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Nathalie MACREZ




39 publication(s) depuis Juin 1997:


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Abstract:
T-type Ca(2+) channel family includes three subunits Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3 and have been shown to control burst firing and intracellular Ca(2+) concentration ([Ca(2+)](i)) in neurons. Here, we investigated whether Ca(V)3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca(V)3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca(V)3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca(2+)](i) oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around -61mV. [Ca(2+)](i) oscillations were critically dependent on Ca(2+) influx through Ca(V)3.1 channels and did not involve Ca(2+) release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca(V)3.1 channels. The trigger of the oscillations was the Ca(V)3.1 window current. This current induced continuous [Ca(2+)](i) increase at -60mV that depolarized the cells and triggered MPOs. Shifting the Ca(V)3.1 window current potential range by increasing the external Ca(2+) concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I(h)) was not required to induce MPOs, but when expressed together with Ca(V)3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca(V)3.1 window current is critical in triggering intrinsic electrical and [Ca(2+)](i) oscillations.




09/2008 | can j physiol pharmacol
Acetylcholine evokes an InsP3R1-dependent transient Ca2+ signal in rat duodenum myocytes.
Fritz N, Dabertrand F, Mironneau J, Macrez N, Morel JL

Abstract:
In smooth muscle myocytes, agonist-activated release of calcium ions (Ca2+) stored in the sarcoplasmic reticulum (SR) occurs via different but overlapping transduction pathways. Hence, to fully study how SR Ca2+ channels are activated, the simultaneous activation of different Ca2+ signals should be separated. In rat duodenum myocytes, we have previously characterized that acetylcholine (ACh) induces Ca2+ oscillations by binding to its M2 muscarinic receptor and activating the ryanodine receptor subtype 2. Here, we show that ACh simultaneously evokes a Ca2+ signal dependent on activation of inositol 1,4,5-trisphosphate (InsP3) receptor subtype 1. A pharmacologic approach, the use of antisense oligonucleotides directed against InsP3R1, and the expression of a specific biosensor derived from green-fluorescent protein coupled to the pleckstrin homology domain of phospholipase C, suggested that the InsP3R1-dependent Ca2+ signal is transient and due to a transient synthesis of InsP3 via M3 muscarinic receptor. Moreover, we suggest that both M2 and M3 signalling pathways are modulating phosphatidylinositol 4,5-bisphosphate and InsP3 concentration, thus describing closely interacting pathways activated by ACh in duodenum myocytes.




Abstract:
Two isoforms of the ryanodine receptor subtype 3 (RYR3) have been described in smooth muscle. The RYR3 short isoform (RYR3S) negatively regulates the calcium-induced calcium release mechanism encoded by the RYR2, whereas the role of the full length isoform of RYR3 (RYR3L) was still unclear. Here, we describe RYR-dependent spontaneous Ca(2+) oscillations measured in 10% of native duodenum myocytes. We investigated the role of RYR3 isoforms in these spontaneous Ca(2+) signals. Inhibition of RYR3S expression by antisense oligonucleotides revealed that both RYR2 and RYR3L were able to propagate spontaneous Ca(2+) waves that were distinguishable by frequency analysis. When RYR3L expression was inhibited, the spontaneous Ca(2+) oscillations were never observed, indicating that RYR3S inhibited the function of RYR2. RYR2 expression inhibition led to Ca(2+) oscillations identical to those observed in control cells suggesting that RYR3S did not functionally interact with RYR3L. The presence and frequency of RYR3L-dependent Ca(2+) oscillations were dependent on sarcoplasmic reticulum Ca(2+) content as revealed by long-term changes of the extracellular Ca(2+) concentration. Our study shows that, in native duodenal myocytes, the spontaneous Ca(2+) waves are encoded by the RYR3L alone, which activity is regulated by sarcoplasmic reticulum Ca(2+) loading.




Abstract:
Oscillations of cytosolic Ca2+ levels are believed to have important roles in various metabolic and signalling processes in many cell types. Previously, we have demonstrated that acetylcholine (ACh) evokes Ca2+ oscillations in vascular myocytes expressing InsP3R1 and InsP3R2, whereas transient responses are activated in vascular myocytes expressing InsP3R1 alone. The molecular mechanisms underlying oscillations remain to be described in these native smooth muscle cells. Two major hypotheses are proposed to explain this crucial signalling activity: (1) Ca2+ oscillations are activated by InsP3 oscillations; and (2) Ca2+ oscillations depend on the regulation of the InsP3R by both InsP3 and Ca2+. In the present study, we used a fluorescent InsP3 biosensor and revealed that ACh induced a transient InsP3 production in all myocytes. Moreover, steady concentrations of 3F-InsP3, a poorly hydrolysable analogue of InsP3, and pharmacological activation of PLC evoked Ca2+ oscillations. Increasing cytosolic Ca2+ inhibited the ACh-induced calcium oscillations but not the transient responses and strongly reduced the 3F-InsP3-evoked Ca2+ response in oscillating cells but not in non-oscillating cells. These results suggest that, in native vascular myocytes, ACh-induced InsP3 production is transient and Ca2+ oscillations depend on a Ca2+ modulation of InsP3R2.




01/11/2007 | J Cell Sci
RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle.
Fritz N, Morel JL, Jeyakumar LH, Fleischer S, Allen PD, Mironneau J, Macrez N

Abstract:
Ryanodine receptor subtype 1 (RyR1) has been primarily characterized in skeletal muscle but several studies have revealed its expression in smooth muscle. Here, we used Ryr1-null mice to investigate the role of this isoform in Ca(2+) signaling in urinary bladder smooth muscle. We show that RyR1 is required for depolarization-induced Ca(2+) sparks, whereas RyR2 and RyR3 are sufficient for spontaneous or caffeine-induced Ca(2+) sparks. Immunostaining revealed specific subcellular localization of RyR1 in the superficial sarcoplasmic reticulum; by contrast, RyR2 and RyR3 are mainly expressed in the deep sarcoplasmic reticulum. Paradoxically, lack of depolarization-induced Ca(2+) sparks in Ryr1(-/-) myocytes was accompanied by an increased number of cells displaying spontaneous or depolarization-induced Ca(2+) waves. Investigation of protein expression showed that FK506-binding protein (FKBP) 12 and FKBP12.6 (both of which are RyR-associated proteins) are downregulated in Ryr1(-/-) myocytes, whereas expression of RyR2 and RyR3 are unchanged. Moreover, treatment with rapamycin, which uncouples FKBPs from RyR, led to an increase of RyR-dependent Ca(2+) signaling in wild-type urinary bladder myocytes but not in Ryr1(-/-) myocytes. In conclusion, although decreased amounts of FKBP increase Ca(2+) signals in Ryr1(-/-) urinary bladder myocytes the depolarization-induced Ca(2+) sparks are specifically lost, demonstrating that RyR1 is required for depolarization-induced Ca(2+) sparks and suggesting that the intracellular localization of RyR1 fine-tunes Ca(2+) signals in smooth muscle.




09/2007 | Am J Physiol Cell Physiol
Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium.
Dabertrand F, Fritz N, Mironneau J, Macrez N, Morel JL

Abstract:
Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca(2+) releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.




Abstract:
In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). We investigated the time-dependent effects of AII on both phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3-5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kgamma and that this isoform was involved in the AII-induced stimulation of InsPs accumulation. NA-induced InsPs accumulation depended on PLCbeta1 activation whereas AII-induced InsPs accumulation depended on PLCgamma1 activation. AII-induced PLCgamma1 activation required both tyrosine kinase and PI3Kgamma since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti-PI3Kgamma antibody abolished AII-induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCgamma1 was only detected for long-lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kgamma and tyrosine kinase is a prerequisite for AII-induced stimulation of PLCgamma1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long-lasting contraction induced by AII.




07/2006 | Cell Calcium
Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells.
Dabertrand F, Morel JL, Sorrentino V, Mironneau J, Mironneau C, Macrez N

Abstract:
The ryanodine receptor subtype 3 (RYR3) is expressed ubiquitously but its physiological function varies from cell to cell. Here, we investigated the role of a dominant negative RYR3 isoform in Ca2+ signalling in native smooth muscle cells. We used intranuclear injection of antisense oligonucleotides to specifically inhibit endogenous RYR3 isoform expression. In mouse duodenum myocytes expressing RYR2 subtype and both spliced and non-spliced RYR3 isoforms, RYR2 and non-spliced RYR3 were activated by caffeine whereas the spliced RYR3 was not. Only RYR2 was responsible for the Ca2+-induced Ca2+ release mechanism that amplified Ca2+ influx- or inositol 1,4,5-trisphosphate-induced Ca2+ signals. However, the spliced RYR3 negatively regulated RYR2 leading to the decrease of amplitude and upstroke velocity of Ca2+ signals. Immunostaining in injected cells showed that the spliced RYR3 was principally expressed near the plasma membrane whilst the non-spliced isoform was revealed around the nucleus. This study shows for the first time that the short isoform of RYR3 controls Ca2+ release through RYR2 in native smooth muscle cells.




Abstract:
The precise contribution of T-type Ca2+ channels in generating action potentials (APs), burst firing and intracellular Ca2+ signals needs further elucidation. Here, we show that CaV3.3 channels can trigger repetitive APs, generating spontaneous membrane potential oscillations (MPOs), and a concomitant increase in the intracellular Ca2+ concentration ([Ca2+]i) when overexpressed in NG108-15 cells. MPOs were dependent on CaV3.3 channel activity given that they were recorded from a potential range of -55 to -70 mV, blocked by nickel and mibefradil, as well as by low external Ca2+ concentration. APs of distinct duration were recorded: short APs (sAP) or prolonged APs (pAP) with a plateau potential near -40 mV. The voltage-dependent properties of the CaV3.3 channels constrained the AP duration and the plateau potential was supported by sustained calcium current through CaV3.3 channels. The sustained current amplitude decreased when the resting holding potential was depolarized, thereby inducing a switch of AP shape from pAP to sAP. Duration of the [Ca2+]i oscillations was also closely related to the shape of APs. The CaV3.3 window current was the oscillation trigger as shown by shifting the CaV3.3 window current potential range as a result of increasing the external Ca2+ concentration, which resulted in a corresponding shift of the AP threshold. Overall, the data demonstrate that the CaV3.3 window current is critical in triggering intrinsic electrical and [Ca2+]i oscillations. The functional expression of CaV3.3 channels can generate spontaneous low-threshold calcium APs through its window current, indicating that CaV3.3 channels can play a primary role in pacemaker activity.




Abstract:
In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.