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J Nargeot

Publications and source records attributed to J Nargeot.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes.

Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca2+ channels by their insensitivity to dihydropyridines and omega-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and omega-Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca2+ channel, named BI [Mori Y. et al. (1991) Nature 350:398-402] or rbA [Starr T. V. B. et al. (1991) Proc Natl Acad Sci USA 88:5621-5625], strongly reduced the expressed Ba2+ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca2+ channel activity was increased by direct intraoocyte injection of cAMP. This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant.

Animals↗
PubMed1993

Two high-voltage-activated, dihydropyridine-sensitive Ca2+ channel currents with distinct electrophysiological and pharmacological properties in cultured rat aortic myocytes.

In smooth muscle cells, essentially two distinct types of voltage-gated Ca2+ channels have been shown, on the basis of their distinct electrophysiological and pharmacological properties, to coexist. Here we report that, in addition to a dihydropyridine (DHP)-sensitive, low-voltage-activated Ba2+ current (IBa,LVA), two types of high-voltage-activated Ba2+ currents with distinct waveforms were recorded in whole-cell clamped aortic myocytes; these were referred to as IBa,HVA1 and IBa,HVA2. They were investigated in cells where no IBa,LVA was detectable. IBa,HVA1 had a slow, monoexponential decay. In contrast, the decay of IBa,HVA2 was much faster and biexponential. In addition, IBa,HVA2 had more negative ranges of activation and steady-state inactivation than IBa,HVA1 and was more sensitive to the DHP antagonist nicardipine (concentrations for half maximum inhibition 0.2 microM and 2 microM, respectively). When using the physiological ion Ca2+ as the charge carrier, the decay of HVA1 currents was not altered, whereas both time constants of HVA2 current decay were accelerated five-fold. Moreover, permeability ratios (ICa/IBa) were also significantly different (0.2 and 0.6 for HVA1 and HVA2 respectively). IBa,HVA1 and IBa,HVA2 are consistent either with the existence and activation of two functionally distinct subtypes of the so-called "DHP-sensitive L-type" Ca2+ channel or with different gating behaviours of a single type of channel. Potentially, they may serve distinct biological functions and constitute distinct targets for neurotransmitters and drugs.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Functional properties of a neuronal class C L-type calcium channel.

The rat brain class C calcium channel alpha 1 subunit cDNA, rbC-II, was subcloned into a vertebrate expression vector and transient expression was assayed following nuclear injection into Xenopus oocytes. Whole cell recordings showed that rbC-II currents (recorded with 40 mM Ba2+ as the charge carrier) had variable activation rates and minimal inactivation over an approximately 700 msec depolarizing step pulse. The pharmacological properties of the rbC-II current were consistent with those of an L-type calcium channel, being sensitive to dihydropyridines (10 microM nifedipine blocked approximately 85% of the current, 10 microM Bay K 8644 enhanced the current between 2- and 10-fold) and not affected by the N- and P-type calcium channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, respectively. Coexpression of rbC-II with cloned rat neuronal calcium channel alpha 2 and beta subunits resulted in several changes to the electrophysiological properties of the rbC-II current including, an increased whole cell peak current, an increased rate of activation and a hyperpolarizing shift in the voltage dependence of activation. Taken together with results showing that the neuronal class D alpha 1 subunit also encodes an L-type calcium channel [Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B. and Harpold M. M. (1992a) Neuron 8: 71-84], these results indicate that the mammalian nervous system expresses two distinct genes encoding L-type calcium channels.

Animals↗
PubMed1993

Repriming of L-type calcium currents revealed during early whole-cell patch-clamp recordings in rat ventricular cells.

1. The establishment of the whole-cell patch-clamp recording configuration (WCR) revealed a type of inhibition to which L-type Ca2+ channels were subject in static rat ventricular myocytes before obtaining the WCR. 2. Immediately after membrane disruption (< 10 s), the Ca2+ current (ICa) was absent but gradually increased in amplitude to reach its final waveform (amplitude and kinetics) 2-3 min after the WCR was reached. 3. Three distinct phases (P) were identified. First, no inward but an outward current, blocked (1-2 min) by Cs+ dialysing from the patch pipette (P1), was recorded. Second, overlapping with (P1), ICa increased dramatically to reach a maximum peak amplitude within 2-3 min (P2). Concomitantly, its rate of decay, initially monoexponential and slow, became biexponential owing to the appearance of a fast component of inactivation (P3). Complete interconversion between slow and fast components sometimes occurred. 4. Changes in current waveform were not related to voltage loss or series resistance variation, and suppression of an outward current (P1) was unlikely to account for P2 and P3. 5. The run-up of ICa was independent of the nature of the permeating ions, the membrane holding potential, depolarization, rate of stimulation, the intracellular Ca2+, ATP, Mg2+, Cs+ and the pH of the pipette solution. Since large Ca2+ currents were recorded using the perforated patch technique, the run-up of ICa is not explained by the wash-out of an inhibitory endogenous macromolecule during cell-pipette exchanges. 6. Pharmacological manipulations, including the use of Ca(2+)-Ba(2+)-EGTA and exposure of the cells to isoprenaline and/or Bay K 8644 prior to recording, did not alter the mechanism primarily responsible for build-up. Unrepriming of channel activity was required before these modulations could be effective. 7. Currents could however be instantly augmented when cells were extracellularly superfused during the run-up step. The wash-out of an inhibitory agent originating in the cell itself (such as H+, NH4+ and lactate) and accumulating in the extracellular microenvironment of the cells seems unlikely. Rather, we suggest that pressure-induced mechanostimulation may be involved in the restoration of Ca2+ channel activity.

Animals↗
PubMed1993

Molecular cloning of three isoforms of the L-type voltage-dependent calcium channel beta subunit from normal human heart.

We have cloned and sequenced cDNAs for three isoforms of the L-type voltage-dependent calcium channel beta subunit isolated from a normal human heart cDNA library. One of these subunits, designated beta a, hybridized with a 3.4-kb message, and the other two, designated beta b and beta c, hybridized with a message of approximately 1.9 kb. The presence of both of these latter two messages in human heart was confirmed by polymerase chain reaction methodology. Considering the differences between beta a and beta b/beta c, we find it likely that these messages may be encoded by two different gene sequences, beta a and beta b/beta c, and that the beta b/beta c sequence can be alternatively spliced in the 209-260 region. The data suggest that the human heart presents a different pattern of beta subunit expression from that found in the rat and rabbit heart.

Amino Acid Sequence↗
PubMed1992

Electrophysiological characterization of a TTX-sensitive sodium current in native Xenopus oocytes.

We have studied a fast inward current expressed in oocytes from one Xenopus laevis. This current was characterized as a sodium current. It was activated by depolarizations to -50 mV or higher, peaked within 3-5 ms, and then decayed following a mono-exponential timecourse. When clamped at different holding potentials, the current displayed voltage-dependent inactivation with a V0.5 of -51 mV. The channel responsible for this Na+ entry was blocked by tetrodotoxin with a K0.5 of 8 nM, and was resistant to block by lidocaine at concentrations up to 100 microM. The pharmacological similarities between neuronal and oocyte sodium channels suggest that the two channels share a conserved structure.

Animals↗
PubMed1992

Differential expression of voltage-gated Ca(2+)-currents in cultivated aortic myocytes.

The expression of different types of Ca(2+)-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca(2+)-currents were identified as either low- or high voltage-activated (ICa,LVA or ICa,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca(2+)-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus, both LVA and HVA Ca(2+)-current types contribute to Ca(2+)-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.

Animals↗
PubMed1992

Endogenous Xenopus-oocyte Ca-channels are regulated by protein kinases A and C.

Calcium entry into Xenopus oocyte occurs mainly through voltage-dependent calcium channels. These channels were characterized as belonging to a particular type of calcium channel insensitive to dihydropyridines, omega-conotoxin, and Agelenopsis aperta venom, but blocked by divalent cations (Co, Cd, Ni). Intracellular injection of cAMP, or bath application of phorbol ester, induced a marked increase in calcium current amplitude and a slowing of the inactivation time-course. Despite their different pharmacology, endogenous calcium channels, like cardiac or neuronal calcium channels, could be thus regulated by protein kinases A and C.

Animals↗
PubMed1992

Cyclic AMP-dependent modulation of cardiac Ca channels expressed in Xenopus laevis oocytes.

Cyclic AMP-dependent modulation of cardiac L-type voltage-dependent Ca channel (VDCC) has been probed in Xenopus laevis oocytes injected with poly(A+) RNA from rat heart. A 2 to 3 fold increase of the Ba current amplitude was routinely obtained upon microinjection of cAMP (50-500 microM). Inhibition of protein kinase A (PKA) dramatically reduced the Ba current amplitude, indicating that cAMP-dependent modulation plays an important role in maintaining the basal activity of expressed Ca channels. Moreover, the effects of the DHP agonist Bay K 8644 on kinetic properties of expressed Ba current (IBa,C) were dependent on PKA activation. The results suggest that most expressed cardiac L-type VDCCs are phosphorylated and demonstrate that reconstitution in Xenopus oocytes is a suitable approach to address how phosphorylation regulates VDCC activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Protein kinase C regulation of cardiac calcium channels expressed in Xenopus oocytes.

L-Type cardiac Ca2+ channels expressed in Xenopus oocyte were studied following rat heart ribonucleic acid, messenger (mRNA) injection. We demonstrate that exogenous Ca2+ channels are sensitive to intracellular regulation by protein kinase C (PKC). This was performed by using two types of PKC activators [phorbol esters and a structural analogue of diacyl-glycerol (DAG)] and a specific peptidic inhibitor. Ca2+ channel modulation resulted in an initial increase of the inward current, without any modification of the voltage-dependent properties, and a second delayed phase, specifically observed with phorbol esters, characterized by a progressive decrease in current amplitude. Concomitantly, a reduction of membrane capacitance, reflecting a reduction of the total membrane surface area, was observed. We suggest that this phenomenon underlies the irreversible decrease of the expressed Ba2+ current via sequestration of Ca2+ channels and/or PKC. We also demonstrate that regulation of cardiac mRNA-directed Ca2+ channels by PKC activators was strictly dependent on intracellular Ca2+ concentration, and was partially additive with cyclic-adenosine-monophosphate-(cAMP) dependent regulation.

Animals↗
PubMed1992

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.

Animals↗
PubMed1992

Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine4 receptors.

In various species, including humans, 5-hydroxytryptamine (5-HT) has been shown to exert positive chronotropic and inotropic cardiac effects through different types of receptors. The goal of the present study was to investigate the regulation by 5-HT of voltage-gated Ca2+ channels in human atrial myocytes and to characterize the receptor involved. Cardiomyocytes isolated enzymatically and mechanically were voltage-clamped using the whole-cell configuration of the patch-clamp technique. Extracellular perfusion of 5-HT increased Ca2+ current (ICa) amplitude with a EC50 (0.1 microM) similar to that observed with isoprenaline. The effects of 5-HT were blocked by the addition of protein kinase A inhibitor in the pipette. In addition, the effects of 5-HT, isoprenaline, and intracellular cAMP on ICa were not additive. These results support the hypothesis that the inotropic effect of 5-HT in human atrial myocytes is related to an increase of ICa via an elevation of intracellular cAMP levels and stimulation of cAMP-dependent protein kinase. The effects of 5-HT were not blocked by antagonists of 5-HT1 (methiothepin), 5-HT2 (ketanserin), or 5-HT3 (ICS 205-930 at a low concentration) receptors. The benzamide derivatives renzapride and zacopride and the azabicyclobenzimidazolone derivative BIMU 8 increased ICa, but less efficiently than did 5-HT or 5-methoxytryptamine. Moreover, ICS 205-930 at high concentrations (greater than 1 microM) completely antagonized the effects of 5-HT. Thus, the pharmacology of the 5-HT receptor involved in an increase of ICa in human atrial myocytes resembles that recently described for the 5-HT4 receptor. In atrial myocytes dissociated from rat, rabbit, guinea pig, or frog, 5-HT at high concentrations had no effect on Ca2+ currents, suggesting that the distribution of 5-HT4 receptors in cardiac tissues is species dependent.

Animals↗
PubMed1991

Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons.

The effects of dihydropyridines (DHPs) normally considered to be specific for L-type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/- 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L-type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Voltage-dependent regulation of L-type cardiac Ca channels by isoproterenol.

The beta-adrenergic cascade is important for the regulation of voltage-dependent Ca channels by phosphorylation. Here we report that isoproterenol (ISO) profoundly alters the voltage-dependent properties of L-type Ca channels studied in rat ventricular cells. ISO (1 microM) shifted both threshold and maximal activation of Ba current (IBa) towards more negative potentials (approx. 10 mV). An equivalent shift was observed in the steady-state voltage-dependent inactivation curve. As a consequence, the potentiation induced by ISO on IBa was greater for weak depolarizations and from negative holding potentials (Vh). We have excluded that the contribution of minor uncompensated series resistances, the activation of Cl currents or changes in junction potential during the experiments account for these effects. In addition, ISO had a dual effect on IBa decay depending on the voltage step (acceleration below, slowing above -10 mV). In conclusion, it is postulated that the voltage dependence of the potentiating effects of ISO on Ca channels activity may ensure a selective regulation among heart tissues with different membrane resting potentials.

Animals↗
PubMed1991

Dihydropyridines interact with calcium-independent potassium currents in embryonic mammalian sensory neurons.

Early embryonic sensory neurons have two K currents resembling delayed rectifier and transient K currents of mature neurons. However, in contrast to those of adult neurons, the embryonic currents can hardly be separated either by electrophysiological or pharmacological methods, limiting their characterisation at these developmental stages. Using the whole-cell recording technique, we found that dihydropyridines (DHPs) inhibit the noninactivating component of the Ca-independent K currents of 13-day mouse embryo dorsal-root ganglion (DRG) cells. The inhibitory effect of nicardipine began around 0.5 microM and was nearly complete at 5 microM while Na currents were not altered. This effect was reversible and voltage-dependent. The same results were obtained using another DHP Ca antagonist, nimodipine, whereas Bay K 8644, a DHP Ca agonist, had no effect. Kinetic properties of the DHP-insensitive K current have been described and compared with those of transient K currents found in differentiated neurons. These results suggest that both Ca and K channels have DHP sites, possibly homologous, at this developmental stage. The DHP inhibition of Ca-independent K channels provides a new tool with which to study K channels both at a molecular level and during DRG development.

Animals↗
PubMed1991

Functional expression of P2Y purinoceptor in Xenopus oocyte injected with brain mRNA.

Xenopus oocytes injected with embryonic guinea-pig brain mRNA expressed functional P2Y purinoceptors. Extracellular ATP stimulated in a dose-dependent manner a delayed Ca(2+)-dependent Cl- current component. Analysis of the interactions of ATP with compounds that affect Ca2+ fluxes through the plasma membrane or Ca2+ release from internal stores indicates that ATP raises [Ca2+]i by a mechanism that involves activation of voltage-dependent Ca2+ channels, which leads to influx of extracellular Ca2+ into the cells, as well as release of Ca2+ from intracellular stores. Since this phenomenon was not found in control oocytes, it is suggested that brain mRNA encoded for a newly synthesized Ca(2+)-release process stimulated by purinoceptor activation. This mechanism could be largely involved in the short-term regulation of intracellular Ca2+ level involved in ATP neuromodulation functions.

Adenosine Triphosphate↗