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First published online 8 November 2005
doi: 10.1242/jcs.02657

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In smooth muscle, FK506-binding protein modulates IP₃ receptor-evoked Ca²⁺ release by mTOR and calcineurin

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

View larger version (16K): [in a new window]   Fig. 1. Co-immunoprecipitation of IP3R and FKBP12 and the presence of mTOR in colonic smooth muscle. IP3R1 (the major isoform present; data not shown) was immunoprecipitated from solubilised colonic smooth muscle. (A,B) Immunoblots were probed with rabbit anti-IP3R1 and rabbit anti-FKBP12 antibodies for the presence of (A) IP3R1 and (B) FKBP12, respectively. Lane 1 in each panel shows the immunoprecipitated protein from colon. Lane 2 shows the relevant positive control (10 µg solubilised supernatant for IP3R or 50 ng recombinant FKBP12). Arrows on the right indicate the position of molecular-mass markers run in parallel to indicate protein migration on the gel. The detection of a band at 12 kDa (B) indicates that FKBP12 is present and associated with IP3R1 in these myocytes. These data are representative of four experiments. (C) FKBP12-IP3R1 association in colon is disrupted by FK506 and rapamycin. IP3R1 was immunoprecipitated from solubilised colon. Immunoprecipitates were probed for the presence of IP3R1 (C) and FKBP12 (D). Lanes 1-4 in each panel are the immunoprecipitated protein from colon, lane 5 is antibody alone (negative control), lane 6 is solubilised colon protein plus IgG sepharose without antibody (negative control) and lane 7 the relevant positive control [solubilised supernatant (C) and recombinant FKBP12 (D)]. The detection of a band in untreated control preparations at 12 kDa (D, lanes 3 and 4) indicates that FKBP12 is present and associated with IP3R1 (n=2). This FKBP12-IP3R1 association was disrupted by the addition of FK506 (D, lane 1, 20 µM) and rapamycin (D, lane 2, 20 µM), each of which reduced the FKBP12 signal. Arrows indicate the position of molecular-mass markers run in parallel to indicate protein migration on the gel. (E) mTOR is present in colonic myocytes. Immunoblots were probed with the anti-mTOR antibody (lane 1). Lane 2 shows the molecular-mass marker to show protein migration on the gel. (F, G) Co-immunoprecipitation of IP3R1 and calcineurin B from solubilised colonic smooth muscle. Immunoprecipitations were performed as above using rabbit anti-IP3R1 antibody, and immunoblots probed for the presence of IP3R1 (F) and calcineurin B (G). Lane 1 in each panel is the immunoprecipitated protein from colon, lane 2 is solubilised preparation plus protein G without antibody, lane 3 antibody alone, lane 4 the positive control (10 µg solubilised supernatant in each case). Arrows indicate the position of molecular-mass markers run in parallel. The detection of a band at 19 kDa indicates that calcineurin B is present and associated with IP3R1. These data are representative of six experiments. (H, I) Co-immunoprecipitation of IP3R1 and calcineurin A from solubilised colonic smooth muscle. Immunoprecipitations were performed as above using rabbit anti-IP3R1 antibody and immunoblots were probed for the presence of IP3R1 (H) and calcineurin A (I). Lane 1 in each panel is the immunoprecipitated protein from colon, lane 2 the solubilised preparation plus protein G without antibody, lane 3 the antibody alone and lane 4 the positive control (10 µg solubilised supernatant in each case). Arrows on the right indicate the position of molecular-mass markers run in parallel. The detection of a band at 55 kDa indicates that calcineurin A is present and associated with IP3R1. This data is representative of two experiments.

View larger version (14K): [in a new window]   Fig. 2. Effects of rapamycin on IP3- and caffeine-evoked Ca2+ increases in voltage-clamped single colonic myocytes. (A) Photolysed caged IP3 () increased [Ca2+]c as indicated by F/F0. Rapamycin (10 µM, n=7) significantly reduced (P<0.05) the IP3-evoked [Ca2+]c transients. (B) Rapamycin (10 µM, n=10, P<0.01) significantly increased the Ca2+ rise evoked by caffeine (CAF, 10 mM) following activation of RyR.

View larger version (13K): [in a new window]   Fig. 3. Effects of the mTOR inhibitors RAD001 and LY294002 on IP3-evoked Ca2+ increases in voltage-clamped single colonic myocytes. The mTOR inhibitors each inhibited IP3-evoked Ca2+ increases. Photolysed caged IP3 () increased [Ca2+]c as indicated by F/F0. The IP3-evoked [Ca2+]c transient was significantly decreased by each inhibitor. (A) RAD001, 10 µM, n=8, P<0.05. (B) LY294002, 20 µM, n=8, P<0.05).

View larger version (14K): [in a new window]   Fig. 4. Effects of FK506 on IP3-evoked Ca2+ increases in voltage-clamped single colonic myocytes. Photolysed caged IP3 () significantly increased [Ca2+]c as indicated by F/F0. FK506 (10 µM, n=11, P<0.05) potentiated the IP3-evoked [Ca2+]c transients.

View larger version (15K): [in a new window]   Fig. 5. Effects of the calcineurin inhibitors cypermethrin and okadaic acid on IP3-evoked Ca2+ increases in voltage-clamped single colonic myocytes. Photolysed caged IP3 () increased [Ca2+]c as indicated by F/F0. The IP3-evoked [Ca2+]c transient was significantly increased by each inhibitor. (A) Cypermethrin 10 µM, n=12, P<0.001; (B) okadaic acid 5 µM, n=8, P<0.05.

View larger version (26K): [in a new window]   Fig. 6. Effect of FK506 on IP3-evoked Ca2+ increases following calcineurin inhibition in voltage-clamped single colonic myocytes. Following pre-treatment with the calcineurin inhibitors (A) CiP (100 µM), (B) okadaic acid (5 µM) or (C) cypermethrin (10 µM), FK506 (10 µM) did not increase the IP3-evoked [Ca2+]c transient produced by photolysed caged IP3 () as it had done in the absence of inhibitors (cf. Fig. 4).

View larger version (27K): [in a new window]   Fig. 7. IP3R and FKBP12 do not co-immunoprecipitate in aortic smooth muscle and neither rapamycin nor FK506 alter IP3-evoked Ca2+ increases in voltage-clamped single aortic myocytes. (A) Similar amounts of FKBP12 protein are expressed in aortic and colonic smooth muscle. Colon and aorta were each hand-homogenised as described in Materials and Methods, and solubilised supernatants from each homogenate was assayed for FKBP12 protein expression. Proteins (10 µg total) from each tissue were separated using SDS-PAGE and immunoblots were probed for the presence of FKBP12 (lanes 1 and 4). Migration and signal intensity were compared with those obtained from recombinant FKBP12 (50 ng) run alongside as positive controls (lanes 2 and 3). The blot is representative of seven and four identical experiments, in colonic and aortic smooth muscles, respectively. (B,C) IP3R1 was immunoprecipitated from solubilised aortic smooth muscle. Immunoblots were probed with rabbit anti-IP3R1 and rabbit anti-FKBP12 antibodies for the presence of (B) IP3R1 and (C) FKBP12, respectively. The first lane in each panel shows the immunoprecipitated protein from aorta, lane 2 the solubilzed preparation plus protein G without antibody, lane 3 the antibody alone and lane 4 the relevant positive control (10 µg solubilised supernatant for IP3R or 50 ng recombinant FKBP12). Arrows on the right indicate the position of molecular mass markers run in parallel to indicate protein migration on the gel. The absence of a band at the 12 kDa level (C) indicates that FKBP12 is not associated with IP3R1 in these myocytes. These data are representative of three experiments. (D,E) Photolysed caged IP3 () increased [Ca2+]c as indicated by F/F0. Neither (D) rapamycin (10 µM, n=3) nor (E) FK506 (10 µM; n=6) significantly altered the IP3-evoked [Ca2+]c transients (P>0.05).