Publications by authors named "Jean-Luc Morel"

23 Publications

  • Page 1 of 1

Effects of centrifugation and whole-body vibrations on blood-brain barrier permeability in mice.

NPJ Microgravity 2020 7;6. Epub 2020 Jan 7.

4CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.

Modifications of gravity levels induce generalized adaptation of mammalian physiology, including vascular, brain, muscle, bone and immunity functions. As a crucial interface between the vascular system and the brain, the blood-brain barrier (BBB) acts as a filter to protect neurons from pathogens and inflammation. Here we compare the effects of several protocols of hypergravity induced by centrifugation and whole-body vibrations (WBV) on BBB integrity. The immunohistochemistry revealed immunoglobulin G (IgG) extravasation from blood to hippocampal parenchyma of mice centrifuged at 2 ×  during 1 or 50 days, whereas short exposures to higher hypergravity mimicking the profiles of spaceflight landing and take-off (short exposures to 5 × ) had no effects. These results suggest prolonged centrifugation (>1 days) at 2 ×  induced a BBB leakage. Moreover, WBV were similarly tested. The short exposure to +2 ×  vibrations (900 s/day at 90 Hz) repeated for 63 days induced IgG extravasation in hippocampal parenchyma, whereas the progressive increase of vibrations from +0.5 to +2 ×  for 63 days was not able to affect the IgG crossing through the BBB. Overall, these results suggest that the BBB permeability is sensitive to prolonged external accelerations. In conclusion, we advise that the protocols of WBV and centrifugation, proposed as countermeasure to spaceflight, should be designed with progressively increasing exposure to reduce potential side effects on the BBB.
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http://dx.doi.org/10.1038/s41526-019-0094-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946672PMC
January 2020

Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells.

PLoS Pathog 2019 09 30;15(9):e1007921. Epub 2019 Sep 30.

Univ. Bordeaux, INSERM, Bordeaux Research in Translational Oncology, BaRITOn, U1053, Bordeaux, France.

Humans are frequently exposed to bacterial genotoxins involved in digestive cancers, colibactin and Cytolethal Distending Toxin (CDT), the latter being secreted by many pathogenic bacteria. Our aim was to evaluate the effects induced by these genotoxins on nuclear remodeling in the context of cell survival. Helicobacter infected mice, coculture experiments with CDT- and colibactin-secreting bacteria and hepatic, intestinal and gastric cells, and xenograft mouse-derived models were used to assess the nuclear remodeling in vitro and in vivo. Our results showed that CDT and colibactin induced-nuclear remodeling can be associated with the formation of deep cytoplasmic invaginations in the nucleus of giant cells. These structures, observed both in vivo and in vitro, correspond to nucleoplasmic reticulum (NR). The core of the NR was found to concentrate ribosomes, proteins involved in mRNA translation, polyadenylated RNA and the main components of the complex mCRD involved in mRNA turnover. These structures are active sites of mRNA translation, correlated with a high degree of ploidy, and involve MAPK and calcium signaling. Additional data showed that insulation and concentration of these adaptive ribonucleoprotein particles within the nucleus are dynamic, transient and protect the cell until the genotoxic stress is relieved. Bacterial genotoxins-induced NR would be a privileged gateway for selected mRNA to be preferably transported therein for local translation. These findings offer new insights into the context of NR formation, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage but also earlier in response to genotoxic bacteria.
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http://dx.doi.org/10.1371/journal.ppat.1007921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824578PMC
September 2019

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

Neurobiol Aging 2017 10 24;58:201-212. Epub 2017 Jun 24.

University Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France; CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France. Electronic address:

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca-release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca signals in PS1dE9 mutant mice.
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http://dx.doi.org/10.1016/j.neurobiolaging.2017.06.015DOI Listing
October 2017

Changes in C57BL6 Mouse Hippocampal Transcriptome Induced by Hypergravity Mimic Acute Corticosterone-Induced Stress.

Front Mol Neurosci 2016 26;9:153. Epub 2016 Dec 26.

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293Bordeaux, France; Centre National de la Recherche Scientifique, Institut des Maladies Neurodégénératives, UMR 5293Bordeaux, France.

Centrifugation is a widely used procedure to study the impact of altered gravity on Earth, as observed during spaceflights, allowing us to understand how a long-term physical constraint can condition the mammalian physiology. It is known that mice, placed in classical cages and maintained during 21 days in a centrifuge at 3G gravity level, undergo physiological adaptations due to hypergravity, and/or stress. Indeed, an increase of corticosterone levels has been previously measured in the plasma of 3G-exposed mice. Corticosterone is known to modify neuronal activity during memory processes. Although learning and memory performances cannot be assessed during the centrifugation, literature largely described a large panel of proteins (channels, second messengers, transcription factors, structural proteins) which expressions are modified during memory processing. Thus, we used the Illumina technology to compare the whole hippocampal transcriptome of three groups of C57Bl6/J mice, in order to gain insights into the effects of hypergravity on cerebral functions. Namely, a group of 21 days 3G-centrifuged mice was compared to (1) a group subjected to an acute corticosterone injection, (2) a group receiving a transdermal chronic administration of corticosterone during 21 days, and (3) aged mice because aging could be characterized by a decrease of hippocampus functions and memory impairment. Our results suggest that hypergravity stress induced by corticosterone administration and aging modulate the expression of genes in the hippocampus. However, the modulations of the transcriptome observed in these conditions are not identical. Hypergravity affects the hippocampus transcriptome and probably modifies its activity. Hypergravity induced changes in hippocampal transcriptome were more similar to acute injection than chronic diffusion of corticosterone or aging.
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http://dx.doi.org/10.3389/fnmol.2016.00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5183579PMC
December 2016

TRPP2 modulates ryanodine- and inositol-1,4,5-trisphosphate receptors-dependent Ca2+ signals in opposite ways in cerebral arteries.

Cell Calcium 2015 Nov 30;58(5):467-75. Epub 2015 Jul 30.

Univ. Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France. Electronic address:

TRPP2 is a cationic channel expressed in plasma membrane and in sarcoplasmic reticulum. In several cell lines, TRPP2 is described as a reticulum Ca(2+) leak channel but it also interacts with ryanodine and inositol 1,4,5-trisphosphate (InsP3) receptors to inhibit and increase the release of Ca(2+) stores, respectively. TRPP2 is known to be expressed in vascular smooth muscle cells, however its function in Ca(2+) signals remains poorly described in native cells, principally because the pharmacology is not developed. TRPP2 was expressed in cerebral arteries. Triptolide evoked Ca(2+) responses in a Ca(2+)-free solution as well as permeabilized arteries. This Ca(2+) signal was inhibited in presence of antisense oligonucleotide and siRNA directed against TRPP2 and antibody directed against the first loop of TRPP2. The partial inhibition of TRPP2 expression increased both the caffeine-evoked Ca(2+) responses and in vivo contraction. It also decreased the InsP3-evoked Ca(2+) responses. Finally, aging affected the regulations in which TRPP2 is engaged, whereas the triptolide-evoked Ca(2+) response was not modified. Taken together, our results have shown that TRPP2 is implicated in triptolide-induced Ca(2+) release from intracellular Ca(2+) stores. TRPP2 functionally interacts with both ryanodine and InsP3 receptors. These interactions were not similar in adult and old mice.
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http://dx.doi.org/10.1016/j.ceca.2015.07.003DOI Listing
November 2015

Blood brain barrier precludes the cerebral arteries to intravenously-injected antisense oligonucleotide.

Eur J Pharmacol 2015 Jan 13;747:141-9. Epub 2014 Dec 13.

Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France. Electronic address:

Alternative splicing of the ryanodine receptor subtype 3 (RyR3) produces a short isoform (RyR3S) able to negatively regulate the ryanodine receptor subtype 2 (RyR2), as shown in cultured smooth muscle cells from mice. The RyR2 subtype has a crucial role in the control of vascular reactivity via the fine tuning of Ca(2+) signaling to regulate cerebral vascular tone. In this study, we have shown that the inhibition of RyR3S expression by a specific antisense oligonucleotide (asRyR3S) was able to increase the Ca(2+) signals implicating RyR2 in cerebral arteries ex vivo. Moreover, we tried to inhibit the expression of RyR3S in vivo. The asRyR3S was complexed with JetPEI and injected intravenously coupled with several methods known to induce a blood brain barrier disruption. We tested solutions to induce osmotic choc (mannitol), inflammation (bacteria lipopolysaccharide and pertussis toxin), vasoconstriction or dilatation (sumatriptan, phenylephrine, histamine), CD73 activation (NECA) and lipid instability (Tween80). All tested technics failed to target asRyR3 in the cerebral arteries wall, whereas the molecule was included in hepatocytes or cardiomyocytes. Our results showed that the RyR3 alternative splicing could have a function in cerebral arteries ex vivo; however, the disruption of the blood brain barrier could not induce the internalization of antisense oligonucleotides in the cerebral arteries, in order to prove the function of RYR3 short isoform in vivo.
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http://dx.doi.org/10.1016/j.ejphar.2014.11.027DOI Listing
January 2015

Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats.

Pflugers Arch 2014 Aug;466(8):1517-28

Microgravity induces a redistribution of blood volume. Consequently, astronauts' body pressure is modified so that the upright blood pressure gradient is abolished, thereby inducing a modification in cerebral blood pressure. This effect is mimicked in the hindlimb unloaded rat model. After a duration of 8 days of unloading, Ca2+ signals activated by depolarization and inositol-1,4,5-trisphosphate intracellular release were increased in cerebral arteries. In the presence of ryanodine and thapsigargin, the depolarization-induced Ca2+ signals remained increased in hindlimb suspended animals, indicating that Ca2+ influx and Ca2+-induced Ca2+ release mechanism were both increased. Spontaneous Ca2+ waves and localized Ca2+ events were also investigated. Increases in both amplitude and frequency of spontaneous Ca2+ waves were measured in hindlimb suspension conditions. After pharmacological segregation of Ca2+ sparks and Ca2+ sparklets, their kinetic parameters were characterized. Hindlimb suspension induced an increase in the frequencies of both Ca2+ localized events, suggesting an increase of excitability. Labeling with bodipy compounds suggested that voltage-dependent Ca2+ channels and ryanodine receptor expressions were increased. Finally, the expression of the ryanodine receptor subtype 1 (RyR1) was increased in hindlimb unloading conditions. Taken together, these results suggest that RyR1 expression and voltage-dependent Ca2+ channels activity are the focal points of the regulation of Ca2+ signals activated by vasoconstriction in rat cerebral arteries with an increase of the voltage-dependent Ca2+ influx.
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http://dx.doi.org/10.1007/s00424-013-1387-9DOI Listing
August 2014

Effect of aging on calcium signaling in C57Bl6J mouse cerebral arteries.

Pflugers Arch 2013 Jun 14;465(6):829-38. Epub 2012 Dec 14.

Institut des Maladies Neurodégénératives, UMR 5293, Universite Bordeaux, 33076, Bordeaux Cedex, France.

In cerebral arteries, alterations of vascular reactivity have been observed but not well molecularly characterized. Therefore, we have hypothesized that cerebrovascular reactivity could be modified by aging via a modification of Ca(2+) signaling in smooth muscle cells. Ca(2+) signals and gene expression implicated in contraction have been measured in posterior and middle cerebral arteries from young (2-3 months) and old (20-22 months) C57Bl6/J mice. Aging induced a decrease of KCl- and caffeine-induced contraction as well as a decrease of the amplitudes and an increase of the durations of KCl- and caffeine-induced Ca(2+) signals. These results could be linked with the decrease of gene expression coding for Cav1.2, RyR2, SERCA2, PLB, STIM1, TRIC-B, and the increase of FKBP12.6 and TPCN1 gene expression. Finally, aging induced a modification of InsP3 subtype expression pattern responsible for a modification of the InsP3 affinity to activate Ca(2+) signals. These results show that aging induces a decrease of contractility correlated with modifications of the expression of genes encoding Ca(2+) signaling toolkit. Globally, the amplitude of Ca(2+) signals was decreased, whereas their duration was increased by a defection of Ca(2+) store refilling.
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http://dx.doi.org/10.1007/s00424-012-1195-7DOI Listing
June 2013

Learning on Jupiter, learning on the Moon: the dark side of the G-force. Effects of gravity changes on neurovascular unit and modulation of learning and memory.

Front Behav Neurosci 2012 24;6:64. Epub 2012 Sep 24.

Université de Bordeaux Bordeaux, France ; Centre National de la Recherche Scientifique Unité Mixte de Recherche 5293, Institut des Maladies Neurodégénératives Talence, France.

On earth, gravity vector conditions the development of all living beings by physically imposing an axis along which to build their organism. Thus, during their whole life, they have to fight against this force not only to maintain their architectural organization but also to coordinate the communication between organs and keep their physiology in a balanced steady-state. In space, astronauts show physiological, psychological, and cognitive deregulations, ranging from bone decalcification or decrease of musculature, to depressive-like disorders, and spatial disorientation. Nonetheless, they are confronted to a great amount of physical changes in their environment such as solar radiations, loss of light-dark cycle, lack of spatial landmarks, confinement, and obviously a dramatic decrease of gravity force. It is thus very hard to selectively discriminate the strict role of gravity level alterations on physiological, and particularly cerebral, dysfunction. To this purpose, it is important to design autonomous models and apparatuses for behavioral phenotyping utilizable under modified gravity environments. Our team actually aims at working on this area of research.
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http://dx.doi.org/10.3389/fnbeh.2012.00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449275PMC
October 2012

The 22nd ion channel meeting, september 2011, france.

Channels (Austin) 2012 May-Jun;6(3):149-53. Epub 2012 May 1.

Neurobiologie des Canaux Ioniques, INSERM-Université de la Méditerranée, Marseille, France.

The 22(nd) Ion Channel Meeting was organized by the French Ion Channel Society (Association Canaux Ioniques) from the 25(th) to the 28(th) of September 2011 on the French Riviera (Giens). This year again, more than one hundred researchers from France, Europe and extra-European countries gathered to present and discuss their recent advances and future challenges in the ion channels and transporters field. The scientific committee organized a plenary lecture and five thematic symposia by inviting international researchers to present their recent outstanding work on themes as diverse as muscular channelopathies, regulation of channels by extracellular matrix, receptor-channels interactions, localization and distribution of ion channels, their involvement in the cell life and death, and finally how they participate in the evolution and adaptability of cellular excitability. These presentations are summarized in this meeting report. Two sessions of oral communications selected from submitted abstracts and two poster sessions were also organized to present the ongoing work of young researchers worldwide.
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http://dx.doi.org/10.4161/chan.20795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431583PMC
December 2012

Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension.

J Appl Physiol (1985) 2012 Feb 17;112(3):471-80. Epub 2011 Nov 17.

Universite de Bordeaux, Institut des Maladies Neurodégénératives, Bordeaux, France.

Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression.
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http://dx.doi.org/10.1152/japplphysiol.00733.2011DOI Listing
February 2012

The 20th ion channel meeting: September 2009, France.

Channels (Austin) 2010 Jul-Aug;4(4):329-33. Epub 2010 Jul 22.

Inserm, U975, Centre de Recherche de L'Institut du Cerveau et de la Moelle Épinière, UPMC Université Paris 06, UMR_S975, CNRS UMR7225, AP-HP, Groupe Hospitalier de la Pitié-Salpêtrière Paris, France.

The French Ion Channel society has existed since 1989 and its main goal is to annually organize a scientific meeting. This meeting, which gathers young and senior French scientists, provides a great opportunity for exchange and interaction among the ion channel research community. Additionally, for many years, the French ion channel meeting has attracted a significant number of scientists from different European countries, promoting the discussion of new insights and advances, as well as aiding in the establishment of collaborations. In this report, we summarize the five symposia selected for their novelty and importance in human channelopathies, neuroplasticity, ion channel regulations, intracellular ion channels and plant physiology.
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http://dx.doi.org/10.4161/chan.4.4.12777DOI Listing
March 2011

Comparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes.

Eur J Pharmacol 2010 Feb 26;628(1-3):36-41. Epub 2009 Nov 26.

Department of Pharmacology, UVM College of Medicine, B-333 Given Building, Burlington, VT 05405-0068, USA.

In Duchenne muscular dystrophy, a stop-codon mutation in the dystrophin gene induces an impairment of skeletal and smooth muscles contraction. In duodenum from mdx mouse, the disease model, the decrease of contractility was linked with the decrease of calcium signals encoded by ryanodine receptor subtype 2. Aminoglycoside and antisense oligonucleotide strategies were investigated to restore calcium signalling in the mdx mouse. Mdx mice were treated by intraperitoneal injection of gentamycin or 2-O-methyl antisense ribonucleotide directed against exon 23 of dystrophin for 2 weeks. The efficiency of both therapeutic strategies was determined by the level of dystrophin protein expression. The physiological effects of both treatments on ryanodine receptor expression and function were followed by RT-PCR, western blot and calcium measurements. Fourteen days after injection of gentamycin or anti-dystrophin antisense, the expression of dystrophin was recovered in skeletal muscle from treated mdx mice. In duodenum cells, RT-PCR and western blot indicated that the expression of ryanodine receptor subtype 2 was similar in treated mice than in control mice in association with the recovery of caffeine-induced Ca(2+) response. No significant difference was observed in the ryanodine subtype 3-dependent spontaneous Ca(2+) oscillations in untreated and treated mice. Conclusions - these results may help to explain the efficiency of aminoglycoside and anti-dystrophin antisense treatments in smooth muscle. Both treatments could be an interesting therapeutic option to restore smooth muscle contraction in patients with Duchenne muscular dystrophy.
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http://dx.doi.org/10.1016/j.ejphar.2009.11.034DOI Listing
February 2010

The decrease of expression of ryanodine receptor sub-type 2 is reversed by gentamycin sulphate in vascular myocytes from mdx mice.

J Cell Mol Med 2009 Sep 11;13(9B):3122-30. Epub 2009 Feb 11.

Université de Bordeaux, CNRS, Centre Neurosciences Intégratives et Cognitives, Unité Mixte de Recherche, Talence Cedex, France.

The mdx mouse, a model of the human Duchenne muscular dystrophy, displays impaired contractile function in skeletal, cardiac and smooth muscles. We explored the possibility that ryanodine receptor (RYR) expression could be altered in vascular muscle. The three RYR sub-types were expressed in portal vein myocytes. As observed through mRNA and protein levels, RYR2 expression was strongly decreased in mdx myocytes, whereas RYR3 and RYR1 expression were unaltered. The use of antisense oligonucleotide directed against RYR sub-types indicated that caffeine-induced Ca(2+) response and Ca(2+) spark frequency depended on RYR2 and RYR1. In mdx mice, caffeine-induced Ca(2+) responses were decreased in both amplitude and maximal rate of rise, and the frequency of Ca(2+) sparks was also strongly decreased. The gentamycin treatment was able to increase both the expression of RYR2 and the caffeine-induced Ca(2+) response to the same level as that observed in wild-type mice. Taken together, these results confirm that both RYR1 and RYR2 are required for vascular Ca(2+) signalling and indicate that inhibition of RYR2 expression may account for the decreased Ca(2+) release from the SR in mdx vascular myocytes. Finally, we suggest that gentamycin can restore the Ca(2+) signalling in smooth muscle from mdx mice by increasing RYR2 and dystrophin expression. These results may help explain the reduced efficacy of contraction in vascular myocytes of mdx mice and Duchenne muscular dystrophy-afflicted patients. Gentamycin treatment could be a good therapeutic tool to restore the vascular function.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00718.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516471PMC
September 2009

Acetylcholine evokes an InsP3R1-dependent transient Ca2+ signal in rat duodenum myocytes.

Can J Physiol Pharmacol 2008 Sep;86(9):626-32

Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1139/y08-067DOI Listing
September 2008

Full length ryanodine receptor subtype 3 encodes spontaneous calcium oscillations in native duodenal smooth muscle cells.

Cell Calcium 2008 Aug 18;44(2):180-9. Epub 2008 Jan 18.

Centre de Neurosciences Intégratives et Cognitives, CNRS UMR5228, Universités de Bordeaux, avenue des facultés, 33405 Talence, France.

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.
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http://dx.doi.org/10.1016/j.ceca.2007.11.009DOI Listing
August 2008

Acetylcholine-induced Ca2+ oscillations are modulated by a Ca2+ regulation of InsP3R2 in rat portal vein myocytes.

Pflugers Arch 2008 May 20;456(2):277-83. Epub 2007 Nov 20.

Molecular Neurobiology, Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1007/s00424-007-0379-zDOI Listing
May 2008

RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle.

J Cell Sci 2007 Nov 9;120(Pt 21):3784-91. Epub 2007 Oct 9.

CNRS UMR 5017, Laboratoire de Signalisation et Interactions Cellulaires, Université Bordeaux 2, Bordeaux, France.

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.
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http://dx.doi.org/10.1242/jcs.009415DOI Listing
November 2007

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium.

Am J Physiol Cell Physiol 2007 Sep 27;293(3):C848-54. Epub 2007 Jun 27.

Centre de Neurosciences Intégratives et Cognitives, UMR5228 CNRS, Université Bordeaux 1 and Université Bordeaux 2, Ave. des Facultés, Talence 33405, France.

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.
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http://dx.doi.org/10.1152/ajpcell.00069.2007DOI Listing
September 2007

Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells.

Cell Calcium 2006 Jul 4;40(1):11-21. Epub 2006 May 4.

Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

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.
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http://dx.doi.org/10.1016/j.ceca.2006.03.008DOI Listing
July 2006

Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes.

J Cell Sci 2005 May 3;118(Pt 10):2261-70. Epub 2005 May 3.

Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

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.
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http://dx.doi.org/10.1242/jcs.02344DOI Listing
May 2005

Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes.

J Biol Chem 2004 May 25;279(20):21287-93. Epub 2004 Feb 25.

Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine- and depolarization-induced calcium responses were inhibited, whereas acetylcholine- and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeine- and depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.
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http://dx.doi.org/10.1074/jbc.M311124200DOI Listing
May 2004

Crucial role of type 2 inositol 1,4,5-trisphosphate receptors for acetylcholine-induced Ca2+ oscillations in vascular myocytes.

Arterioscler Thromb Vasc Biol 2003 Sep 31;23(9):1567-75. Epub 2003 Jul 31.

Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, Bordeaux, France.

Objective: The aim of this study was to correlate the expression of InsP3R subtypes in native vascular and visceral myocytes with specific Ca2+-signaling patterns.

Methods And Results: By Western blot and immunostaining, we showed that rat portal vein expressed InsP3R1 and InsP3R2 but not InsP3R3, whereas rat ureter expressed InsP3R1 and InsP3R3 but not InsP3R2. Acetylcholine induced single Ca2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca2+ oscillations. By correlating Ca2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP3R1 and InsP3R2 whereas nonoscillating vascular cells expressed only InsP3R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca2+-ATPases, Ca2+ influx, and mitochondrial Ca2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP3R subtypes, we showed that acetylcholine-induced Ca2+ oscillations were specifically blocked by the anti-InsP3R antibody. These data were supported by antisense oligonucleotides targeting InsP3R2, which selectively inhibited Ca2+ oscillations.

Conclusions: Our results suggest that in native smooth muscle cells, a differential expression of InsP3R subtypes encodes specific InsP3-mediated Ca2+ responses and that the presence of the InsP3R2 subtype is required for acetylcholine-induced Ca2+ oscillations in vascular myocytes.
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http://dx.doi.org/10.1161/01.ATV.0000089013.82552.5DDOI Listing
September 2003