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First published online 9 September 2003
doi: 10.1242/jcs.00713

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Cyclic ADP-ribose increases Ca²⁺ removal in smooth muscle

Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK

View larger version (23K): [in a new window]   Fig. 1A. Depolarization- and InsP3-evoked [Ca2+]c increases in myocytes. (Aa) Depolarization to +10 mV (from –70 mV) (v) activated ICa (iv) and increased [Ca2+]c as represented by the intensity of colour changes in the frames (ii) and fluorescence traces (F/F0; iii). The cell [Ca2+]c images in the frames in (ii) were taken before (A), during (B,C) and after (D-F) the depolarization, and show the [Ca2+]c changes occurring throughout the cell. The precise times at which the images were obtained are indicated by their respective letters above the Ca2+ line traces in (ii). The fluorescence ratio (F/F0) changes plotted against time (iii) come from 1 pixel wide lines (536 nm) across the cell at 10 µm intervals. The positions of the lines are indicated in (i) (right-hand panel), although the lines drawn are wider than 1 pixel to facilitate visualization. A bright-field image of the cell and position of the patch pipette is shown (i, left-hand panel). (Ab) Similar parameters were measured in the same cell after photolysis of caged InsP3 () which also increased [Ca2+]c (ii,iii).

View larger version (20K): [in a new window]   Fig. 1B. Depolarization- and caffeine but not cADPR-evoked [Ca2+c] increases in myocytes. (Ba) Depolarization (iv) activated ICa (iii) and increased [Ca2+]c (i,ii). (i) Images of the [Ca2+]c changes occurring throughout the cell before (A), during (B,C) and after (D-F) the depolarization, the times indicated by their respective letters above the plot of [Ca2+]c against time in (ii). The changes in fluorescence ratio (F/F0) with time (ii) are again from 1-pixel-wide lines (536 nm) across the cell at 10µm intervals. The positions of the lines are indicated in (Bbi) right-hand panel; the lines drawn are wider than 1 pixel to facilitate visualization. A bright field image of the cell and position of the patch pipette is shown (Bbi, left-hand panel). (Bb) cADPR ( 50 µM) did not increase [Ca2+]c. (Bc) caffeine (CAF 10 mM, v) remained effective in this respect. The delay in onset of the caffeine response probably reflects the time required for the concentration of caffeine to build up between the puffer pipette and the cell.

View larger version (8K): [in a new window]   Fig. 2. cADPR increased [Ca2+]c in sea urchin eggs. cADPR (; 1 mM in the injection solution, giving a concentration in the cell of 22 µM) released Ca2+ in sea urchin eggs. The flash artefact was removed for clarity.

View larger version (38K): [in a new window]   Fig. 3. Depolarisation but not cADPR contracted single colonic myocytes. Photolysed cADPR ( 50 µM) failed to increase F/F0 (C) or to contract colonic myocytes [P>0.05 (Di)]. Depolarisation to +10 mV [holding potential –70 mV (A)] induced an inward ICa (B and expanded timescale inset) and a Ca2+ transient (C). Following the increase in [Ca2+]c (F/F0), (Dii) shows the mean changes in cell length produced by depolarization (P<0.05) and (Di) the failure of cADPR to induce contraction (n=10-18, P>0.05). Alignment of time with the videotaped contractile behaviour of the cell (E) was achieved by cell illumination with a flash lamp (Ei) or an LED triggered by depolarisation (Eii).

View larger version (12K): [in a new window]   Fig. 4. cADPR did not increase the amplitude of depolarization-evoked Ca2+ transients. Depolarization (–70 to 0 mV, Aii,Bii,Cii) evoked Ca2+ transients; their amplitudes (expressed in F/F0 units) were not significantly affected by cADPR (; 50 µM, Ai, Bi, Ci) at various time intervals (A, 2 seconds; B, 5 seconds; C, 10 seconds) beforehand; that is, cADPR did not cause CICR following Ca2+ influx.

View larger version (25K): [in a new window]   Fig. 5. Caffeine increased the amplitude of depolarization-evoked Ca2+ transients. Caffeine (500 µM in the bathing solution) reversibly increased the amplitude of depolarization-evoked [–70 mV to 10 mV (B)] Ca2+ transients (A). The amount of Ca2+ entering the cell by depolarization (i.e. the `calculated' increase in [Ca2+]c) (red line) under control conditions (Di), with caffeine (Dii) and after caffeine washout (Diii) was compared with the `measured' increase in [Ca2+]c (blue line) as determined from the Ca2+ transient in the same cell. The time courses of the `measured' and `calculated' Ca2+ increases (see Materials and Methods) were similar. There was a greater increase in the `measured' Ca2+ value for a similar `calculated' Ca2+ value in caffeine than in its absence (control). Caffeine removal partially restored the relationship between the `calculated' and `measured' increases in [Ca2+]c. A-D are components of the same experiment. These results show that these concentrations of caffeine cause CICR after depolarization-evoked ICa.

View larger version (21K): [in a new window]   Fig. 6. cADPR did not affect the amplitude of submaximal caffeine responses. cADPR (; 50 µM) photolysed 1 second beforehand did not significantly alter the amplitude of submaximal responses to caffeine [third and subsequent responses (B)]. In summarized results (A; n=10), asterisks indicate a significant difference between maximal (first two from left) and submaximal responses to caffeine. *P<0.01.

View larger version (24K): [in a new window]   Fig. 7. cADPR failed to modulate STOCs. cADPR (; 50 µM) had no significant effect on the amplitude or frequency of STOCs (ii and expanded time base i) evoked by membrane depolarization from –70 mV to –20 mV (iv) or on [Ca2+]c (iii). By contrast, caffeine (10 mM, v) increased [Ca2+]c and evoked a transient outward current after which STOCs were suppressed, presumably as a result of store depletion (iii). There was no change in the probability of a STOC occurring (PSTOC; vi) before (100 seconds) and after (150 seconds) cADPR (vi; n=7; P>0.05).

View larger version (42K): [in a new window]   Fig. 8. FKBP12 and FKBP12.6 detection in whole brain and colon and the effects of recombinant FKPB12.6 on Ca2+ release and cADPR. (A) immunoblots were probed with SA168 antiserum (Ai) and C-19 polyclonal antibody (Aii), respectively. Lanes 1 and 2 were loaded with recombinant FKBP12 and FKBP12.6, respectively (200 ng each), lanes 3-5 with 25, 50 and 75 µg, respectively, total brain homogenate, and lanes 6-8 with the same amounts of total homogenate from colon. FKBP12 and/or FKBP12.6 were detected in brain and colon by SA168 as shown by bands at the 12 kDa level. The detection of bands at the 12 kDa level by C-19 suggests that only FKBP12 is present in myocytes (n=3). (B) Recombinant FKBP12.6 (1 ng ml–1 via the patch pipette) significantly reduced the magnitude of STOCs (i) evoked by membrane depolarization (iii, –70 mV to –30 mV, P<0.05) but cADPR () remained ineffective in altering [Ca2+]c or STOCs. (C) There was no significant change in STOC frequency or amplitude over a comparable timescale in separate time-matched control experiments in the absence of FKBP12.6 (Ci expanded time base, ii). (D) Lanes 1-3, shows 100 µg loads of total brain homogenate, lane 4 shows recombinant FKBP12 (200 ng) and lane 5 shows FKBP12.6 (200 ng; Di). Brain homogenate was treated with either FK506 (20 µM; lane 2) or cADPR (50 µM; lane 3) and subjected to electrophoresis. The results are representative of three independent experiments. Developed immunoblots were subjected to densitometric analysis (optical density, O.D.) and results normalized to the standard amount of recombinant FKBP12 loaded on each gel. (ii) Summary of experiments, control and FK506 (n=4) cADPR (n=3; *P<0.05).

View larger version (17K): [in a new window]   Fig. 9. cADPR increased the rate of Ca2+ removal following depolarization and inferred plasma membrane Ca2+ pump activity. (A) Dialysing the cell with free cADPR (300 µM; Ab,c) increased the rate of [Ca2+]c decline following depolarization (–70 mV to +10 mV) compared with controls [Aa,c; n=40 (control), n=17 (free cADPR)]. (B) The effects of cADPR on the sarcolemma Ca2+ pump. The sarcolemma Ca2+ pump activity was obtained by inhibiting removal occurring via mitochondria, SR and Na+-Ca2+ exchange. By subtracting from the rates of decline in cADPR, the rates in control (minus those occurring when removal by mitochondria, SR and Na+-Ca2+ exchange were inhibited), an inferred Ca2+ pump rate was obtained. This was increased by cADPR (*P<0.05; **P<0.01).

View larger version (16K): [in a new window]   Fig. 10. The rates of Ca2+ removal following depolarization- and InsP3-induced increases in [Ca2+]c were reduced by 8-bromo cADPR. (A) The membrane permeable cADPR antagonist, 8-bromo cADPR (20 µM, added to the bath), significantly slowed the rate of Ca2+ removal following the increases in [Ca2+]c (ai and bi) produced either by InsP3 (, ai,iii; n=6) or depolarization (–70 mV to +10 mV, bi,iii; n=6). (B) Inhibition of Ca2+ removal mechanisms [ii; mitochondria, SR Ca2+ pump and Na+-Ca2+ exchange using CCCP and oligomycin (OL) Na+-free bathing solution (NaF) and thapsigargin (TG) together] significantly slowed the rate of decline following depolarization-evoked (–60 mV to +10 mV) [Ca2+]c increases. Thereafter, 8-bromo-cADPR was added (iii) in the continued presence of the removal inhibitors; this increased the baseline [Ca2+]c and slowed further the rate of [Ca2+]c decline (notice the increased time base on iii) (summarised in Bb) (*P<0.05; n=5-7 for each data point).