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First published online 3 May 2005
doi: 10.1242/jcs.02344

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

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

View larger version (40K): [in a new window]   Fig. 1. Ca2+ oscillations evoked by acetylcholine (ACh) in duodenum myocytes. Dependence on M2 muscarinic receptors and ryanodine receptors. (A) Typical Ca2+ oscillations induced by 1 µM ACh in an intact cell and a cell permeabilized with 10 µg/ml saponin. (B) Compiled data from intact cells showing the effects of ryanodine (10 µM, 30 minutes), thapsigargin (50 nM, 1 minute), RU360 (1 µM, 30 minutes) and of a superfusion with a Ca2+-free EGTA-containing solution (30 seconds) on the amplitude of the first Ca2+ peak (left) and the percentage of oscillating cells (right). (C) Compiled data from intact cells showing the effects of methoctramine (1 and 10 µM, 10 minutes) and 4-DAMP (0.1 and 1 nM, 10 minutes) on the amplitude of the first Ca2+ peak (left) and the percentage of oscillating cells (right). (D) Compiled data from permeabilized cells showing the effect of heparin (1 mg/ml), and heparin in presence of 10 µM methoctramine or 10 nM 4DAMP on the amplitude of the first Ca2+ peak (left) and the percentage of oscillating cells (right). F/F0 is the fluorescence ratio during a response to that at rest. Heparin was applied 5 minutes before ACh application. Data are means ± s.e.m. with the number of tested cells indicated in parentheses. Myocytes were loaded with 2 µM Fluo4-AM (stars indicate P<0.05).

View larger version (24K): [in a new window]   Fig. 2. Effects of 8Br-cADPR and ADP-ribosyl cyclase inhibitors on ACh-induced Ca2+ oscillations. (A) Typical ACh-induced Ca2+ response obtained in a myocyte permeabilized with 10 µg/ml saponin in control conditions and in the presence of 20 µM 8Br-cADPR for 5 minutes. (B) Compiled data showing the effects of 8Br-cADPR (20 µM) in presence of 10 nM 4DAMP for 5 minutes on the amplitude of the first Ca2+ peak of ACh-induced Ca2+ responses (left) and on the percentage of oscillating permeabilized cells (right). (C) Compiled data showing the effects of ZnCl2 (2 mM, 5 minutes) or NGD+ (2 mM, 15 minutes) on the amplitude of the first Ca2+ peak of ACh-induced Ca2+ responses (left) and the percentage of oscillating permeabilized cells (right). (D) Compiled data showing the effects of an anti-CD38 antibody (1 µg/ml, 5 minutes) on the amplitude of ACh-induced Ca2+ responses (left) and the percentage of oscillating permeabilized cells. Cells were permeabilized with 100 µg/ml saponin. The inactivated antibody was obtained by heating (90°C, 20 minutes). Data are means ± s.e.m. with the number of tested cells indicated in parentheses. Myocytes were loaded with 2 µM Fluo4-AM (stars indicate P<0.05).

View larger version (30K): [in a new window]   Fig. 3. Effects of ACh on ADP-ribosyl cyclase activity and activation of Ca2+ oscillations by cADPR. (A) Typical fluorescence signal (ex=300 nm; em=420 nm) of a permeabilized cell induced by 1 µM ACh in control conditions and in presence of methoctramine (10 µM, 3 minutes). (B) Compiled data showing the effects of methoctramine (10 µM), 4DAMP (10 nM), anti-CD38 antibody (1 µg/ml), ZnCl2 (2 mM) on the amplitude of ACh-induced fluorescent signals and the effects of GTPS (100 µM). Myocytes were superfused with a permeabilization solution (100 µg/ml saponin) containing 100 µM NGD+ and 100 µM ATP. All experiments were made within 10 minutes following application of permeabilization solution. (C) Typical Ca2+ signals induced by cADPR (100 nM) in intact or permeabilized cells. (D) Compiled data from permeabilized cells showing the effects of heparin (1 mg/ml, 5 minutes), ryanodine (10 µM, 10 minutes), 8Br-cADPR (50 µM, 5 minutes) and rapamycin (10 µM, 10 minutes) on the amplitude of the first Ca2+ peak of cADPR-induced Ca2+ responses (left) and the percentage of oscillating cells (right). Myocytes were loaded with 2 µM Fluo4-AM and permeabilized with 10 µg/ml saponin. Data are means ± s.e.m. with the number of tested cells indicated in parentheses (stars indicate P<0.05).

View larger version (50K): [in a new window]   Fig. 4. Expression of RYR subtypes in duodenum myocytes. (A) cDNA fragments of RYR subtypes were amplified from duodenum myocytes. cDNA fragments were separated on a 2% agarose gel, visualized by staining with ethidium bromide and shown as a contrasted image. Molecular size standard is 174-HinfI scale. (B) Compiled data of total fluorescence (white bars) and non-specific fluorescence (hatched bars) observed in cells with the anti-RYR1 (1:1000), anti-RYR2 (1:500) and anti-RYR3 (1:1000) antibodies. Data are means ± s.e.m. with the number of tested cells indicated in parentheses (stars indicate P<0.05). AU, arbitrary units. (C) Typical confocal sections showing immunostaining obtained with anti-RYR1 (1:1000), anti-RYR2 (1:500) and anti-RYR3 (1:1000) antibodies. Images were contrasted with Confocal Assistant 4.2 software. The same correction factors were applied on all the images. (D) Compiled data of specific fluorescence observed in non-injected cells (white bars) and injected cells (hatched bars). Three days after injection of antisense oligonucleotides, myocytes were stained with anti-RYR1 (left bars), anti-RYR2 (middle bars) and anti-RYR3 (right bars) antibodies. Data are means ± s.e.m. with the number of tested cells indicated in parentheses (stars indicate P<0.05). AU, arbitrary units.

View larger version (18K): [in a new window]   Fig. 5. Effects of antisense oligonucleotides directed against the RYR subtypes on Ca2+ oscillations induced by caffeine, ACh and cADPR. Compiled data showing the amplitude of Ca2+ responses evoked by 10 mM caffeine (A), 1 µM ACh (B), 100 nM cADPR (C) in non-injected cells (white bars) and cells injected with 10 µM asRYR antisense oligonucleotides (black bars). Results in A and B were obtained in intact cells and results in C were obtained in permeabilized cells. Data are means ± s.e.m. with the number of tested cells indicated in parentheses. Myocytes were loaded with 2 µM Fluo4-AM and used 3 days after oligonucleotide injection (stars indicate P<0.05).

View larger version (16K): [in a new window]   Fig. 6. Effects of antisense oligonucleotides directed against the RYR subtypes on Ca2+ oscillations induced by rapamycin. (A) Typical Ca2+ oscillations induced by 10 µM rapamycin on permeabilized cells. (B) Compiled data showing the amplitude of Ca2+ responses evoked by 10 µM rapamycin in non-injected cells (white bar) and cells injected with 10 µM asRYR antisense oligonucleotides (black bars). (C) Percentage of oscillating cells in the same conditions as in B. Data are means ± s.e.m. with the number of tested cells indicated in parentheses. Myocytes were loaded with 2 µM Fluo4-AM and used 3 days after oligonucleotide injection (stars indicate P<0.05).

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