Abstract
the calcium-sensitive fluorescent probe chlorotetracycline was used to monitor calcium movement in human platelets. The chlorotetracycline fluorescence signal is a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. Experiments perturbing the system with the calcium ionophore A23187 shows that the level of free internal calcium in the organelle depends upon the cytoplasmic level, which, in turn, depends upon the passive permeability of the plasma membrane. Chlorotetracycline in the cytoplasmic compartment does not respond to changes in the cytoplasmic calcium concentration, which is held in the micromolar to submicromolar range by an extrusion system. The calcium concentration in the cytoplasmic compartment can be directly manipulated by the calcium ionophore A23187 and is measured in parallel experiments with Quin 2, a high-affinity indicator. The calcium transport systems of the organelles are shown to be less susceptible to short circuit by A23187. Analysis shows that mitochondrial uptake is slow (ti/2 = 20 minutes), produces a large increase in chlorotetracycline fluorescence, and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid (t1/2 = 2 minutes), produces a smaller increase in chlorotetracycline fluorescence, is inhibited by trifluoperazine, and is less sensitive to A23187. The Km is estimated as 1 /*m or lower. Studies show that the chlorotetracycline technique is useful for the monitoring of calcium uptake and release by the platelet organelles, and suggests that the Quin-2/chlorotetracycline technique will be useful as a diagnostic of both physiological and pathological activation mechanisms.
