Localized Ca2+ uncaging induces Ca2+ release through IP3R in smooth muscle
Abstract
Aim:Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle.
Methods:Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm×0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 μmol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm.
Results:Local uncaging of caged Ca2+was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments.
Conclusion:(1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.
Keywords:
Methods:Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm×0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 μmol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm.
Results:Local uncaging of caged Ca2+was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments.
Conclusion:(1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.