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

Abstract

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 approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 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 approximately 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) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.

pH-dependent Intraluminal Organization of Mucin Granules in Live Human Mucous/Goblet Cells

“…6). These results are consistent with the existence of an intragranular condensed mucin matrix and its subsequent expansion upon exocytosis, as proposed in the ion exchangetriggered phase transition model of granule exocytosis (29,30,68,69).…”

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

“…Together, these data indicate that the absence of CgA markedly reduces the ability of the vesicular matrix to bind and retain CA, resulting in a shortening of the period for each releasing event. The results obtained also provide direct evidences on the implication of the vesicle matrix in the control of the kinetics of exocytosis (Schroeder et al, 1996;Marszalek et al, 1997;Borges et al, 2000).…”

Mucin Granule Intraluminal Organization

“…Matrix swelling had three major features: (i ) it was massive (i.e., the matrix increased its volume many times); (ii) it had a kinetic similar to the swelling of condensed artificial polymer gels, including a high Hill coefficient; and (iii) it could be arrested to some extent by increasing the extracellular concentrations of Ca 2ϩ and H ϩ , suggesting that these two cations were critical for the mechanism of mucin intragranular condensation. Indeed, it has been shown that the mucin granule lumen is rich in Ca 2ϩ and H ϩ (37,38) In view of these properties, and similar observations in other regulated secretory cell systems (39)(40)(41) 37). Disruption of Ca 2ϩ shielding would result in a rapid increase of repulsive forces among mucin sialic and sulfate groups, and eventually the decondensation and hydration of the granule matrix (14,37).…”