of I(CaL) are regulated by the diastolic membrane potential in rat and human cardiomyocytes. This regulation, which associates slowing of I(CaL) inactivation with reduced SR Ca(2+) release and underlies facilitation of Ca(2+) channels activity, may have profound physiological relevance for catecholamines enhancement of Ca(2+) influx. It is impaired in failing hearts, possibly due to lowered SR Ca(2+) release.

Adrenergic beta-Agonists↗
PubMed2000

Facilitation of the L-type calcium current in rabbit sino-atrial cells: effect on cardiac automaticity.

OBJECTIVE: The L-type Ca(2+) current (I(Ca,L)) contributes to the generation and modulation of the pacemaker action potential (AP). We investigated facilitation of I(Ca,L) in sino-atrial cells. METHODS: Facilitation was studied in regularly-beating cells isolated enzymatically from young albino rabbits (0.8-1 kg). We used the whole-cell patch-clamp technique to vary the frequency of the test depolarizations evoked at -10 mV or the conditioning diastolic membrane potential prior to the test pulse. RESULTS: High frequencies (range 0.2-3.5 Hz) slowed the decay kinetics of I(Ca,L) evoked from a holding potential (HP) of -80 mV in 68% of cells resulting in a larger Ca(2+) influx during the test pulse. The amount of facilitation increased progressively between 0.2 and 3.0 Hz. When the frequency was changed from 0.1 to 1 Hz, the averaged increase in the time integral of I(Ca,L) was 27+/-7% (n=22). Application of conditioning voltages between -80 and -50 mV induced similar facilitation of I(Ca,L) in 73% of cells. The maximal increase of Ca(2+) entry occurred between -60 and -50 mV, and was on average 38+/-14% for conditioning prepulses of 5 s in duration (n=15). Numerical simulations of the pacemaker activity showed that facilitation of I(Ca,L) promotes stability of sino-atrial rate by enhancing Ca(2+) entry, thus establishing a negative feedback control against excessive heart rate slowing. CONCLUSION: Facilitation of I(Ca,L) is present in rabbit sino-atrial cells. The underlying mechanism reflects modulation of I(Ca,L) decay kinetics by diastolic membrane potential and frequency of depolarization. This phenomenon may provide an important regulatory mechanism of sino-atrial automaticity.

Animals↗
PubMed2000

Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels.

The voltage gated calcium channel family is a major target for a range of therapeutic drugs. Mibefradil (Ro 40-5967) belongs to a new chemical class of these molecules which differs from other Ca2+ antagonists by its ability to potently block T-type Ca2+ channels. However, this molecule has also been shown to inhibit other Ca2+ channel subtypes. To further analyze the mechanism governing the Ca2+ channel-Mibefradil interaction, we examined the effect of Mibefradil on various recombinant Ca2+ channels expressed in mammalian cells from their cloned cDNAs, using Ca2+ as the permeant ion at physiological concentration. Expression of alpha1A, alpha1C, and alpha1E in tsA 201 cells resulted in Ca2+ currents with functional characteristics closely related to those of their native counterparts. Mibefradil blocked alpha1A and alpha1E with a Kd comparable to that reported for T-type channels, but had a lower affinity (approximately 30-fold) for alpha1C. For each channel, inhibition by Mibefradil was consistent with high-affinity binding to the inactivated state. Modulation of the voltage-dependent inactivation properties by the nature of the coexpressed beta subunit or the alpha1 splice variant altered block at the Mibefradil receptor site. Therefore, we conclude that the tissue and sub-cellular localization of calcium channel subunits as well as their specific associations are essential parameters to understand the in vivo effects of Mibefradil.

Alternative Splicing↗
PubMed2000

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion.

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.

Aging↗
PubMed2000

[Is atypical sodium current related to arterial pathophysiology?].

Primary cultured human coronary myocytes, derived from patients with end-stage heart failure (NYHA, classes III and IV) caused by an ischemic disease and undergoing heart transplantation, express a voltage-gated tetrodotoxin-sensitive sodium current (INa). This current has atypical electrophysiological and pharmacological properties and regulates intracellular sodium ([Na+]i) and calcium ([Ca2+]i). Our work is aimed at identifying its role and regulation of expression during pathophysiology. We currently investigate whether INa is expressed in vascular smooth muscles cells (VSMCs) isolated from either healthy or diseased (atheromatous) arteries in human and, in parallel, in pig, rabbit and rat. Cells were enzymatically isolated, primary cultured and macroscopic INa were recorded using the whole cell patch clamp technique. We found that INa is expressed in VSMCs grown from human aortic (90%; n = 48) and pulmonary (44%; n = 16) arteries and in the human aortic cell line HAVSMC (94%; n = 27). INa was also detected in pig coronary (60%; n = 25) and rabbit aortic (47%; n = 15) VSMCs, but not in rat aortic myocytes (n = 30). These different INa were activated at similar range of potentials (approximately -45 mV), had similar sensitivity to tetrodotoxin (IC50 around 5 nM) and similar density (2 to 4 pA/pF). Their expression was related to cell dedifferentiation in vitro. However, INa was observed more frequently in human myocytes derived from diseased arteries (ischemic cardiopathy) than in those derived from healthy tissues (dilated cardiopathy). In conclusion, INa may contribute to increase the basal arterial contractility and play a role in pathological situations including hypertension.

Action Potentials↗
PubMed1999

Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells.

The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca(2+) channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (alpha(1S)) and cardiac (alpha(1C)) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nM all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca(2+) channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca(2+) channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (alpha(1C)) L-type Ca(2+) channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca(2+) channels in mature cardiac cells.

Animals↗
PubMed1999

Regulation of Ca2+ homeostasis by atypical Na+ currents in cultured human coronary myocytes.

Primary cultured human coronary myocytes (HCMs) derived from ischemic human hearts express an atypical voltage-gated tetrodotoxin (TTX)-sensitive sodium current (I(Na)). The whole-cell patch-clamp technique was used to study the properties of I(Na) in HCMs. The variations of intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were monitored in non-voltage-clamped cells loaded with Fura-2 or benzofuran isophthalate, respectively, using microspectrofluorimetry. The activation and steady-state inactivation properties of I(Na) determined a "window" current between -50 and -10 mV suggestive of a steady-state Na+ influx at the cell resting membrane potential. Consistent with this hypothesis, the resting [Na+]i was decreased by TTX (1 micromol/L). In contrast, it was increased by Na+ channel agonists that also promoted a large rise in [Ca2+]i. Veratridine (10 micromol/L), toxin V from Anemonia sulcata (0.1 micromol/L), and N-bromoacetamide (300 micromol/L) increased [Ca2+]i by 7- to 15-fold. This increase was prevented by prior application of TTX or lidocaine (10 micromol/L) and by the use of Na(+)-free or Ca(2+)-free external solutions. The Ca(2+)-channel antagonist nicardipine (5 micromol/L) blocked the effect of veratridine on [Ca2+]i only partially. The residual component disappeared when external Na+ was replaced by Li+ known to block the Na+/Ca2+ exchanger. The resting [Ca2+]i was decreased by TTX in some cells. In conclusion, I(Na) regulates [Ca2+]i in primary cultured HCMs. This regulation, effective at baseline, involves a tonic control of Ca2+ influx via depolarization-gated Ca2+ channels and, to a lesser extent, via a Na+/Ca2+ exchanger working in the reverse mode.

Adult↗
PubMed1999

Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

Alternative Splicing↗
PubMed1998

Heart rate as a determinant of L-type Ca2+ channel activity: mechanisms and implication in force-frequency relation.

Early studies in enzymatically isolated animal cardiomyocytes indicated that voltage-gated "L-type" Ca2+ currents (ICaL) can be upregulated following an increase of the frequency of activation. Recently, we evidenced a similar regulation of ICaL in human cardiomyocytes from both left and right ventricles and atria over a physiopathological range of stimulations (between 0.5 and 5 Hz). This regulation, enhanced by the beta-adrenergic stimulation, may be involved in the frequency-dependent potentiation of cardiac contractile force in the human healthy myocardium. We show here that the frequency-dependent regulation of ICaL is controlled by the level of phosphorylation, as well as dephosphorylation, of the Ca2+ channels. It was enhanced following activation of the protein kinase A activated by intracellular cyclic AMP (cAMP). Therefore, we anticipate that all agents stimulating cAMP production will favor this process, which was demonstrated here by activating 5HT-4 receptors using serotonin. Alternatively, it was also enhanced by the phosphatase inhibitor okadaic acid which prevents Ca2+ channels dephosphorylation. Alteration or abnormal modulation by beta-adrenergic receptor stimulation of the frequency-dependent facilitation of ICaL may partly explain the altered force-frequency relation described in heart failure.

Biomechanical Phenomena↗
PubMed1998

Ca2+ currents in compensated hypertrophy and heart failure.

Transmembrane voltage-gated Ca2+ channels play a central role in the development and control of heart contractility which is modulated by the concentration of free cytosolic calcium ions (Ca2+). Ca2+ channels are closed at the normal membrane resting potential of cardiac cells. During the fast upstroke of the action potential (AP), they are gated into an open state by membrane depolarisation and thereby transduce the electrical signal into a chemical signal. In addition to its contribution to the AP plateau, Ca2+ influx through L-type Ca2+ channels induces a release of Ca2+ ions from the sarcoplasmic reticulum (SR) which initiates contraction. Because of their central role in excitation-contraction (E-C) coupling, L-type Ca2+ channels are a key target to regulate inotropy [1]. The role of T-type Ca2+ channels is more obscure. In addition to a putative part in the rhythmic activity of the heart, they may be implicated at early stages of development and during pathology of contractile tissues [2]. Despite therapeutic advances improving exercise tolerance and survival, congestive heart failure (HF) remains a major problem in cardiovascular medicine. It is a highly lethal disease; half of the mortality being related to ventricular failure whereas sudden death of the other patients is unexpected [3]. Although HF has diverse aetiologies, common abnormalities include hypertrophy, contractile dysfunction and alteration of electrophysiological properties contributing to low cardiac output and sudden death. A significant prolongation of the AP duration with delayed repolarisation has been observed both during compensated hypertrophy (CH) and in end-stage HF caused by dilated cardiomyopathy (Fig. 1A) [4-8]. This lengthening can result from either an increase in inward currents or a decrease in outward currents or both. A reduction of K+ currents has been demonstrated [6,9]. Prolonged Na+/Ca2+ exchange current may also be involved [9]. In contrast, there is a large variability in the results concerning Ca2+ currents (ICa). The purpose of this paper is to review results obtained in various animal models of CH and HF with special emphasis on recent studies in human cells. We focus on: (i) the pathophysiological role of T-type Ca2+ channels, present in some animal models of hypertrophy; (ii) the density and properties of L-type Ca2+ channels and alteration of major physiological regulations of these channels by heart rate and beta-adrenergic receptor stimulation; and (iii) recent advances in the molecular biology of the L-type Ca2+ channel and future directions.

Adrenergic beta-Agonists↗
PubMed1998

Antisense depletion of beta-subunits fails to affect T-type calcium channels properties in a neuroblastoma cell line.

Voltage-gated calcium channels can be classified into high voltage activated (HVA) and low voltage activated (LVA or T-type) subtypes. The molecular diversity of HVA channels primarily results from different genes encoding their pore-forming alpha1 subunits. These channels share a common structure with an alpha1 subunit associated with at least two regulatory subunits (beta, alpha2-delta). Any of the six alpha1-related channels identified to date are regulated in their functional properties through an interaction with the ancillary beta-subunit. By contrast, the diversity and the molecular identity of LVA or T-type calcium channels have yet to be defined. Whether LVA channels are modulated by a beta-subunit, like HVA channels, is unknown. To address this issue, we have used an antisense strategy to inhibit beta-subunit expression in the NG 108-15 neuroblastoma cell line. Differentiated NG 108-15 cells express both LVA and HVA channels. We found that LVA currents were unaffected when cells were incubated with beta-antisense, while HVA currents were drastically decreased. Since LVA Ca channel currents in NG 108-15 cells are not regulated by beta-subunits, it is reasonable to postulate that the pore-forming subunit(s) of these channels lacks an interaction domain with a beta-subunit (AID). This molecular feature, which is common to various T-type channels, indicates further that LVA calcium channels belong to a channel family structurally distant from HVA channels.

Animals↗
PubMed1997

Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit.

The modulation of voltage-dependent Ca2+ channels at presynaptic nerve terminals is an important factor in the control of neurotransmitter release and synaptic efficacy. Some terminals contain multiple Ca2(+)-channel subtypes (N and P/Q), which are differentially regulated by G-protein activation and by protein kinase C (PKC)-dependent phosphorylation. Regulation of channel activity by crosstalk between second messenger pathways has been reported although the molecular mechanisms underlying crosstalk have not been described. Here we show that crosstalk occurs at the level of the presynaptic Ca2(+)-channel complex. The alpha1 subunit domain I-II linker, which connects the first and second transmembrane domains, contributes to the PKC-dependent upregulation of channel activity, while G-protein-dependent inhibition occurs through binding of Gbetagamma to two sites in the I-II linker. Crosstalk results from the PKC-dependent phosphorylation of one of the Gbetagamma binding sites which antagonizes Gbetagamma-induced inhibition. The results provide a mechanism for the highly regulated and dynamic control of neurotransmitter release that depends on the integration of multiple presynaptic signals.

Amino Acid Sequence↗
PubMed1997

Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

Voltage-gated Ca2+ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca2+-channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L- (but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that (a) L-type Ca2+ channels are the major pathway for voltage-gated Ca2+ entry in human coronary myocytes; (b) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and (c) the expression of T-type Ca2+ channels in culture may be triggered by cell proliferation.

Adult↗
PubMed1997

Assignment of human genes for beta 2 and beta 4 subunits of voltage-dependent Ca2+ channels to chromosomes 10p12 and 2q22-q23.

We have used human beta 2 and beta 4 cDNA probes to map the genes encoding two isoforms of the regulatory beta subunit of voltage-activated Ca2+ channels, viz. CACNB2 (beta 2) and CACNB4 (beta 4), to human chromosomes 10p12 and 2q22-q23, respectively, by fluorescence in situ hybridization. The gene encoding the beta 2 protein, first described as a Lambert-Eaton myasthenic syndrome (LEMS) antigen in humans, is found close to a region that undergoes chromosome rearrangements in small cell lung cancer, which occurs in association with LEMS. CACNB2 (beta 2) and CACNB4 (beta 4) genes are members of the ion-channel gene superfamily and it should now be possible to examine their loci by linkage analysis of ion-channel-related disorders. To date, no such disease-related gene has been assigned to 10p12 and 2q22-q23.

Calcium Channels↗
PubMed1997

Molecular basis of the diversity of calcium channels in cardiovascular tissues.

Voltage-dependent calcium (Ca2+) channels control a variety of physiological functions, such as excitation-contraction coupling in cardiac and smooth muscle. secretion of hormones and release of neurotransmitters. Studies on dissociated or cultured cells enabled us to compare their electrophysiological and pharmacological properties and their regulation in various tissues. Molecular genetics has provided a structural basis with which to observe the functional diversity of Ca2+ channels, which are composed of several subunits (alpha 1, alpha 2-delta, beta, gamma). Structure-function experiments, using expression in Xenopus oocytes, were designed to explain the molecular basis underlying this functional diversity. Six genes have been identified encoding the pore subunit (alpha 1) which determines the basic profile, i.e. the pharmacology of any Ca2+ channel. However, using a reconstitution model, the auxiliary subunits, but mainly beta subunits, for which four genes and several variants have been isolated, are able to modify the level of expression and the properties of a Ca2+ current directed by an alpha 1 subunit. Our structure-function studies are mainly designed to investigate the functional consequences of alpha 1-beta interaction on electrophysiological and pharmacological properties, especially in the case of cardiovascular Ca2+ channels. These studies should lead to a better understanding of the molecular basis underlying the differences between cardiac and vascular Ca2+ channels and also their implication in pathophysiology. Functional expression of the various combinations of subunit isoforms and identification of the precise oligomeric structure of voltage-dependent Ca2+ channels in specific cell types should help in the development of new therapeutic drugs.

Animals↗
PubMed1997

A novel tetrodotoxin-sensitive Na+ current in cultured human coronary myocytes.

Voltage-gated Na+ currents (INaS) are usually not found in arterial smooth muscle. We enzymatically isolated myocytes from the media of left coronary arteries of heart transplant patients with ischemic cardiopathy. Using the whole-cell voltage-clamp technique (20 degrees C to 22 degrees C), we detected no INa in any of the freshly isolated myocytes. In contrast, when the cells were grown in culture, we could record a large INa. This INa was characterized by a biexponential decay comprising a fast inactivating and sustained components that could not be separated by their electrophysiological and pharmacological properties. INa activated at depolarizations positive to -50 mV, was maximal at 0 mV, and was available from relatively low resting membrane potentials (half-inactivation at -46 mV). INa was modulated by several ligands known to bind selectively at different sites of Na+ channels. It was blocked with high affinity by tetrodotoxin (IC50, approximately 10 nmol/L) and local anesthetics (bupivacaine and lidocaine; IC50, approximately 100 nmol/L) and by Cd2+ (IC50, approximately 300 mumol/L). INa was modulated by Na+ channel agonists such as toxin AsV from Anemonia sulcata and veratridine, which slowed current kinetics dramatically. In conclusion, human coronary myocytes in culture can express an atypical tetrodotoxin-sensitive INa with a large sustained component, which is expected to contribute to massive Na+ influx into these cells. Phenotypic modulation of the expression of this INa may be related to cell dedifferentiation and proliferation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗