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Nathalie MACREZ

39 publication(s) depuis Juin 1997:

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06/08/2004 | circ res
Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate.
Le Blanc C, Mironneau C, Barbot C, Henaff M, Bondeva T, Wetzker R, Macrez N

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.

05/2004 | curr mol med
Local Ca2+ signals in cellular signalling.
Macrez N, Mironneau J

Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.

01/02/2002 | J Physiol
Identification and function of ryanodine receptor subtype 3 in non-pregnant mouse myometrial cells.
Mironneau J, Macrez N, Morel JL, Sorrentino V, Mironneau C

Subtype 3 of the ryanodine receptor (RYR3) is a ubiquitous Ca2+ release channel which is predominantly expressed in smooth muscle tissues and certain regions of the brain. We show by reverse transcription-polymerase chain reaction (RT-PCR) that non-pregnant mouse myometrial cells expressed only RYR3 and therefore could be a good model for studying the role of endogenous RYR3. Expression of RYR3 was confirmed by Western blotting and immunostaining. Confocal Ca2+ measurements revealed that in 1.7 mM extracellular Ca2+, neither caffeine nor photolysis of caged Ca2+ were able to trigger any Ca2+ responses, whereas in the same cells oxytocin activated propagated Ca2+ waves. However, under conditions of increased sarcoplasmic reticulum (SR) Ca2+ loading, brought about by superfusing myometrial cells in 10 mM extracellular Ca2+, all the myometrial cells responded to caffeine and photolysis of caged Ca2+, indicating that it was possible to activate RYR3. The caffeine-induced Ca2+ responses were inhibited by intracellular application of an anti-RYR3-specific antibody. Immunodetection of RYR3 with the same antibody revealed a rather homogeneous distribution of fluorescence in confocal cell sections. In agreement with these observations, spontaneous or triggered Ca2+ sparks were not detected. In conclusion, our results suggest that under conditions of increased SR Ca2+ loading, endogenous RYR3 may contribute to the Ca2+ responses of myometrial cells.

12/10/2001 | circ res
Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.
Macrez N, Mironneau C, Carricaburu V, Quignard JF, Babich A, Czupalla C, Nurnberg B, Mironneau J

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

31/08/2001 | J Biol Chem
Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.
Quignard JF, Mironneau J, Carricaburu V, Fournier B, Babich A, Nurnberg B, Mironneau C, Macrez N

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

06/04/2001 | J Biol Chem
Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes.
Mironneau J, Coussin F, Jeyakumar LH, Fleischer S, Mironneau C, Macrez N

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).

1. Previous data have shown that activation of beta(3)-adrenoceptors stimulates vascular L-type Ca(2+) channels through a G alphas-induced stimulation of the cyclic AMP/PKA pathway. The present study investigated whether beta-adrenergic stimulation also uses the G beta gamma/PI3K/PKC pathway to modulate L-type Ca(2+) channels in rat portal vein myocytes. 2. Peak Ba(2+) current (I(Ba)) measured using the whole-cell patch clamp method was maximally increased by application of 10 microm isoprenaline after blockade of beta(3)-adrenoceptors by 1 microM SR59230A. Under these conditions, the isoprenaline-induced stimulation of I(Ba) was reversed by ICI-118551 (a specific beta(2)-adrenoceptor antagonist) but not by atenolol (a specific beta(1)-adrenoceptor antagonist). The beta(2)-adrenoceptor agonist salbutamol increased I(Ba), an effect which was reversed by ICI-118551 whereas the beta(1)-adrenoceptor agonist dobutamine had no effect on I(Ba). 3. Application of PKA inhibitors (H-89 and Rp 8-Br-cyclic AMPs) or a PKC inhibitor (calphostin C) alone did not affect the beta(2)-adrenergic stimulation of I(Ba) whereas simultaneous application of both PKA and PKC inhibitors completely blocked this stimulation. 4. The beta(2)-adrenergic stimulation of L-type Ca(2+) channels was blocked by a pre-treatment with cholera toxin and by intracellular application of an anti-G alphas antibody (directed against the carboxyl terminus of G alphas). In the presence of H-89, intracellular infusion of an anti-Gss(com) antibody or a beta ARK(1) peptide as well as a pre-treatment with wortmannin (a PI3K inhibitor) blocked the beta(2)-adrenergic stimulation of I(Ba). 5. These results suggest that the beta(2)-adrenergic stimulation of vascular L-type Ca(2+) channels involves both G alphas and G beta gamma subunits which exert their stimulatory effects through PKA and PI3K/PKC pathways, respectively.

01/07/2000 | Biochem J
Ca(2+) signals mediated by Ins(1,4,5)P(3)-gated channels in rat ureteric myocytes.
Boittin FX, Coussin F, Morel JL, Halet G, Macrez N, Mironneau J

Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors.

05/05/2000 | J Biol Chem
Gbeta 5gamma 2 is a highly selective activator of phospholipid-dependent enzymes.
Maier U, Babich A, Macrez N, Leopoldt D, Gierschik P, Illenberger D, Nurnberg B

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.

04/2000 | Br J Pharmacol
Beta-3 adrenergic stimulation of L-type Ca(2+) channels in rat portal vein myocytes.
Viard P, Macrez N, Coussin F, Morel JL, Mironneau J

1. The effects of beta(3)-adrenergic stimulation were studied on the L-type Ca(2+) channel in single myocytes from rat portal vein using the whole-cell mode of the patch-clamp technique. 2. Reverse transcription-polymerase chain reaction showed that beta(1)-, beta(2)- and beta(3)-adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non-selective beta(1)- and beta(2)-adrenoceptor antagonist) and SR59230A (a beta(3)-adrenoceptor antagonist) were needed to inhibit the isoprenaline-induced increase in L-type Ca(2+) channel current. 3. L-type Ca(2+) channels were stimulated by CGP12177A (a beta(3)-adrenoceptor agonist with potent beta(1)- and beta(2)-adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A-induced stimulation of Ca(2+) channel current was blocked by SR59230A, cyclic AMP-dependent protein kinase inhibitors, H-89 and Rp 8-Br-cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19-31 peptide. This stimulation was mimicked by forskolin and 8-Br-cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the beta(3)-adrenoceptor-induced stimulation was maintained after withdrawal of the agonist. 4. The beta(3)-adrenoceptor stimulation of L-type Ca(2+) channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti-Galpha(s) antibody. This stimulation was unaffected by intracellular infusion of an anti-Gbeta(com) antibody and a betaARK(1) peptide. 5. These results show that activation of beta(3)-adrenoceptors stimulates L-type Ca(2+) channels in vascular myocytes through a Galpha(s)-induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels.