Kinetics of release of serotonin from isolated secretory granules .1. Amperometric detection of serotonin from electroporated granules

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance similar to 100 mu m) and positioned adjacent (<1 mu m) to a carbon fiber microelectrode. A short (similar to 30 mu s) high-intensity voltage pulse (electric field of similar to 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with similar to 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) congruent to 21r(2)) and reflects the time required for membrane rupture. The average t(1/2)(spike) of the amperometric spikes was found to be congruent to 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.