Modulation of exocytosis by estrogen, altered membrane composition, and osmolarity

This thesis is concerned with exocytotic plasticity.; Constant potential amperometry is one method to monitor exocytosis from single cells in culture with sub millisecond time resolution. By placing a carbon fiber against the surface of an excitable cell, electroactive neuromotransmitters released by the cell (such as dopamine) can be quantitatively oxidized to produce a small current that is proportional to the amount secreted.; One such manipulation, a collaboration with Yoshiko Niimura, demonstrates the effects of increasing various types of phospholipids on exocytosis. When cells are incubated with phosphatidylcholine (PC), phosphatidylethanolamine (PE), or sphingomyelin (SM), changes in the quantal size and/or vesicle fusion kinetics are observed. PC slowed and PE accelerated the expulsion of neurotransmitter from vesicles. Phosphatidylserine (PS) increased the number of stimulated amperometric peaks. Electron micrographs of PS-treated cells indicated a larger fraction of vesicles "docked" to the plasma membrane might underlie the enhanced rate of secretion. Together these data suggest that differences in membrane composition affect exocytosis and might be involved in cell function related to plasticity.; Plasma membrane tension also appears to affect exocytosis. In collaboration with Leslie Sombers, we examined how reducing tension in the plasma membrane by incubating PC12 cells in hypertonic saline caused the vesicle-to-plasma membrane fusion pore to stabilize, yielding amperometric peaks with a longer and larger foot portion. This foot amplification was more pronounced when cells were loaded with the dopamine precursor, L-(3,4)-dihydroxyphenylalanine (L-DOPA), a treatment that caused vesicular volume and vesicular membrane tension to increase. These data lend support to the hypothesis that differences in membrane tension between the vesicle and plasma membrane induce a more stable fusion pore conformation.; Electron microscopic measurements of L-DOPA-treated cells revealed the halo of large dense core vesicles (LDCVs) contained most of the newly loaded DA. Likewise, when exocytosis was measured following treatment in hyperosmotic solutions, most of the loaded DA that leaked through the stabilized fusion pore appeared to derive from the halo.; A third, unexpected effect of combining L-DOPA incubation with hyperosmotic saline was to convert a substantial portion (15.3%) of LDCVs in PC12 cells into multicored vesicles. This phenomenon provides evidence that exchange of membranes between LDCVs is possible under some conditions, and this lipid exchange might be involved in regulating vesicle size and composition.; Estrogen has been identified in the brain at higher concentrations than even the amounts found in reproductive organs. In addition, several estrogen receptors are expressed in brain areas such as the hypothalamus (controls the reproductive system) and the hippocampus (involved in learning and memory) and it seems likely that estrogen acts as both a hormone in the body and a neuromodulator in the brain. Using PC12 and GT1-7 cell cultures to model what might happen when neurons are exposed to estrogen, it appears that estrogen (specifically 17-beta-estradiol) can suppress exocytosis by blocking voltage-gated calcium ion channels, inhibiting calcium release from ryanodine-receptor-regulated intracellular calcium stores, and even promote exocytosis by opening voltage-gated calcium ion channels. The timing of the exposure to estrogen and subsequent observation period seems to be important in determining whether the effect of estrogen appears as an increase or decrease exocytosis. Estrogen consistently prevents other stimuli from promoting exocytosis, but at the same time causes exocytosis during and shortly after it is applied to cells. While this dual action complicates interpretation of the results, it has been confirmed using changes in cytosolic calcium as an alternate measurement. (Abstract shortened by UMI.)...