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First published online 9 October 2007
doi: 10.1242/jcs.009415

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

Nicolas Fritz^1,*, Jean-Luc Morel^1,2, Loice H. Jeyakumar³, Sidney Fleischer³, Paul D. Allen⁴, Jean Mironneau¹ and Nathalie Macrez^1,2,

¹ CNRS UMR 5017, Laboratoire de Signalisation et Interactions Cellulaires, Université Bordeaux 2, Bordeaux, France
² Université de Bordeaux1, CNIC, CNRS UMR 5228, Talence, France
³ Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
⁴ Department of Anaesthesia Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA

View larger version (20K): [in this window] [in a new window]   Fig. 1. RyR subtype expression in WT and RyR1–/– mouse embryonic detrusor muscle. Total proteins from urinary bladder (100 µg per lane), skeletal muscle (50 µg per lane), cardiac muscle (50 µg per lane) and brain (50 µg per lane) were separated on SDS-PAGE and analyzed by western blot with anti-RyR1, anti-RyR2 and anti-RyR3 antibodies. Skeletal and cardiac muscles were chosen as positive control tissues for the anti-RyR1 and anti-RyR2 antibodies, respectively, and brain extracts were used as positive control for the anti-RyR3 antibody. Similar results were obtained in five independent experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Spontaneous and triggered Ca2+ events in WT and RyR1–/– urinary bladder myocytes. (A-C) Typical confocal line-scan images of (A) spontaneous, (B) caffeine-activated or (C) depolarization-activated Ca2+ events in WT (left) and RyR1–/– (right) myocytes. Caffeine (10 mM) was applied near the cell by pressure ejection from a glass pipette while depolarizing pulses of 50 mV, 100 mseconds from a holding potential of –40 mV were applied to whole-cell patch-clamped myocytes. Fluo-4 fluorescence averaged from a 2-µm region indicated by the bar on the line-scan image is illustrated under each image. Myocytes were loaded with Fluo4-AM and when patch-clamped, 50 µM Fluo-4 was added to the intracellular medium. Spatio-temporal parameters of these Ca2+ events are given in Table 1.

View larger version (8K): [in this window] [in a new window]   Fig. 3. Statistical analysis of Ca2+ sparks in WT and RyR1–/– urinary bladder myocytes. Given is the percentage of cells that produce spontaneous Ca2+ sparks or Ca2+ sparks activated by 10 mM caffeine or membrane depolarization (depolarizing pulses of 50 mV, 100 mseconds from holding potential –40mV) in WT and RyR1–/– myocytes. The number of cells that produce sparks versus the total number of cells tested in each condition is indicated in parentheses.

View larger version (27K): [in this window] [in a new window]   Fig. 4. RyR2 and RyR3 expression levels in WT and RyR1–/– mouse urinary bladder. Semi-quantitative western-blot analysis of the expression levels of RyR2 and RyR3 compared with the expression of Ins(1,4,5)PR3 (InsP3R3) in WT and RyR1–/– mouse urinary bladder (100 µg per lane). (A) Immunoblots showing the expression of RyR2, RyR3 and Ins(1,4,5)PR3 in WT and RyR1–/– mouse urinary bladder. (B) Average of RyR-Ins(1,4,5)PR3 band luminescence intensity ratios for RyR2 and RyR3 obtained in three independent experiments.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Intracellular localization of RyR subtypes in WT and RyR1–/– myocytes. (A-C) Immunostainings performed in WT and RyR1–/– urinary bladder myocytes with specific antibodies against (A) RyR1, (B) RyR2 and (C) RyR3 and Alexa Fluor 488-coupled secondary antibodies. (Left panels) Transmission images of each immmunostained cell was performed to locate the plasma membrane. The cell outline (dotted) was then reported on the fluorescent confocal image of the cell (middle panel) and fluorescence profiles (a-f) depicting subcellular localization of RyR were drawn for each confocal image (right panel). Edges of cells were reported on fluorescence profiles as dotted lines. Representative pictures of n=10-15 cells in each conditions.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Changes in FKBP12 and FKBP12.6 expression in embryonic RyR1–/– mouse urinary bladder. (A) Semi-quantitative RT-PCR analysis of FKBP12 and FKBP12.6 mRNA in control and RyR1–/– mouse myocytes. (B) Bar graph showing a significant diminution of FKBP12:GAPDH ratio in RyR1–/– mouse myocytes in six different RNA extractions tested in triplicate. (C) Western blot analysis of FKBP12, FKBP12.6 and -actin expression in WT and RyR1–/– mouse myocytes. (D) Bar graph of FKBP12 and FKBP12.6 fluorescence area per µg of protein obtained from four mice of each strain. *P<0.05.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Effect of rapamycin on RyR-dependent Ca2+ signaling WT and RyR1–/– urinary bladder myocytes. (A) Effects of rapamycin (1 µM) on 10 mM caffeine-induced Ca2+ responses in WT urinary bladder myocytes and in RyR1–/– mouse myocytes. (B-D) Ca2+ responses induced by (B) 100 µM and (C) 50 µM rapamycin in WT or in (D) RyR1–/– myocytes (D). Rapamycin was applied near the cell by pressure ejection from a glass pipette at the time indicated on the line-scan image. *P<0.05.

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