Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of two spectrally well-defined fluorescent molecules in a small detection volume at the single-molecule level. To expand the capability of FCCS measurement, we constructed a chimera that consisted of tandem fused fluorescent proteins with three colors for the simultaneous evaluation of the caspase-3 and caspase-9 activities. Using FCCS, we found the apoptosis-induced caspase-3 and caspase-9 activation to be detectable by decreases in the cross-correlation amplitudes both in vitro and in living cells. Here we report the simultaneous detection of caspase-3 and caspase-9 activation in living cells.
Myocardial intracellular Ca2+ and membrane potential oscillations were studied in isolated guinea pig and rat pulmonary veins via immunohistochemical, confocal microscopic and electrophysiological analyses. The myocardial layer was present between the smooth muscle layer and the adventitia, and was more developed in the guinea pig than in the rat. Intracellular Ca2+ oscillations were observed in both species, which were inhibited by ryanodine. Spontaneous Ca2+ waves were observed to propagate along the longitudinal axis of the cell or as a spiral rotating around a subcellular core; the propagation velocity of these Ca2+ waves was similar to that reported in atrial and ventricular cardiomyocytes. Spontaneous action potentials were present in about 35% and 4% of the preparations from the guinea pig and rat, respectively. In quiescent preparations from the guinea pig, noradrenaline induced a slow depolarization of the resting membrane potential, followed by constant repetitive generation of action potentials, which were inhibited by ryanodine. In quiescent preparations from the rat, noradrenaline induced an initial hyperpolarization and a subsequent depolarization of the resting membrane potential. This was followed by generation of automatic action potentials which occurred in repetitive bursts. Ryanodine either completely abolished or reduced the duration of action-potential bursts. These results indicate that the pulmonary vein myocardium generates automatic electrical activity under adrenergic influence, which is probably triggered by intracellular Ca2+ oscillations. The differences in firing patterns between the guinea pig and rat may be due to differences in the hyperpolarizing mechanisms.