1 Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
2 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
* Author for correspondence (e-mail: jean-luc.morel{at}umr5017.u-bordeaux2.fr)
Accepted 21 February 2005
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Summary |
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Key words: Cyclic ADP-ribose, Ryanodine receptors, Ca2+ oscillations, Acetylcholine, Smooth muscle
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Introduction |
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In smooth muscle, it is generally accepted that acetylcholine (ACh) activates Ca2+ release by M3 muscarinic receptor stimulation and an InsP3-dependent mechanism (Thomas and Ehlert, 1994; Morel et al., 1997
), although two studies have suggested that M1 or M2 muscarinic receptors may be involved in Ca2+ signalling (Ge et al., 2003
; White et al., 2003
). In the present study, we show that ACh induces Ca2+ oscillations by stimulation of RYR2 through a cADPR/M2 muscarinic receptor pathway. Both cADPR and ACh-induced Ca2+ oscillations also require FKBP12.6 interaction with RYR2.
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Materials and Methods |
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Cell permeabilization
To permeabilize myocytes, physiological solution was replaced by a solution containing 140 mM KCl, 20 mM Hepes, 0.5 mM MgCl2 and 10 µg/ml saponin, pH 7.4 with NaOH. When 100 µg/ml saponin was used, the solution was supplemented with 100 µM ATP. Cell permeabilization was estimated by application of tetramethylrhodamine isothiocyanate (TRITC)-coupled phalloidin. In cells incubated in the presence of 100 and 10 µg/ml saponin, the TRITC fluorescence increased to a maximum value in 1.0±0.5 minute (n=20) and 6.2±1.4 minutes (n=20), respectively, and remained steady in the continuous presence of saponin, whereas no TRITC fluorescence was detected in intact cells for 20 minutes (data not shown). Both caffeine- and ACh-induced Ca2+ responses similar in amplitude and kinetics were obtained in this solution for 15-20 minutes, suggesting that the leakage of fluo-4 out of the cells was not significant during this time period.
RT-PCR reactions
Total RNA was extracted from duodenum myocytes using the RNA preparation kit from Epicentre (Madison, WI, USA), following the instructions of the supplier. The RNA concentration was determined at DO260nm with an Eppendorf biophotometer (Eppendorf, Le Pecq, France). The reverse transcription reaction was performed on 50 ng of RNA using Sensiscript-RT kit (Qiagen, Hilden, Germany). PCR conditions were described previously (Morel et al., 2004). PCR were performed with a thermal cycler (Eppendorf). Amplification products were separated by electrophoresis (2% agarose gel) and visualized by ethidium bromide staining. Gels were photographed with EDAS120 and analysed with KDS1D 2.0 software (Kodak Digital Science, Paris, France). Sense (s) and antisense (as) primer pairs specific for RYRl, RYR2 and RYR3 were described previously (Coussin et al., 2000
).
Microinjection of oligonucleotides
Phosphorothioate antisense oligonucleotides (denoted with the prefix `as') used in the present study were described previously (Coussin et al., 2000). Briefly, oligonucleotides were designed on the known cloned sequences deposited in the GenBank database with DNAstar (Lasergene software). Oligonucleotides were injected into the nuclei of myocytes by a manual injection system with femtotips II (Eppendorf) as previously described (Macrez-Lepretre et al., 1997
). Myocytes were then cultured for 2 to 4 days in culture medium. For physiological experiments, the glass slides were transferred into the perfusion chamber.
Cytosolic Ca2+ measurements
Cells were incubated in physiological solution containing 2 µM fluo-4 acetoxymethylester (AM) for 20 minutes at 37°C. The cells were then washed and allowed to cleave the dye to the active fluo-4 compound for 10 minutes. Images were acquired using the image series or line-scan mode of a confocal Bio-Rad MRC 1024ES (Bio-Rad, Paris, France) connected to a Nikon Diaphot microscope. Excitation light was delivered by a 25 mW argon ion laser (Ion Laser Technology, Salt Lake City, UT, USA) through a Nikon Plan Apo x60, 1.4 NA objective lens. Fluo-4 was excited at 488 nm, and emitted fluorescence was filtered and measured at 540±30 nm. At the setting used to detect fluo-4 fluorescence, the resolution of the microscope was 0.4x0.4x1.5 µm (x-, y- and z-axis). Image series consist of images of the same confocal section of the cell taken at 1.2 second intervals. To analyse variation of fluorescence, regions of interest (ROI), used for each frame of the series, were drawn around each myocyte. The fluorescence value was divided by the fluorescence of the first frame (baseline) and reported as F/F0. Image processing and analysis were performed using Lasersharp 2000 (Bio-Rad) software and IDL software (RSI, Boulder, CO, USA), respectively. ACh, cADPR and caffeine were applied by pressure ejection from a glass pipette for the period indicated in the figures. All experiments were carried out at 26±1°C.
Immunostaining of RYRs
Myocytes were washed with PBS, fixed with 4% (vol/vol) formaldehyde and 0.05% glutaraldehyde for 10 minutes at room temperature, and permeabilized in PBS containing 3% FCS and 1 mg/ml saponin for 20 minutes. Cells were incubated with PBS, saponin (1 mg/ml) and anti-RYR antibodies overnight at 4°C. Then, cells were washed (4x 5 minutes) and incubated with the appropriate secondary Alexa Fluor 488 antibody for 45 minutes at room temperature. After washing in PBS, cells were mounted in Vectashield (AbCys, Paris, France). Images of the stained cells were obtained with a confocal microscope, and fluorescence was estimated by grey level analysis using IDL software (RSI) in 0.5 µm confocal sections. On each cell, fluorescence measurement was acquired from a z-series analysis (20±5 sections) using Lasersharp software (Bio-Rad) and expressed by unit volume. Cells were compared by keeping acquisition parameters (such as grey scale, exposure time, iris aperture, gain and laser power) constant.
ADP-ribosyl cyclase activity measurement
To evaluate the activity of ADP-ribosyl cyclase, we used the ability of the cyclase to catalyse the cyclization of nicotinamide guanine dinucleotide (NGD+) in cyclic GDP ribose (cGDPR), a fluorescent compound. NGD+ was excited at 300 nm and emitted fluorescence was filtered and measured at 420 nm (Graeff et al., 1994). Permeabilized cells were incubated in the presence of 100 µM NGD+ and the emitted fluorescence was collected with a CoolSnap HQ charge-coupled device camera (Roper Scientific, Evry, France). Images were acquired as described by LeBlanc et al. (LeBlanc et al., 2004
). The signal was processed by correcting each fluorescence image for background fluorescence. Averaged frames were collected every 0.5 seconds. To analyse variation of fluorescence, the sum of the fluorescence of each myocyte was integrated and this value of fluorescence (F) was divided by the fluorescence of the first image (F0) and reported as F/F0. Measurements were made at 26°C±1°C.
Chemicals and drugs
Fluo-4 acetoxymethylester (Fluo4-AM) was from Teflab (Austin, TX, USA). Alexa Fluor-labelled secondary antibodies were from Molecular Probes (Leiden, The Netherlands). Caffeine was from Merck (Nogent sur Marne, France). Ryanodine was from Calbiochem (Meudon, France). Medium M199, streptomycin, penicillin and collagenase were from Invitrogen (Cergy Pontoise, France). Anti-CD38 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4DAMP) and methoctramine were from RBI (Natik, MA, USA). All primers and phosphorothioate antisense oligonucleotides were synthesized by and purchased from Eurogentec (Seraing, Belgium). cADPR was from Amersham Biosciences (Orsay, France). All other chemicals were from Sigma. The rabbit anti-RYR3-specific antibody was directed against the deduced amino acid sequence, residues 4326-4336 (11 amino acids), of rabbit RYR3 (Jeyakumar et al., 1998). The rabbit anti-RYR2-specific antibody was directed against the deduced amino acid sequence, residues 1344-1365 (22 amino acids), of rabbit RYR2 (Jeyakumar et al., 2001
). The rabbit anti-RYR1-specific antibody was directed against the deduced amino acid sequence, residues 4476-4486 (11 amino acids), of rabbit RYR1 (Jeyakumar et al., 2002
).
Data analysis
Data are expressed as means ± s.e.m.; n represents the number of tested cells. Significance was tested by means of Student's t-test. P values <0.05 were considered as significant.
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Results |
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M2 and M3 muscarinic receptor-dependent transduction pathways
Duodenum myocytes express both M2 and M3 muscarinic receptors (Kuemmerle and Makhlouf, 1995; Morel et al., 1997
). Application of 4DAMP, an inhibitor of M3 muscarinic receptors in the nanomolar range (0.1-10 nM), significantly decreased the amplitude of ACh-induced Ca2+ responses but did not modify the percentage of intact cells that showed Ca2+ oscillations (Fig. 1C). Application of methoctramine, a selective inhibitor of the M2 muscarinic receptor in the micromolar range (1-10 µM), decreased the frequency of Ca2+ oscillations before decreasing the amplitude of ACh-induced Ca2+ responses. In the presence of 10 µM methoctramine, only 27% of cells triggered Ca2+ oscillations during ACh application (55% in control conditions, Fig. 1C).
These results prompted us to identify the Ca2+ signalling pathways activated by each muscarinic receptor by using inhibitors of InsP3 and cADPR pathways on permeabilized cells. In the presence of heparin, application of 10 nM 4DAMP was unable to modify the heparin-resistant Ca2+ response whereas 10 µM methoctramine totally abolished it (Fig. 1D). These results suggest that M3 and M2 muscarinic receptor subtypes activate two different pathways and that only the M2 muscarinic receptor-activated pathway may generate Ca2+ oscillations.
Involvement of cADPR in ACh-induced Ca2+ oscillations
ACh has been reported to activate the cADPR pathway by binding to M2 muscarinic receptor (White et al., 2003) and it has been proposed that cADPR may induce Ca2+ release through RYR activation (reviewed by Guse, 2004
). A selective and competitive inhibitor of cADPR binding sites, 8Br-cADPR, was applied (20 µM) 5 minutes before application of 1 µM ACh to permeabilized duodenum myocytes (Fig. 2A). In the presence of 8Br-cADPR, the amplitude of the first Ca2+ peak was significantly reduced while Ca2+ oscillations were practically suppressed (Fig. 2B). Application of methoctramine did not modify the 8Br-cADPR-resistant Ca2+ response (n=20) whereas 10 nM 4DAMP (Fig. 2B) and 1 mg/ml heparin (n=8) abolished the response. These results indicate that distinct signalling pathways are activated by ACh: the first one involves the M3 muscarinic receptor subtype and InsP3Rs and the second one involves the M2 muscarinic receptor subtype and cADPR.
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ADP-ribosyl cyclase activity and cADPR-induced Ca2+ oscillations
As previously reported in biochemical studies on subcellular fractions (Graeff et al., 1994; Sternfeld et al., 2003
), we used the cyclization of NGD+ into cGDPR (a fluorescent compound) to evaluate the ADP-ribosyl cyclase activity. In single permeabilized duodenum myocytes, application of 1 µM ACh for 30 seconds induced an increase in fluorescence detected at 420 nm (Fig. 3A) whereas application of a solution without ACh did not modify the fluorescence profile for 5 minutes. Both ADP-ribosyl cyclase inhibitors (ZnCl2 and the anti-CD38 antibody) and methoctramine (10 µM) inhibited the ACh-induced fluorescence signal whereas 4DAMP (1 nM) had no effect (Fig. 3B). Also the application of GTP
S (100 µM) induced an increase in fluorescence similar to that evoked by ACh application.
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Expression of RYR subtypes
To investigate more precisely the function of RYRs in ACh-induced Ca2+ responses, expression of RYR subtypes was examined by RT-PCR and immunostaining using specific antibodies. RT-PCR revealed that RYR1, RYR2 and RYR3 are potentially expressed (Fig. 4A). Immunodetection of RYR subtypes was performed with specific anti-RYR1, anti-RYR2 and anti-RYR3 antibodies. Specificity and absence of cross-reactivity of these antibodies have been previously described (Jeyakumar et al., 1998; Jeyakumar et al., 2001
; Jeyakumar et al., 2002
). Immunodetection in confocal sections of duodenum myocytes of primary antibody binding sites was revealed with the anti-rabbit Alexa Fluor 488 secondary antibody and the specificity was attested by the use of available antigenic peptides. Non-specific fluorescence (NSF) was determined when specific anti-RYR subtype antibody was pre-incubated with its antigenic peptide 1 hour before application of the immunostaining protocol. When the cell fluorescence obtained with the anti-RYR subtype antibody was higher than NSF (twofold increase), the cell was considered to be immunopositive and specific fluorescence could be estimated. Fig. 4C illustrates typical immunostainings obtained in duodenum myocytes. Measurements of cell fluorescence (Fig. 4B) revealed a specific staining with the anti-RYR1, anti-RYR2 and anti-RYR3 antibodies.
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The Ca2+ responses evoked by 10 mM caffeine were strongly decreased 3 days after injection of 10 µM asRYR1 plus 10 µM asRYR2 (asRYR1+2; Fig. 5A). In asRYR1-injected cells and in asRYR2-injected cells, the caffeine-induced Ca2+ responses were significantly reduced whereas injection of asRYR3 potentiated the Ca2+ responses (Fig. 5A). In the same cells, ACh was tested 3 minutes after caffeine application. It can be noted that the amplitude of the ACh-induced Ca2+ responses in myocytes cultured for 3 days was similar to that obtained within 4-30 hours of culture (Fig. 1B,C). Injection of asRYR1+2, asRYR1 or asRYR2 strongly decreased the amplitude of ACh-induced Ca2+ responses whereas injection of asRYR3 induced higher Ca2+ responses (Fig. 5B). In contrast, the effects of RYR subtype inhibition on Ca2+ oscillations revealed that oscillations were never observed in asRYR1+2-injected cells whereas the proportion of oscillating cells was slightly affected in asRYR1-injected cells compared to asRYR2-injected cells (44% and 10%, respectively, versus 53% in non-injected cells). In asRYR3-injected cells, the proportion of oscillating cells reached 70% of tested cells. Similarly, the frequency of Ca2+ oscillations was slightly diminished by injection of asRYR1 (4.6±0.8 oscillations/minute), clearly inhibited by injection of asRYR2 (3.0±1.1 oscillations/minute) and increased by injection of asRYR3 (6.2±1.1 oscillations/minute) when compared to non-injected cells (5.2±1.0 oscillations/minute).
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The cADPR-induced Ca2+ oscillations were differentially affected by inhibition of RYR subtypes. In asRYR1-injected cells, the amplitude of cADPR-induced Ca2+ responses was not significantly modified (Fig. 5C) and oscillations were obtained in 55% of tested cells. In contrast, the cADPR-induced Ca2+ responses were strongly decreased in both asRYR1+2 and asRYR2-injected cells (Fig. 5C) and Ca2+ oscillations were never observed. As seen with ACh and caffeine application, the cADPR-induced Ca2+ response was increased in asRYR3-injected cells (Fig. 5C) as well as the number of oscillating cells (65% of tested cells).
Several studies have reported that cADPR may modify the interactions between FKBP12/12.6 and RYRs (Noguchi et al., 1997; Tang et al., 2002
). Therefore, we investigated the effects of 10 µM rapamycin, which induces uncoupling between FKBP12/12.6 and RYRs in permeabilized cells. The amplitude of rapamycin-induced Ca2+ oscillations was similar to that obtained with cADPR (Fig. 6A) but the number of oscillating cells was smaller (41% of tested cells). As observed with cADPR, both amplitude of rapamycin-induced Ca2+ responses and number of oscillating cells were not modified in asRYR1-injected cells, strongly decreased in both asRYR2- and asRYR1+2-injected cells and increased in asRYR3-injected cells (Fig. 6B,C). In the continuous presence of rapamycin (10 µM for 10 minutes), caffeine induced a reduced Ca2+ response whereas cADPR was unable to induce any response (Fig. 3D), suggesting that rapamycin and cADPR might act on the same protein, i.e. FKBP12/12.6.
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Discussion |
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In different cell types, it has been proposed that Ca2+ oscillations can be supported by different Ca2+ channels or Ca2+ stores: InsP3Rs (Harootunian et al., 1991; Miyakawa et al., 1999
; Morel et al., 2003
) or RYRs (Morel et al., 1996
) and mitochondria (Rizzuto et al., 2000
). Activation of Ca2+ influx is another trigger for Ca2+ oscillations via membrane potential oscillations (Sneyd et al., 2004
). Our results showed that in duodenum myocytes, Ca2+ influx was not needed to generate ACh-induced Ca2+ responses or to sustain Ca2+ oscillations since these responses were obtained in intact cells superfused in Ca2+-free EGTA-containing solution and in saponin-permeabilized cells. By pharmacological inhibition of a mitochondria Ca2+ uniporter, we also excluded the mitochondria as a Ca2+ buffer for these Ca2+ oscillations. Therefore, our results support the idea that the ACh-induced Ca2+ oscillations depend on a Ca2+ release from the sarcoplasmic reticulum. The distinct roles of RYRs and InsP3Rs in Ca2+ responses were illustrated by using heparin (an inhibitor of InsP3Rs), which decreased the first Ca2+ peak of ACh-induced responses without causing changes in Ca2+ oscillation generation and frequency, and ryanodine, which decreased both Ca2+ response amplitude, and generation and frequency of Ca2+ oscillations. RT-PCR and immunostainings indicate that InsP3R1 is the only InsP3R subtype expressed in rat duodenum myocytes (J.-L.M., unpublished data). In addition, expression of InsP3R1 alone is not sufficient to induce Ca2+ oscillations, as previously reported (Miyakawa et al., 1999
; Morel et al., 2003
). An important finding in this paper is that Ca2+ oscillations are totally dependent on RYR2 activation. Although involvement of RYRs has been proposed in Ca2+ oscillations, the RYR subtype responsible for Ca2+ oscillations has not been previously identified. In vascular myocytes, the function of RYR2 and RYR1 subtypes in Ca2+ release has been studied by using antisense oligonucleotides targeting the RYR subtypes (Coussin et al., 2000
). With the same antisense strategy, we showed that, in duodenum myocytes, RYR2 was the trigger for ACh-induced Ca2+ oscillations whereas RYR1 participated in the Ca2+ response amplitude probably through a CICR mechanism.
Although activation of M2 muscarinic receptors is classically known to inhibit adenylyl cyclase activity (Peralta et al., 1988) or to modify membrane potential by inhibiting Ca2+-activated K+ channel (Kotlikoff et al., 1992
), it has been recently proposed that M2 muscarinic receptors may induce Ca2+ signals by activation of the cADPR pathway (White et al., 2003
) or by stimulation of a voltage-dependent Ca2+ channel (Cav1.2b) via the phosphatidylinositol 3-kinase/PKC pathway (Callaghan et al., 2004
). Activation of the cADPR pathway by ACh in duodenum myocytes is shown by: (1) inhibition of Ca2+ oscillations by application of the cADPR competitive antagonist (8Br-cADPR), (2) inhibition of ACh-induced Ca2+ oscillations by inhibitors of ADP-ribosyl cyclase (ZnCl2, anti-CD38 antibody) and (3) detection of ADP-ribosyl cyclase activity by fluorescence experiments as the enzyme cyclizes NGD+ (non fluorescent) to produce cGDPR, a fluorescent compound (Graeff et al., 1994
). This method has been used successfully in microsomes and cellular homogenates from bovine chromaffin cells (Morita et al., 1997
), rat vascular myocytes (de Toledo et al., 2000
) and human myometrium (Chini et al., 2002
). We were able to detect ADP-ribosyl cyclase activity in single permeabilized cells under the same conditions that we used for Ca2+ measurements. ACh induced a fluorescence signal corresponding to an increase of ADP-ribosyl cyclase activity. With this method, we showed that ACh activated the cyclase by binding to the M2 muscarinic receptor and that this stimulatory effect was inhibited by ZnCl2 and the anti-CD38 antibody. These results are in agreement with data reporting an ADP-ribosyl cyclase activity in rat duodenum (de Toledo et al., 2000
), and its activation by ACh in adrenal chromaffin cells (Morita et al., 1997
).
RYRs, FKBP12/12.6, Ca2+ pumps (SERCA and plasma membrane Ca2+ pumps) and InsP3Rs have been reported to be the main cADPR targets in different cells types (Guse, 2004) and modulation by cADPR of Ca2+ oscillations has been shown in porcine tracheal muscle (Prakash et al., 1998
). In duodenum myocytes, we showed that (1) cADPR induced Ca2+ oscillations similar to those evoked by ACh; (2) in the presence of cADPR, the amplitude of caffeine-induced Ca2+ responses was significantly decreased, suggesting that cADPR preferentially induced Ca2+ release; (3) cADPR-induced Ca2+ oscillations were not modified after inhibition of InsP3Rs by heparin, indicating no participation of InsP3Rs; (4) cADPR induced Ca2+ oscillations in permeabilized cells that were similar to those obtained in intact cells, excluding a role for the plasma membrane Ca2+ pumps; (5) partial inhibition of SERCA by thapsigargin did not affect the Ca2+ oscillation mechanism, suggesting that SERCA activation was not the trigger for Ca2+ oscillations. This is in contrast to previous data suggesting that cADPR may modulate SERCA activity. For example, cADPR has been reported to increase the rate of decline of Ca2+ responses following membrane depolarizations in colonic myocytes (Bradley et al., 2003
) and both the frequency of Ca2+ sparks and amplitude of caffeine-induced Ca2+ responses in cardiomyocytes (Lukyanenko et al., 2001
). In both cases, cADPR was unable to induce a Ca2+ signal by itself. Moreover, the cADPR effects on Ca2+ pumps are obtained with higher cADPR concentrations (1-10 µM) than those used to activate RYR-dependent Ca2+ release (1-100 nM), as reported by Guse (Guse, 2004
). We also showed that (6) cADPR-induced Ca2+ responses were inhibited by ryanodine and the anti-RYR2 antisense oligonucleotide, suggesting that RYR2 could be the target of cADPR although the cADPR receptor has not been identified so far.
In pancreatic cells, various agonists stimulate cADPR production (Sternfeld et al., 2003) and cADPR may bind to FKBP12.6, a RYR-associated protein (Noguchi et al., 1997
). cADPR and FK506, an activator of FKBP12/12.6, have similar effects, which are not additive (Ozawa, 2004
). In arterial myocytes, activation of purified RYRs by cADPR has been shown to be inhibited by FK506 (Tang et al., 2002
) and cADPR is unable to activate Ca2+ release in tracheal myocytes from FKBP12.6 knockout mice (Wang et al., 2004
). In duodenum myocytes, we found that rapamycin (an inhibitor of FKBP12/12.6/RYR interactions, analogue to FK506) inhibited cADPR-induced Ca2+ oscillations, suggesting that the effects of cADPR on Ca2+ release involved FKBP. In smooth muscle, FKBP12.6 is associated with RYR2 but not with other RYR isoforms or InsP3Rs (Wang et al., 2004
) and modulating interactions between FKBP12.6 and RYR2 have been reported (Tang et al., 2002
). Using an antisense strategy, we showed that in duodenum myocytes, only the anti-RYR2 antisense oligonucleotide inhibited the rapamycin-induced Ca2+ oscillations, supporting the idea that the FKBP12 involved in the cADPR pathway is coupled to RYR2. In addition, in the continuous presence of rapamycin, cADPR was ineffective suggesting that both compounds interacted at the same protein level. Consequently, we can propose that in duodenum myocytes cADPR induces activation of RYR2 to produce Ca2+ oscillations through an uncoupling between RYR2 and FKBP12.6. RYR3 has also been proposed as a possible target for cADPR in T-lymphocytes and Jurkat cells (Kunerth et al., 2004
). However, this possibility can be discarded in duodenum myocytes, since inhibition of RYR3 resulted in a stimulation of Ca2+ release in response to ACh, cADPR and rapamycin. An increase in Ca2+ spark frequency has been previously reported in vascular myocytes of RYR3 knockout mice (Löhn et al., 2001
). The spliced variants described by Jiang et al. (Jiang et al., 2003
) may offer an explanation for this observation since one of these spliced variant may heteromerize with the other RYR3 variants or RYR2 and inhibit their function. Therefore, in our experiments, inhibition of all the RYR3 variants may account for an increase in Ca2+ signals dependent on RYR2.
In conclusion, this study proposes for the first time a selective signalling pathway responsible for ACh-induced Ca2+ oscillations in rat duodenum myocytes. This pathway involves the M2 muscarinic receptor, resulting in activation of ADP-ribosyl cyclase and subsequent production of cADPR. Interactions between cADPR and FKBP12.6 may lead to activation of RYR2 and the secondary recruitment of RYR1 via a CICR mechanism. This signalling pathway may be important for the duodenum peristalsis, which is under vagal parasympathetic control.
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Acknowledgments |
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References |
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---|
Arnaudeau, S., Boittin, F. X., Macrez, N., Lavie, J. L., Mironneau, C. and Mironneau, J. (1997). L-type and Ca2+ release channel-dependent hierarchical Ca2+ signalling in rat portal vein myocytes. Cell Calcium 22, 399-411.[CrossRef][Medline]
Berridge, M. J., Lipp, P. and Bootman, M. D. (2000). The versatility and universality of calcium signaling. Nat. Rev. Mol. Cell. Biol. 1, 11-21.[CrossRef][Medline]
Boittin, F. X., Macrez, N., Halet, G. and Mironneau, J. (1999). Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes. Am. J. Physiol. 277, C139-C151.[Medline]
Bradley, K. N., Currie, S., MacMillan, D., Muir, T. C. and McCarron, J. G. (2003). Cyclic ADP-ribose increases Ca2+ removal in smooth muscle. J. Cell Sci. 116, 4291-4306.
Callaghan, B., Koh, S. D. and Keef, K. D. (2004). Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol 3-kinase, protein kinase C, and c-Src. Circ. Res. 94, 626-633.
Chini, E. N., Chini, C. C., Barata da Silva, H. and Zielinska, W. (2002). The cyclic-ADP-ribose signaling pathway in human myometrium. Arch. Biochem. Biophys. 407, 152-159.[CrossRef][Medline]
Coussin, F., Macrez, N., Morel, J. L. and Mironneau, J. (2000). Requirement of ryanodine receptor subtypes 1 and 2 for Ca2+-induced Ca2+ release in vascular myocytes. J. Biol. Chem. 275, 9596-9603.
de Toledo, F. G., Cheng, J., Liang, M., Chini, E. N. and Dousa, T. P. (2000). ADP-Ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation. Circ. Res. 86, 1153-1159.
Fill, M. and Copello, J. A. (2002). Ryanodine receptor calcium release channels. Physiol. Rev. 82, 893-922.
Ge, Z. D., Zhang, D. X., Chen, Y. F., Yi, F. X., Zou, A. P., Campbell, W. B. and Li, P. L. (2003). Cyclic ADP-ribose contributes to contraction and Ca2+ release by M1 muscarinic receptor activation in coronary arterial smooth muscle. J. Vasc. Res. 40, 28-36.[CrossRef][Medline]
Graeff, R. M., Walseth, T. F., Fryxell, K., Branton, W. D. and Lee, H. C. (1994). Enzymatic synthesis and characterizations of cyclic GDP-ribose. J. Biol. Chem. 269, 30260-30267.
Guse, A. H. (2004). Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR). Curr. Mol. Med. 4, 239-248.[CrossRef][Medline]
Harootunian, A. T., Kao, J. P., Paranjape, S. and Tsien, R. Y. (1991). Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3. Science 251, 75-78.[Medline]
Higashida, H., Yokoyama, S., Hashii, M., Taketo, M., Higashida, M., Takayasu, T., Ohshima, T., Takasawa, S., Okamoto, H. and Noda, M. (1997). Muscarinic receptor-mediated dual regulation of ADP-ribosyl cyclase in NG108-15 neuronal cell membranes. J. Biol. Chem. 272, 31272-31277.
Jeyakumar, L. H., Copello, J. A., O'Malley, A. M., Wu, G. M., Grassucci, R., Wagenknecht, T. and Fleischer, S. (1998). Purification and characterization of ryanodine receptor 3 from mammalian tissue. J. Biol. Chem. 273, 16011-16020.
Jeyakumar, L. H., Ballester, L., Cheng, D. S., McIntyre, J. O., Chang, P., Olivey, H. E., Rollins-Smith, L., Barnett, J. V., Murray, K., Xin, H. B. et al. (2001). FKBP binding characteristics of cardiac microsomes from diverse vertebrates. Biochem. Biophys. Res. Commun. 281, 979-986.[CrossRef][Medline]
Jeyakumar, L. H., Gleaves, L. A., Ridley, B. D., Chang, P., Atkinson, J., Barnett, J. V. and Fleischer, S. (2002). The skeletal muscle ryanodine receptor isoform 1 is found at the intercalated discs in human and mouse hearts. J. Muscle Res. Cell. Motil. 23, 285-292.[CrossRef][Medline]
Ji, G., Feldman, M. E., Greene, K. S., Sorrentino, V., Xin, H. B. and Kotlikoff, M. I. (2004). RYR2 proteins contribute to the formation of Ca2+ sparks in smooth muscle. J. Gen. Physiol. 123, 377-386.
Jiang, D., Xiao, B., Li, X. and Chen, S. R. (2003). Smooth muscle tissues express a major dominant negative splice variant of the type 3 Ca2+ release channel (ryanodine receptor). J. Biol. Chem. 278, 4763-4769.
Kaftan, E., Marks, A. R. and Ehrlich, B. E. (1996). Effects of rapamycin on ryanodine receptor/Ca2+-release channels from cardiac muscle. Circ. Res. 78, 990-997.
Kotlikoff, M. I., Kume, H. and Tomasic, M. (1992). Muscarinic regulation of membrane ion channels in airway smooth muscle cells. Biochem. Pharmacol. 43, 5-10.[CrossRef][Medline]
Kuemmerle, J. F. and Makhlouf, G. M. (1995). Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca2+-induced Ca2+ release in intestinal longitudinal muscle. J. Biol. Chem. 270, 25488-25494.
Kunerth, S., Langhorst, M. F., Schwarzmann, N., Gu, X., Huang, L., Yang, Z., Zhang, L., Mills, S. J., Zhang, L. H., Potter, B. V. et al. (2004). Amplification and propagation of pacemaker Ca2+ signals by cyclic ADP-ribose and the type 3 ryanodine receptor in T cells. J. Cell Sci. 117, 2141-2419.
LeBlanc, C., Mironneau, C., Barbot, C., Henaff, M., Bondeva, T., Wetzker, R. and Macrez, N. (2004). Regulation of vascular L-type calcium channels by phosphatidylinositol 3, 4, 5-trisphosphate. Circ. Res. 95, 300-307.
Lee, H. C. (2004). Multiplicity of Ca2+ messengers and Ca2+ stores: a perspective from cyclic ADP-ribose and NAADP. Curr. Mol. Med. 4, 227-237.[CrossRef][Medline]
Löhn, M., Jessner, W., Furstenau, M., Wellner, M., Sorrentino, V., Haller, H., Luft, F. C. and Gollasch, M. (2001). Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells. Circ. Res. 89, 1051-1057.
Lukyanenko, V., Györke, I., Wiesner, T. F. and Györke, S. (2001). Potentiation of Ca2+ release by cADP-ribose in the heart is mediated by enhanced SR Ca2+ uptake into the sarcoplasmic reticulum. Circ. Res. 89, 614-622.
Macrez-Lepretre, N., Kalkbrenner, F., Schultz, G. and Mironneau, J. (1997). Distinct functions of Gq and G11 proteins in coupling alpha1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes. J. Biol. Chem. 272, 5261-5268.
McCall, E., Li, L., Satoh, H., Shannon, T. R., Blatter, L. A. and Bers, D. M. (1996). Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes. Circ. Res. 79, 1110-1121.
Miyakawa, T., Maeda, A., Yamazawa, T., Hirose, K., Kurosaki, T. and Iino, M. (1999). Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J. 18, 1303-1308.
Morel, J. L., Macrez-Lepretre, N. and Mironneau, J. (1996). Angiotensin II-activated Ca2+ entry-induced release of Ca2+ from intracellular stores in rat portal vein myocytes. Brit. J. Pharmacol. 118, 73-78.[Medline]
Morel, J. L., Macrez, N. and Mironneau, J. (1997). Specific Gq protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes. Brit. J. Pharmacol. 121, 451-458.[Medline]
Morel, J. L., Fritz, N., Lavie, J. L. and Mironneau, J. (2003). Crucial role of type 2 inositol 1,4,5-trisphosphate receptors for acetylcholine-induced Ca2+ oscillations in vascular myocytes. Arterioscler. Thromb. Vasc. Biol. 23, 1567-1575.
Morel, J. L., Rakotoarisoa, L., Jeyakumar, L. H., Fleischer, S., Mironneau, C. and Mironneau, J. (2004). Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes. J. Biol. Chem. 279, 21287-21293.
Morita, K., Kitayama, S. and Dohi, T. (1997). Stimulation of cyclic ADP-ribose synthesis by acetylcholine and its role in catecholamine release in bovine adrenal chromaffin cells. J. Biol. Chem. 272, 21002-21009.
Noguchi, N., Takasawa, S., Nata, K., Tohgo, A., Kato, I., Ikehata, F., Yonekura, H. and Okamoto, H. (1997). Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes. J. Biol. Chem. 272, 3133-3136.
Ozawa, T. (2004). Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506. Biochem. Biophys. Acta 1693, 159-166.[CrossRef][Medline]
Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J. and Capon, D. J. (1988). Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature 334, 434-437.[CrossRef][Medline]
Prakash, Y. S., Kannan, M. S., Walseth, T. F. and Sieck, G. C. (1998). Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle. Am. J. Physiol. 274, C1653-C1660.[Medline]
Rizzuto, R., Bernardi, P. and Pozzan, T. (2000). Mitochondria as all-round players of the calcium game. J. Physiol. 529, 37-47.
Sneyd, J., Tsaneva-Atanasova, K., Yule, D. I., Thompson, J. L. and Shuttleworth, T. J. (2004). Control of calcium oscillations by membrane fluxes. Proc. Natl. Acad. Sci. USA 101, 1392-1396.
Sternfeld, L., Krause, E., Guse, A. H. and Schulz, I. (2003). Hormonal control of ADP-ribosyl cyclase activity in pancreatic acinar cells from rats. J. Biol. Chem. 278, 33629-33636.
Tang, W. X., Chen, Y. F., Zou, A. P., Campbell, W. B. and Li, P. L. (2002). Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle. Am. J. Physiol. Heart Circ. Physiol. 282, H1304-H1310.
Thomas, E. A. and Ehlert, F. J. (1994). Pertussis toxin blocks M2 muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M3-mediated contractions and phosphoinositide hydrolysis. J. Pharmacol. Exp. Ther. 271, 1042-1050.[Abstract]
Wang, Y. X., Zheng, Y. M., Mei, Q. B., Wang, Q. S., Collier, M. L., Fleischer, S., Xin, H. B. and Kotlikoff, M. I. (2004). FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells. Am. J. Physiol. Cell. Physiol. 286, C538-C546.
White, T. A., Kannan, M. S. and Walseth, T. F. (2003). Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle. FASEB J. 17, 482-484.
Xiao, B., Masumiya, H., Jiang, D., Wang, R., Sei, Y., Zhang, L., Murayama, T., Ogawa, Y., Lai, F. A., Wagenknecht, T. and Chen, S. R. (2002). Isoform-dependent formation of heteromeric Ca2+ release channels (ryanodine receptors). J. Biol. Chem. 277, 41778-41785.
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