Influence of cholesterol on catecholamine release from the fusion pore of large dense core chromaffin granules.

Changes in cellular cholesterol can affect exocytosis, but the influence of cholesterol in fusion pore kinetics is unclear. Using carbon fiber amperometry, we monitored quantal catecholamine release from rat chromaffin cells. To bypass any possible effect of cholesterol perturbation on ion channels or the colocalization of voltage-gated Ca(2+) channels with sites of exocytosis, exocytosis was stimulated via uniform elevation of cytosolic [Ca(2+)] (with whole-cell dialysis of a Ca(2+)-buffered solution). Under this condition, alterations of cellular cholesterol affected neither the mean number of amperometric events triggered per cell nor their quantal size and the kinetics of their main spike (which reflects the rapid release during and after rapid fusion pore dilation). In contrast, the reduction of cellular cholesterol shortened the "prespike foot" signals (which reflect the leakage of catecholamine via a semi-stable fusion pore) and reduced the proportion of "stand-alone foot" signals (which reflect the release via a flickering fusion pore that may close before it dilates significantly), whereas an oversupply of cholesterol had opposite effects. Acute extraction of cholesterol from the cytosol (via whole-cell dialysis of a cholesterol extractor) also shortened the prespike foot signals and reduced the proportion of stand-alone foot signals, but acute extracellular application of cholesterol extractor or "soluble" cholesterol had no effect. Our data raise the possibility that cholesterol molecules, particularly those in the cytoplasmic leaflet, helps to constrain the narrow waistline of a semi-stable fusion pore while it is flickering or before it starts to dilate rapidly.

Differential regulation of multiple populations of granules in rat adrenal chromaffin cells by culture duration and cyclic AMP.

We employed carbon fiber amperometry to measure the amount of catecholamine released from individual granules (i.e. the quantal size, Q) of rat chromaffin cells. The distribution of Q1/3 of amperometric events could be reasonably described by the summation of at least three Gaussians, suggesting that rat chromaffin cells contained at least three distinct populations of granules, with a small, medium or large modal Q. After 3 days of culture, the mean cellular Q reduced by approximately 14%, which did not arise from a uniform percentage decrease in the Q of every granule. Instead, the rundown involved a > 11% decrease in the proportional release from large Q granules and a > 19% decrease in the modal Q-value of small Q granules. In contrast, when cells were cultured with dibutyryl-cAMP (dBcAMP) for 3 days, their mean cellular Q increased by approximately 38% (relative to time-matched controls). This increase in Q was not associated with any shift in the proportional release from the three populations of granules. Instead, cAMP increased the average amount of catecholamines released from all three populations of granules. Our data raise the possibility that distinct populations of granules in rat chromaffin cells can be regulated either differentially or uniformly.