Modulation of vesicular catecholamine release from rat PC12 cells

Abstract

Intercellular communication is of vital importance for the nervous system, since the nervous system is the main coordinating system in animals. Nerve cell communication is initiated by the release of chemical messengers, neurotransmitters, from the presynaptic nerve cell. The neurotransmitters, such as catecholamines like dopamine, are stored in specialized vesicles. These vesicles are emptied in the synaptic cleft upon stimulation, a process called exocytosis. The nature and strength of the presynaptic signal is determined by the type of neurotransmitter released, the number of vesicles that fused and the amount of neurotransmitter released per vesicle. The development of new and more sensitive techniques to measure neurotransmitter release, in combination with recent developments in genetics and biochemistry, increased our knowledge on the presynaptic mechanisms of neurotransmission. Although the central nervous system is a main target for many toxic substances and drugs, little is known about the neurotoxicology of the presynaptic response. The research described in this thesis was conducted to gain insight into the mechanisms of modulation of neurotransmitter release by endogenous factors and toxic substances. The model system used in the studies described in this thesis was the rat phaeochromocytoma PC12 cell, which is commonly used as an in vitro model for both neurosecretory and neuronal cells. The results described in Chapter 2 provide a detailed characterization of this model system. Vesicular catecholamine release has been measured using carbon fiber microelectrode amperometry, which allows for the detection of exocytotic events at millisecond resolution and with a detection threshold of ~15 zeptomole (zepto = 10-21), or ~9000 dopamine molecules. Vesicular catecholamine release from PC12 cells is limited because of a slow rate of vesicle cycling. In addition, exocytosis is also limited by Ca2+ channel inactivation. Furthermore, it was shown that catecholamines are released from a heterogeneous population of vesicles. To overcome these limitations PC12 cells were differentiated with dexamethasone into a more chromaffin-like cell type. The differentiated PC12 cells were used to study the effects of the heavy metal Pb2+, the neurotoxic organic solvent toluene and three types of PCBs. The effect of Pb2+ on exocytosis is described in Chapter 3 and Chapter 4. Using PC12 cells permeabilized by ionomycin, it was shown that Pb2+ induces vesicular catecholamine release, with a much higher potency than Ca2+, through a direct interaction with the exocytotic machinery. In addition, Pb2+ exerts its effect on the exocytotic machinery even in the absence of Ca2+. The results obtained with amperometry were combined with Ca2+- and Pb2+-imaging experiments to determine the intracellular concentrations of Ca2+ and heavy metal ions simultaneously using the fluorescent dye Indo-1 in confocal laser scanning microscopy. It was demonstrated that PC12 cells contain a considerable Pb2+ buffering capacity. Partial saturation of this high-affinity buffer with Pb2+ is sufficient to evoke exocytosis. In addition, it was shown that modulation of PKC, CaM kinase II, calcineurin, calmodulin and synaptotagmin affects vesicular neurotransmitter release. It appeared that the frequency of basal vesicular catecholamine release is mainly modulated by the activity of PKC and calcineurin. Although PKC is a highly sensitive target for Pb2+, Pb2+-induced vesicular release is mainly modulated by CaM kinase II, which thus provides a novel and plausible target for the direct intracellular actions of Pb2+ leading to vesicular catecholamine release. In Chapter 5 the effect of the neurotoxic organic solvent toluene on vesicular catecholamine release are described. Toluene concentration-dependently increases the basal frequency of exocytosis, due to an increase in intracellular Ca2+ concentration. The increase in basal exocytosis, which occurs at neurotoxicological relevant concentration, is caused by enhanced influx of extracellular Ca2+ through high voltage-activated Ca2+ channels and is not due to a direct effect of toluene on the exocytotic machinery. The results of Chapter 6 demonstrate that acute (<15 min) exposure to PCB 4 and PCB 126 enhances the basal frequency of vesicular neurotransmitter release, whereas the amount of neurotransmitter secreted per vesicle is unaffected. PCB 128 does neither affect the release frequency, nor the amount of neurotransmitter secreted per vesicle. Subchronic (3 days) exposure to PCB 4, PCB 126 or PCB 128 did not affect any of the parameters investigated. The effects of PCBs on neurotransmitter release previously found using cell populations cannot be explained by the slight enhancement of basal release. In addition, the effects on basal release frequency are observed at PCB concentrations apparently ineffective in changing dopamine transport or tyrosine hydroxylase activity. The dexamethasone-differentiated PC12 cells provide a suitable model system to distinguish distinct mechanisms leading to modulation of neurotransmitter release. PCBs, as well as toluene and Pb2+, have the potential to increase the frequency of basal catecholamine release. Although elevated levels of dopamine and some other neurotransmitters, like glutamate, are generally believed to affect the development, maintenance, and survival of neurons, it remains difficult to predict functional consequences of elevated levels of these neurotransmitters in vivo. Therefore, the ultimate challenge might just be to translate the observed in vitro effects on quantal neurotransmitter release into functional consequences for neurotransmission in general and for nervous system function in vivo.