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First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01136

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Requirement of ryanodine receptors for pacemaker Ca²⁺ activity in ICC and HEK293 cells

Masahiro Aoyama^1,*, Aki Yamada^4,*, Jing Wang², Susumu Ohya⁴, Shinji Furuzono⁴, Takayo Goto⁴, Shingo Hotta⁴, Yasushi Ito¹, Tatsuaki Matsubara³, Kaoru Shimokata¹, S. R. Wayne Chen⁵, Yuji Imaizumi^4, and Shinsuke Nakayama^2,

¹ Department of Physiological Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
² Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
³ Department of Metabolic Diseases, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
⁴ Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
⁵ Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

View larger version (28K): [in a new window]   Fig. 5. RT-PCR examination for RyR1 to 3. (A) Control amplicons for RyR1 to 3 and GAPDH extracted from brain, heart and skeletal muscle. Amplification, 35 cycles. (B) RT-PCR examinations of NTC (no template control) and RNA samples obtained from c-Kit-immunopositive cells and smooth muscle cells. Amplification, 45 cycles. (C) RT-PCR examinations for FKBP12 and FKBP12.6. Amplification, 45 cycles. In B, expression of c-kit was also examined. The numbers in the right of each gel indicate the size marker (bp).

View larger version (43K): [in a new window]   Fig. 1. Effects of ryanodine on [Ca2+]i oscillations in the presence of nifedipine. Single or multiple [Ca2+]i oscillation sites are seen in A or B, respectively. Aa1 to 3 show pseudo-colour ratio images of the fluo-3 fluorescent intensity recorded at lines 1 to 3 (indicated in Ab) from a cultured cell cluster preparation in the presence of 2 µM nifedipine. The fluo-3 fluorescence was used as an index of [Ca2+]i activity. Each image was normalised by the fluorescent intensity of the initial image (acquired at t=0 in the first or third trace of Ab). The panels Aa4 and Aa5 show c-Kit-immunostaining and transmission images, respectively, obtained from the same cell cluster. Bar, 100 µm. Note that the square region in Aa1 showing [Ca2+]i oscillations indicates c-Kit-immunopositivity. The traces in Ab show changes in [Ca2+]i (fluo-3 intensity: Ft/F0) in the square region. After observing control [Ca2+]i oscillations in the presence of 2 µM nifedipine (left trace), 1 µM ryanodine was applied. The second (middle) trace of [Ca2+]i oscillations was obtained 5 minutes after 1 µM ryanodine application. Subsequently, the ryanodine concentration was increased to 10 µM. The third (right) trace in Ab was obtained 5 minutes after increasing the ryanodine concentration. The three panels in Ba are pseudo-colour ratio images of [Ca2+]i activity obtained from another cell cluster preparation showing multiple oscillation sites. Images Ba1 to Ba3 were acquired at lines 1 to 3 in Bb. In Bb the blue and red traces indicate changes in [Ca2+]i in the square regions of x [=Ft(x)/F0] and y [=Ft(y)/F0] in panel Ba1. After observing control [Ca2+]i oscillation in the presence of nifedipine (2 µM) (left trace in Bb), 10 µM ryanodine was applied to the extracellular solution. The second (middle) and third (right) traces were obtained 2 and 8 minutes after the application of ryanodine, respectively. In Bc, Ft(y)/F0 (y-axis) is plotted against Ft(x)/F0 (x-axis). Each graph Bd indicates the cross-correlation function between Ft(x)/F0 and Ft(y)/F0. Note, [Ca2+]i oscillations in x and y are well correlated even when significantly impaired.

View larger version (36K): [in a new window]   Fig. 2. Caffeine reversibly suppresses [Ca2+]i oscillations in ICCs. Panels A1 to 3 show [Ca2+]i images recorded at lines (1) to (3) in B, respectively. Horizontal bar=100 µm. Traces in B indicate the time course of the changes in [Ca2+]i (Ft/F0) in the square regions indicated in A(1): blue line for (x) and red line for (y). After observing control [Ca2+]i oscillations in the presence of 2 µM nifedipine (Ba), 10 mM caffeine was applied. Bb and Bc were recorded after 3 minutes and 5 minutes, respectively. Bd shows recovery of [Ca2+]i oscillations 5 minutes after washout of caffeine in the presence of nifedipine. In C and D, after observing control [Ca2+]i oscillations (a), effects of 30 µM tetracaine and 10 µM FK506 were examined, respectively. The trace Cb was recorded 5 minutes after tetracaine application, while Db was 2 minutes after FK506 application.

View larger version (19K): [in a new window]   Fig. 3. Examples of the effects of InsP3 receptor blockers. The traces in A and B were obtained from regions showing [Ca2+]i oscillations in different cell cluster preparations. In A, after observing control [Ca2+]i oscillations in the presence of nifedipine (2 µM) (Aa), 10 µM 2APB was applied. The trace Ab was recorded 3 minutes after 10 µM 2APB application. Blue and red lines indicate changes in [Ca2+]i measured in two different regions at a distance of 50 µm. This distance is comparable to that for regions x and y shown in Fig. 1Ba1 and Fig. 2A1. In B, after observing a control in the presence of nifedipine (Ba), 10 µM xestospongin C was applied. The trace Bb was obtained 2 minutes after the application of xestospongin C.

View larger version (67K): [in a new window]   Fig. 4. Immunohistochemistry for c-Kit and ryanodine receptors. Smooth muscle (including the myenteric plexus) of mouse small intestine was double-labelled with anti-c-Kit antibody (A: ACK2 with Alexa 488, red) and anti-ryanodine receptor antibody (B: C34 with Alexa 594, green). C shows a merged image. Note that network-like cells were mostly stained in yellow, indicating that ICCs contain ryanodine receptors.

View larger version (53K): [in a new window]   Fig. 6. Spontaneous Ca2+ oscillations were detected in HEK293 cells transfected with RyR3. (Aa) The time-course of [Ca2+]i changes in HEK293 cell expressing RyR3 (black line) and a non-expressing cell (red line). Cells were sequentially treated with 1, 2, 5 and 10 mM caffeine and 1 µM ACh. (Ab) Ca2+ images in cells loaded with fura-2AM were obtained during resting conditions and in the presence of 1 mM caffeine or 1 µM ACh. The bottom-right panel in Ab indicates the immunofluorescence staining of RyR. It is noted that cells responding to 1 mM caffeine were stained with immunofluorescent RyR3 antibody. Bars in Ab, 20 µm. (B) The time courses of spontaneous [Ca2+]i oscillations (a) were obtained from cells (1 and 2) as indicated in a series of confocal images (b). The Ca2+ images were obtained every 3 seconds, and cells were loaded with fluo-4 for 15 minutes. (C) Confocal images following fluo-4 loading (a) and staining with BODIPY FL-X ryanodine (b) in HEK293 cells transfected with RyR3. (a) Cells were loaded with fluo-4. [Ca2+]i oscillations were then recorded in four indicated cells (1-4) among the 19 in this frame. (b) After recording [Ca2+]i oscillation, cells were stained with BODIPY FL-X ryanodine for 5 minutes and washed. Among the 19 cells in the frame, 12 cells, including all four cells exhibiting [Ca2+]i oscillations were well stained with BODIPY FL-X ryanodine. (c) The time courses of [Ca2+]i oscillations of four cells in the frame.

View larger version (36K): [in a new window]   Fig. 7. Pharmacological characterisation of spontaneous Ca2+ oscillations in HEK293 cells transfected with RyR3. In A-F, effects of the following drugs or treatments were examined: (A) 10 µM ryanodine; (B) 10-50 µM tetracaine; (C) 20 µM FK506 after transient application of caffeine; (D) 100 µM 2APB; (E) removal of external Ca2+; (F) application of 40 µM SK&F96365.

View larger version (39K): [in a new window]   Fig. 8. Images of spontaneous Ca2+ release in HEK293 cells expressing RyR3. (Aa) Sequence of seven confocal Ca2+ images of fluo-4 fluorescence in HEK293 cells expressing RyR3. The images in Aa were obtained at the time indicated by dashed vertical lines in Ab. (Ab) F and F0 are fluorescence intensity at a certain time and at resting conditions, respectively. F/F0 in circular spots (2 µm in diameter) at the point indicated by x, y and z, and that over the whole image of the part of the cell were measured and plotted against time. The black line indicates the change in F/F0 over the whole area of the cell. Bar in panel Aa, 10 µm. (B) Images of Ca2+ sparks (a) and F/F0 in the areas x, y and z in the same cell.

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